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https://f1000research.com/articles/6-2045/v1
|
23 Nov 17
|
{
"type": "Research Article",
"title": "Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour",
"authors": [
"Jashdeep Bhattacharjee",
"Barun Das",
"Alaknanda Mishra",
"Preeti Sahay",
"Pramod Upadhyay",
"Jashdeep Bhattacharjee",
"Barun Das",
"Alaknanda Mishra",
"Preeti Sahay"
],
"abstract": "Background: Magnetic sorting of cells, based on microbead conjugated antibodies (Abs), employs positive as well as negative immunomagnetic separation methods, for isolation of a specific cell population. These microbeads are suggested to be nontoxic, biodegradable carriers conjugated to various antibodies. Isolation of cells through positive selection involves the attachment of antibody conjugated microbeads to the cells of interest, followed by their isolation in the presence of a strong magnetic field to obtain higher purity. Negative selection involves attachment of microbead conjugated antibodies to all other cell populations except the cells of interest, which remain untagged. In the present study, we compared the two methods for their effect on functional and immunophenotypic behavior of isolated CD14+ monocytes. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from healthy volunteers by density gradient centrifugation. Human blood derived monocytes were isolated through positive selection and negative selection, making use of the appropriate monocyte isolation kit. Monocytes were then stimulated with lipopolysaccharide (LPS) and their activation and proliferation capacity were examined. The degradation or dissociation of cell-bound microbeads was also investigated. Results: We observed an impaired LPS sensitivity as well as poor activation and proliferation capacity upon stimulation by LPS in positively sorted CD14+ monocytes as compared to negatively sorted CD14+ monocytes. The attached microbeads did not degrade and remained attached to the cells even after 6 days of culture. Conclusions: Our results suggest that positively sorted CD14+ cells exhibit hampered functionality and may result in inaccurate analysis and observations in downstream applications. However, these cells can be used for immediate analytical procedures.",
"keywords": [
"immune-magnetic cell sorting",
"lipopolysaccharide sensitivity",
"CD14+ve monocytes"
],
"content": "Introduction\n\nMagnetic sorting is a common technique used to obtain a highly pure population of cells of interest from a mixed population of cells, making use of microbead conjugated antibodies against the cell surface antigen. Positive sorting involves the tagging of cells with magnetic microbead conjugated antibodies, followed by isolation of the labeled cells by placing them in a magnetic field. After positive sorting, cells that have microbead conjugated antibodies on their surface can be conveniently analyzed using flow cytometry (Füchslin et al., 2010; Miltenyi et al., 1990; Pei et al., 1998). Negative sorting involves the labeling of all cells, except the cells of interest, by incubating them in a cocktail of magnetic microbead conjugated antibodies and subsequently removing them by placing them in a magnetic field.\n\nCluster of differentiation 14 (CD14) are specific markers used to identify monocytic populations, and they act as a coreceptors for LPS (Guha & Mackman, 2001). It has been suggested that positive magnetic sorting of CD14 does not trigger any signal transduction pathways or alter its functionality, since CD14 lacks a cytoplasmatic domain and such cells are reported to function in a restricted manner (Tomlinson et al., 2013). Observations made with microbead labeled cells during in vitro (Füchslin et al., 2010; Horgan et al., 2009; Semple et al., 1993) and in vivo experiments (Ribaut et al., 2008; Safarík & Safaríková, 1999) suggested that cells post microbead attachment do not reflect the true picture and undergo altered behaviour.\n\nWe investigated the immunophenotypic behaviour and molecular characteristics of monocytes after both positive and negative sorting, by analyzing their response and proliferation to stimuli like LPS. The biodegradation profile of the attached microbeads from the CD14+ cells was also investigated.\n\n\nMethods\n\nThe investigation was approved (project serial number: IHEC/#52/10) by the Institutional human ethics committee of the National Institute of Immunology, New Delhi-67, India.\n\nExperiments were performed at the National Institute of Immunology, New Delhi. 20 ml of peripheral blood was collected from five healthy volunteers aged between 25–30 years, after obtaining their written informed consent. Blood was collected more than once from some of the volunteers and there was a minimum gap of three months between two successive sample collections. The peripheral blood mononuclear cells (PBMCs) were isolated from blood by density gradient centrifugation using HiSepTM LSM 1077 (Himedia, Mumbai; India). The obtained PBMCs were washed thrice with Dulbecco’s phosphate buffered saline (Himedia, Mumbai; India) and counted using the trypan blue dye exclusion method with a hemocytometer (Rohem Industries Pvt Ltd, India).\n\nHuman blood derived monocytes were sorted using anti-human CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), as per manufacturer’s protocol. Similarly, monocytes were isolated by negative sorting using the monocyte isolation Kit II (Miltenyi Biotec, Bergisch Gladbach; Germany) according to manufacturer’s protocol.\n\nThe positively sorted CD14 positive cells were re-suspended in RPMI 1640 medium (Himedia, Mumbai; India) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Beit-Haemek Israel) and 1X antibiotic-antimycotic solution containing streptomycin sulphate, penicillin and amphotericin-B (Himedia, Mumbai; India), and plated at a density of 4 × 106 cells per well in 6 well low adherence plates (Corning, Tewksbury; USA). The cells were periodically harvested by gentle scrapping and passed through a magnetic column. Cells with and without bound microbeads (obtained in the flowthrough) were counted using a hemocytometer (Rohem Industries Pvt Ltd, India).\n\nMonocytes separated either by positive or negative selections were re-suspended in RPMI 1640 media supplemented with 10% FBS and 1X antibiotic-antimycotic solution. 1 million cells/ well were plated in a 24 well cell culture plate (Corning, Tewksbury, USA) and placed in a humidified CO2 incubator (ShelLab, Cornelius; USA) at 5% CO2/ 37°C for 24 hours. The cells were examined for adherence and thereafter stimulated with 1 ml complete RPMI media containing 500ng/ml of LPS (Sigma, St. Louis; USA). Fresh media containing 500ng/ml of LPS was replaced at each time point (8h, 16h, 24h) for supernatant collection.\n\nThe supernatants collected at various time points were analysed for the presence of pro- and anti-inflammatory cytokines; IL-8, IL-10, TGF-β1 and RANTES, using cytometric bead array (CBA) (BD Biosciences, San Jose; USA) as per manufacturer’s protocol. The data was recorded using BD FACSVerse (BD Biosciences, San Jose; USA) and was analysed using FCAP Array software v3.0 (BD Biosciences, San Jose; USA). The assay samples were appropriately diluted to match the detection range of the CBA kit.\n\nThe magnetic sorted monocytes were plated at a density of 1 million cells per well in a 24 well plate. After allowing the cells to adhere for 24 hours, RPMI 1640 media supplemented with 10% FBS and 1% antibiotic-antimycotic solution containing 500 ng/ml of LPS was added to the respective wells. The culture plates were then placed in Cell-IQ (CM Technologies, Tampere; Finland) and specific fields were focussed using 10X objective magnification. Time lapse microscopy was performed and analysed for 48 hours at 30 minutes interval using live cell imaging and software (Cell IQ Analyser, Finland).\n\n\nResults\n\nSorted monocytes incubated with LPS were examined for secretion of various pro- and anti-inflammatory cytokines. The levels of IL-8, IL-10, TGF-β1 and RANTES at different time points after positively and negatively isolated CD14+ monocytes were incubated with LPS were analyzed. The secretion of IL-8 was observed to be at its maximum at 8 hours in negatively sorted monocytes and at 16 hours in positively sorted monocytes (Figure 1A). The IL-8 level in negatively sorted CD14+ cells was 6 times higher than positively sorted CD14+ cells. A similar pattern was observed for secretion of RANTES (Figure 1C) and TGF-β1 (Figure 1D), though the differences were not very pronounced. It was of significance to observe the reversed pattern for an anti-inflammatory cytokine, IL-10 (Figure 1B).\n\nGreater secretion of the pro-inflammatory cytokines in negatively sorted CD14+ monocytes upon activation was observed, during proliferation of activated monocytes. Figure 2 shows the variation in the average number of cells per field (487μm×364μm) for positively and negatively sorted cells. Further, the progression of proliferation is shown in Video V1 for negatively sorted cells and Video V2 for positively sorted cells. Alongside Figure 2, the videos show that negatively sorted cells proliferated rapidly and extensively upon activation, and the maximum number of cells was reached after 16 hours. However, there was no clearly defined time point of maximum number of cells reached by positively sorted cells.\n\nTo examine the degradation of microbeads, PBMCs were labeled with anti-human CD14 antibody conjugated microbeads and sorted under a magnetic field. The sorted cells were maintained in a culture and the numbers of cells with and without microbeads were counted via flow cytometric analysis. Results of three independent samples are shown in Figure 3.\n\nThese results show the variation in percentage of cells collected in the ‘flow through’ of column placed in magnetic field. These were the cells from which the microbeads were either degraded or detected and cells were without microbeads. In all the three cultures we examined, the percentage of cells without microbeads was different but there was hardly any change in these percentages when the culture was continued for 6 days. This suggests that in typical culture conditions the Ab-microbeads remain bound with cells for many days.\n\n\nDiscussion\n\nMagnetic cell sorting for the separation of large numbers of cells according to specific cell surface markers is a technique that is commonly used (Adams et al., 2008). It is a common notion that magnetic beads are biodegradable, do not activate cells and do not affect downstream application. We have however observed that the activation and proliferation of positively sorted CD14+ cells is impaired compared to the negatively sorted cells.\n\nThe activation of monocytes by LPS is known to occur through surface CD14, which is an LPS sensing receptor. Surface CD14 plays a crucial role; it binds and transfers LPS to the surface via TLR4:MD2 complex to enable its recognition. The LPS stimulation of monocytes activates several intracellular signaling pathways which in turn activates a variety of transcription factors ultimately leading to induction of many genes encoding inflammatory cytokines (Guha & Mackman, 2001). In short, CD14 is involved in the LPS-induced release of IL-8, which is an important pro-inflammatory cytokine (He et al., 2013).\n\nAfter positively sorting CD14+ monocytes from PBMCs, the surface CD14 molecules on monocytes are blocked by anti-CD14 microbeads and these CD14+ surface sites can no longer mediate the stimulation by LPS and the positively sorted CD14+ monocytes may show impaired stimulation by LPS.\n\nIn two experiments, identical numbers of CD14+ monocytes isolated by positive and negative sorting were stimulated with LPS and their activation and proliferation was monitored. Figure 1 shows that upon stimulation by LPS the negatively sorted CD14+ monocytes secreted enormous amount of IL-8 almost instantaneously and they exhibited acute proliferation (Figure 2). This is in accordance with the observation that IL-8 transcript is highly expressed in LPS-stimulated monocytes (Standiford et al., 1992; Suzuki et al., 2000).\n\nThe positively sorted CD14+ monocytes responded only after 24 hours, and their level of stimulation was impaired and the cells did not proliferate. This delayed and reduced stimulation by LPS is due to the CD14 independent receptors which function to direct LPS mediated cytokine secretion under conditions where the CD14 dependent pathway is blocked or non-functional (Lynn et al., 1993). There are a few LPS-associated cell surface proteins which are distinct from CD14 and these surface proteins too can bind TLRs to initiate a response (Triantafilou et al., 2001). Our results suggest that the density of these surface proteins is low compared to CD14 as lesser stimulation was observed when CD14 surface groups were blocked by Abs and secondly, the delayed stimulation indicate that most likely a different pathway was followed for their activation.\n\nFurther, the secretion of somewhat higher amount of IL-10 by positively sorted CD14+ monocytes only suggest the absence of highly inflammatory conditions upon LPS activation. The levels of RANTES and TGF-β1 also indicate that the LPS activation of monocytes via CD14 independent receptors produces unique results which could be very different from common experimental situation.\n\nIn one such related report, the, human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated to macrophages (Neu et al., 2013). The phagocytosis of Listeria monocytogenes (Lm) by GM-CSF-derived macrophages (GM-M) was markedly influenced by the method used for isolation of monocytes. The GM-M derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-M generated from positively selected monocytes displayed high phagocytosis of Lm. The paper concludes that macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages. In yet another report (Elkord et al., 2005) it was demonstrated that the human dendritic cells generated from positively isolated monocytes by anti-CD14-coated microbeads show impaired induction by LPS.\n\nSimilar findings have been made by examining the gene expression profiles of CD8+ T cells, B cells and monocytes isolated using positive selection, negative selection and FACS (Beliakova-Bethell et al., 2014).\n\nIn these reports it was not investigated further why the positively isolated monocytes were not suitable and had poor cytokines production upon stimulation. Our data suggests that in these experiments, the positively isolated monocytes were tagged permanently with anti-CD14 molecules attached with microbeads. The positively isolated CD14+ monocytes are identical with monocytes whose surface CD14 molecules have been blocked by Abs and these monocytes are known to behave differently (Delirezh et al., 2013; Elkord et al., 2005; Kim & Kim, 2014). In this context, diverse outcome could be observed when cells positively isolated by antibody bound microbeads were used for extended culture work (Govers et al., 2012; Greish et al., 2012; Lapenna et al., 2013; Meinhardt et al., 2012).\n\nThere are two important findings from these experiments; positively isolated CD14+ monocytes have impaired LPS sensitivity and magnetic beads used in positive isolation do not degrade within days. These conclusions suggest that for most experiments, positively isolated cells are usable for analysis purpose only and should not be used for any further culture experiments.\n\n\nData availability\n\nDataset 1: Raw data corresponding to the results shown in Figure 1. DOI, 10.5256/f1000research.12802.d182356 (Bhattacharjee et al., 2017a)\n\nDataset 2: Raw data corresponding to the results shown in Figure 2. DOI, 10.5256/f1000research.12802.d182357 (Bhattacharjee et al., 2017b)\n\nDataset 3: Raw data corresponding to the results shown in Figure 3. DOI, 10.5256/f1000research.12802.d182358 (Bhattacharjee et al., 2017c)",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work has been supported by the core grant received from the Department of Biotechnology, Government of India, to the National Institute of Immunology, New Delhi.\n\n\nAuthor details\n\nThis work was carried out at: National Institute of Immunology, New Delhi, India. The first author, Jashdeep Bhattacharjee, has now moved to: Division of Gastroenterology, Hepatology and Nutrition, Children’s Hospital Los Angeles.\n\n\nSupplementary material\n\nSupplementary Video V1: The progression of proliferation after stimulation by LPS in negatively sorted CD14+ monocytes.\n\nClick here to access the data.\n\nSupplementary Video V2: The progression of proliferation after stimulation by LPS in positively sorted CD14+ monocytes.\n\nClick here to access the data.\n\n\nReferences\n\nAdams JD, Kim U, Soh HT: Multitarget magnetic activated cell sorter. Proc Natl Acad Sci U S A. 2008; 105(47): 18165–18170. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeliakova-Bethell N, Massanella M, White C, et al.: The effect of cell subset isolation method on gene expression in leukocytes. Cytometry A. 2014; 85(1): 94–104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBhattacharjee J, Das B, Mishra A, et al.: Dataset 1 in: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour. F1000Research. 2017a. Data Source\n\nBhattacharjee J, Das B, Mishra A, et al.: Dataset 2 in: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour. F1000Research. 2017b. Data Source\n\nBhattacharjee J, Das B, Mishra A, et al.: Dataset 3 in: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour. F1000Research. 2017c. Data Source\n\nDelirezh N, Shojaeefar E, Parvin P, et al.: Comparison the effects of two monocyte isolation methods, plastic adherence and magnetic activated cell sorting methods, on phagocytic activity of generated dendritic cells. Cell J. 2013; 15(3): 218–223. PubMed Abstract | Free Full Text\n\nElkord E, Williams PE, Kynaston H, et al.: Human monocyte isolation methods influence cytokine production from in vitro generated dendritic cells. Immunology. 2005; 114(2): 204–212. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFüchslin HP, Kötzsch S, Keserue HA, et al.: Rapid and quantitative detection of Legionella pneumophila applying immunomagnetic separation and flow cytometry. Cytometry A. 2010; 77(3): 264–274. PubMed Abstract | Publisher Full Text\n\nGovers C, Berrevoets C, Treffers-Westerlaken E, et al.: Magnetic-activated cell sorting of TCR-engineered T cells, using tCD34 as a gene marker, but not peptide-MHC multimers, results in significant numbers of functional CD4+ and CD8+ T cells. Hum Gene Ther Methods. 2012; 23(3): 213–224. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreish S, Abogresha N, Abdel-Hady Z, et al.: Human umbilical cord mesenchymal stem cells as treatment of adjuvant rheumatoid arthritis in a rat model. World J Stem Cells. 2012; 4(10): 101–109. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuha M, Mackman N: LPS induction of gene expression in human monocytes. Cell Signal. 2001; 13(2): 85–94. PubMed Abstract | Publisher Full Text\n\nHe W, Qu T, Yu Q, et al.: LPS induces IL-8 expression through TLR4, MyD88, NF-kappaB and MAPK pathways in human dental pulp stem cells. Int Endod J. 2013; 46(2): 128–136. PubMed Abstract | Publisher Full Text\n\nHorgan K, Shaw S, Boirivant M: Immunomagnetic purification of T cell subpopulations. Curr Protoc Immunol. 2009; Chapter 7: Unit7.4. PubMed Abstract | Publisher Full Text\n\nKim D, Kim JY: Anti-CD14 antibody reduces LPS responsiveness via TLR4 internalization in human monocytes. Mol Immunol. 2014; 57(2): 210–215. PubMed Abstract | Publisher Full Text\n\nLapenna A, B-Lynch C, Kapeni C, et al.: A simple model system enabling human CD34+ cells to undertake differentiation towards T cells. PLoS One. 2013; 8(7): e69572. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLynn WA, Liu Y, Golenbock DT: Neither CD14 nor serum is absolutely necessary for activation of mononuclear phagocytes by bacterial lipopolysaccharide. Infect Immun. 1993; 61(10): 4452–4461. PubMed Abstract | Free Full Text\n\nMeinhardt K, Kroeger I, Abendroth A, et al.: Influence of NK cell magnetic bead isolation methods on phenotype and function of murine NK cells. J Immunol Methods. 2012; 378(1–2): 1–10. PubMed Abstract | Publisher Full Text\n\nMiltenyi S, Müller W, Weichel W, et al.: High gradient magnetic cell separation with MACS. Cytometry. 1990; 11(2): 231–238. PubMed Abstract | Publisher Full Text\n\nNeu C, Sedlag A, Bayer C, et al.: CD14-dependent monocyte isolation enhances phagocytosis of listeria monocytogenes by proinflammatory, GM-CSF-derived macrophages. PLoS One. 2013; 8(6): e66898. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPei R, Wang G, Tarsitani C, et al.: Simultaneous HLA Class I and Class II antibodies screening with flow cytometry. Hum Immunol. 1998; 59(5): 313–322. PubMed Abstract | Publisher Full Text\n\nRibaut C, Berry A, Chevalley S, et al.: Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species. Malar J. 2008; 7: 45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSafarík I, Safaríková M: Use of magnetic techniques for the isolation of cells. J Chromatogr B Biomed Sci Appl. 1999; 722(1–2): 33–53. PubMed Abstract\n\nSemple JW, Allen D, Chang W, et al.: Rapid separation of CD4+ and CD19+ lymphocyte populations from human peripheral blood by a magnetic activated cell sorter (MACS). Cytometry. 1993; 14(8): 955–960. PubMed Abstract | Publisher Full Text\n\nStandiford TJ, Strieter RM, Allen RM, et al.: IL-7 up-regulates the expression of IL-8 from resting and stimulated human blood monocytes. J Immunol. 1992; 149(6): 2035–2039. PubMed Abstract\n\nSuzuki T, Hashimoto S, Toyoda N, et al.: Comprehensive gene expression profile of LPS-stimulated human monocytes by SAGE. Blood. 2000; 96(7): 2584–2591. PubMed Abstract\n\nTomlinson MJ, Tomlinson S, Yang XB, et al.: Cell separation: Terminology and practical considerations. J Tissue Eng. 2013; 4: 2041731412472690. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTriantafilou K, Triantafilou M, Dedrick RL: A CD14-independent LPS receptor cluster. Nat Immunol. 2001; 2(4): 338–345. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "28324",
"date": "12 Dec 2017",
"name": "Nadejda Beliakova-Bethell",
"expertise": [
"Reviewer Expertise Virology",
"immunology",
"cell separation and flow cytometry",
"analysis of gene expression"
],
"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 assesses monocyte function following isolation of these cells from total peripheral blood mononuclear cells (PBMC) using positive or negative immunomagnetic selection. It addresses an important topic, because cell isolation procedure may affect cell function and skew conclusions obtained from in vitro studies of immune cells isolated from blood. Despite a limited assessment of functions and isolation methods, this study should have an impact on selection of monocyte isolation protocol when these cells are intended for investigation of specific responses, which this study tested. The study design is appropriate and conclusions drawn are adequately supported by the results. The paper may be improved, however, by providing more details in methods, raw data and discussion, and by careful revision of citations as detailed below:\nMethods: Cytometic bead array: there are several cytometric bead arrays available for human samples (e.g. “human inflammatory cytokine kit”, “human chemokine kit”). Which one was used? Legend for Figure 1 indicates that 5 samples were pooled. It would be helpful to provide details on how the pooling was done (the entire samples, equal volumes, equal proportions?) and what dilutions were made for the assay.\nLive cell imaging: It appears that information in methods contradicts the data shown in Figure 2. According to the figure and supplied raw data, imaging was performed over a course of over 76 hours, while methods section states “48 hours”.\nRaw data: Data for Figure 1: it might be easier to understand the data if columns were labeled by cytokine and sample (e.g. IL8 negative selection, IL8 positive selection, etc. as opposed to IL8B, IL8C, etc.) Data for Figure 2: were 36 fields for each sample for each time point individually counted, as the information on the figure indicates? Raw data appears to have only the averages. Was the distribution of counts tight, or was there high variation? Including raw data for individual field counts might be more appropriate.\nDiscussion: Another option to positively isolate monocytes is to use anti-CD33 instead of anti-CD14 coated beads. This may be more appropriate in studies that aim to measure response to LPS, when positive selection is preferred (e.g. in cases of limited sample from which sequential separation of different lymphocyte subsets is desired). It might be worthy to discuss this option.\nCitations: Several citations throughout the paper appear to be inaccurate; for example, in Introduction some citations on altered behavior of cells following microbead attachment: (1) Safarik and Safarikova is a method review, and Horgan et al. is a protocol; neither studies cell behavior; (2) Semple et al. observed no functional changes in limited tests they did; the difference that they report pertains to CD19+ vs CD19- cells, not positively isolated vs negatively isolated cells; (3) Fuchslin et al. labeled bacterial cells for their quantification in water; and Ribaut et al. labeled parasites for their isolation from infected blood for research purposes; the relevance of these two citations for the present study may be questionable. In Discussion, Adams et al. describe a special case of MACS for multitarget sort, and may not be appropriate for referencing commonality of magnetic sorting. While the results from the present study are consistent with gene expression studies by Beliakova-Bethell et al. in terms of effects of positive selection, the reference to Beliakova-Bethell et al. is made following description of Neu et al. and Elkord et al. studies, stating that the findings were similar. This is not accurate because Neu et al. and Elkord et al. performed functional assessment following cell separation, while Beliakova-Bethell et al. lysed the cells immediately after isolation and did not measure cell function. Instead of the statement of similarity of findings, it might be worth pointing out that positive selection affects monocytes the most both in the short term and in the long term.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3283",
"date": "22 Dec 2017",
"name": "Pramod Upadhyay",
"role": "Author Response",
"response": "Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically Methods: Cytometic bead array: there are several cytometric bead arrays available for human samples (e.g. “human inflammatory cytokine kit”, “human chemokine kit”). Which one was used? Legend for Figure 1 indicates that 5 samples were pooled. It would be helpful to provide details on how the pooling was done (the entire samples, equal volumes, equal proportions?) and what dilutions were made for the assay. We used Cytometric Bead Array (CBA) Soluble Protein Flex Set and equal volumes of the samples were pooled. The CBA assay was performed on culture supernatants without any dilution. These details have been included in the revised manuscript. Live cell imaging: It appears that information in methods contradicts the data shown in Figure 2. According to the figure and supplied raw data, imaging was performed over a course of over 76 hours, while methods section states “48 hours”. Live cell imaging: The imaging was performed till 76 hours and it was a mistake to mention this as 48 hours in the method section, we thank you for pointing out this anomaly and this has been corrected in the revised manuscript. Raw data: Data for Figure 1: it might be easier to understand the data if columns were labeled by cytokine and sample (e.g. IL8 negative selection, IL8 positive selection, etc. as opposed to IL8B, IL8C, etc.) Suggested changes have been made. Data for Figure 2: were 36 fields for each sample for each time point individually counted, as the information on the figure indicates? Raw data appears to have only the averages. Was the distribution of counts tight, or was there high variation? Including raw data for individual field counts might be more appropriate. During the live cell imaging experiment images were logged and later cells were counted using the Cell-IQ Analyser software. The protocol for cell counting in the analysis software was assembled to finally provide the average number of cell count in the selected fields. The cell counts from individual fields of various images were embedded in the analysis software, thought it was possible to export these tables but due to very large number of data files we focussed only on the averages. Discussion: Another option to positively isolate monocytes is to use anti-CD33 instead of anti-CD14 coated beads. This may be more appropriate in studies that aim to measure response to LPS, when positive selection is preferred (e.g. in cases of limited sample from which sequential separation of different lymphocyte subsets is desired). It might be worthy to discuss this option. This is a good suggestion keeping in mind that the CD14 is an endogenous ligand for CD33 in monocyte-derived immature dendritic cells. It has been reported (Hiroshi Nakada et al.; J Biol Chem. 2014 Sep 5; 289(36): 25341–25350) that when monocyte-derived immature dendritic cells were stimulated with LPS in the presence of anti-CD33Ab, the production of IL-12 and phosphorylation of NF-κB decreased significantly. Most likely the positively isolated monocytes using CD33-Ab beads would behave in a similar manner. This possibility has been discussed in the Discussion section of the revised manuscript. Citations: Several citations throughout the paper appear to be inaccurate; for example, in Introduction some citations on altered behavior of cells following microbead attachment: We once again thank the reviewer for thoroughly examining the citations; the list of references has been appropriately revised. The differences between the findings of Beliakova-Bethell et al. and Neu et al. & Elkord et al. have been incorporated in the revised manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/6-2045
|
https://f1000research.com/articles/6-1998/v1
|
13 Nov 17
|
{
"type": "Case Report",
"title": "Case Report: A rare case of prosthetic valve infective endocarditis caused by Aerococcus urinae",
"authors": [
"Muhammad Adeel",
"Saman Tariq",
"Hisham Akthar",
"Ahmed Zaghloul",
"Corina Iorgoveanu",
"Carina Dehner",
"Muhammad Adeel",
"Saman Tariq",
"Hisham Akthar",
"Ahmed Zaghloul",
"Corina Iorgoveanu"
],
"abstract": "Infective endocarditis (IE) is a serious and life threatening cardiac condition, most commonly caused by staphylococci, streptococci, enterococci and rarely by HACEK organisms (Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella corrodens and Kingella). Here, we present a case of IE caused by Aerococcus urinae in a 75-year-old man with a bioprosthetic aortic valve. Aerococcus urinae is a gram-positive, catalase negative microorganism, and is usually an isolate of complicated urinary tract infections in the elderly male population. It is associated with high morbidity and mortality. Awareness of this organism as a cause of IE is important, since failure to recognize the condition may lead to adverse clinical outcomes and significant complications with even fatal outcome, as in this case.",
"keywords": [
"infective endocarditis",
"prosthetic valve endocarditis",
"Aerococcus urinae"
],
"content": "Introduction\n\nThe diagnosis of infective endocarditis (Habib et al.) is based on a number of factors, including patient history, physical examination, as well as diagnostic tools (blood cultures, chest X-ray and echocardiography) (Durack et al., 1994), (Lukes et al., 1993). Risk factors for IE include advanced age (> 60 years), male gender, history of intravenous drug use, poor dentition, structural or valvular heart disease and presence of prosthesis. It is most commonly caused by Staphylococcus aureus, Streptococcus viridans, and enterococci, and rarely by HACEK (Sharara et al., 2016) organisms. Here, we describe a rare case of IE secondary to Aerococcus urinae, a gram-positive, catalase negative coccus that grows in clusters. It is associated with high mortality and neurological complications (Ebnöther et al., 2002).\n\n\nCase report\n\nA 75-year-old Caucasian man presented to his local hospital with malaise, fever and nausea for 5 days. He had a bio prosthetic aortic valve replacement for mixed aortic valve disease 12 years ago, further significant past medical history included placement of a permanent pacemaker for complete heart block, right total hip replacement, hypertension and benign prostatic hyperplasia (BPH). The patient had no history of smoking, alcohol consumption or illicit drug use. The patient had no recent surgeries or dental work and the review of systems was unremarkable. The physical exam revealed vital parameters of HR 97 bpm regular, BP 134/87, temperature of 101.5°F, respiratory rate of 18 per minute and oxygen saturation of 96% on room air. On precordial auscultation a systolic and a diastolic murmur were heard in aortic area, mild bi-basal crepitation, but no JVD or peripheral edema. The rest of the physical exam was unremarkable. His labs showed a normal white cell count (WCC) of 9.9 × 106/L, but his C-reactive protein (CRP) was elevated to 214.9 (normal <5mg/L) with a stable haemoglobin (11.2 g/dl), further labs were unremarkable. His mid-stream urine showed WCC < 20; red cell count (RCC) of 20-50 and it grew mixed organisms, all considered part of the normal flora. Chest X-ray, CT scan of the brain, thorax, abdomen and pelvis did not show any significant cause of sepsis.\n\nThe patient was empirically commenced on IV piperacillin-tazobactam and vancomycin for sepsis treatment. His blood cultures grew Aerococcus urinae sensitive to penicillin within 24 hours of admission.\n\nA trans-thoracic echocardiogram showed mild aortic regurgitation and mitral regurgitation with no clear vegetation, however, trans-esophageal echocardiogram (TOE) showed a moderate aortic regurgitation due to a large mobile vegetation on the bio-prosthetic aortic valve with normal left ventricular function, no peri-valvular abscess was noted (See Image 1a and 1b).\n\n1A: Transesophageal echocardiogram (TEE), mid-esophageal view showing mobile echo density on prosthetic aortic valve. 1B: Transesophageal echocardiogram (TEE), mid-esophageal view enlarged to show mobile echo density on prosthetic aortic valve.\n\nClinical presentation, echocardiographic findings and positive blood cultures fulfilled Duke’s criteria (Hoen et al., 1996) for IE. Patient was managed as prosthetic aortic valve endocarditis from Aerococcus urinae with IV amoxicillin 2 grams every 4 hours, and gentamicin 1 mg/kg twice daily as per local guidelines. Antibiotic therapy for 6 weeks in total with early surgery for prosthetic valve replacement was planned (Truninger et al., 1999).\n\nDespite prompt initiation of appropriate antibiotic treatment and intensive clinical monitoring, the patient failed to improve this hospitalization and developed a large pulmonary edema and progressive aortic regurgitation, and died before definitive surgery. As per family’s wishes, an autopsy was not performed.\n\n\nDiscussion\n\nAerococcus urinae is a gram-positive, catalase negative coccus which grows in clusters. It is mostly associated with urinary tract infections in elderly men, especially in the setting of structural abnormalities e.g. BPH, urethral strictures and nephrolithiasis. It has been associated with culture negative infective endocarditis (Slany et al., 2007). It is reported to be sensitive to penicillins/cephalosporins and resistant to sulfonamides and aminoglycosides (Skov et al., 2001). There are less than 20 reported cases of IE caused by Aerococcus urinae worldwide.\n\nDespite being sensitive to common antibiotics, prosthetic valve endocarditis (PVE) secondary to Aerococcus urinae can be difficult to manage with antibiotic therapy alone, and often requires surgical intervention (Wang et al., 2007). The indications for surgical intervention for PVE include severe prosthetic dysfunction, severe heart failure, large persistent vegetation and abscess or peri-valvular involvement (Habib et al., 2005). The presence of vegetation on the valve created a consistent source of bacteria that could embolize and can serve as a source of sepsis.\n\nThis case highlights the importance of source control by expediting prosthesis removal in presence of overt symptoms of worsening cardiac failure and worsening prosthesis dysfunction (regurgitation in this case), as medical therapy alone may not be sufficient to effectively treat Aerococcus urinae IE despite appropriate sensitivities. Early identification is crucial and can be life-saving. The main problem is current diagnostic testing for microorganisms – whereas 16s sequencing would be the most time-efficient method, it’s rarely done, as the expertise is limited and costs are high. Recently, there is good evidence for the use of MALDI-TOF (Senneby et al., 2013), (Senneby et al., 2016) due to increased detection rates, even in direct comparison to 16s sequencing.\n\nIn conclusion, Aerococcus urinae used to be a rare cause of IE but rates have been increasing significantly within the last 10 years. Therefore establishing a concise and broadly acknowledged protocol from diagnosis up to patient management is critical.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient. Permission was also granted from a next of kin for publication of the manuscript.",
"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\nDurack DT, Lukes AS, Bright DK: New criteria for diagnosis of infective endocarditis: utilization of specific echocardiographic findings. Duke Endocarditis Service. Am J Med. 1994; 96(3): 200–209. PubMed Abstract | Publisher Full Text\n\nEbnöther C, Altwegg M, Gottschalk J, et al.: Aerococcus urinae endocarditis: case report and review of the literature. Infection. 2002; 30(5): 310–313. PubMed Abstract | Publisher Full Text\n\nHabib G, Tribouilloy C, Thuny F, et al.: Prosthetic valve endocarditis: who needs surgery? A multicentre study of 104 cases. Heart. 2005; 91(7): 954–959. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoen B, Béguinot I, Rabaud C, et al.: The Duke criteria for diagnosing infective endocarditis are specific: analysis of 100 patients with acute fever or fever of unknown origin. Clin Infect Dis. 1996; 23(2): 298–302. PubMed Abstract | Publisher Full Text\n\nLukes AS, Bright DK, Durack DT: Diagnosis of infective endocarditis. Infect Dis Clin North Am. 1993; 7(1): 1–8. PubMed Abstract\n\nSenneby E, Göransson L, Weiber S, et al.: A population-based study of aerococcal bacteraemia in the MALDI-TOF MS-era. Eur J Clin Microbiol Infect Dis. 2016; 35(5): 755–762. PubMed Abstract | Publisher Full Text\n\nSenneby E, Nilson B, Petersson AC, et al.: Matrix-assisted laser desorption ionization-time of flight mass spectrometry is a sensitive and specific method for identification of aerococci. J Clin Microbiol. 2013; 51(4): 1303–1304. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSharara SL, Tayyar R, Kanafani ZA, et al.: HACEK endocarditis: a review. Expert Rev Anti Infect Ther. 2016; 14(6): 539–545. PubMed Abstract | Publisher Full Text\n\nSkov R, Christensen JJ, Korner B, et al.: In vitro antimicrobial susceptibility of Aerococcus urinae to 14 antibiotics, and time-kill curves for penicillin, gentamicin and vancomycin. J Antimicrob Chemother. 2001; 48(5): 653–658. PubMed Abstract | Publisher Full Text\n\nSlany M, Freiberger T, Pavlik P, et al.: Culture-negative infective endocarditis caused by Aerococcus urinae. J Heart Valve Dis. 2007; 16(2): 203–205. PubMed Abstract\n\nTruninger K, Attenhofer Jost CH, Seifert B, et al.: Long term follow up of prosthetic valve endocarditis: what characteristics identify patients who were treated successfully with antibiotics alone? Heart. 1999; 82(6): 714–720. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang A, Athan E, Pappas PA, et al.: Contemporary clinical profile and outcome of prosthetic valve endocarditis. JAMA. 2007; 297(12): 1354–1361. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "28819",
"date": "11 Dec 2017",
"name": "Magnus Rasmussen",
"expertise": [
"Reviewer Expertise endocarditis"
],
"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 describes a case of prosthetic valve infective (IE) caused by Aerococcus urinae with fatal outcome. The number of case reports on this condition is increasing and it is not immediately obvious that another case with poor outcome is helpful. This case, however, has an important learning point in that a patient with prosthetic valve endocarditis, in resource-rich settings, must be treated in a centre where acute cardiac surgery can be performed or near such a centre. It is of less importance if the causative bacterium in this case were A. urinae or any other bacterium. I list my major concerns and minor points below:\n\nMajor concerns 1. The prognosis of A. urinae IE is not poor. Many cases with fatal outcome have been published but in the only population-based survey1 demonstrate a relatively favourable prognosis compared to other pathogens. The risk with case reports is a publication-bias where only dramatic cases are published. The case series should be quoted and is the only reliable source on information on A. urinae IE. A poor prognosis is claimed in the abstract, introduction and discussion. This claim must be modified based on the findings by Sunnerhagen et al. 2. A diagnosis of IE is established through the Dukes criteria, I suggest a reference to Li is given2. Symptoms and chest X-ray are irrelevant for the diagnostic process. Please modify introduction. 3. In the case description it is twice stated that the patient has sepsis. Sepsis-3 criteria are not fulfilled. Please rephrase. 4. The cultures grew A. urinae. How was the species determination performed? How many cultures? MIC for ampicillin and gentamycin should be given. 5. It is claimed that TEE demonstrates a “moderate aortic regurgitation due to a large mobile vegetation”. It is important if the regurgitation was paravalvular or through the valves. Maximum size of the vegetation is also crucial since this is important for establishing the indication for operation. Left ventricular function should also be commented on as well as if there were signs of vegetations on the pacemaker cable. 6. It is claimed that ampi+genta was commenced according to local guidelines. For which bacterial species are these guidelines meant. The use of aminoglycosides in this condition is controversial1. 7. How was “progressive aortic regurgitation verified? Could pacemaker failure have played a role? 8. Why was the patient not moved for emergency surgery when he deteriorated? This seem like an avoidable fatality! 9. In the discussion it is claimed that there are only 20 reports. This is not true1. Cases up until 2015 are summarized in a review3. 10. Surgical intervention is claimed to be common in the discussion with a quote to Wang. Please read and quote Sunnerhagen instead1. Surgery is relatively rarely needed. 11. “Large persistent vegetation” is claimed as an indication for surgery. Large is enough. 12 “The presence of vegetation on the valve created a consistent source of bacteria that could embolize and can serve as a source of sepsis.” This statement has nothing to do with the current case and should be omitted. 13 “The main problem is current diagnostic testing for microorganisms– whereas 16s sequencing would be the most time-efficient method, it’s rarely done, as the expertise is limited and costs… and so on” This is irrelevant for the case since the reason to operate is not dependent on microbiological diagnostics. Irrespective of the causative pathogen this patient would have been saved by timely heart surgery. 14. The claim “In conclusion, Aerococcus urinae used to be a rare cause of IE but rates have been increasing significantly within the last 10 years.” Lacks support and should be deleted. A. urinae has been increasingly REPORTED as a cause of IE but incidence is likely unchanged. 15. In discussing Duke criteria in the case presentation one must keep in mind that A. urinae in 2/2 cultures (4/4 bottles) only fulfill Duke criteria if the cultures were taken with\n\nMinor comments 1. I suggest another title. Something like “fatal case of A. urinae prosthetic valve endocrditis.” 2. Why mention HACEK in the abstract? Those organisms are exceedingly rare and for example much less common than betaheamolytic strep. 3. In case presentation spell out JVD. 4. “Stable haemoglobin”- what is meant. Are the authors referring to repeated measurements?\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": "3418",
"date": "20 Feb 2018",
"name": "Carina Dehner",
"role": "Author Response",
"response": "1) Abstract:- It is always important to also having focus on more rare etiologies of IE. In the abstract it is stated that the mortality rate is high. This is suggested to be modified to: Initial descriptions of collections of IE cases with A. urinae demonstrated a high morbidity and mortality rate, whereas a recent Swedish epidemiological study could not retrieve this.New reference to the introduction was added to highlight the better outcome 2) Introduction:- Dukes criteria should be mentioned. - New references added to mention Duke’s criteria or its modifications - There is not a species named Streptococcus viridans.Correction made· Recent diagnostic improvements should be included, especially MALDI-TOF mass spectrometry.Added in abstract- a gram-positive, catalase-negativeCorrection made 3) Case description: Specific description of PM electrode findings should be given.Included in description, PM lead was not involved.Microbiological data are very scarce. Blood-culture system and number of positive bottles should be given. Likewise identification criteria and susceptibility methods and results, including MIC values of relevant antibiotics should be given.This is now added to the case description4) Discussion: 16S is slang: it should be partial 16S rRNA gene sequencing analysisCorrection made"
}
]
},
{
"id": "29616",
"date": "22 Jan 2018",
"name": "Jens J Christensen",
"expertise": [
"Reviewer Expertise Clinical 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\nA fatal case of IE caused by Aerococcus urinae, in a 75-year-old man with a bioprosthetic aortic valve is presented and discussed. Very precise and covering comments have been given by reviewer 1. Microbiological data should be examined thouroughly and extended. Language correction seems indicated. The following comments can be added.\n1)\n\nAbstract:\nIt is always important to also having focus on more rare etiologies of IE. In the abstract it is stated that the mortality rate is high. This is suggested to be modified to: Initial descriptions of collections of IE cases with A. urinae demonstrated a high morbidity and mortality rate, whereas a recent Swedish epidemiological study could not retrieve this.\n\n2)\n\nIntroduction:\nDukes criteria should be mentioned.\n\nThere is not a species named Streptococcus viridans. Recent diagnostic improvements should be included, especially MALDI-TOF mass spectrometry. a gram-positive, catalase-negative\n\n3)\n\nCase description:\nSpecific description of PM electrode findings should be given. A more detailed disease timespan is desirable. Microbiological data are very scarce. Blood-culture system and number of positive bottles should be given. Likewise identification criteria and susceptibility methods and results, including MIC values of relevant antibiotics should be given. A thorough microbiological examination of the manuscript seems indicated Aerococcus urinae should only be fully written the first time\n\n4)\n\nDiscussion\n16S is slang: it should be partial 16S rRNA gene sequencing analysis\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? No",
"responses": [
{
"c_id": "3417",
"date": "20 Feb 2018",
"name": "Carina Dehner",
"role": "Author Response",
"response": "1. The prognosis of A. urinae IE is not poor. Many cases with fatal outcome have been published but in the only population-based survey1 demonstrate a relatively favourable prognosis compared to other pathogens. The risk with case reports is a publication-bias where only dramatic cases are published. The case series should be quoted and is the only reliable source on information on A. urinae IE. A poor prognosis is claimed in the abstract, introduction and discussion. This claim must be modified based on the findings by Sunnerhagen et al.A new reference was added to the introduction about favourable outcome to IE caused by Aerococcus urinae2. A diagnosis of IE is established through the Dukes criteria, I suggest a reference to Li is given2.Symptoms and chest X-ray are irrelevant for the diagnostic process. Please modify introduction. A new reference was added to diagnose IE based on Dukes criteria or their modifications3. In the case description it is twice stated that the patient has sepsis. Sepsis-3 criteria are not fulfilled. Please rephrase. Rephrased4. The cultures grew A. urinae. How was the species determination performed? How many cultures? MIC for ampicillin and gentamycin should be given. MICs for ampicillin and gentamicin added, description added about blood cultures5. It is claimed that TEE demonstrates a “moderate aortic regurgitation due to a large mobile vegetation”. It is important if the regurgitation was paravalvular or through the valves. Maximum size of the vegetation is also crucial since this is important for establishing the indication for operation. Left ventricular function should also be commented on as well as if there were signs of vegetations on the pacemaker cable. Transesophgeal echo description expanded, commented on LV and pacemaker lead.6. It is claimed that ampi+genta was commenced according to local guidelines. For which bacterial species are these guidelines meant. The use of aminoglycosides in this condition is controversial1. Hospital guidelines for suspected/possible IE were followed7. How was “progressive aortic regurgitation verified? Could pacemaker failure have played a role? Repeat transthoracic echo showed worsening of regurgitation; this is added to the case8. Why was the patient not moved for emergency surgery when he deteriorated? This seem like an avoidable fatality! Deterioration was sudden and rapid, arrangements were made but patient died before the surgery 9. In the discussion it is claimed that there are only 20 reports. This is not true1. Cases up until 2015 are summarized in a review3. Updated number of Aerococcus urinae IE reported 10. Surgical intervention is claimed to be common in the discussion with a quote to Wang. Please read and quote Sunnerhagen instead1. Surgery is relatively rarely needed. Reference added to show that surgery in most cases is not indicated11. “Large persistent vegetation” is claimed as an indication for surgery. Large is enough. Modified12 “The presence of vegetation on the valve created a consistent source of bacteria that could embolize and can serve as a source of sepsis.” This statement has nothing to do with the current case and should be omitted. Omitted 13 “The main problem is current diagnostic testing for microorganisms– whereas 16s sequencing would be the most time-efficient method, it’s rarely done, as the expertise is limited and costs… and so on” This is irrelevant for the case since the reason to operate is not dependent on microbiological diagnostics. Irrespective of the causative pathogen this patient would have been saved by timely heart surgery. This is kept; we wish our readers to know that improved methods of isolation are important and could help with management14. The claim “In conclusion, Aerococcus urinae used to be a rare cause of IE butrates have been increasing significantly within the last 10 years.” Lacks support and should be deleted. A. urinae has been increasingly REPORTED as a cause of IE but incidence is likely unchanged. This is now added that that increase in reported cases is due to better isolation methods. 15. In discussing Duke criteria in the case presentation one must keep in mind that A. urinae in 2/2 cultures (4/4 bottles) only fulfill Duke criteria if the cultures were taken with Agreed"
}
]
}
] | 1
|
https://f1000research.com/articles/6-1998
|
https://f1000research.com/articles/6-1335/v1
|
07 Aug 17
|
{
"type": "Review",
"title": "What do we know about grant peer review in the health sciences?",
"authors": [
"Susan Guthrie",
"Ioana Ghiga",
"Steven Wooding",
"Ioana Ghiga",
"Steven Wooding"
],
"abstract": "Background: Peer review decisions award >95% of academic medical research funding, so it is crucial to understand how well they work and if they could be improved. Methods: This paper summarises evidence from 105 relevant papers identified through a literature search on the effectiveness and burden of peer review for grant funding. Results: There is a remarkable paucity of evidence about the overall efficiency of peer review for funding allocation, given its centrality to the modern system of science. From the available evidence, we can identify some conclusions around the effectiveness and burden of peer review. The strongest evidence around effectiveness indicates a bias against innovative research. There is also fairly clear evidence that peer review is, at best, a weak predictor of future research performance, and that ratings vary considerably between reviewers. There is some evidence of age bias and cronyism. Good evidence shows that the burden of peer review is high and that around 75% of it falls on applicants. By contrast, many of the efforts to reduce burden are focused on funders and reviewers/panel members. Conclusions: We suggest funders should acknowledge, assess and analyse the uncertainty around peer review, even using reviewers’ uncertainty as an input to funding decisions. Funders could consider a lottery element in some parts of their funding allocation process, to reduce both burden and bias, and allow better evaluation of decision processes. Alternatively, the distribution of scores from different reviewers could be better utilised as a possible way to identify novel, innovative research. Above all, there is a need for open, transparent experimentation and evaluation of different ways to fund research. This also requires more openness across the wider scientific community to support such investigations, acknowledging the lack of evidence about the primacy of the current system and the impossibility of achieving perfection.",
"keywords": [
"peer review",
"grant awarding",
"funding allocation",
"grant reviewing"
],
"content": "Introduction\n\nHealth research has contributed enormously to society, but it is also expensive. This has led to increasing demands to understand and improve how research is supported. Most effort has focused on evaluating impacts of research, on society and the economy. Funders are gathering evidence of impact using online survey platforms such as Researchfish in the UK, and national assessment frameworks including Excellence for Research in Australia (ERA).\n\nMuch less work has focussed on understanding how research is selected for support. Peer review is used to allocate the vast majority of competitive research funding internationally. Therefore it is crucial to understand whether peer review is effective and efficient - whether it can fairly, reliably allocate research funding without bias. In this study, we carried out a rapid evidence assessment which asked whether the peer review process lives up to these aspirations.\n\nThe research was commissioned by the Canadian Institutes of Health Research (CIHR) to support an ongoing review of CIHR’s peer review system, particularly the Peer Review Expert Panel which was convened to review the design and adjudication processes of CIHR’s investigator-initiated research programmes.\n\n\nMethods\n\nWe identified relevant literature through five routes:\n\n1. Google Scholar search using the search terms below, for publications from 2009 onwards. We reviewed the top 500 search results for each query.\n\nSearch terms:\n\n– ‘Grant peer review’\n\n– ‘Grant review’ AND ‘panel’\n\n– (‘Peer review’ AND ‘funding application’) OR (‘peer review’ AND proposal) OR (‘peer review’ AND funding) OR (‘peer review’ AND award) or (‘peer review’ AND ‘reviewer bias’)\n\n2. Grey literature: we searched the websites of major funding bodies and other academic bodies (e.g. learned societies) that we expected to have published relevant research (Table 1).\n\n3. Searching the Cochrane publication list for systematic reviews on grant peer review. This did not identify any relevant reviews conducted since 2009.\n\n4. An initial set of publications already known to the authors and sponsors of the work.\n\n5. Snowballing: from the reference lists of publications identified following screening.\n\nSome elements of our strategy were focused on evidence from the health sciences (particularly grey literature), but our wider searches, including Google Scholar, were not restricted by field of research.\n\nPublications were initially screened on title, and abstract (where available). Studies needed to include empirical consideration of the effectiveness and/or burden of grant review processes. Studies were excluded on the basis of being:\n\n– Purely descriptive, describing a specific peer review process.\n\n– Focused on wider concerns around the funding process, with no (or only tangential) reference to the peer review process in particular.\n\n– Focused on manuscript peer review rather than peer review for funding purposes.\n\n– From 2008 or earlier.\n\n– Reviews, with no additional synthesis or analysis, summarising work from before 2008, or studies already identified and included individually.\n\nIf studies were relevant full text was retrieved and an Excel spreadsheet was used to capture key information on the study and its conclusions.\n\nWe identified 105 studies for inclusion. Table 2 summarises the range of studies identified.\n\nQuality of evidence was rated on a scale of 1–4 based on GRADE (Guyatt et al., 2008)1. We aggregated the overall strength of the evidence for each area of criticism based on the scale in Box 1.\n\n1. Assumptions: Intuitive assumptions and widely shared beliefs prevail\n\n2. Suggestive: There is insufficient evidence to draw a clear conclusion (but the evidence is at least suggestive)\n\n3. Conflicting: There are conflicting results from well-conducted studies\n\n4. Agreement: A number of well-conducted studies agree\n\n5. Compelling: Systematic reviews are compelling.\n\nWhen synthesising our findings, we also drew on our previous review of the topic (Ismail et al., 2009)\n\n\nResults\n\nWe summarise our findings in Table 3 with each discussed in detail below.\n\nThe meaning of ‘best’ science is not fixed. What constitutes the ‘best’ science will vary, however it may include research that is innovative, interdisciplinary and applied. This section considers biases against any particular type of research and whether peer review is a good predictor of future success.\n\nPeer review is probably anti-innovation. Braben (2004) has suggested that supporting highly innovative research is important because it drives technological change and economic growth – an idea increasingly embraced by research funders. NIH has expressed concern at falling numbers of innovative or risky applications, suggesting ‘competitive pressures have pushed researchers to submit more conservative applications’ (Kaplan, 2005; Scarpa, 2006). Low success rates may have exacerbated the situation, inducing ‘conservative, short-term thinking in applicants, reviewers, and funders’ (Alberts et al., 2014). On the other hand, a system is necessary to distinguish between innovative research and that grounded in ‘reckless speculation’ (Hackett & Chubin, 2003). Although ‘innovative research’ and ‘high-risk research’ are often conflated, they are not necessarily synonymous, here we include both aspects of innovation.\n\nInnovative proposals may have less preceding work supporting them, and hence receive less praise from reviewers (RIN, 2010; Spier, 2002). This lack of preceding work requires less risk-averse mind-set from the reviewer (Spier, 2002). Innovative proposals from young researchers may suffer a ‘double disadvantage’: lacking previous work, both because of their novelty and the researcher’s shorter track record.\n\nThe challenge of supporting innovation is not new, in 1977, Thomas Kuhn wrote of an ‘essential tension’ between originality and tradition. These tensions were also included in a 2006 UK Treasury report which noted ‘the UK is still susceptible to a charge of risk aversion, as classic peer review criteria emphasise tests of scholarship over potential impact’ (Treasury, 2006, p. 16). Empirical evidence of this problem come from recent work identifying lower scoring of novel proposals, even controlling for factors such as proposal quality, further this deficit could not be explained by the novel proposals being less feasible (Boudreau et al., 2012; Boudreau et al., 2016).\n\nRisk aversion may also affect the preparation of applications: Fang & Casadevall (2009) suggested that falling success rates lead to conservatism because of the perceived increased risk associated with innovative proposals.\n\nApproaches to these problems include using reviewers with different cognitive biases for different schemes – specifically targeting specialists in translational or high-risk, innovative research (Langfeldt, 2001). This approach has been used (though not evaluated) in NIH’s high-risk, high-reward Pioneer awards (Gewin, 2012).\n\nMaking ‘innovation’ an assessment criteria is another approach (Lindner et al., 2016; Luukkonen, 2012). Views on this are mixed, some suggesting panels lack the expertise to assess innovation (Costello, 2010), whilst others see the approach as effective (Spiegel, 2010). Analysis of NIH application scores suggests that those for innovation are closely correlated with overall scores (Lindner et al., 2016).\n\nOther analysis (Giraudeau et al., 2011; Linton, 2016), suggests that disagreement among scoring could be used to identify innovative research – high disagreement being taken as an indicator of work with high potential but also high risk. Similarly, Lee (2015) suggests combating conservatism by increasing the weight given to criteria – such as innovation – which are typically underweighted by reviewers.\n\nAn approach that sidesteps the issue is to select researchers purely on their merit, regardless of the research they plan to conduct. Researchers then have freedom to pursue new and novel ideas and work flexibly, as opportunities arise (e.g. by the MacArthur Fellows programme4).\n\nFinally, Holliday & Robotin (2010) suggest that a Delphi process (a structured deliberative process) could be used to assess the merits of research ‘in situations where the available scientific evidence is limited and if review panels have widely divergent opinions’. The process was also found to be efficient and flexible from a time perspective.\n\nIt is not clear if peer review treats interdisciplinary research fairly. Critics argue interdisciplinary research is disadvantaged because (1) interdisciplinary proposal reviews may have to combine multiple distinct understandings of ‘quality’ – undermining the strength of the review (Feller, 2006), and (2) it is more difficult to identify ‘peers’ to review such work. This latter challenge is exacerbated by the standard structure of peer review processes in which only a few reviewers examine each proposal in detail, or at the initial stages, reducing the breadth of reviewing expertise further (Gluckman, 2012).\n\nA study on the US National Science Foundation (NSF) revealed that, in interdisciplinary studies at least, peer review favours ‘research that is performed by academics, in the sciences, and that falls completely within the reviewers’ own domain of expertise’ (Porter & Rossini, 1985, p. 37). With interdisciplinary teams it can be hard to isolate the contribution of each researcher, which can reduce the investigators chance of getting further funding by ‘weakening’ their track record (Cooksey, 2006a)\n\nThere has been limited further work in this area since 2009. Increasing the size of the review panel and broadening the range of expertise and disciplines present has been suggested as a way to address these problems. However, this increases burden and can only work if the role of the initial in-depth reviewer(s) is diminished (Gluckman, 2012).\n\nIt is not clear if peer review fairly assesses applied research. The Cooksey Report on health research funding in the UK noted that peer review ‘can in some instances inhibit programmes in translational and applied health research’ (Cooksey, 2006b). The report suggested that one reason for this inhibition was because peer review prevented the iterative development of research projects where funder and researcher worked together. Cooksey also suggested that because applied researchers publish in specialist (i.e. lower-impact) journals, they received less credit for publications than basic researchers. Including research users and considering the likely impact of research as part of the funding process may address these concerns. In our 2009 review, we noted the Canadian Health Services Research Foundation pioneering work through the use of ‘merit review panels’ to evaluate proposals, combining members from both academic and wider user/policy communities. This approach has now spread to other major funders, notably NIHR. Considering impact at the application stage – an approach criticised for disadvantaging innovative research – is likely to be beneficial when reviewing research closer to application.\n\nThe evidence around peer review’s bias against applied research is not strong and has changed little since 2009. It is hard to know what criteria individual reviewers apply, as studies are hampered by methodological problems and funders are reluctant to release scores from peer review panels (Feller, 2006). While several studies have examined how reviewers assess proposals in the humanities and social sciences (Guetzkow et al., 2004; Mansilla, 2006), work in the natural sciences is lacking. A study of NIH shows the success rate of clinical research proposals is marginally less than those for laboratory research (Kotchen et al., 2004). This is in line with a recent CIHR study showing that health services and policy research applications were less successful than biomedical research applications (Tamblyn et al., 2016).\n\nPeer review is at best only a weak predictor of future performance. Work by Fang & Casadevall suggests peer review can ‘winnow’ out bad research proposals (Fang & Casadevall, 2012). However, recent studies from several NIH Institutes and the Netherlands have challenged the idea that peer review can effectively select the best research. Studies comparing percentile application rankings with the research’s subsequent bibliometric performance found no association (Danthi et al., 2014; Danthi et al., 2015; Doyle et al., 2015; Fang et al., 2016; Kaltman et al., 2014; van den Besselaar & Sandström, 2015). Two further such studies found that grant review outcomes only weakly predict bibliometric performance (Lauer et al., 2015; Reinhart, 2009). Bibliometric analyses are by no means perfect measures of performance – only capturing a proxy of academic performance (Belter, 2015). Nonetheless, the findings suggest that peer review assessment is, at best, a crude predictor of performance.\n\nUsing an alternative metric, Galbraith et al. showed that peer reviewers’ opinions were only weakly predictive of the commercial success of early stage technologies in small businesses (Galbraith et al., 2010).\n\nFang & Casadevall (2012) comment that, while reviewers can usually identify the top 20–30 per cent of grant applications, going further to identify the top 10 per cent is ‘impossible without a crystal ball or time machine’ (p.898).\n\nIf peer review is reliable, the judgements of different peer reviewers on the same proposal should be highly correlated. The grounds for the continuing use of peer review would be severely undermined if systematic unreliability were demonstrated. Funders have been criticised for not making sufficient efforts to measure and monitor the reliability of assessments across reviewers (Fang & Casadevall, 2009). In this section, we consider two concerns surrounding peer review, namely individual reviews and overall consistency of decisionmaking – and how they might be addressed.\n\nIt is clear that ratings vary considerably between reviewers. Single-rater reliabilities5 are not encouraging, but have been hampered by the methodological difficulties of modelling the complex interactions between reviewers in multi-stage peer review processes. In particular, the work of Jayasinghe et al. (2003) demonstrates a single-rater reliability correlation of just 0.21 for the humanities and social sciences, and an even lower correlation of 0.19 for the sciences. Similarly, Fogelholm et al. found an inter-rater reliability of around 0.23 for medical research (Fogelholm et al., 2012). In contrast, two studies have found a higher level of agreement between reviwers. The first study which built in some of the complexities of the peer review process, found a dependent reliability6 rating for individual peer reviewers of 0.80. The second study on the review process for Marie Curie Actions (a major EU funding stream) measured inter-rater reliability based on the average deviation in scores between raters, and found a high level of agreement (Pina et al., 2015).\n\nStrikingly, the chance of improvements from initial ratings during panel discussion is virtually nil (e.g. from ‘no award’ or ‘possible award’ to ‘award’). This suggests that initial triage of applications may be preferable to re-rating rounds (Bornmann, et al.).\n\nIncreasing diversity of background and discipline of peer reviewers also reduces rating consistency. Lobb et al. (2013) identified a low intra-class correlation coefficient (0.12) when comparing reviewers from a research, practice or policy background. They also noted that the level of agreement among experts from different disciplines was considerably lower than that among adjudicators of the same discipline, meaning that the presence of several practitioners from the same discipline area could have the potential to skew funding outcomes, depending on the wider makeup of the panel. This suggests that peer review processes may not work well for transdisciplinary teams integrating both academic and non-academic experts. Taking a different perspective, Reinhart found that although the global intra-class correlation coefficient was 0.41, there were considerable differences between fields, for example, biology (0.45) versus medicine (0.20) (Reinhart, 2009).\n\nThere is conflicting evidence on whether peer review can achieve acceptable levels of decisionmaking consistency. Existing studies offer mixed judgements on the reliability of grant peer review. Bornmann identified a threshold of 80–90 per cent as the expectation for agreement for this kind of decisionmaking (Bornmann et al., 2008). Two early studies (Cole et al., 1981; Hodgson, 1997) we noted in 2009 found reliability rates across funding boards of 75 and 73 per cent respectively for funding decisions which they felt was a satisfactory level of agreement. More recent evidence is mixed. The most recent study comparing the outcome of two independent panels found an agreement rate of 83 per cent (Clarke et al., 2016), whilst a previous study in 2012 was less favourable, showing agreement levels of 65–69 per cent (Fogelholm et al., 2012).\n\nGraves et al. (2011) examined the variability of panel members’ individual scores and calculated how this translates into the variability of overall proposal ranking, and hence funding decisions. They found that such variability could affect the outcome for 29 per cent of the proposals considered, and that variability differed widely between panels. Abdoul et al. have suggested that scoring variability might be partially explained by differences in reviewer behaviour, such as the time taken to do the assessment, assessment methods, and variation in the relative weighting of different criteria by different reviewers (Abdoul et al., 2012).\n\nRecent studies focusing more on the impact of panel meetings have shown very limited effects on improving consistency and reliability. Fogelholm et al. (2012) suggested that mean reviewer scores prior to the panel meeting were similar to the panel consensus score. The authors concluded that using the mean reviewers’ scores was a practical and economical alternative. Similarly, although Pina et al. (2015) identified both a subset of panels and subset of proposals with high levels of disagreement, where consensus meetings improved agreement, across the whole population they could not detect an overall improvement in agreement.\n\nIn contrast, Martin et al. (2010) found meeting discussions had an important effect in more than 13 per cent of applications in their analysis of a sample of standard (R01) NIH research grant applications.\n\nTwo funders have experimented with, and evaluated, virtual peer review both by teleconference and through the use of Second Life, a virtual world. NIH estimated that using Second Life telepresence, peer review could cut panel costs by one third (Bohannon, 2011). Pier et al. (2015) compared videoconference and face-to-face panels. They set up one videoconference and three face-to-face panels modelled on NIH review procedures, concluding that scoring was similar between face-to-face and videoconference panels. Both the Bohannon and Pier studies of virtual panels noted that participants valued the social aspects of meeting in person and preferred the face-to-face arrangements.\n\nGallo et al. (2013) examined four years of peer review discussions, two years face-to-face and two years teleconferencing. They found minimal differences in merit score distribution, inter-rater reliability or reviewer demographics. They also noted that panel discussion, of any type, only affects the funding decision for around 10 per cent of applications relative to original scores.\n\nApproaches to improve reliability have been tried. The NIH peer review self-study suggested some possible improvements to the peer review process to combat low reliability, focusing principally on better training for reviewers (NIH, 2008). NIH suggested such training should focus on: (1) emphasising the strengths (rather than weaknesses) of research proposals; (2) focusing on the potential impact of research; (3) reviewing the merit of the proposal and not re-writing it; (4) recognising the problem of implicit bias in study sections; (5) using benchmark applications during panel meetings to provide review guidelines; and (6) pointing out potential bias towards lesser known applicant organisations.\n\nRecent work by Sattler et al. (2015) has evaluated the effect this type of brief training programme. The study found inter-rater reliability increased from 0.61 to 0.89, and the amount of time spent reviewing also increased, for both new and experienced reviewers.\n\nIf inconsistency stems from discrepancies in review quality (which is by no means clear), it might be feasible to evaluate the quality of reviews, although this approach has its own challenges – for example, what is a ‘good’ review? If a review is not consistent with other review does that intrinsically make it ‘bad’? It could be the outlier picking up on the true potential of an innovative application. However, this approach is used by many funders, as shown in a report by the European Science Foundation (2011) which found in a survey of European research funders that more than half (60 per cent) evaluate the quality of all reviews as standard practice using a range of criteria (e.g. completeness, level of substantiation, appropriateness, comprehensibility, timeliness and usefulness), and may return the review to a reviewer or reject the review. Organisations felt that review quality was higher where these checks were made, but noted little difference quality between cases where all reviews are evaluated versus just a sample. However, no data was available to assess these suggestions, and no empirical analysis had been carried out. Adding such an evaluation process clearly adds to the burden of the process.\n\nIs peer review fair? Having considered the evidence suggesting that consensus on peer review decisions is rare, what factors might underlie the observed discrepancies? To what extent is peer review open to the same allegations of bias that plague science more widely, particularly around gender, race, intellectual school or institutional affiliation? A recent study (Day, 2015) has shown that low levels of passive bias as well as individual cases of significant active bias among reviewers can have significant impacts on the outcomes of a grant peer review process. In this section we consider the potential for bias in peer review across four main areas: gender, age, cronyism and cognitive particularism.\n\nBias could occur at various places in the peer review process. While bias on the part of the peer reviewers themselves (such as sexism or racism) has received considerable attention in the literature, funding competitions can be biased through eligibility and award selection criteria. Such criteria may be prejudiced against early career researchers or innovative research – although there is no strong evidence that this occurs. In addition wider systemic biases may mean that the number of applications received is lower from particular groups.\n\nBlinding of applications provides a defence against the most obvious abuses by reviewers – rejecting proposals on the grounds of race, gender, institutional affiliation and so forth (Lee et al., 2012). A study from South Korea by Lee et al. (2000) demonstrated a significant bias in sighted proposal evaluation towards those from particular research departments, senior researchers, and those already academically recognised. This is reinforced by a review of studies by the NSF, which found only ‘a weak correlation’ between panel ratings of blinded short version and unblinded full versions of the same applications (Bhattacharjee, 2012). While some funding bodies now routinely attempt to anonymise proposals before passing them on to reviewers, there is some dispute as to whether anonymisation is truly possible. Some authors contend that some degree of identification is always possible from anonymised research proposals (Bhattacharjee, 2012)\n\nThere is a substantial body of conflicting evidence on whether peer review is gender biased. The evidence on gender bias is inconclusive. Studies suggesting bias include an important study of the grant peer review system of the Swedish Medical Research Council strongly suggested that reviewers were unable to judge scientific merit independently of gender (Wenneras & Wold, 1997). These findings were supported by a subsequent meta-analysis of 21 studies on this topic, which found that grant applications submitted by men were 7 per cent more likely to be approved than those submitted by women (Bornmann et al., 2007).7 Furthermore, recent studies have also found evidence of gender bias (Jang et al., 2016; Kaatz et al., 2014; Kaatz et al., 2015; Tamblyn et al., 2016; van der Lee & Ellemers, 2015; Volker & Steenbeek, 2015). For example, van der Lee & Ellemers (2015) reported a 4 per cent ‘loss’ of women during the grant review process for awards to early career scientists by the Netherlands Organization for Scientific Research (NWO). In a review of research on gender bias by Kaatz et al. (2014), women generally have lower rates of publication and lower success rates for high-status research awards than do men.\n\nOn the other hand, a review of the gender bias literature by Ceci & Williams (2011) showed that the weight of evidence suggests that peer review is fair across gender, with all smaller-scale studies analysed, along with all but one of the large-scale studies, failing to replicate Wenneras & Wold’s findings. And even for the remaining large-scale study the findings were reversed by a reanalysis. The lack of gender bias has been supported by several subsequent studies (Marsh et al., 2011; Mutz et al., 2012; Reinhart, 2009; Turner et al., 2014; Van Arensbergen et al., 2013).\n\nThere is a small conflicting evidence base on whether peer review is biased by age. Although review processes that partly rely on the previous publications or funding successes of the applicant may be biased against early career researchers, Jayasinghe et al. (2001); Jayasinghe et al. (2003) found that the age of the applicants did not directly impact upon grant success, a findings supported by Reinhart (2009). However, this finding is directly contradicted by a study comparing sighted and blinded reviews of research grant proposals in South Korea (Lee et al., 2000). A subsequent study, also based in South Korea (Jang et al., 2016), found that evaluation scores and selection success rates decline with age.\n\nConcerns about age bias are closely tied to concerns about bias against early career researchers, who may be disadvantaged through lacking preliminary results or a substantial portfolio of work. The challenges of providing adequate support for early career researchers is widely recognised (Bazeley, 2003) and was raised in a 2008 NIH review which identified significant decreases early career success rates which could not be accounted for by variations in application quality (NIH, 2008). Similar concerns were also noted by Spiegel (2010), who showed that the average age researchers won their first full NIH project grant awards (R01) had been steadily increasing. Since then, the NIH has introduced measures aimed at equalising success rates for new and established investigators for new (not renewal) applications.\n\nThere is evidence that peer review suffers from cronyism. Cronyism is a concern for many major funders, who have detailed conflict of interest processes in place to counter the presence or perception of such biases. However, (Wenneras & Wold, 1997) show that prior affiliation with a reviewer considerably increased a researcher’s chances of funding, Similarly, a large-scale study of applications to the National Science Foundation of Korea found that applications reviewed by previous or current affiliates were more likely to be successful (Jang et al., 2016). A review of NSF proposals by (Bhattacharjee, 2012), found that panel assessments of full proposals and shorter anonymised versions of the same proposals showed weak correlations.\n\nLuukkonen (2012) notes that panel debate may fail to counter crude forms of cronyism since panels often cover a wide area of research, and each specific area is only represented by a few experts, so the other members may defer to the experts’ knowledge. Members of funding panels may also benefit directly from their membership. One study noted that panel members submit more applications, and have more grant awards (van den Besselaar, 2012). The challenge in this area is separating factors such as good researchers who submit more applications being selected to join panels or having a better sense of what makes a good application, from nepotism.\n\nThere is conflicting evidence on whether peer review demonstrates cognitive particularism (favouring your own field or way of thinking). The idea that reviewers and panel members will favour proposals in their own fields or that align with their ways of thinking has been termed ‘cognitive particularism’ (Travis & Collins, 1991). Fang & Casadevall (2009), suggest that ‘reviewer biases favour topics well understood and appreciated by the [funding panel]’ (p.930). Travis & Collins (1991) found that reviewers tend to favour proposals supporting their own school of thought, and argues that this is likely to have a much bigger impact on the direction of science than institutional bias or cronyism identified by other studies (Langfeldt, 2006; Wenneras & Wold, 1997). Research by Li (2015) suggests the same. Work by Wang & Sandström (2015) suggests that ‘cognitive distance’ may influence reviewer decisions in a more complicated way, with reviewers more likely to favour applications in areas they are either very familiar with, or completely unfamiliar with. Other studies find that reviewers are more critical of applications in areas of their own expertise (Boudreau et al., 2016; Gallo et al., 2016).\n\nA number of studies suggest that studies in molecular biology are more likely to be successful in comparison to other fields of bioscience. (Bornmann & Daniel, 2006) found a slight statistical effect and further studies reveal that peer-reviewed grant proposals in molecular biology tend to have a better chance of receiving grant funding than proposals in other bioscience fields (Kotchen et al., 2004; Taylor, 2001).\n\nThere is also dispute about how to resolve this potential problem Alberts et al. (2014) suggests that such effects could be countered by broadening ‘the range of scientific problems judged by each group and include[ing] a diversity of fields on each panel’, suggesting that ‘senior scientists with a wide appreciation for different fields can play important roles by counteracting the tendency of specialists to overvalue work in their own field’ (p.5777). However, Li (2015) advises caution, noting that though evaluators may be biased in favour of projects in their own area, they are also likely to be better able to assess the quality of those projects, and the benefits of this expertise may well outweigh any possible biases.\n\nThere is suggestive evidence that the peer review process slows, and hinders, the progress of research. In some cases such as an emerging epidemic the time taken by peer review could reduce the number of people benefiting from the research, such slowing of the research process could also reduce the economic viability of a new product, (e.g. Agres, 2005; Cures, 2005; Daniels, 2004; Roy, 1985). The many stages of grant peer review can take from 9 to 18 months from submission to funding. It is less clear how often this time significantly hinders the progress of science. In the health sciences, research is one of many steps in develop new treatments and practices (Hanney et al., 2015). Research suggests that the time required for translation of research from initial idea to adopted practice is around 17 years, so peer review may be a relatively small contributor, however any one translation pathway may have multiple stages of peer review (Morris et al., 2011).\n\nThere is good evidence that peer review has the support of most major scientific stakeholders. Though criticism of the peer review process abounds, empirical evidence, though limited, indicates that support for peer review amongst the academic community remain strong (Bornmann, 2011; Wooding & Grant, 2003). The dominance of peer review across funding systems internationally suggests it has the confidence of institutional stakeholders. A recent review of literature about the NIH peer review processes found a firm belief in the transparency and objectivity of peer review amongst grant reviewers (Miner, 2011).\n\nIn contrast an emerging body of literature suggests traditional academic peer review may not be appropriate for all types of research. A recent study on indigenous research showed the competitive nature of peer review was counterproductive and that peer review did not have the confidence of relevant stakeholders (Street et al., 2009). Similar concerns have been expressed about the assessment of community engagement proposals (Ahmed & Palermo, 2010).\n\nThe burden of peer review is increasing. In a survey of 28 biomedical research funding organisations across 19 countries (Schroter et al., 2010), declined review requests, late reports and administrative burden were the most frequently mentioned challenges, and all organisations reported an increase in burden in the previous five years (although they reported that the quality of reviews had remained the same). A study by the Royal Society of New Zealand reported a similar increase in the difficulty of recruiting senior reviewers (Gluckman, 2012).\n\nThe burden of the peer review system is high and falls primarily on the applicants. The overall monetised cost of the peer review system, including application preparation, has been estimated to account for as much as 20–35 per cent of the allocated budget (Gluckman, 2012). Graves et al. (2011) report that the monetised costs of the application system for NHMRC are $14,000 per grant, whilst extrapolating RCUK (Research Councils UK, 2006) estimates suggests that the costs of the application process are 10–17 per cent of the total cost of research. An evaluation of the CIHR Operating Open Grants Program (OOGP) found the application cost of OOGP grants to be Can$14,000 (Peckham et al., 2012). When providing congressional testimony individual researchers have estimated that as much as 60 per cent of their time is devoted to seeking funding (Fang & Casadevall, 2009).\n\nBurden on applicants. The bulk of the resources consumed by the peer review process are in the writing and reviewing of applications. RCUK work showed the distribution of monetised burden was 74 per cent in application production, 21 per cent in reviewing process (including time of reviewers, panel membership and modifying proposals), and 5 per cent in Research Council costs and payments to reviewers (Research Councils UK, 2006).\n\nMore recent work by Graves et al. (2011) used a small survey of NHMRC researchers to estimate that the burden fell even more heavily on the applicants, assigning a split of 85 per cent for application production, 9 per cent for reviewing and 5 per cent for administration. Barnett et al. (2015) reinforced this conclusion with a larger survey of 285 applicants who had submitted 632 proposals to four health services research funding rounds from May 2012 to November 2013, at the Australian Centre for Health Services Innovation. A review by the New Zealand Royal Society made a similar estimate of the burden shouldered by the applicants – pegging it at 80 per cent (Gluckman, 2012).\n\nIn contrast two studies of the Natural Sciences and Engineering Research Council (NSERC) of Canada peer review process and came to strikingly different conclusions. Gordon & Poulin (2009) estimated the cost of the NSERC system, including application preparation, review and administration costs at Can$44m. They suggest this money could alternatively provide all researchers in the field with an annual baseline grant of Can$30,000. However, Roorda (2009) takes issue with Gordon and Poulin’s assumptions suggesting they have overestimated costs by a factor of 23. The correct answer appears to be in between – there is disagreement about how the costs should be allocated and neither side provides a justification of their estimates of the time spent on grant preparation (the key driver).\n\nHerbert et al. (2013) suggest burden on NHMRC applicants could be reduced by simplifying the application process (currently 80–120 page applications). Other examples of funding agencies reducing the length and complexity of applications include NIH did cut the length of their applications for R01s8 from 25 pages to 12 in 2009, although there were calls to make the application even shorter (Fang & Casadevall, 2009).\n\nBarnett et al. (2015) examined the effect of reducing the complexity of the application. Surprisingly, they found that reducing application complexity slightly increased preparation time. They suggest that this may be because researchers allocate a fixed fraction of their time to application preparation. Theoretical work by Geard & Noble (2010) using agent based modelling found that applicants devote ‘excessive’ time to proposal preparation (Geard & Noble, 2010). Barnett et al. (2015) examined four rounds of a funding scheme in Australian which significantly shortened the application (to 1,200 words). Qualitative feedback was positive, suggesting it took seven days to develop an application, but generalisability is limited. The level of effort devoted to application preparation is all the more striking given Herbert et al.’s (2013) finding that increased effort did not translate into increased success rates.\n\nA few qualitative studies have examined the burden of the system on particular groups of researchers and the wider implications on researchers’ quality of life. A survey of 215 NHMRC applicants concluded that the ‘impact of preparing grant proposals for a single annual deadline is stressful, time consuming and conflicts with family responsibilities’ (p.1), although it did not quantify the effects or time taken (Herbert et al., 2014). A study of early career investigators applying for funding at CIHR identified the application process as burdensome and noted the decrease in success rates for open operating grants from 30 per cent in 2005–2006 to 15 per cent in 2014–2015 (Association of Canadian Early Career Health Researchers, 2016).\n\nThe institutional costs of application preparation were examined by the US Government Accountability Office (GAO) in 2016, which concluded that pre-award requirements for applicants to develop and submit detailed documentation for grant proposals, and increased prescriptiveness of certain requirements, had increased universities’ workload and costs, but the study (GAO, 2016) did not quantify these increases\n\nBurden on reviewers and panel members. Time invested by reviewers and panel members is consistently identified as the second-highest monetised cost of peer review, making up about 15 per cent of the burden. Two types of studies carried out in this area have both aimed at optimising the process, balancing the trade-off between burden and quality to achieve efficiency.\n\nThe first study approach trialled simplified processes for grant review to test how much time they save and whether they affected funding decisions (Herbert et al., 2015) – particular the use of a shortened application form and smaller review panels. They found the simplified processes achieved agreement with the current award system of close to 75 per cent (which they suggested was the ‘acceptable’ threshold based on a review of previous surveys), at estimated savings of 33–78 per cent of review costs.\n\nThe second study used statistical techniques to estimate the optimum number of reviewers (Snell, 2015) trading off improved reproducibility with additional reviewer burden. They found that five reviewers were optimal; similar work by Graves et al. (2011) on a different funding scheme found 11 reviewers was the most effective number.\n\nIn addition to experimental changes there are examples of funding agency policy changes that have been examined. The NSF changed its review procedures in 2012 to reduce burden by introducing triage on short preliminary applications with a 75 per cent cull rate, with annual rather than six-monthly applications. The General Accountability Office has praised the system and it reduces administrative burden on programme officers. However, because several changes happened simultaneously, it is not clear whether this is because of the triaging. It also resulted in reduced success rates, partly because of more applications (perhaps because they were easier to write but also because of funding reductions (Mervis, 2016).\n\nOne of the drivers of the burden on funders is identifying appropriate reviewers for each proposal. Mervis (2014) reports on a radical experiment at NSF where applicants reviewed each other’s grants (each applicant completing seven reviews), consequently reducing this burden to zero. To guard against applicants marking their competitors down, they were rewarded for scores that aligned with the other reviewers. The pilot allowed the number of reviews per proposal to be increased from three or four to seven and the reviews provided were more detailed. Because of the additional reviews, NSF was able to dispense with panel discussion, thus saving administrative costs.\n\n\nDiscussion\n\nIn this section we summarise our findings: firstly, on the availability of evidence, considering the scope and coverage of the existing literature; secondly, on what that the evidence shows, and finally, highlighting the implications for health research funders.\n\nQuestions about the effectiveness and burden of peer review can be addressed at two levels. At a high level, does peer review support valuable science? And at a lower level, can the design of peer review systems be improved to increase effectiveness and reduce burden?\n\nIt is clear that the current system of funding has produced significant benefits for society, suggesting that peer review supports valuable science. However, whether peer review is demonstrably better than any other system is impossible to judge with certainty because of the lack of comparators: no funding agencies have made significant use of alternative systems.\n\nMoving to the lower level, considering comparisons between or research on peer review systems, there is only a very small number of robust, well-conducted studies. Much of the literature identified is anecdotal in nature and we found no systematic reviews, underlining the fragility of the evidence base. However, we did identify a series of robust, high-quality studies that have been carried out since our last review in 2009. Despite this new work it is still true that most studies examine the peer review process of one particular funder in one particular context, rather than looking across funders or contexts, and few go beyond process measures to judge effectiveness.\n\nThis persistent lack of evidence about the allocation of the ‘inputs’ to research is all the more striking given the advances in understanding the outputs and outcomes of research through research impact assessment over the last decade.\n\nThe central problem when assessing peer review is the lack of an absolute standard or ‘ground truth’ to judge against. There will be uncertainty in all peer review decisions - it is, after all, predicting the future. And there is evidence suggesting it is not a particularly good predictor, at least for bibliometric performance. At present most funders do not capture, use, or even acknowledge this uncertainty, despite clear evidence of inconsistency in peer review ratings and mixed evidence on the reproducibility of panel decisions.\n\nThese is good evidence that peer review suffers from biases. The strongest evidence is of a bias against innovation and although a range of improvements have been suggested, none have been robustly evaluated. There is some evidence peer review is influenced by cognitive distance and suffers from cronyism and suggestive evidence that there are age biases. Considerable work has been done on gender bias, with conflicting results, which illustrates the challenges of accounting for biases outside the scope of the peer review process, for example through eligibility or the culture of the wider scientific system.\n\nThough the problem of burden is widely recognised, funders’ considerations often focus on their own and reviewers’ burden as these are more immediately visible (and costly) to them. However, it is clear that the burden largely falls on applicants (rather than reviewers or panel members).\n\nFalling success rates across many funders compound the burden on applicants. One way to address these challenges could be to reduce the complexity of the application process, with evidence suggesting similar decisions can be made with much shorter applications and less information. However, small decreases in application length do not seem to translate into application preparation time so such changes would need to be carefully evaluated.\n\nDespite the plethora of comment pieces criticising the peer review system, there is no empirical evidence suggesting whether peer review has more or less support among key stakeholders than it did in 2009.\n\nImproving effectiveness. This section outlines our reflections on ideas for improving peer review processes.\n\nWe feel the uncertainty in peer review - clear in the inconsistency of ratings and weak predictive power in terms of future academic performance - should be acknowledged, captured and used to improve decision making and for analysis. Reviewers should be asked both for their rating of the proposal and a measure of their confidence in this rating - some smaller funders, such as the Villum and Velux Foundations in Denmark, are starting to implement such systems. Funders could also analyse levels of disagreement between reviewers, which may be an indicator of innovative research (Linton, 2016), or take a portfolio approach selecting projects scoring highly across different criteria, including innovation (Lee, 2015).\n\nA second approach is to acknowledge the difficulty of predicting the future and introduce an explicit element of randomness into the allocation system. This could be done to differing extents – from completely random allocation of funding to the use of a lottery system within set groups of applicants. Fang & Casadevall (2016) propose a two-stage system, in which the best applications are identified and then a smaller percentage are funded using a lottery. Avin (2015) proposes using two thresholds, above the higher threshold all applications are funded and below the lower threshold all applications are rejected, applications between the two thresholds are funded at random, effectively blurring the funding line.\n\nA lottery approach should reduce biases in decision making since the selection from the fundable pool is random; however, applicant eligibility restrictions/selection for the lottery could reintroduce bias. Selecting into a fundable pool requires less fine-grained decisions addressing concerns about the reliability of peer review. The use of lottery systems is a promising, but politically challenging idea, so far is has only been used in very limited cases, such as the Explorer Grants offered by the Health Research Council of New Zealand9, and as such we think it merits further empirical research (Barnett, 2016).\n\nOther approaches to address bias include blinding of reviewers (e.g. Lee et al., 2012), though the feasibility of this is debated ((Bhattacharjee, 2012). Other funders have also used training approaches to address bias (e.g. CIHR) and to improve quality of reviews (e.g. NIH, 2008) and there is limited evidence that the approach could reduce the discrepancies between reviewers (Sattler et al., 2015).\n\nReducing Burden. Applicant burden should be considered as a priority compared to reviewer and administrative burden as it represents around 75% of the system burden. This can be addressed by reducing the level of burden or increasing the value unsuccessful applicants receive by applying. Changes to reduce burden need to be carefully evaluated as there is evidence that even significant reductions in application length/complexity may not reduce applicant burden as much as expected. An alternative approach is to make the process more valuable for the applicants. Reviewer and panel feedback may be one way to do this.\n\nTechnology provides ways to reduce the time burden of the peer review process for panel members and funders - for example by eliminating travel - and does not appear to significantly affect the outcomes. However, face-to-face discussion of applications brings other side-benefits, including social interaction and network formation, other research suggests these side-benefits may be important to the progress of science and hence may need to be supported in other ways if peer review is done remotely.\n\nImproving the evidence base. It remains striking how little robust evidence is available about peer review as a method for grant allocation. Given the centrality of the peer review process in the current science funding system, there is a need for better evidence, not only on the overall effectiveness of peer review but also to help improve the design of peer review processes. We suggest three fruitful areas for investigator are the links between the peer review process and the wider context of science funding; the social processes of peer review and panel meetings.\n\nSystem impacts affect the peer review process, and peer review changes affect the system, so both need to be considered together to understand the dynamic behaviour of the overall research process. All of the studies we identified considered aspects of the peer review system in isolation – for example tracking success rates or reviewer burden. However, system changes such as decreased funding, or changes in researcher demographics, often happen alongside changes to the peer review system.\n\nEven in the fairly barren landscape of evidence we explored, it was startling that we could find no studies examining the social processes that occur during panel discussions – a central part of the peer review process. Such studies will clearly be challenging and require the cooperation of funders working in concert, but we feel are essential to understand how to optimise one of the fundamental processes of science.\n\nAt a more mundane level, funders should be more willing to experiment with, evaluate and publish results from evaluations of alternative approaches. Through our conversations with funders it appears that where analysis is carried out it is often not published, partly because of the extreme sensitivity around funding allocation procedures. Funders are not the only ones who need to take a more reflective approach: they will need the support of the wider scientific community to support such investigations, and acknowledge the lack of evidence about the primacy of the current system and the impossibility of achieving perfection.\n\n\nConclusions\n\nMany criticisms of the peer review system reflect conflicts between the needs of stakeholders. Researchers look to peer review to uphold research standards and promote the ‘best’ science, while politicians and funders use it to provide accountability for spending (Viner et al., 2004). This tension requires peer review to both protect the identities of reviewers while appearing transparent to applicants; to be innovative yet assure quality; to be based on human judgement yet free of human biases (Hackett & Chubin, 2003).\n\nWe think that current dissatisfaction with the peer review process is amplified by falling success rates, so it is important to remember that the concerns around peer review are heavily influenced by funding policy and the size of research budgets.\n\nAs a society, if we are to improve how we use our research funds, we need a better understanding of the peer review process. When making changes, funders should: build in before and after comparisons; strive to make data available for analysis; openly publish studies of their processes and work together on comparative analysis.\n\nWe need to overcome the reluctance of funders and scientists to acknowledge the uncertainties intrinsic to allocating research funding, and encourage them to experiment with peer review and other allocation processes.\n\n\nNotes\n\n1GRADE is an internationally accepted system for the assessment of evidence quality. GRADE offers four levels of evidence quality: high, moderate, low, and very low. Randomised trials begin as high-quality evidence and observational studies as low-quality evidence, and studies may be downgraded as a result of limitations in study design or implementation, imprecision of estimates, variability in results, indirectness of evidence, or publication bias. Equally, quality may be upgraded based on a very large magnitude of effect or if all plausible biases would reduce an apparent effect (Guyatt et al., 2008).\n\n4As of 5 January 2017: https://www.macfound.org/programs/fellows/\n\n5Defined as ‘the correlation between two independent assessors of the same submissions across a large number of different submissions’ (Jayasinghe et al., 2003, p.280).\n\n6In a multi-stage review process, the assessor at each evaluation stage will know the score given to a particular research proposal at the previous stage. This particular study assessed the reliability of grant peer review processes by determining the proportion of those applications for which the dependent ratings on the same proposal did not change from the first to the second and third stage.\n\n7Bornmann et al. (2007) are clear, however, that the reasons for this observed discrepancy are not known. This is important because aggregation effects over a range of fields of study may – as the authors acknowledge –create strong statistical effects implying gender bias. The authors also suggest that future improvements to the model will need to take into account the cohort of application, since the study described here covered publications produced over the period 1979–2004, and there have been significant changes to reduce gender bias in science and science funding over this period.\n\n8The Research Project Grant (R01) is the original and historically oldest grant mechanism used by NIH. The R01 provides support for health-related research and development based on the mission of the NIH. R01s can be investigator-initiated or can solicited via a Request for Applications.\n\n9As of 5 January 2017: http://www.hrc.govt.nz/funding-opportunities/researcher-initiated-proposals/explorer-grants",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis report was produced with funding from the Canadian Institutes of Health Research.\n\n\nAcknowledgements\n\nThe authors acknowledge the Performance and Accountability Branch at the Canadian Institutes of Health Research for funding this study, and Sarah Viehbeck, Shevaun Corey, Kwadwo Bosompra, Michael Goodyer and David Peckham in that Branch for their input and advice on the development of the work.\n\nThe views expressed in this report are those of the authors and do not necessarily reflect those of the Canadian Institutes of Health Research.\n\nWe would also like to thank our RAND Europe quality assurance reviewers Catherine Lichten and Gavin Cochrane for their helpful comments and suggestions.\n\n\nReferences\n\nAbdoul H, Perrey C, Amiel P, et al.: Peer review of grant applications: criteria used and qualitative study of reviewer practices. PLoS One. 2012; 7(9): e46054. 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}
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[
{
"id": "24827",
"date": "09 Aug 2017",
"name": "Adrian Barnett",
"expertise": [
"Reviewer Expertise Statistics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very useful review of an area that is vitally important for science and that is constantly being examined by funding agencies. It included some papers that I had not read, but there were a few additional papers that I thought could be included (detailed below).\nThe results once again highlight the incredible lack of studies in this area. The paper ends with some sensible recommendations, including the need for funders to experiment more and make their data available to researchers.\nI was surprised that some of the more innovative solutions to funding peer review were not included, specifically using prediction markets 1 and using the “wisdom of the crowd”2.\nWhy was 2009 chosen as the time threshold? Is it because that was the year of the previous review?\nThis paper should be included in the discussion on interdisciplinary research: Interdisciplinary research has consistently lower funding success, Lindell Bromham, Russell Dinnage & Xia Hua. Nature 534, 684–687 doi:10.1038/nature18315 3. This paper agrees with the other two mentioned, as there are lower success rates for applications with more cross-disciplinary researchers.\n\nOne force against “cognitive particularism” is that strict conflict of interest rules from funding bodies can often rule out reviewers with the greatest knowledge, particularly in small fields or small countries. This study may touch on this issue: Abdoul H et al, Non-Financial Conflicts of Interest in Academic Grant Evaluation: A Qualitative Study of Multiple Stakeholders in France, PLoS ONE, 7/4: e35247.4\nIn terms of using technology in the review process, some researchers have suggested that videos may produce more reliable peer reviewer ratings and take less time to prepare: Doran MR, Lott WB, Doran SE. Trends Biochem Sci. 2014 Apr;39(4):151-3. doi: 10.1016/j.tibs.2014.01.004. Multimedia: a necessary step in the evolution of research funding applications5.\nMinor comments\nIntroduction, 1st paragraph. As an Australian researcher I would argue that the ERA has not really measured research quality, rather it has simply measured research output. Maybe you could say “Funders have attempted to gather evidence…”\n\nThe “>95%” figure in the abstract feels about right, but is there a reference for this?\n\nFor the Google search terms, “fellowship” could also have been added, so “Fellowship OR funding”.\n\nThe link to this paper did not work: The Novelty Paradox & Bias for Normal Science: Evidence from Randomized Medical Grant Proposal Evaluations\n\nPage 6, “when reviewing research closer to application”, I didn’t understand this.\n\nPage 7, “only affects the funding decision for around 10 per cent of applications relative to original scores” but that could still be an important percentage, particularly if it’s those near the funding line\n\nPage 9, “found that panel assessments of full proposals and shorter anonymised versions of the same proposals showed weak correlations” do you need to add, “implying that knowledge of the applicants influences the score”. Although as well as a change in blinding there was also a change in the size of the application, so it may be hard to conclude anything about cronyism here.\n\nFootnote 9, the NZ Health Research Council has been using random allocation for this scheme since at least April 2015\n\nI agree that giving more feedback would improve the value of the process (page 12), but our experience with the NHMRC is that this also opens them up to appeals which can take a lot of time for their staff. This doesn’t mean that we shouldn’t try, and giving frank feedback was a feature of a funding scheme we designed (already cited paper “Streamlined research funding using short proposals and accelerated peer review: an observational study”).\n\nThis paper may be of interest: Scientometrics, July 2016, Volume 108, Issue 1, pp 263–288, The consequences of competition: simulating the effects of research grant allocation strategies.6\n\nThis parody of a grant application may be useful for the section on peer reviewers being biased against innovative proposals: doi:10.1097/ede.0000000000000453 “John Snow’s Grant Application”7\n\nThis paper examined the costs of applying for NIH funding and could be included in the section on the costs to applicants: Nursing Outlook, Volume 63, Issue 6, November–December 2015, Pages 639-649, Time and costs of preparing and submitting an NIH grant application at a school of nursing, https://doi.org/10.1016/j.outlook.2015.09.0038\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? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "3522",
"date": "27 Mar 2018",
"name": "Steven Wooding",
"role": "Author Response",
"response": "Thank you for your review and helpful comments. We have made some revisions to the paper in response to these suggestions. In particular, we have added most of the references you suggest to the paper and made some clarifications where required. We have also updated methods with the reason for the 2009 cut off and run searches using the term ‘fellowship’ as suggested, which added two additional papers to the review. Responses to main specific points raised are as follows: · Prediction markets and “wisdom of the crowds”: These approaches are complete alternatives to peer review, rather than refinements, and hence are beyond the scope of the review. The wisdom of the crowds approach is for ex post evaluation rather than ex ante – it simulated REF2014 assessments. · 2009 time threshold: Yes, this is because it was the date of the previous review. · Paper by Bromham et al: Added · Conflict of interest rules: Interesting point, but we don’t have any evidence which would enable us to comment on this in detail. The study by Abdoul et al you note mainly concerns the awareness that reviewers have of conflicts of interests rather than the effects of these conflicts in terms of excluding knowledgeable reviewers · Use of videos: Added a note that this approach has been suggested but not yet evaluated. · Minor comments also all addressed as appropriate within the revised paper."
}
]
},
{
"id": "24828",
"date": "14 Aug 2017",
"name": "Aled Morgan Edwards",
"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 nice and comprehensive review of the studies that assess the strengths and weaknesses of the peer review process.\nAlthough there are no new conclusions, it does reveal or highlight the three major issues that plague our mechanism to dole out research funding and the use of peer review.\n\nThe first is fundamental - without a clear and agreed upon definition of what constitutes the \"best\" science, or the \"best\" outcomes, it will be impossible to know whether the grant adjudication process is meeting, or can ever meet, its objectives.\n\nSecond, the paper highlights what is often overlooked, and this is the real cost in time of writing funding proposals. Although scientists often manufacture a narrative that writing grants is good for them (for example, making you catch up on the literature), the real costs to the system are rarely factored in.\n\nI think this paper did a nice job in highlighted this issue.\nFinally, I would have liked a greater discussion on what is an amazing disconnect.\n\nAll the empirical evidence highlights the deficiencies of the process to allocate grant funding. It is clear that it is neither scientifically founded, nor evidence-based.\n\nYet one of the only strongly supported aspects of the peer review process is that is has the strong support of the community. I find this fascinating.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "3521",
"date": "27 Mar 2018",
"name": "Steven Wooding",
"role": "Author Response",
"response": "Thank you for your review and comments. Reflecting on the disconnect you highlight in the final paragraph above we have added a sentence to the paper commenting on this and noting it as a topic that might warrant further investigation."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1335
|
https://f1000research.com/articles/7-284/v1
|
06 Mar 18
|
{
"type": "Case Report",
"title": "Case Report: Dermoscopic features of oral lichen planus - the evolution of mucoscopy",
"authors": [
"Sidharth Sonthalia",
"Sangeeta Varma",
"Abhijeet Kumar Jha",
"Deepak Jakhar",
"Feroze Kaliyadan",
"Sangeeta Varma",
"Abhijeet Kumar Jha",
"Deepak Jakhar",
"Feroze Kaliyadan"
],
"abstract": "Dermoscopy, a non-invasive technique for cutaneous diagnosis is being increasingly studied in various disorders of the skin, nails and scalp. However, it has been under-utilized for the diagnosis and characterization of mucosal disorders. The dermoscopic characterization of cutaneous lichen planus and its variants has been well documented with Wickham’s striae constituting the hallmark of the condition. However, the dermoscopic features of oral lichen planus with hand-held or videodermoscopy remain to be elucidated. We present the case of a young adult man who presented with asymptomatic white lacy lesions over a bluish-black background over the tongue, patchy hyperpigmentation of the buccal mucosa and gingivae, and longitudinal melanonychia involving some nails. History of intake of any drugs preceding the lesions, smoking, chewing of betel nut and dental implants was negative. Family history was non-contributory. There were no cutaneous lesions suggestive of lichen planus. Mucoscopy (dermoscopy of the mucosa, oral in this case) and onychoscopy were done followed by biopsy from the tongue that confirmed the diagnosis of lichen planus. Oral mucoscopy of the tongue revealed a tri-colored pattern with structureless veil-like grey-white areas (modified Wickham’s striae), well-demarcated red glossy erosions, and violaceous-to-brown clods. Additionally, vascular pattern of dotted and linear to curved vessels along the borders of leukoplakia-like areas and erosions were observed. Onychoscopy confirmed lichen planus-associated melanonychia. Dermoscopy also proved useful in conveniently ruling out other disorders typified by mucosal and nail pigmentation such as Laugier Hunziker syndrome and drug-induced changes. Although direct oral microscopy has been used in defining features of oral lichen planus, to the best of our knowledge this case is the first report on mucoscopy or dermoscopy of oral lichen planus",
"keywords": [
"dermoscopy",
"mucoscopy",
"lichen planus",
"oral",
"mucosal",
"leukoplakia",
"veil-like",
"speckled-pearly erosion",
"dotted",
"linear",
"curvilinear",
"vessels",
"clods",
"brown"
],
"content": "Introduction\n\nDermoscopy has unleashed opportunities of exploring structures and features of the skin invisible to the unaided eye. Inflammoscopy, i.e. dermoscopy of inflammatory dermatoses has sufficiently advanced to the point of facilitating dermoscopic differentiation between plaque psoriasis, eczema and pityriasis rosea1.\n\nWickham striae (WS) characterized by white crossing streaks are the dermoscopic hallmark of cutaneous LP1–4. A background of dull red color, and vessels of mixed morphology (dotted and linear) represent additional dermoscopic findings of LP1,5. There is paucity of data on dermoscopy of mucosal, especially oral lichen planus (OLP), which is encountered in more than one-third cases of cutaneous LP, with an estimated global prevalence of 0.5–2.2%6–8.\n\n\nCase details\n\nA 19-year-old Indian gentleman was evaluated for asymptomatic patchy pigmentation over multiple finger and toe nails, the tongue, and buccal cavity, noticed eighteen months back. There was no history of preceding trauma, drug intake, soreness of mouth, or dental procedures or amalgam filling. He denied addictions like smoking or chewing of betel nut or tobacco. Current and past medical history were unremarkable. There was no history of parental consanguinity, familial nail pigmentation or any familial pigmentary disorder. Examination of oral mucosa revealed poor oral hygiene. The dorsum of the tongue revealed violaceous to dark grey discoloration extending onto the ventral surface, interspersed with white reticular lesions and focal tiny bright red erosions (Figure 1). Buccal mucosae revealed brown colored macules with focal presence of white reticular lesions. Lingual papillae projections appeared blunted in the discolored central area. Although mild desquamative gingivitis with gingival hyperpigmentation were appreciable, the lips were spared with no visible freckling (Figure 2). Examination of nails revealed longitudinal melanonychia of multiple fingers and toe nails (Figure 3). Relevant hematological and biochemical investigations ruled out hepatitis, dyslipidemia, diabetes and thyroid disorder.\n\n\nDermoscopic features\n\nVideo dermoscopy (EScope; polarized mode, ×20) of the dorsum of the tongue revealed blunting of papillae (Figure 4), in contrast to the preserved papillary pattern observed in the peripheral portion (Figure 5). The affected area displayed a tri-color pattern constituted by – 1) structureless veil-like grey-white to bluish-white areas, 2) bright red slightly depressed areas, and 3) interspersed violaceous-to-brown clods. A few foci of specked-pearly white structures were also observed (Figure 4). Dotted and linear to curvilinear vessels were visible at the junction of the white and red areas. Dermoscopy from the surrounding normal-appearing areas of the tongue dorsum revealed the typical fungiform lingual papillae (Figure 5). Dermoscopy from buccal mucosa only revealed diffusely spread violaceous clods. Onychoscopy revealed multiple 3–4 mm wide uniformly pigmented parallel linear bands of pigmentation with pseudo-Hutchinson sign (Figure 6).\n\n\nInvestigations and diagnosis\n\nA 10% KOH smear from the oral mucosa was negative for candidiasis. Histopathology revealed irregular acanthosis, basal layer vacuolization, necrotic keratinocytes, moderately dense interface dermatitis .and pigment incontinence (Figure 7). A final diagnosis of erosive oral lichen planus was made.\n\n\nDiscussion\n\nThe dermoscopic features of cutaneous LP are typified by the presence of a dull red background, white crossing streaks of WS (multiple patterns), and mixed pattern of dotted and linear vessels at the periphery of the lesions1–5. OLP may occur in isolation, or in association with cutaneous and/or nail LP. Buccal mucosa and tongue are most commonly affected, followed by gums and labial mucosa9. In contrast to the well documented dermoscopic features of cutaneous LP, lichen planus pigmentosus, nail lichen planus and lichen planopilaris10, the dermoscopic characterization of OLP is almost non-extant. To the best of our knowledge, there is a single case report of dermoscopy of LP involving the lower lip11. Drogoszewska et al. in their study, employed direct oral microscopy, a non-invasive diagnostic technique based on the principles of dermoscopy and colposcopy, to describe the in vivo picture of erosive OLP. The purpose of this study was to evaluate the role of the technique as a guide to selecting optimal biopsy site to reveal dysplastic changes12. In their study, Drogoszewska et al. described the typical ‘direct microscopic’ picture of erosive OLP as bi-colored consisting of planar to minimally elevated, dull white, hyperkeratotic leukoplakia-like areas (LLA) lesions, and well-demarcated, bright red and glossy erosions with a smooth moist surface, present adjacent to the LLAs12. Further, they reported subepithelial capillaries to be invisible within the lesions. In the current case, three different colors and patterns were observed by video dermoscopy of OLP – veil-like structureless greyish-white areas, bright red well-demarcated erosions, and interspersed violaceous-to-brown colored clods. The latter are suggestive of sub-epithelial pigment incontinence. Thus a tri-colored, pattern was observed.\n\nThe pattern and appearance of WS was different in oral mucosal LP compared to the pattern typical of cutaneous LP. In cutaneous LP, WS most commonly present as white streaks in a reticular pattern, although other patterns have been reported including circular, radial streaming, linear, globular, veil-like, leaf venation, and starry sky/white dots10,13,14. In the current case, WS presented as – veil-like structureless grey-white to bluish white areas, and specked-pearly pattern in few foci. It is interesting to note, that such modified appearance of WS has also been reported at another mucosal site, the vulva. In the dermoscopic evaluation of 10 women with vulvar LP, Borghi et al. reported that WS in more than half the patients gave a similar veil-like structureless grey-white to blue-white appearance15. They also observed white homogenous areas in 50% patients15.\n\nIn our experience, LP involving the cutaneous aspect of the lip displays the typical WS, whereas the mucosal aspect shows WS resembling LLAs. Dotted and linear to curvilinear vessels were visible at the junction of the white and red areas, akin to the vascular pattern observed in dermoscopy of cutaneous LP. The fourth feature from the tongue lesion was blunting of lingual papillae. This feature may depend on the morphological sub-type of OLP.\n\nThe onychoscopic findings of uniform-colored 3–4 mm broad bands of longitudinal melanonychia and the pseudo-Hutchinson’s sign stemming from hyperpigmentation of the nail bed and matrix reflecting through the transparent nail folds may be seen in LP, with other common reported causes being racial pigmentation, Laugier-Hunziker syndrome (LHS), and drug-induced melanonychia16.\n\n\nConclusion\n\nWe suggest that a tri-colored pattern constituted by modified WS with a veil-like grey-white to bluish-white structureless morphology (or LLAs) and focal speckled-pearly appearance, red erosions, and violaceous-to-brown clods, in addition to dotted and linear to curved vessels along the junction of LLAs and erosions are characteristic of OLP. Last but not the least, akin to the evolution of other sub-specialties of dermoscopy (trichoscopy, inflammoscopy, entomodermoscopy, onychoscopy etc.), mucoscopy needs to be explored more to extend the versatility of dermoscopy for diagnosis of mucosal disorders.\n\n\nData availability\n\nNo data is associated with this article.\n\n\nConsent\n\nWritten informed consent for publication of the clinical details and clinical images was obtained from the patient himself.",
"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\nLallas A, Zalaudek I, Argenziano G, et al.: Dermoscopy in general dermatology. Dermatol Clin. 2013; 31(4): 679–94. PubMed Abstract | Publisher Full Text\n\nVázquez-López F, Manjón-Haces JA, Maldonado-Seral C, et al.: Dermoscopic features of plaque psoriasis and lichen planus: new observations. Dermatology. 2003; 207(2): 151–6. PubMed Abstract | Publisher Full Text\n\nZalaudek I, Argenziano G: Dermoscopy subpatterns of inflammatory skin disorders. Arch Dermatol. 2006; 142(6): 808. PubMed Abstract | Publisher Full Text\n\nVazquez-Lopez F, Palacios-Garcia L, Gomez-Diez S, et al.: Dermoscopy for discriminating between lichenoid sarcoidosis and lichen planus. Arch Dermatol. 2011; 147(6): 1130. PubMed Abstract | Publisher Full Text\n\nLallas A, Kyrgidis A, Tzellos TG, et al.: Accuracy of dermoscopic criteria for the diagnosis of psoriasis, dermatitis, lichen planus and pityriasis rosea. Br J Dermatol. 2012; 166(6): 1198–205. PubMed Abstract | Publisher Full Text\n\nBoyd AS, Neldner KH: Lichen planus. J Am Acad Dermatol. 1991; 25(4): 593–619. PubMed Abstract | Publisher Full Text\n\nPindborg JJ, Mehta FS, Daftary DK, et al.: Prevalence of oral lichen planus among 7639 Indian villagers in Kerala, South India. Acta Derm Venereol. 1972; 52(3): 216–220. PubMed Abstract\n\nBhonsle RB, Pindborg JJ, Gupta PC, et al.: Incidence rate of oral lichen planus among Indian villagers. Acta Derm Venereol. 1979; 59(3): 255–257. PubMed Abstract\n\nEisen D: The clinical manifestations and treatment of oral lichen planus. Dermatol Clin. 2003; 21(1): 79–89. PubMed Abstract | Publisher Full Text\n\nFriedman P, Sabban EC, Marcucci C, et al.: Dermoscopic findings in different clinical variants of lichen planus. Is dermoscopy useful? Dermatol Pract Concept. 2015; 5(4): 51–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYeo IK, Kim HK, Kim DH, et al.: Oral Lichen Planus for Whom Dermoscopy Was Used as an Adjuvant Diagnostic Tool. Korean J Dermatol. 2012; 50(2): 167–170. Reference Source\n\nDrogoszewska B, Chomik P, Polcyn A, et al.: Clinical diagnosis of oral erosive lichen planus by direct oral microscopy. Postepy Dermatol Alergol. 2014; 31(4): 222–228. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGüngör Ş, Topal IO, Göncü EK: Dermoscopic patterns in active and regressive lichen planus and lichen planus variants: a morphological study. Dermatol Pract Concept. 2015; 5(2): 45–53. PubMed Abstract | Free Full Text\n\nLitaiem N, Mansour Y, Jones M, et al.: Dermoscopic signs of lichen planus. BMJ Case Rep. 2016; 2016: pii: bcr2015213923. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBorghi A, Corazza M, Minghetti S, et al.: Preliminary study on dermoscopic features of vulvar lichen planus: new insights for diagnosis. J Eur Acad Dermatol Venereol. 2016; 30(6): 1063–1065. PubMed Abstract | Publisher Full Text\n\nJefferson J, Rich P: Melanonychia. Dermatol Res Pract. 2012; 2012: 952186. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "31604",
"date": "15 Mar 2018",
"name": "Balachandra S. Ankad",
"expertise": [
"Reviewer Expertise Dermoscopy"
],
"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\nDear Authors,\nVery good manuscript. In introduction, mention full form of LP. Figure number 4; bluish-white areas are labeled by yellow arrows but mentioned as yellow areas. Kindly change it.\nComments\nIt is a good manuscript describing and characterizing dermoscopic patterns in oral lichen planus. Authors have detailed each dermoscopic pattern seen in the case. Discussion is authentic and covered the necessary aspects of it.\nStrengths of manuscript\nVery good dermoscopic image. Authors have correlated dermoscopic and histopathologic changes very well. Description of dermoscopic image is appropriate.\nWeakness of manuscript\nIt is a report of single case, hence needs more validation of dermoscopic findings.\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": "31603",
"date": "16 Mar 2018",
"name": "Sukesh MS",
"expertise": [
"Reviewer Expertise Dermoscopy and trichoscopy"
],
"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 enumerates the dermoscopic findings of oral lichen planus, which is described less in literature. The images, descriptions of each dermoscopic finding, histopathological correlation and discussion is aptly written. Wickham striae seen as veil-like structureless grey-white to bluish white areas, and specked-pearly pattern are highlighting dermoscopic features described here , for oral lichen planus. These findings need to be validated on larger studies.\nShort comings - 1] Mention of how the videodermoscope was used / modified to examine the buccal mucosa either though a description or through an image , would have been more informative 2] Correction of description of image 4 - 'Yellow areas' to 'yellow arrows' ; needs 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": []
}
] | 1
|
https://f1000research.com/articles/7-284
|
https://f1000research.com/articles/7-399/v1
|
26 Mar 18
|
{
"type": "Research Article",
"title": "The beta-adrenergic agonist zilpaterol hydrochloride may predispose feedlot cattle to cardiac remodeling and dysfunction",
"authors": [
"Joseph M. Neary",
"Franklyn B. Garry",
"Daniel H. Gould",
"Timothy N. Holt",
"R. Dale Brown",
"Franklyn B. Garry",
"Daniel H. Gould",
"Timothy N. Holt",
"R. Dale Brown"
],
"abstract": "Background: The aim of this study was to address producer concerns that the β2-adrenergic agonist zilpaterol hydrochloride, a bovine growth promotant, predisposes cattle to cardiac disease and death. Our objectives were to evaluate the effect of zilpaterol on cardiac function, morphology, and risk of myocardial injury. Methods: A prospective, case-control study was conducted on one feedlot in northern Colorado using convenience sampling of Angus-based steers (n = 80). Pulmonary arterial pressures (mean, systolic, and diastolic) were measured. Plasma cardiac troponin I was measured in a sub-sample of steers that were followed to slaughter (n = 31). The carcass, left ventricle plus inter-ventricular septum, and right ventricle were weighed. Results: Relative to controls, steers fed zilpaterol hydrochloride had an adjusted left ventricle and septum that was 185 g heavier (95 % CI: 19, 350 g; P = 0.03), a diastolic pulmonary arterial pressure that was 10 mm Hg greater (95 % CI: 3, 17 mm Hg; P = 0.004), and a greater concentration of cardiac troponin I (P = 0.01), a biomarker of myocardial injury. Furthermore, left ventricular mass tended to be positively and deleteriously associated with diastolic pulmonary arterial pressure in steers fed zilpaterol (P = 0.08) but not controls (P = 0.28). Conclusions: Our findings suggest that zilpaterol hydrochloride induced sufficient left ventricular hypertrophy to cause impaired left ventricular relaxation or diastolic dysfunction and myocardial injury. In conclusion, these results support concerns that, in the feedlot studied, zilpaterol hydrochloride predisposes cattle to cardiac disease.",
"keywords": [
"feedlot",
"heart",
"morbidity",
"mortality",
"health"
],
"content": "Introduction\n\nIn the United States, two β-adrenergic agonists (βAA) are approved for the purpose of growth promotion in cattle fed in confinement for slaughter: ractopamine hydrochloride1, a predominantly β1AA, and zilpaterol hydrochloride2, a predominantly β2AA. In 2011, approximately 57% of cattle in U.S. feedlots were fed a βAA3. Beta-adrenergic agonists are synthetic, structural, and functional analogs of the endogenously produced compound epinephrine (adrenaline). They have the favorable effect of inducing skeletal muscle hypertrophy while decreasing adiposity4.\n\nChronic β-adrenoreceptor stimulation, however, may also cause left ventricular hypertrophy5, a strong, independent risk factor for death in humans6–8. Moreover, a large observational study concluded that 40 to 50% of deaths among feedlot cattle treated with the β2 adrenergic receptor-selective agonist zilpaterol hydrochloride (Zilmax®, Merck Animal Health, Summit, NJ, USA) were attributable to the drug9. The etiology of this increased mortality risk has not been investigated, but anecdotal reports suggest that cattle fed β2AA are at increased risk of heart failure. This is biologically plausible; mortality associated with chronic use of β2AA use in humans has been primarily attributed to myocardial disease10,11.\n\nThe aim of this study was to address the concerns that zilpaterol hydrochloride may predispose cattle to heart failure. An in-situ, prospective, case-control study was performed at one feedlot in northern Colorado, U.S.A., to evaluate the effect of zilpaterol hydrochloride on bovine cardiopulmonary physiology, cardiac morphology, and the risk of myocardial injury.\n\n\nMethods\n\nThe Colorado State University Animal Care and Use Committee approved of this project (Protocol 13-4451A). Permission to conduct the study at the feedlot and abattoir was granted by the owners prior to the start of sampling. All efforts were taken to ameliorate animal suffering by minimizing the time that steers were restrained for the purposes of this study.\n\nBlack-hided, Angus-based steers that were within 30 days of slaughter were studied in one feedlot located 1,440 m above sea level in northern Colorado (Table 1). The sample size was limited to 80 due to voluntary market withdrawal of the drug during the course of the study. Cattle were procured from the western U.S. through the auction market system and directly from producers. On arrival at the feedlot the cattle weighed approximately 300 kg. They were vaccinated against bacterial and viral pathogens, injected with an anthelmintic, implanted with a hormone growth promotant, and given an ear tag with a unique identification number. The cattle were harvested approximately 180 days after arrival and were managed per standardized protocols developed by specialists in the areas of feedlot animal health, nutrition, and husbandry. Steers were fed zilpaterol hydrochloride per the label directions: 7.6 g/ton zilpaterol (8.3 ppm on a 100% dry matter basis), to provide 60 to 90 mg zilpaterol hydrochloride per head per day. Steers were fed zilpaterol for 21 days followed by a 7-day withdrawal period, rather than the minimum 3-day period required. Feed and water were always available.\n\nSteers were chosen from each of 9 and 11 different pens, respectively. Control steers (CS) were selected that had the same, or a similar, slaughter date to zilpaterol treated steers (ZS). Steers were chosen from within each pen in such a way as to minimize animal disturbance. Steers were moved up to the chute individually, or in pairs, so that they spent minimal time within the curved alley. Steers were restrained in a chute for sampling and measurements. The ZS were studied at days 7 (n = 10), 8 (n = 6), 15 (n = 4), and 21 (n = 17) of β2AA exposure. In total, 35 apparently healthy CS and 28 apparently healthy ZS were sampled. Sixteen of the 80 steers studied, 8 ZS and 8 CS, were treated for respiratory disease at various times while at the feedlot. These animals were not included in the statistical analyses.\n\nThe sampling dates (Table 1) were determined by the schedule availability of investigators and feedlot personnel. Sampling was conducted between 8:00 am and 11:00am. In the original study design, approximately equal numbers of ZS and CS were to be sampled each day. This did not occur, however, for two reasons: first, zilpaterol hydrochloride was voluntarily withdrawn from the market on August 16th, 2013; and second, on August 29th sampling was called off by the feedlot manager after 2 ZS became highly stressed in the alley and refused to exit. Their rectal temperatures reached 42.2°C (108 °F) (Quicktemp Digital Thermometer, Agripro Enterprises, Iowa Falls, Iowa, USA). The steers were cooled with alcohol poured over the skin and, after approximately 1 hour, walked forwards out of the chute; consequently, only 11 healthy ZS, and 2 ZS and 2 CS that had been treated for respiratory disease, were studied on that day. Following the withdrawal of the drug from the market, only CS were available for further study.\n\nA full description of the equipment, materials, and facilities required for PAP testing is provided elsewhere12. In brief, an 8.9 cm (3.5 inch) 12-gauge needle was inserted into the jugular vein through which flexible, saline-filled catheter/polyethylene tubing was threaded. The catheter tip was then fed through the right atrium and ventricle and into the pulmonary artery. A pressure transducer connected the catheter to an oscilloscope (BM5Vet, Bionet America Inc., Tustin, CA, USA). The position of the catheter was determined from the pressure waveform on the oscilloscope. The jugular vein, right atrium, right ventricle, and pulmonary artery have distinct pressure waveforms12. Mean, systolic, and diastolic pressures were recorded after the pulmonary pressure waveforms had stabilized. Diastolic PAP is hemodynamically influenced by left ventricular chamber pressures shortly prior to contraction, and therefore provides an indirect measure of end-diastolic left ventricular filling pressure13. Pulmonary arteriolar wedge pressure is a more direct measure of left ventricular end-diastolic pressure. Wedge pressures are, however, more difficult and time-consuming to obtain in cattle because the fluid-filled catheters used are not balloon-tipped; consequently, to minimize the time that cattle spent in the chute, arteriolar wedge pressures were not collected.\n\nCardiac troponin I analyses were performed on a single day using plasma samples that had been obtained from all steers that were followed to slaughter. The plasma had been stored at -80° C and thawed at room temperature prior to analysis (cTnI, i-STAT 1, Abaxis, Union City, CA).\n\nIn the original study design, all cattle at the feedlot that died within the late feeding period were to be evaluated postmortem throughout the duration of the study. Due to an insufficient number of feedlot personnel, however, postmortem data were not collected. Cardiac dimension data were, therefore, collected at slaughter from the remaining 2 cohorts of steers: the 11 ZS studied on August 29th, 2013, and slaughtered on September 5th; and the 20 CS studied on October 21st and slaughtered on October 29th, 2013. Only steers that were healthy at the time of sampling and had not been treated for disease while at the feedlot were followed to slaughter.\n\nThe steers were slaughtered at a nearby USDA-approved facility. The individual animal identification number of every animal in the study remained with the carcass and heart throughout the slaughter process. This allowed the hot carcass weight (HCW, mass of the unchilled carcass after removal of the head, hide, and viscera) to be paired with cardiac mass. After inspection by a USDA veterinarian, the heart was removed from the processing line, placed in a plastic bag, and stored in ice.\n\nThe atria were dissected from the ventricles at the level of the atrioventricular valves and the ventricles subsequently divided into a left ventricle plus inter-ventricular septum (LVS) and right ventricular free wall (RV). The ventricles were weighed using a scale with precision to 0.045 kg (0.1 lb). All cardiac tissue was dissected and weighed within 3-hours of slaughter.\n\nData were analyzed using statistical software (STATA version 12.1, Stata Corporation, College Station, Texas, USA). Statistics are provided as an adjusted average (mean or median) ± 95% confidence interval of the average. The animal served as the experimental unit. Only steers that were apparently healthy at the time of testing and had not been treated for any disease while at the feedlot were included in statistical analyses. Steers that had been treated, or needed treatment, for disease were not included in any statistical analyses due to the small sample size.\n\nBetween-group statistical differences in PAP (3 variables) were assessed using quantile regression for nonparametric data while controlling for pen-level clustering (‘qreg2’ command)14. In subsequent analyses, pen-level clustering was not a concern because ZS and CS that were followed to slaughter belonged to the same 2-pens. Differences in right and left ventricular masses were assessed using linear regression analyses. A Wilcoxon rank-sum test (Mann-Whitney U test) was performed for evaluation of cardiac troponin I due to the non-parametric distribution of residual error about the mean (‘ranksum’ command). Finally, the effect of left ventricular mass per unit of HCW on diastolic PAP was evaluated, stratified by the feeding of zilpaterol. Left ventricular mass per unit of HCW was chosen as the dependent variable rather than LVS because of the potential collinearity between LVS and HCW. A linear regression analysis was performed for ZS and, due to lack of normality of the standardized residuals, quantile regression was performed for CS (‘qreg’ command). Evaluations of model fit included graphical and statistical assessments of residual error normality (Shapiro-Wilk test), heteroskedasticity (Breusch-Pagan/Cook-Weisberg test), and linearity.\n\n\nResults\n\nThe results of this study indicate that, in the feedlot studied, steers fed the βAA zilpaterol hydrochloride had greater left ventricular hypertrophy and plasma cardiac troponin I, a biomarker of myocardial injury, than controls. The mass of the left ventricle tended to be positively associated with diastolic PAP in ZS, but there was no association between left ventricular mass and diastolic PAP in CS.\n\nDiastolic PAP was 10 mm Hg greater in ZS than CS (95% CI: 3, 17 mm Hg; P = 0.004). Mean and systolic PAP were not statistically different (P > 0.20). Mean PAP was 47 mm Hg (95% CI: 44, 50 mm Hg) in both ZS and CS. Systolic PAP was 80 mm Hg (95% CI: 76, 84 mm Hg) in CS and 82 mm Hg (95% CI: 73, 90 mm Hg) in ZS.\n\nCattle treated for respiratory disease had greater pressures than healthy cattle. Mean PAP was 57 (95% CI: 36, 78 mm Hg) in CS and 61 (95% CI: 33, 89 mm Hg) in ZS. Systolic PAP was 95 mm Hg (95% CI: 60, 130 mm Hg) in CS and 93 mm Hg (95% CI: 52, 134 mm Hg) in ZS. Diastolic PAP was 20 mm Hg (95% CI: 4, 36 mm Hg) in CS and 37 (95% CI: 16, 58 mm Hg) in ZS.\n\nSteers fed zilpaterol hydrochloride had hypertrophy of the LVS. Steers fed zilpaterol had an adjusted LVS mass that was 185 g (95% CI: 19, 350 g) heavier than CS (P = 0.03). Right ventricular mass did not differ between ZS and CS (P = 0.33). The HCW of ZS was 403 kg (95% CI: 376, 429 kg) and 418 kg (95% CI: 400, 437 kg) in CS (P = 0.29). The adjusted mean mass of the RV was 588 g (95% CI: 526, 650 g) in CS and 535 g (95% CI: 450, 619 g) in ZS. The adjusted mean mass of the LVS was 1.512 kg (95% CI: 1.419, 1.605 kg) in CS and 1.697 kg (95% CI: 1.570, 1.824 kg) in ZS.\n\nThe mass of the LVS per unit of HCW tended to be associated with diastolic PAP in ZS (P = 0.08) but not CS (P = 0.28) (Figure 1A). In ZS, a 0.1 kg increase in LVS per 100 kg of HCW was associated with a 9 mm Hg (95% CI: -1, 19 mm Hg) increase in diastolic PAP.\n\nLeft ventricular hypertrophy in steers fed zilpaterol hydrochloride tended to be positively associated with diastolic PAP. Cardiac troponin I concentrations were significantly greater in steers fed zilpaterol hydrochloride. The two steers with the greatest left ventricular hypertrophy had the greatest diastolic pulmonary arterial pressures (A) and the greatest cardiac troponin I concentrations (B).\n\nPlasma cardiac troponin I (cTnI) concentrations were significantly greater in ZS than CS (P = 0.01). The greatest concentrations were observed in the two steers with the most hypertrophied left ventricles (Figure 1B). In CS, the concentrations of cTnI ranged from 0.00 to 0.02 ng/mL; 5% (1 of 20 steers) had a cTnI concentration of 0.02 ng/mL. In ZS, the concentrations of cTnI ranged from 0.00 to 0.04 ng/mL; 36% (4 of 11 steers) had a cTnI concentrations of 0.02 ng/mL or greater. The complete results are as follows: among the 20 CS sampled, 14 (70%) had a cTnI of 0.00 ng/mL, 5 (25%) had a cTnI of 0.01 ng/mL, and 1 (5%) had a cTnI of 0.02 ng/mL; among the 11 ZS sampled, 3 (27%) had a cTnI of 0.00 ng/mL, 4 (36%) had a cTnI of 0.01 ng/mL, 2 (18%) had a cTnI of 0.02 ng/mL, 1 (9%) had a cTnI of 0.03 ng/mL, and 1 (9%) had a cTnI of 0.04 ng/mL.\n\n\nDiscussion\n\nThe findings of this study indicate that, in the feedlot studied, the β2AA zilpaterol hydrochloride is associated with left ventricular hypertrophy and myocardial injury. Furthermore, the tendency in our study for left ventricular mass to be positively and deleteriously associated with diastolic PAP in steers fed zilpaterol hydrochloride suggests that βAA-induced left ventricular hypertrophy likely caused diastolic dysfunction.\n\nLeft ventricular hypertrophy is a product of chronic β-adrenoreceptor activation and is a major risk factor for cardiovascular morbidity and mortality in humans and animals5,15. Two major sequelae of ventricular hypertrophy pertinent to our study are diastolic dysfunction16 and myocardial ischemia17. Diastolic dysfunction is defined as failure of the myofibrils to return to their resting length and ventricular filling is slow or incomplete unless atrial pressure increases16. A hallmark of diastolic dysfunction is an increase in left ventricular end-diastolic pressure16. End-diastolic pressure is positively correlated with diastolic PAP, even when pulmonary vascular resistance or heart rate is elevated13,18; consequently, the positive association between left ventricular mass and diastolic pulmonary arterial pressure in steers fed zilpaterol, but not controls, suggests that the myocardial hypertrophic effects of zilpaterol induced diastolic dysfunction. This is not unreasonable: the myocardial hypertrophic action of βAA, such as isoprenaline, are used for the study of diastolic dysfunction in animal models of heart failure19–21.\n\nHypertrophy also predisposes to myocardial ischemia17. This may explain why steers fed zilpaterol had significantly greater cardiac troponin I concentrations than controls, the latter having concentrations similar to those reported in healthy cows22. Intriguingly, the two steers with the greatest cardiac troponin I concentrations also had the greatest left ventricular hypertrophy. This may have been coincidental, but the research literature suggests otherwise. Left ventricular hypertrophy in humans is a strong predictor of death from cardiovascular disease6. Without histological evaluation of the myocardial tissue, however, we cannot say if the statistical difference observed was biologically relevant.\n\nMyocardial injury has, to our knowledge, not been previously associated with zilpaterol in cattle. A recent study of 30-Angus steers found no histologic evidence of apoptosis associated with the βAA zilpaterol and ractopamine23. Myocardial necrosis and fibrosis have, however, been reported in association with ßAA in other species24,25. The myocardial injury observed in association with zilpaterol in our study could have been exacerbated by poor air quality since the inhalation of particulate matter may exacerbate cardiopulmonary injury induced by ßAA26,27. This may, in part, explain the seasonal variation observed in mortality risk associated with zilpaterol hydrochloride9.\n\nThe health implications of diastolic dysfunction in cattle fed βAA may be extensive: diastolic dysfunction in humans increases the risk of pulmonary edema28 and all-cause mortality29,30. Given that the findings of our study suggest that β2AA-induced left ventricular hypertrophy caused diastolic dysfunction, and that an increased in all-cause mortality has been associated with the feeding of βAA to cattle9, studies to evaluate the effect of ßAA on cardiac function and morphology are needed. Suitable echocardiographic measures of diastolic ventricular function include transmitral filling velocity, ventricular relaxation velocity, and pulmonary vein flow. Pulmonary arteriolar wedge pressures would provide a more accurate measure of left ventricular end-diastolic pressure than diastolic PAP. Balloon floatation catheters are superior to the saline-filled polyethylene tubing typically used for PAP measurement12 because arteriolar occlusion is necessary for accurate wedge pressure measurement.\n\nA limitation of our study design is that it was necessary to forfeit external validity to maintain internal validity. This allowed us to successfully address the aim of this study. To achieve internal validity, cattle were not randomized into treatment groups because, in the feedlot studied, the feeding of zilpaterol was at the discretion of the feedlot manager. Concerned that zilpaterol may increase the risk of death, particularly among cattle with a history of disease, the feedlot manager only fed zilpaterol to cattle in pens with a low incidence of morbidity and mortality. Given that respiratory disease predisposes cattle to heart failure31, any confounding, if present, should have biased the results towards the null. Large, randomized studies are necessary to validate our findings because our results cannot be extrapolated to other feedlots and the number of cattle studied was small.\n\nIn conclusion, our results raise concerns that, in the feedlot studied, zilpaterol hydrochloride predisposes cattle to cardiac disease. Healthy cattle fed the βAA zilpaterol hydrochloride had left ventricular hypertrophy that was positively associated with diastolic PAP and myocardial injury.\n\n\nData availability\n\nAll raw data underlying this study is available from Harvard Dataverse under a CC0 Public Domain Dedication: http://dx.doi.org/10.7910/DVN/4IKGIN32",
"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\nHealth and Human Services, F. a. D. A. w: Freedom of information summary. Original new animal drug application. NADA 141– 221 ractopamine hydrochloride (OPTAFLEXX 45) type A medicated article for beef cattle. 2003. Reference Source\n\nHealth and Human Services, F. a. D. A. w: Freedom of information summary. Original new animal drug application. NADA 141– 258 ZILMAX (zilpaterol hydrochloride) type A medicated article for cattle fed in confinement for slaughter. 2006.\n\nUSDA: Feedlot 2011 “Part I: Management Practices on U.S. Feedlots with a Capacity of 1,000 or More Head.” USDA–APHIS–VS–CEAH–NAHMS. Fort Collins, CO, 2011.\n\nJohnson BJ, Smith SB, Chung KY: Historical Overview of the Effect of β-Adrenergic Agonists on Beef Cattle Production. Asian-Australas J Anim Sci. 2014; 27(5): 757–766. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOsadchii OE: Cardiac hypertrophy induced by sustained beta-adrenoreceptor activation: pathophysiological aspects. Heart Fail Rev. 2007; 12(1): 66–86. PubMed Abstract | Publisher Full Text\n\nBrown DW, Giles WH, Croft JB: Left ventricular hypertrophy as a predictor of coronary heart disease mortality and the effect of hypertension. Am Heart J. 2000; 140(6): 848–856. PubMed Abstract | Publisher Full Text\n\nHaider AW, Larson MG, Benjamin EJ, et al.: Increased left ventricular mass and hypertrophy are associated with increased risk for sudden death. J Am Coll Cardiol. 1998; 32(5): 1454–1459. PubMed Abstract | Publisher Full Text\n\nSundstrom J, Lind L, Arnlöv J, et al.: Echocardiographic and electrocardiographic diagnoses of left ventricular hypertrophy predict mortality independently of each other in a population of elderly men. Circulation. 2001; 103(19): 2346–2351. PubMed Abstract | Publisher Full Text\n\nLoneragan GH, Thomson DU, Scott HM: Increased mortality in groups of cattle administered the β-adrenergic agonists ractopamine hydrochloride and zilpaterol hydrochloride. PLoS One. 2014; 9(3): e91177. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAu DH, Curtis JR, Every NR, et al.: Association between inhaled beta-agonists and the risk of unstable angina and myocardial infarction. Chest. 2002; 121(3): 846–851. PubMed Abstract | Publisher Full Text\n\nSuissa S, Hemmelgarn B, Blais L, et al.: Bronchodilators and acute cardiac death. Am J Respir Crit Care Med. 1996; 154(6 Pt 1): 1598–1602. PubMed Abstract | Publisher Full Text\n\nHolt TN, Callan RJ: Pulmonary arterial pressure testing for high mountain disease in cattle. Vet Clin North Am Food Anim Pract. 2007; 23(3): 575–596, vii. PubMed Abstract | Publisher Full Text\n\nFalicov RE, Resnekov L: Relationship of the pulmonary artery end-diastolic pressure to the left ventricular end-diastolic and mean filling pressures in patients with and without left ventricular dysfunction. Circulation. 1970; 42(1): 65–73. PubMed Abstract | Publisher Full Text\n\nParente P, Santos Silva J: Quantile Regression with Clustered Data. Journal of Econometric Methods. 2016; 5(1): 1–15. Publisher Full Text\n\nZierhut W, Zimmer HG: Significance of myocardial alpha- and beta-adrenoceptors in catecholamine-induced cardiac hypertrophy. Circ Res. 1989; 65(5): 1417–1425. PubMed Abstract | Publisher Full Text\n\nGaasch WH, Zile MR: Left ventricular diastolic dysfunction and diastolic heart failure. Annu Rev Med. 2004; 55: 373–394. PubMed Abstract | Publisher Full Text\n\nDuncker DJ, Bache RJ: Effect of chronotropic and inotropic stimulation on the coronary pressure-flow relation in left ventricular hypertrophy. Basic Res Cardiol. 1997; 92(4): 271–286. PubMed Abstract | Publisher Full Text\n\nBouchard RJ, Gault JH, Ross J Jr: Evaluation of pulmonary arterial end-diastolic pressure as an estimate of left ventricular end-diastolic pressure in patients with normal and abnormal left ventricular performance. Circulation. 1971; 44(6): 1072–1079. PubMed Abstract | Publisher Full Text\n\nTakaki M: Cardiac mechanoenergetics for understanding isoproterenol-induced rat heart failure. Pathophysiology. 2012; 19(3): 163–170. PubMed Abstract | Publisher Full Text\n\nMa X, Song Y, Chen C, et al.: Distinct actions of intermittent and sustained β-adrenoceptor stimulation on cardiac remodeling. Sci China Life Sci. 2011; 54(6): 493–501. PubMed Abstract | Publisher Full Text\n\nBerzingi C, Chen F, Finkel MS: p38 MAP kinase inhibitor prevents diastolic dysfunction in rats following HIV gp120 injection in vivo. Cardiovasc Toxicol. 2009; 9(3): 142–150. PubMed Abstract | Publisher Full Text\n\nVarga A, Angelos JA, Graham TW, et al.: Preliminary Investigation of Cardiac Troponin I Concentration in Cows with Common Production Diseases. J Vet Intern Med. 2013; 27(6): 1613–1621. PubMed Abstract | Publisher Full Text\n\nFrese DA, Reinhardt CD, Bartle SJ, et al.: Effect of ractopamine hydrochloride and zilpaterol hydrochloride on cardiac electrophysiologic and hematologic variables in finishing steers. J Am Vet Med Assoc. 2016; 249(6): 668–677. PubMed Abstract | Publisher Full Text\n\nYaeger MJ, Mullin K, Ensley SM, et al.: Myocardial toxicity in a group of greyhounds administered ractopamine. Vet Pathol. 2012; 49(3): 569–573. PubMed Abstract | Publisher Full Text\n\nBurniston JG, Ng Y, Clark WA, et al.: Myotoxic effects of clenbuterol in the rat heart and soleus muscle. J Appl Physiol (1985). 2002; 93(5): 1824–1832. PubMed Abstract | Publisher Full Text\n\nCarll AP, Haykal-Coates N, Winsett DW, et al.: Particulate matter inhalation exacerbates cardiopulmonary injury in a rat model of isoproterenol-induced cardiomyopathy. Inhal Toxicol. 2010; 22(5): 355–368. PubMed Abstract | Publisher Full Text\n\nChiarella SE, Soberanes S, Urich D, et al.: β2-Adrenergic agonists augment air pollution-induced IL-6 release and thrombosis. J Clin Invest. 2014; 124(7): 2935–2946. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLamont RF: The pathophysiology of pulmonary oedema with the use of beta-agonists. BJOG. 2000; 107(4): 439–444. PubMed Abstract | Publisher Full Text\n\nRedfield MM, Jacobsen SJ, Burnett JC Jr, et al.: Burden of systolic and diastolic ventricular dysfunction in the community: appreciating the scope of the heart failure epidemic. JAMA. 2003; 289(2): 194–202. PubMed Abstract | Publisher Full Text\n\nKane GC, Karon BL, Mahoney DW, et al.: Progression of left ventricular diastolic dysfunction and risk of heart failure. JAMA. 2011; 306(8): 856–863. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeary JM, Booker CW, Wildman BK, et al.: Right-Sided Congestive Heart Failure in North American Feedlot Cattle. J Vet Intern Med. 2016; 30(1): 326–334. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeary J: Raw data: Growth promotion with the beta-adrenergic agonist zilpaterol hydrochloride may predispose feedlot cattle to cardiac remodeling and dysfunction. Harvard Dataverse, V1, UNF:6:tWTwcydm4r5SFm+ILXs0Vw==. 2018. Data Source"
}
|
[
{
"id": "32480",
"date": "03 Apr 2018",
"name": "H. Kirk Hammond",
"expertise": [
"Reviewer Expertise Translational physiology",
"cardiovascular physiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and unusual study that examines LV mass in steers after administration of zilpaterol (Z).\n\nProblems Background/Introduction\nWere all animals on “hormone growth promotant” to increase growth rates/skeletal muscle mass?\nWhat hormone? Is this hormone the more likely cause of increased skeletal muscle mass, rather than Z? Need to sort this out in the intro. A possible flaw is that one cannot sort out what deleterious effects are due to the hormone vs Z - ideally a third group on neither hormone or Z would be required. Not asking you to re-do the study, but this confounding element needs discussion.\n\nMethods\nPA Pressures. Not clear how the pulmonary pressures were measured. Please include: anesthesia, body position, how the pressure calibrations were made (for example, “0” at what level of the body and in what position.\n\nStatistics. I was frustrated that the authors did not simply provide the mean ±SD of each of the 2 groups with a simple Student’s t-test to examine for group differences.\nPlease provide such data (and p values) for LV weight, LV/BW ratio, TnI, and pulmonary pressures (PAS, PAD.PA mean), HR\n\nResults\nThe findings are: increased LV mass, and increased PA pressures (although a between-group simple comparison for significance is required to be certain of this; see #3). The TnI data are quite weak and likely showed no group difference and should be de-emphasized. If blood is available, CK-MB might provide helpful support if it shows a group difference.\n\nThe absence of histological examination of the heart is a major omission in this study and should be emphasized in the discussion.\n\nDiscussion\nThe findings (if confirmed, see point 3,4) are increased LV weight and increased pulmonary pressure. The demonstration of heart failure or diastolic dysfunction was not established, so the authors should be more circumspect on their confidence that the animals have diastolic dysfunction.\n\nLV Assessment. Too bad that in-life echocardiography or diastolic arrest and measurement of LV wall thickness were not assessed. As it stands, we cannot be sure whether the LV mass increase was due to eccentric of concentric LVH, and whether there was a change in EF. This should be emphasized 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? 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? Partly",
"responses": []
},
{
"id": "32894",
"date": "16 Apr 2018",
"name": "Harvey Morgan Scott",
"expertise": [
"Reviewer Expertise Veterimary pathobiology",
"epidemiology",
"food animal production medicine",
"food safety",
"microbial ecology"
],
"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 read the paper with great interest. The authors have uncovered a plausible mechanism by which increased mortality in late feeding period cattle might occur when fed a commercially approved beta-2-adrenergic agonist. The product was withdrawn from the U.S. feeding market during the midst of this study in 2013, disrupting the study itself; however, the work that had occurred to that point provides a compelling glimpse into the potential pathology that was occurring when comparing matched treated and control animals. These are NOT cases and controls (in the traditional sense of a case-control study design); rather, the study is a frequency matched and closed cohort study with the matched control set drawn from non-treated pens of cattle concurrently to the 'exposed' animals (not: cases using epidemiological terminology) and the latter treated as determined by the feedlot manager. Thus, it is not an experimental design, it is an observational design. Also, it is not a case-control study, the closest design that fits is a cohort design (arguably, closed in that animals become at risk once the pen in which they reside is assigned to receive the beta-agonist and the control (unexposed) animals are determined to be in pens not receiving the BAA). An in vivo catheter-based arterial pressure assay was performed in the chutes, as well as a later post-mortem (at slaughter) matched comparison of ventricular wall mass versus carcass weight. The study became rather lop-sided/unbalanced during a particularly hot and stressful period which unfortunately coincided with the product withdrawal from the market. This has left some deficits in the potential statistical power of the study. Nevertheless, the findings are considerable and of interest and it is important that these results be presented with transparency and completeness. To this points I have some concerns (see below)\nThe authors should present the estimates for each of the parameters (plus, n=? animals and variances/standard errors) and for each of the groups. It is not enough to express the differences or ratios since the reader needs to do a comparison with expected norms to determine whether these reflect biological reality. For example, the diastolic pressures are not described (simply their differences) whereas the mean (overall, across diastolic and systolic) and also systolic by itself are presented openly. We need the diastolic pressures by group. We need to know the LVS mass, the carcass weights, etc and not just the ratios and differences between groups. Please also tabulate the cardiac troponin data. These data are intriguing but not easily understood in the narrative presentation. Why should we see the tabulated data for LVS and carcass weight? Because the BAA product increases carcass weight substantially and could affect the ratio independent of the septal mass (likely biasing it towards the null, I think...?; and, the reader needs to know). A carefully crafted table or two would make short work of this and greatly improve the paper and its readability.\nUnless I have overlooked it, Figure 1 neglects to mention in the legend what the solid diamonds and clear circles represent.\nThe authors mention that 8 ZS and 8 CS (exposed and unexposed animals, respectively) were not included in statistical analysis due to previous bouts of respiratory disease. However, later these animals appeared to have been discussed despite this refrain (see paragraph 2 of the section under pulmonary arterial pressures)? I remain confused by this exclusion statement then the statistical comparisons based on pulmonary conditions?\nFull disclosure: I have co-authored previous work identifying increased mortality in cattle fed BAAs (not just the B(2)AA studied in the article) and that work is cited in the paper (citation 9). That study was not mechanistic in nature; rather, it explored mortality experience across many hundreds of thousands of animals. The authors mention the 40-50% attributable fraction in that paper by Loneragan et al (2014), but that number applied also to the other commercially available beta-agonist (so both products, not just the products studied here). It is important to note that the attributable fraction applied only during the period in which the product was fed (last 21-28 days); in other words, earlier mortality due to respiratory disease and other calfhood diseases are not included in the denominator in Loneragan et al (2014) but the sentence almost implies that the 40-50% AF in that paper was across the entire feeding period (it is not). It is very important that we consider that animals dying in the late feeding period (where all the feed costs have accrued and the excessive protein waste is greatest) are accorded appropriate and clear mortality attribution.\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? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/7-399
|
https://f1000research.com/articles/7-378/v1
|
26 Mar 18
|
{
"type": "Method Article",
"title": "An unsupervised disease module identification technique in biological networks using novel quality metric based on connectivity, conductance and modularity",
"authors": [
"Raghvendra Mall",
"Ehsan Ullah",
"Khalid Kunji",
"Michele Ceccarelli",
"Halima Bensmail",
"Ehsan Ullah",
"Khalid Kunji",
"Michele Ceccarelli",
"Halima Bensmail"
],
"abstract": "Disease processes are usually driven by several genes interacting in molecular modules or pathways leading to the disease. The identification of such modules in gene or protein networks is the core of computational methods in biomedical research. With this pretext, the Disease Module Identification (DMI) DREAM Challenge was initiated as an effort to systematically assess module identification methods on a panel of 6 diverse genomic networks. In this paper, we propose a generic refinement method based on ideas of merging and splitting the hierarchical tree obtained from any community detection technique for constrained DMI in biological networks. The only constraint was that size of community is in the range [3, 100]. We propose a novel model evaluation metric, called F-score, computed from several unsupervised quality metrics like modularity, conductance and connectivity to determine the quality of a graph partition at given level of hierarchy. We also propose a quality measure, namely Inverse Confidence, which ranks and prune insignificant modules to obtain a curated list of candidate disease modules (DM) for biological network. The predicted modules are evaluated on the basis of the total number of unique candidate modules that are associated with complex traits and diseases from over 200 genome-wide association study (GWAS) datasets. During the competition, we identified 42 modules, ranking 15th at the official false detection rate (FDR) cut-off of 0.05 for identifying statistically significant DM in the 6 benchmark networks. However, for stringent FDR cut-offs 0.025 and 0.01, the proposed method identified 31 (rank 9) and 16 DMIs (rank 10) respectively. From additional analysis, our proposed approach detected a total of 44 DM in the networks in comparison to 60 for the winner of DREAM Challenge. Interestingly, for several individual benchmark networks, our performance was better or competitive with the winner.",
"keywords": [
"Disease Module Identification",
"Biological Networks",
"Community Detection",
"GWAS",
"Modularity",
"Conductance",
"PPI",
"Co-expression"
],
"content": "Motivation & background\n\nA variety of genomic data has been used to construct biological networks. Biological networks are scale free by nature1 and it is well-known that scale-free networks exhibit community like structure2–5. Community like structure in networks is equivalent to presence of a high degree of modularity5. In biological networks, the modules often comprise of genes or proteins that are involved in the same biological functions. Network module identification methods, commonly known as community detection4–9 and graph partitioning methods10–12, attempt to reveal these functional units2,13,14 which is key to derive biological insights from genomic networks15–18. However, the performance of different community detection methods using diverse parameter settings to uncover biologically relevant modules in myriad networks remain poorly understood because there has been no community effort to transparently evaluate module identification methods on common benchmarks and across diverse types of genomic networks. Thus, it is very difficult to objectively compare the strengths and limitations of alternative approaches. Evaluation of module identification methods typically relied either on random graphs13, which do not allow for assessment of biological relevance of modules, or on pre-annotated functional gene sets18 (e.g., gene ontology or molecular pathway databases such as KEGG), which are still primarily incomplete and biased towards well-studied pathways.\n\nTo address these issues, an open community DREAM challenge enabling comprehensive and rigorous assessment of module identification methods across a broad range of gene and protein networks was initiated. The task in sub-challenge 1 was to identify functional modules in 6 individual benchmark networks s.t. the module size satisfied the constraint: 3 ≤ modul esize ≤ 100. The predicted modules were evaluated based on data from disease-relevant genome-wide association studies (GWAS). GWAS have successfully identified thousands of genetic loci associated with a broad range of complex traits and diseases. The variants are mapped to genes allowing to ask whether specific network modules are enriched in these genes19. The DREAM challenge organizers employed a comprehensive collection of over 200 GWAS datasets, thereby, covering a broad spectrum of functional units, many of which have not been annotated previously.\n\nIn this paper, we focus on sub-challenge 1 where the goal is to predict functional modules for individual anonymized networks across a broad range of gene and protein networks. Our proposed pipeline requires a hierarchical tree from any state-of-the-art hierarchical community detection technique as input. The pipeline first identifies the optimal level of hierarchy using an F-score comprising of quality metrics like conductance13, modularity2 and connectivity1. Then it traverses the hierarchy bottom-up from the optimal level allowing to merge smaller communities based on the weighted connectivity criterion as long as they fit the size constraint. Further, it splits giant connected components (modulesize > 100).\n\nFor each giant connected component, we re-build the hierarchical tree using a linkage based agglomerative hierarchical technique and identify the optimal cut (number of clusters k) using the proposed F-score criterion. Finally, we propose a metric to indicate the confidence in each module among the final set of detected modules and develop a method to automatically select the right confidence threshold to prune less meaningful modules. Figure 1 depicts the proposed pipeline for the constrained disease module identification problem.\n\n\nMethods\n\nThe disease module identification methods were evaluated using 6 benchmark networks. Details of the networks are provided in Table 1.\n\nThere are several preprocessing steps performed before the input network can be processed by the pipeline. The node IDs are mapped to a continuous set of integers starting from 1. If the aforementioned procedure is not performed, the network will end up with several isolated nodes and missing IDs. All the edge-weights in each network are normalized between 0 and 1. The input network are considered weighted and undirected in all our pipeline.\n\nWe experimented with removal of edges with a weight lower than a threshold t = 0.05 but observed that the corresponding results deteriorated. Hence, we recommend keeping all the edges in the network.\n\nIn the initial submission rounds, we ran several out-ofthe-box state-of-the-art community detection techniques including Order Statistics Local Optimization Method (OSLOM)4, Louvain5, Multi-level Hieararical Kernel Spectral Clustering (MHKSC)6,7, Dynamic Tree Cut20 and METIS10. We also tried to use the results obtained from these methods as input to consensus clustering based method PCAgglo21 and ensemble clustering based method Ensemble-Clue22 which are evaluated using complex traits and disease modules in 76 European GWAS datasets.\n\nOSLOM is based on the local optimization of a fitness function expressing the statistical significance of communities with regards to random fluctuations, which is estimated with tools of extreme and order Statistics. The Louvain method is a greedy optimization method that attempts to optimize the modularity of a network partition. MHKSC technique uses a kernel spectral clustering formulation to random walk and exploits the structure of the projections in the eigenspace to automatically determine a set of increasing distance thresholds. It then uses these distance thresholds in a test phase to obtain multiple levels of hierarchy using principles of agglomerative hierarchical clustering. Dynamic Tree Cut method implements novel dynamic branch cutting technique for hierarchical clustering where it detects clusters in a dendogram depending on their shape. They are capable of identifying nested clusters, can identify clusters of various shape and are suitable for automation. METIS is a set of serial programs for multilevel recursive partitioning of the graph to produce fill reducing orderings for sparse matrices. PCAgglo performs logistic PCA on the concatenated node membership matrix formed from k different methods and then agglomerative hierarchical clustering is performed on the principal components. For METIS, Dynamic Tree Cut, PCAgglo and Ensemble-Clue, we selected that level of hierarchy for which the average module size was close to the best as per the exploratory data analysis provided by the DREAM Challenge organizers. The results that we obtained by direct application of out-ofthe-box state-of-the-art community detection methods is depicted in Table 2.\n\nComparison of several out-of-the-box community detection methods along with one consensus and one ensemble based clustering method for disease module identification on 6 different biological networks. Here N represents total number of candidate disease modules and ns represents the number of significant/detected disease modules in the 76 genome wide association study (GWAS) datasets. OSLAM - Order Statistics Local Optimization Method, MHKSC - Multi-level Hieararical Kernel Spectral Clustering.\n\nThe Best of All result were not submitted during the preliminary rounds of the Challenge because the Best of All method depicts the maximum number of enriched modules that can be identified by a simple ‘max’ combination of these techniques at default settings. However, as per our understanding the goal of the challenge is to develop a method or a generic framework which can optimally identify disease modules from various gene and protein interaction networks at different parameter settings. We gained several insights from these preliminary results including:\n\nMethods like OSLOM, MHKSC and PCAgglo generated a set of clusters whose cluster size distribution is nearly power law.\n\nFor most of these methods there were several giant connected components which were ignored due to the strict upper bound constraint on the module size.\n\nFor most of these methods nearly half of the nodes in each network were part of giant connected components that were removed due to size constraint.\n\nMETIS generated uniform sized clusters and included most of the nodes in each network, hence can’t be optimized further.\n\nWe didn’t get more enriched modules from a consensus (PCAgglo) or ensemble (ensemble-clue) based clustering methods.\n\nLet G(V, E) be an undirected graph with n = |V| representing number of nodes and m = |E| representing number of edges. Let S be the set of modules (or a partition of the network), where ns is the number of nodes in a module s ∈ S; ms be the number of edges in s i.e. ms = |(u, v) ∈ E : u ∈ s, v ∈ s| and cs be the number of edges on the boundary of s i.e. cs = |(u, v) ∈ E : u ∈ s, v ∉ s| and d(u) is the degree of node u.\n\nWe provide summary of quality metrics used and definition of proposed quality metrics below:\n\n1. Modularity: Modularity is a global metric which takes value between −1 and 1. It measures the density of links inside communities compared to links between communities. For a weighted graph, modularity of a network partition is defined as: Q(S)=14∑s∈S(ms−E(ms)), where ms−E(ms) is the difference between ms, the number of edges between nodes in s and E(ms), the expected number of such edges in a random graph with identical degree sequence. Modularity value ≤ 0 indicate that the corresponding partition behaves worse than a random partition of the network. Modularity score can only be obtained for graph partitions.\n\n2. Conductance: Conductance is a local quality metric which can defined for each individual community in the network and takes values between 0 and 0.5. It is defined as: CC(s)=ms2ms+cs. It measures the fraction of total volume of edges associated with the nodes in a module s ∈ S pointing outside the cluster. Conductance for a partition S can be calculated by taking an average of the conductance values for all modules s ∈ S.\n\n3. Connectivity: Connectivity is a sub-local quality metric which can be defined for each individual node in the network and can be averaged for all the nodes in a module s (considering connectivity to other nodes in the same community) to get local connectivity metric. It can be averaged for all the modules s ∈ S to obtain the global connectivity CN(S) for a partition S. It was used in 1 to evaluate whether genes perturbed by trait-associated variants are more densely interconnected than expected in complex diseases and generate connectivity enrichment curves. The connectivity matrix K is defined as: K=(I+W^)p; with W^=D−12WD−12, where K is p-step random walk kernel used to define pairwise connectivity between the nodes, I is an identity matrix, W is the weighted adjacency matrix and D is the weighted diagonal degree matrix. We set p = 4 for our biological networks as it allows to capture all meaningful interactions for paths of length ≤ 4. The connectivity of a node i is estimated as CN(vi)=∑jKij, connectivity of module s is CN(s)=∑i,j∈sKijns2, and connectivity of partition S as CN(S)=∑s∈SCN(s)|S|. is Here |⋅| represents the cardinality function.\n\n4. F-score: We need a quality metric which evaluates the quality of a partition using modularity, conductance and connectivity. While modularity captures global information, conductance and connectivity can capture local information. The proposed quality metric is defined as: F(S)=CC(S)Q(S)+CN(S)2Q(S)CN(S).\n\nHigher value of modularity indicates better quality clusters, lower value of conductance leads to good quality communities and higher value of connectivity indicate better quality of modules. We need to maximize modularity and connectivity while minimizing conductance. Hence, we take harmonic mean of modularity and connectivity in the denominator of F-score metric to give importance to both of the quality metrics. Thus, with conductance in the numerator, the minimum value of F-score corresponds to the partition S with best quality cluster. However, if modularity value is ≤ 0 then we set F-score to a very large value which depicts the poor quality of the partition.\n\n5. Inverse Confidence: We need a metric to rank all the modules generated from the proposed framework. We first considered the average connectivity metric CN(s) for a community s. However, the connectivity criterion prefers smaller size modules which tend to be more cliquish than bigger modules. We also considered using the conductance CC(s) of a community s to rank all the modules in partition S. However, conductance value decreases as size of the community increases due to larger volume of the module (which is denominator of CC(⋅)). We propose an inverse confidence metric to rank all the communities in a partition S as: IC(s)=CC(s)CN(s)×ns2. We utilized the Inverse Confidence metric in conjugation with modularity to remove out less meaningful communities as illustrated in Figure 2 and explained within the proposed framework. We finally convert the inverse confidence value of each module into a confidence score as: Conf(s)=1−IC(s)argsmax(IC(s)), where the denominator is used for normalization.\n\nHere we also highlight the optimal inverse confidence threshold value.\n\nWe followed the steps indicated in Figure 1 to build the proposed framework for constrained disease module identification.\n\n1. Given an input network we perform the preprocessing step to create a modified input network where the node IDs are monotonically increasing, edge weights are noramlized, and the network becomes weighted and undirected.\n\n2. Run a state-of-the-art hierarchical community detection technique to generate the hierarchical tree structure.\n\n3. Estimate quality of each level of hierarchy using modularity, conductance and connectivity.\n\n4. Select that level of hierarchy for which the F-score is minimum.\n\n5. For communities of size > 100 go to Step 2 until either the constraint exceeding communities cannot be split further or modularity of resulting cluster memberships becomes very poor.\n\n6. In the merge step, we start with the partition (S) at the best level of hierarchy and traverse the hierarchical tree from that level in a bottom-up fashion. We iteratively merge those communities whose weighted mean connectivity score is less than the connectivity score for a module at next level of hierarchy where the module consists of those previous communities i.e.a. Here p an q are modules at level h − 1 and s is community at level h such that p, q ∈ s. This results in an intermediate partition set or a set of modules.\n\n7. We then consider all the communities s s.t. ns > 100. For each such community s, we consider the sub-graph comprising only the nodes from that community. We transform the corresponding weighted adjacency matrix i.e. Ŵ = W(vi,vj), ∀vi, vj ∈ s into a distance matrix DŴ = 1 − Ŵ. We then build the agglomerative hierarchical tree using the linkage clustering with Ward’s distance.\n\n8. For each community s (ns > 100), once we obtain the agglomerative hierarchical tree, we cut the tree for different values of k i.e. the number of clusters. We evaluate each such partition using the F-score and select that partition which has the minimum positive F-score.\n\n9. Using Steps 6–7 on these bigger modules and the small size communities which satisfy the size constraint, we generate another set of intermediate clusters.\n\n10. We rank this intermediate set of communities using the inverse confidence score i.e. IC(s), ∀s ∈ S. Lower inverse confidence corresponds to higher rank. We now remove all modules whose size exceeds the size constraint i.e. ns ≤ 3 and ns ≥ 100.\n\n11. In this final step, we propose a mechanism to select the best set of modules for evaluation in an automated fashion independent of the network. We can calculate the maximum and minimum value of inverse confidence (IC) from the inverse confidence (IC) scores of all the communities in the intermediate partition S. We iteratively decrease the inverse confidence threshold from maximum to minimum thereby pruning clusters. At each such threshold, we calculate the modularity of the remaining set of partition using the subgraph corresponding to this partition S′ i.e. GS′. We select the threshold where the difference between Q(S′, θ) and Qprev is minimum i.e. argθ min|Q(S′, θ)−Qprev|. Here |⋅| represents the absolute value, Qprev is the modularity of the partition obtained at Step 2 and calculated in Step 3. For the final submission, we consider all the modules in the optimum partition i.e. s ∈ S′ obtained by pruning communities whose IC (s) ≥ θ .\n\n\nResults\n\nFor our final submission, we utilized the method which is the fastest and most suitable for hierarchical graph partitioning i.e. Louvain method5 as we were allowed to make only 1 submission. We formulated a recursive version of Louvain method where communities of size greater than 100 were recursively partitioned. We also designed a constraint satisfying version of MHKSC6,7 and compared its performance with the recursive Louvain method within the proposed generic framework. The evaluation criterion used in the Challenge was the total number of significant modules identified in the 6 benchmark networks on a hold-out set of 104 GWAS datasets at the false discovery rate (FDR) cut-off23 of 0.05 for multiple testing. We compare the results obtained from proposed generic framework using both the Louvain and MHKSC methods with the winners of the DREAM Challenge in Table 3.\n\nHere the proposed generic frameworks are referred as Constrained Louvain and Constrained Multi-level Hieararical Kernel Spectral Clustering (MHKSC) and we use * to represent the winners of the competition. Here N represents total number of candidate disease modules and ns represents the total number of significant disease modules identified in the104 genome wide association study (GWAS) datasets. In the final round of the challenge, we submitted the results corresponding to Constrained Louvain method.\n\nFrom Table 3, we observe that the winners (Double Spectral Clustering and Resolution Adjusted Clustering) perform far better than Constrained Louvain method on the protein-protein interaction networks (Networks 1 and 2) and homology network (Network 6). However, for the signaling, co-expression and the cancer networks (Networks 3, 4 and 5), proposed Constrained Louvain method has comparable performance with the winners of the challenge. To gain a sense of the robustness of the ranking with respect to the final GWAS data, the challenge organizers sub-sampled the hold-out set by drawing 76 GWASs (same number as during the preliminary phase) out of the 104 GWAS datasets. They created 1, 000 subsamples of the hold-out set. The methods were then scored on each subsample (Sub-sampling was done here without replacement.)\n\nThe performance of each competing method t for a given network was compared to the highest scoring method across the sub-samples by the paired Bayes factor Bt i.e. the method with the highest score on this network in the hold-out set (all 104 GWASs) was defined as reference. The score ns(t, b) of method t in subsample b was thus compared with the score ns(ref , b) of the reference method in the same subsample b. The Bayes factor Bt is defined as the number of times the reference method outperforms method t, divided by the number of times method t outperforms or ties the reference method over all subsamples. Methods with Bt < 4 were considered a tie with the reference method (i.e., method t outperforms the reference in more than 1 out of 5 subsamples). For networks 3, 4 and 5, the Bayes factor of proposed Constrained Louvain method was less than 4. This indicates that the proposed generic framework, though not the winner, is useful, generic and robust enough for identification of statistically significant disease modules in biological networks.\n\nWith the availability of the de-anonymized version of the networks along with the scoring tools used during the competition, we were able to perform additional experiments for the Constrained Louvain method. After the challenge, we identified an error in labeling the nodes in the significant disease modules that we submitted for the homology network (Network 6) during the competition. After correcting the labeling error, we identified 2 significant disease modules from Network 6.\n\nMoreover, we performed additional analysis using 5 different FDR cut-offs (multiple testing) for each of the 6 benchmark networks to obtain the trends in the number of significant disease modules identified by the proposed generic framework for these cut-offs. This result is depicted in Figure 3. The FDR cut-off used as evaluation criterion during the competition was 0.05.\n\n\nDiscussion\n\nThe DREAM Challenge organizers made the GWAS datasets along with de-anonymized networks available to the challenge participants. This allowed us to further analyze our results. For each benchmark network, we identified the proteins or genes that make up the significant disease modules.\n\nWe investigated association of identified disease modules with disease/trait of the provided GWAS datasets. We used the official competition FDR cut-off of 0.05 as significance threshold to identify disease modules for each benchmark network. Table 4–Table 9 provides a detailed analysis of the significant modules and their corresponding associated disease (inferred from 104 hold-out GWAS datasets) for Networks 1,2,3,4,5 and 6 respectively. Each module is found to be associated with at least two GWAS datasets of the corresponding disease/trait. Moreover, many modules were found associated with multiple disease/trait of similar nature. For example, as shown in Table 4, module 19 in 1_ppi network is found to be associated with anthropometric traits. This indicates that the identified modules correspond to preserved biological functions of genes/proteins.\n\n\nData availability\n\nThe Challenge datasets for registered participants are available at: https://www.synapse.org/#!Synapse:syn6156761/wiki/400659. Challenge documentation, including the detailed description of the Challenge design, overall results and scoring scripts can be found at: https://www.synapse.org/#!Synapse:syn6156761/wiki/400647.\n\nSource code for the proposed framework is available from: https://github.com/raghvendra5688/DMI/tree/DMI_v1.0\n\nThe archived source code for the proposed framework along with a README file can be found at: https://doi.org/10.5281/zenodo.119742424.",
"appendix": "Competing interests\n\n\n\n‘No competing interests were disclosed’.\n\n\nGrant information\n\n‘The author(s) declared that no grants were involved in supporting this work.’\n\n\nAcknowledgements\n\nWe would like to thank Ms. Kanchan Karnani for helping out with preparing the flowchart (Figure 1).\n\n\nReferences\n\nMarbach D, Lamparter D, Quon G, et al.: Tissue-specific regulatory circuits reveal variable modular perturbations across complex diseases. Nat Methods. 2016; 13(4): 366–370. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNewman ME: Modularity and community structure in networks. Proc Natl Acad Sci U S A. 2006; 103(23): 8577–8582. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJiang J, Wen S, Yu S, et al.: The structure of communities in scale-free networks. Concurr Comp-Pract E. 2017; 29(14): e4040. Publisher Full Text\n\nLancichinetti A, Radicchi F, Ramasco JJ, et al.: Finding statistically significant communities in networks. PLoS One. 2011; 6(4): e18961. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlondel VD, Guillaume JL, Lambiotte R, et al.: Fast unfolding of communities in large networks. J Stat Mech. 2008; 2008(10): P10008. Publisher Full Text\n\nMall R, Langone R, Suykens JA: Multilevel hierarchical kernel spectral clustering for real-life large scale complex networks. PLoS One. 2014; 9(6): e99966. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMall R, Langone R, Suykens JA: Furs: Fast and unique representative subset selection retaining large-scale community structure. Soc Network Anal Min. 2013; 3(4): 1075–1095. Publisher Full Text\n\nMall R, Langone R, Suykens JA: Self-tuned kernel spectral clustering for large scale networks. In Big Data, 2013 IEEE International Conference on. IEEE, 2013; 385–393. Publisher Full Text\n\nMall R, Jumutc V, Langone R, et al.: Representative subsets for big data learning using k-nn graphs. In Big Data (Big Data), 2014 IEEE International Conference on. IEEE, 2014; 37–42. Publisher Full Text\n\nKarypis G, Kumar V: Metis-serial graph partitioning and fill-reducing matrix ordering. 2012. Reference Source\n\nDhillon IS: Co-clustering documents and words using bipartite spectral graph partitioning. In Proceedings of the seventh ACM SIGKDD international conference on Knowledge discovery and data mining. ACM, 2001; 269–274. Publisher Full Text\n\nDhillon IS, Guan Y, Kulis B: Weighted graph cuts without eigenvectors a multilevel approach. IEEE Trans Pattern Anal Mach Intell. 2007; 29(11): 1944–57. PubMed Abstract | Publisher Full Text\n\nFortunato S, Hric D: Community detection in networks: A user guide. Phys Rep. 2016; 659: 1–44. Publisher Full Text\n\nParthasarathy S, Tatikonda S, Ucar D: A survey of graph mining techniques for biological datasets. In Managing and mining graph data. Springer, 2010; 547–580. Publisher Full Text\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\nCalifano A, Butte AJ, Friend S, et al.: Leveraging models of cell regulation and gwas data in integrative network-based association studies. Nat Genet. 2012; 44(8): 841–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMitra K, Carvunis AR, Ramesh SK, et al.: Integrative approaches for finding modular structure in biological networks. Nat Rev Genet. 2013; 14(10): 719–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLangfelder P, Horvath S: WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. , 2008; 9(1): 559. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLamparter D, Marbach D, Rueedi R, et al.: Fast and Rigorous Computation of Gene and Pathway Scores from SNP-Based Summary Statistics. PLoS Comput Biol. 2016; 12(1): e1004714. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLangfelder P, Zhang B, Horvath S: Defining clusters from a hierarchical cluster tree: the Dynamic Tree Cut package for R. Bioinformatics. 2007; 24(5): 719–720. PubMed Abstract | Publisher Full Text\n\nAsur S, Ucar D, Parthasarathy S: An ensemble framework for clustering protein-protein interaction networks. Bioinformatics. 2007; 23(13): i29–i40. PubMed Abstract | Publisher Full Text\n\nHornik K: A clue for cluster ensembles. J Stat Softw. 2005; 14(12): 1–25. Publisher Full Text\n\nBenjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B Met. 1995; 50(1): 289–300. Reference Source\n\nMall R: raghvendra5688/DMI: Disease Module Identification (Version1) (VersionDMIv1.0). Zenodo. 2018. Data Source"
}
|
[
{
"id": "32972",
"date": "08 May 2018",
"name": "Eric E Schadt",
"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 their paper, “An unsupervised disease module identification technique in biological networks using novel quality metric based on connectivity, conductance and modularity”, Mall et al present an approach to identifying modules of genes from different types of networks, where their approach uses a novel quality metric to evaluate the quality of the partitions based on a number of network metrics such as modularity, conductance and connectivity. Through a series of steps defining the module detection pipeline employed by the authors, they identify modules for the different types of networks and assess the “truth” of the module using enrichment metrics based on GWAS findings as defined by the DMI Dream competition in which the authors had submitted their approach and results.\n\nThe approach detailed by the authors seems reasonable, and the idea of the DMI to test a great variety of methods against one another is excellent. The processing of networks to identify biologically meaningful modules is still an important area of research and competitions such as DMI have helped to assess progress and identify best practices.\n\nHowever, while the work presented is potentially interesting, as written the general DMI approach and specific results are difficult to understand and so hard to evaluate in light of this (as detailed below in the specific comments). Further, this paper represents a detailing of one of many approaches that were used in the DMI challenge, and shows results only compared to the winner of the challenge. The approach detailed apparently ranked 15th in the competition, and so was beaten by 14 other approaches. There is no discussion around this, no discussion on why a reader should care to know about one approach that ranked 15th compared to, say, the 19 other approaches that ranked in the top 20 (14 of which would have beaten the described method). There is no motivation provided on why knowledge of the authors’ approach should be considered in light of 14 other approaches that beat it in the DMI competition. Do the authors believe the DMI competition was the best way in which to assess module identification methods and that the field should adopt the top-scoring methods for this as the state of the art? Do the authors believe that for the type of networks such as coexpression where their approach was comparable to the winners, that their approach has broader utility? Where the other top 13 methods similar with respect to performance across network types?\n\nSpecific Comments:\n\nThe paper is somewhat oddly written in that the methods section contain some methods along with some results and generally a failure to really describe the methods employed by DMI to compare methods.Without this the only way to understand the results in the paper is to invest much time going through the challenge description and the results, and so on. That is a huge burden placed on the reader. The components necessary to understand the results given in the paper should be described in the paper, and if there are references that describe fuller details, then that could be summarized in the methods so that the reader understands what was done and where to go for more details.(Further details on what is missing are given in the following comments.) The authors given preliminary experiments done in the methods section, which ostensibly drove some thinking and refining on the approach they ultimately settled on.The preliminary experiments are not really methods, they are more results. And then there is an “insights gained” section in the methods, which again is not really a method but rather detail learnings from these earlier results. While the authors do detail their own module identification process, the way in which the validity of the modules were assessed is not clearly articulated.What were the criteria set forth by DMI?How were the genes identified given a GWAS finding?There is error associated with identifying the vast majority of genes associated with a GWAS finding, so how was this handled?Was an enrichment score used for genes from GWAS being identified in the module?Was it per disease and combined over all diseases?Did effect sizes come into play? Etc.There should at least be a summary of this so that the reader can understand what it means to be able to count a module in the accuracy score for the competition.But there is nothing on this. The results speak to the paired Bayes factor that was used to compare methods, but you can’t really understand the appropriateness of that without have an understanding of the above questions. There is a link to the Synapse platform regarding the challenge, with many scores of pages of material and then a paper posted on biorxiv that provides details on the challenge and the findings.But it is not yet peer reviewed, it does not appear to be published yet, and so all of the missing detail in this present paper simply points to other papers that are not peer reviewed.It’s again a pretty tall order to ask a reviewer to sift through endless pages of material to understand the context of a paper they have been asked to review and to then review on top of that papers upon which the paper they were asked to review is based.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No",
"responses": []
},
{
"id": "34227",
"date": "08 Jun 2018",
"name": "Yunpeng Liu",
"expertise": [
"Reviewer Expertise Transcription regulation",
"network biology",
"cancer 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\nIn this paper, the authors describe a new pipeline for identifying disease modules from large-scale biological networks in the DREAM challenge. The pipeline builds upon off-the-shelf hierarchical community detection methods and first generates an initial partitioning of the network using a given community detection algorithm. It then integrates multiple properties of the network including modularity, conductance and connectivity into an F score to benchmark the partitioning at different levels of the hierarchy and selects the best partitioning. Next the pipeline merges modules in a way that increases connectivity, and resulting modules that exceed size threshold are partitioned again using hierarchical clustering and split at a level corresponding to the F score minimum. After a second round of merging similar to previous steps, a final set of modules are generated and ranked using a score termed inverse confidence. Using known disease-gene associations obtained from GWAS datasets, the authors verified the modules identified from their pipeline with multiple community detection algorithms, and compared performance across difference networks with that of the top team in the challenge. The authors conclude that despite the top performing team scoring highest overall, there are several cases where the number of modules identified in this paper are at least comparable to that from the top scoring method. Additionally, the authors claim that their pipeline is a generic framework for identifying statistically significant disease modules from biological networks. The methodology put forward in this paper seems novel. However, neither the utility of the module identification pipeline nor its generalizability are adequately demonstrated. This is likely due to vagueness in the description of methods and lack of theoretical justification and supporting computational experiments to validate the procedures and scoring metrics devised by the authors. Specific comments are listed in detail below.\nThe description of the module identification framework seems elusive and lacks detail, rendering it unclear whether it is based on sound theoretical foundations. The framework works through a series of merge-split-merge steps that seems to hint at an iterative procedure to refine network partitioning. However, the method stops at the second merging step and discards all modules whose sizes exceed thresholds. The authors need to provide a rationale for this – is this because of empirical results that the modules identified from the pipeline do not change much after these steps and thus do not require further iterations in general? In addition, the paper seems to switch between methods and scoring schemes in different steps of the pipeline, for example the merging step is performed using increasing connectivity as criterion, whereas the splitting step uses a hierarchical clustering with minimum F score as partitioning criterion. At the final step, the authors used minimal change in modularity score as the criterion for setting a cutoff for module significance. These heuristics seem inconsistent with the idea of integrating multiple network properties to assess quality of network partitioning. Without justification for such a design the framework it would not appear convincing enough to be a fair and generalizable pipeline for module identification. The paper only compares their results with that from the top scoring team in the DREAM challenge and finds that there is a subset of networks where their performance is comparable to that of the top scoring method. This is rather inconclusive in terms of the method of comparison as well as the comprehensiveness of the datasets covered. Is the best scoring team’s method (not explained in detail in the paper) applicable to the pipeline that the authors proposed? If so it would be more convincing if the authors could see improvement over the best team’s method by refining modules through their pipeline. Is a single comparison adequate? Can the pipeline improve results from other teams that use hierarchical methods for module identification too? Answers to these questions will help further verify the utility of the proposed pipeline. The authors gained some insight by inspecting results from each of the off-the-shelf methods they tested using their module identification pipeline – different methods behave very differently in terms of module number and size distribution. A question that naturally arises from these observations is what goes into a good module identification method? Instead of using real biological networks, the authors may gain a better view of the performance of these algorithms by benchmarking them against synthetic networks where modules are simulated under different generative models. Because these methods, including the novel pipeline in this paper itself, only takes network topology into consideration, it is important to test if they work well with different network models.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\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? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-378
|
https://f1000research.com/articles/7-375/v1
|
26 Mar 18
|
{
"type": "Brief Report",
"title": "Modelling risk using Bayes theorem of infection by antibiotic-resistant Escherichia coli in rural and urban populations of Ecuador",
"authors": [
"Lenin Javier Ramirez Cando",
"Bence Mátyás",
"Zayda Jaqueline Lozano-Haro",
"Zayda Jaqueline Lozano-Haro"
],
"abstract": "Strains of antibiotic-resistant bacteria have become more and more prevalent. This has attracted the attention of health agencies worldwide, leading to an urgent search for mechanisms to put a stop to this phenomenon. This study focuses on estimating the probability of a person in Ecuador (at potential risk) contracting an infection due to ampicillin-resistant Escherichia coli through the consumption of contaminated water, for which a residence area of people was considered in urban or rural areas. The analysis was carried out using the Bayes Theorem and the results show that in the rural population the probability of contracting an infection of this kind is 8.41% whilst in the urban area the probability is 3.57%. These results show an urgent need to provide safe water sources to the population, as well as to instigate an environmental legislation reform that allows for controlling the release of emerging pollutants, including antibiotics.",
"keywords": [
"Antibiotic resistance",
"Emerging pollutants",
"Risk assessment"
],
"content": "Introduction\n\nFor years, humankind has sought to mitigate the impacts produced by human development. Perhaps one of the most serious and unregulated problems in legislation is that of emerging pollutants (EP). According to Fernández et al. (2016)1 these are common-use chemical compounds of natural or synthetic origin, which, despite not being considered significant in terms of distribution and/or concentration, present a risk to the environment and human health. Within EPs, antibiotics make up a group that is becoming increasingly important and their presence in the environment has become a global public health problem. Concern surrounding the presence of antibiotics in bodies of water and the subsequent increase in bacterial resistance has led the WHO to publish a list of bacteria that have developed capacities for inhibiting widely-used antibiotics. Bacterial resistance according to Rodríguez, A. (2016)2 arises from the excessive and irrational use of antibiotics to treat infections that affect human beings’ bodies. This is due to an independent adaptive selection of the bacteria and the family of a specific strain as in the case of Staphylococcus aureus and its ability to inhibit conventional penicillin. This is not an isolated event; on the contrary, Cabrera, C. et al. (2007)3 maintain that by mutations in the chromosome of certain bacteria or by genetic exchange, bacteria can develop high innate resistance to antibiotics with several mechanisms between species of the same family or between different families. This study will explore the relationship that exists between bacterial resistance to antibiotics as a problem of emerging pollutants and public health for the inhabitants of Ecuador, taking area of residence as the reference.\n\n\nMethods\n\nIn order to estimate the probability of an Ecuadorian contracting an antibiotic-resistant bacterial infection, one must consider the area where the person lives, the presence or absence of Escherichia coli in the main sources of water for human consumption in the country, and the antibiotic to be analysed (in this case ampicillin). The analysis was carried out considering the Bayes Theorem which, according to Marrero, D. (2014)4, is expressed as the conditional probability of a random event ‘A’ given another event ‘B’. Below is its formula, which takes into account that several events of A can be exclusive:\n\nWhere: P(Ai ) are the a priori probabilities.\n\nP(B|Ai ) are the probabilities of B in hypothesis Ai.\n\nP(Ai |B) are the a posteriori probabilities.\n\n∑i=1kP(B|Ai)P(Ai) is the sum of the probabilities of B in the hypothesis Ai times the a priori probabilities.\n\nOur analysis focuses on the occurrence of three events:\n\n(i) the probability of consuming contaminated water, depending on the area where a person lives.\n\nii) the probability that a person who consumes contaminated water will contract an infection due to Escherichia coli.\n\niii) the probability that the contracted infection is resistant to antibiotics, using ampicillin as a reference.\n\n\nResults\n\nIn order to carry out this estimate, data published by the INEN according to Ecuadorians’ areas of residence has been taken as a reference. The data are shown in Table 1:\n\n//Source: INEC, Population census (2010)5.\n\nIn the same way, the INEN provides information regarding water quality (shown in Table 2):\n\nSource: INEC Survey (December, 2016)6.\n\nThereafter, we applied the aforementioned Bayes Theorem to find out the probability that people living in urban or rural areas who consume drinking water may be contaminated with Escherichia coli. For this case:\n\nP(Ai |B) is P(urban|contaminated water)\n\nP(Ai ) is P(urban)&P(rural)\n\nP(B|Ai ) is P(contaminated water|urban)&\n\nP(contaminated water|rural)\n\n\n\n\n\nBy carrying out the same analysis for the rural population, one obtains a 54.81% probability that a person living in the rural area may consume contaminated water (Table 3).\n\nOnce the probability of a person drinking contaminated water is known, it is necessary to find out the probability of contracting an infection due to Escherichia coli by consuming contaminated water. According to Vila, J. et al.(2016)7, in a study of 33 people living in South America in urban and rural areas, there is a 9.1% and 12.2% respectively that they house the aforementioned bacteria.\n\nOnce the probability of contracting an E. coli infection due to contaminated water consumption is identified, it is necessary to find out how many of these infections are resistant to antibiotics, which in our case was ampicillin. Bianchi, V. et al., (2014)8, in a study conducted in the San Juan River in Argentina, showed that the average ampicillin-resistant UFC percentage in urban areas was 73.39% and for rural areas 92.85%. Using the Bayes Theorem for each of the cases described above, we obtained the following results:\n\n\nConclusions\n\nThis study shows that both urban and rural populations are exposed to an antibiotic-resistant infection. However, Ecuador’s rural population is more exposed because the water sources they use are not safe. This draws attention to the necessity of providing safe, clean drinking water to the entire population. Even so, the high standards of water quality that many Ecuadorian cities have does not completely eliminate the risk of contracting antibiotic-resistant infections, thus demonstrating that an urgent legislation reform is required in order to control the release of these types of pollutants into bodies of water.\n\n\nData availability\n\nPopulation and water quality data was obtained from the Instituto Nacional de Estadística y Censos (INEC), Population census (2010): http://www.ecuadorencifras.gob.ec/base-de-datos-censo-de-poblacion-y-vivienda/ and INEC, Survey (2016): http://www.ecuadorencifras.gob.ec/documentos/web-inec/EMPLEO/2017/Indicadores%20ODS% 20Agua,%20Saneamiento%20e%20Higiene/ Presentacion_Agua_2017_05.pdf",
"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\nAlós J: [Antibiotic resistance: A global crisis]. Enfermedadesinfec- ciosas y microbiología clínica. 2015; 33(10): 692– 697. Publisher Full Text\n\nRodríguez A: Resistencia bacteriana en urocultivos, en pacientes del área de hospitalización del hospital un canto a la vida “Padre Carolo”, en el periodo comprendido entre enero 2012 adiciembre 2013. (Tesis de pregrado). Universidad Central del Ecuador, Quito. 2015; Reference Source\n\nCabrera C, Gómez R, Zúñiga A: La resistencia de bacterias a antibióticos, antisépticos y desinfectantes una manifestación de los mecanismos de supervivencia y adaptación. Revista Colombia Médica. 2007; 38(2): 149–158. Reference Source\n\nMarrero D: Introducción a la Estadística Bayesiana. (Tesis de grado). Universidad Autónoma de Barcelona, España. 2014. Reference Source\n\nInstituto nacional de estadísticas y censos: Censo de Población y Vivienda 2010. 2010. Reference Source\n\nInstituto nacional de estadísticas y cen-sos: Medición de los indicadores ODS de Agua, Saneamiento e Higiene (ASH) en el Ecuador. 2016. Reference Source\n\nVila J, Oliveira I, Zboromyska Y, et al.: Diarrea del viajero. Revista Enfermedades infecciosas y microbiología clínica. 2016; 34(9): 579–84. Publisher Full Text\n\nBianchi V, Varela P, Flores D, et al.: Evaluación de Escherichia Coli resistente a antibióticos como especie bioindicadora de contaminación fecal en agua y peces en la cuenca inferior del río San Juan. Revista Natura Neotropicalis. 2014; 45(2): 45–69. Publisher Full Text"
}
|
[
{
"id": "32440",
"date": "28 Mar 2018",
"name": "Jorge Ramírez",
"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 methods in this paper are adequate, however, in probability theory, according to Bayes’ theorem that describes the probability of an event, based on prior knowledge of conditions that might be related to the event. So, in case of this paper I think it is necessary to show data about pollution water made in different areas of Ecuador, for two reason: the first one is to make known the difference between rural and urban area, and the second one, if “this study focused on estimating the probability of a person in Ecuador contracting an infection due to ampicillin-resistant Escherichia coli through the consumption of contaminated water”, it’s necessary to know if the source of water in Ecuador are contaminated with ampicillin-resistant E. coli. So, if the water in Ecuador is contaminated with ampicillin-resistant E. coli, is highly probably that the people of Ecuador contracting an infection, then using Bayes’ theorem, a water quality of water (contaminated with E. coli) can be used to more accurately assess the probability that the contracting an infection, compared to the assessment of the probability of people contracting infection made without knowledge of data of quality of water.\nIn conclusion the paper is correct because the aim is shows that both urban and rural populations are exposed to an antibiotic-resistant infection, and in Ecuador there aren't researched in this area. For this reason I think the worked can be published.",
"responses": [
{
"c_id": "4296",
"date": "18 Dec 2018",
"name": "Bence Mátyás",
"role": "Author Response",
"response": "We are grateful for Dr. Ramirez Robles Jorge Yandry's report,We just wish to add that considering Bayes theorem capacity to aggregate new information, starting with non-known data analysis is an effective way."
}
]
},
{
"id": "32439",
"date": "08 May 2018",
"name": "Andrés Ricardo Izquierdo Romero",
"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 clear, however in the manuscript is cited Fernández et al. (2016), and it is not found in the bibliography. I recommend placing more citations of other investigations where Bayes Theorem is used, for this type of investigation.\n\nThe statistical analysis if applicable, however I recommend placing the calculations to obtain the values that were obtained and discussed, such as:\nProbability of contracting an E. coli infection: 38.08% (people living in urban areas), 61.92% (people living in rural areas) Probability of contracting an infection with a resistant strain: 32.97% (people living in urban areas), 67.03% (people living in rural areas) Probability of contracting an infection resistant to ampicillin due to the consumption of contaminated water: 3.57% (people living in urban areas), 8.41% (people living in rural areas)\n\nI suggest that it is necessary to show the data for water pollution in rural and urban areas of Ecuador, as well as the parameters for water quality according to the area of residence, such as water contamination with ampicillin-resistant E. coli, for estimate the probability of a person in Ecuador contracting an ampicillin-resistant Escherichia coli infection through the consumption of contaminated water, as the manuscript suggests.\nThe data of Vila, J. et al. (2016) can be used statistically, is a small study of 33 people living in South America in urban and rural areas, compared to the urban and rural population in Ecuador, 2010, which is 14,483,499 people, for the determination of the probability that it is Ecuadorian to consume contaminated water. an infection by Escherichia coli. There are data on E. coli infections in contaminated water in Ecuador, to strengthen the results obtained.\nTo determine the probability that an Ecuadorian will consume contaminated water to contract an infection caused by antibiotic-resistant Escherichia coli, used data from Bianchi et al. (2014) of the San Juan River in Argentina, showing that the average percentage of CFU resistant to ampicillin in urban areas was 73.39% and for rural areas 92.85%. These data are comparable with water sources in Ecuador, such as rivers, environmental conditions, etc., to be able to use them. Similarly, it is suggested to know if there are data of E. coli infections resistant to antibiotics in contaminated water in Ecuador.\nIn conclusion, the objective of the work is interesting, however, he suggested that to investigate more real data of Ecuador, to strengthen the obtained results.",
"responses": [
{
"c_id": "4297",
"date": "18 Dec 2018",
"name": "Bence Mátyás",
"role": "Author Response",
"response": "Dear Dr. Andrés Ricardo Izquierdo Romero, thank you for your report. \"The manuscript is clear, however in the manuscript is cited Fernández et al. (2016), and it is not found in the bibliography. I recommend placing more citations of other investigations where Bayes Theorem is used, for this type of investigation.\"authors' reply:We will ask for a minor correction in the article regarding the missing citation. Considering this piece is a research note, we do not think that expanding the article with more citations is necessary.\"I suggest that it is necessary to show the data for water pollution in rural and urban areas of Ecuador, as well as the parameters for water quality according to the area of residence, such as water contamination with ampicillin-resistant E. coli, for estimate the probability of a person in Ecuador contracting an ampicillin-resistant Escherichia coli infection through the consumption of contaminated water, as the manuscript suggests.\"authors' reply:This information is presented in external links only in order to keep the research note brief.\"The data of Vila, J. et al. (2016) can be used statistically, is a small study of 33 people living in South America in urban and rural areas, compared to the urban and rural population in Ecuador, 2010, which is 14,483,499 people, for the determination of the probability that it is Ecuadorian to consume contaminated water. an infection by Escherichia coli. There are data on E. coli infections in contaminated water in Ecuador, to strengthen the results obtained.\"authors' reply:The Bayes theorem allows to add more specific information later in a new analysis.\"To determine the probability that an Ecuadorian will consume contaminated water to contract an infection caused by antibiotic-resistant Escherichia coli, used data from Bianchi et al. (2014) of the San Juan River in Argentina, showing that the average percentage of CFU resistant to ampicillin in urban areas was 73.39% and for rural areas 92.85%. These data are comparable with water sources in Ecuador, such as rivers, environmental conditions, etc., to be able to use them. Similarly, it is suggested to know if there are data of E. coli infections resistant to antibiotics in contaminated water in Ecuador... In conclusion, the objective of the work is interesting, however, he suggested that to investigate more real data of Ecuador, to strengthen the obtained results.\"authors' reply:The Bayes theorem allows to add the specific information later in a new analysis, making analysis deeper, however, this research note tends to offer the first step for a further analysis regarding Bayesian statistics."
}
]
},
{
"id": "47288",
"date": "08 May 2019",
"name": "Imre Vágó",
"expertise": [
"Reviewer Expertise Plant nutrition"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI read with great interest Ramirez and colleagues’ study in which they present an estimation about the probability of contracting infection due to ampicillin-resistant E. coli though the consumption of contaminated water in the urban and rural areas of Ecuador.\nThis publication is a good example for how to obtain valuable and useful knowledge through consistent data analysing and data evaluation. The goal of the study is well described. The manuscript is clear; the used methods in this paper are adequate. However, I would like to share my observations in order to aid readers’ understanding.\nDespite the authors analysing separately the urban and rural populations of Ecuador, the basis of the differentiation is not defined.\n\nIn this paper, the data of urban and rural populations in Ecuador is outdated; it referred to 2010 data (Table 1). I mean, it would be better to use the latest published dataset from 2018, source: https://tradingeconomics.com/ecuador/rural-population-percent-of-total-population-wb-data.html. It states that the total population is 16,624,858 and also the ratio of urban population (64.3%) increased, compared to 2010.\n\nThe ratio of population consuming Escherichia coli-infected water (Table 2) is surprisingly high, especially for the rural population, compared to Europe. Does it really cover reality? Using ampicillin resistance values measured in the Bianchi et al. (2014) study in the water of San Juan River in Argentina, is critical. The distance is about 3600 km, the climate conditions are significantly different. Sorry, I am not aware of the ampicillin application frequency in South America, perhaps this is the reason for my doubts about this data.\n\nLast but not least: there are two typos in the paper. One of them is the missing closing bracket “)” before the equal sign ‘=’ in the Marrero’s equation. The other error is in Table 2; instead of last row (Total, 100, 100) it would be better to create another column, with the identical name and values.\n\nIn conclusion, the results of the research work are proving that the Bayes Theorem is suitable for the evaluation of elaborated data sets. The publication meets all of the scientific requirements; the results have practical relevance and they can be used by the decision makers also. That is why I suggest the presented paper for indexing.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-375
|
https://f1000research.com/articles/7-374/v1
|
26 Mar 18
|
{
"type": "Research Article",
"title": "Preliminary evidence that hydrostatic edema may contribute to the formation of diffuse alveolar damage in a Holstein calf model",
"authors": [
"Joseph M. Neary",
"Dee Church",
"Dee Church"
],
"abstract": "Background: Two notable findings of clinically healthy feedlot cattle suggest they may have pulmonary hydrostatic edema during the finishing phase of production: increased pulmonary arterial wedge pressures and pulmonary venous hypertrophy. The goal of this study was to determine if increased pulmonary arterial wedge pressure (PAWP) in a Holstein calf could lead to diffuse alveolar damage consistent with the early, exudative phase of acute interstitial pneumonia of feedlot cattle. Methods: Six male Holstein dairy calves were given daily subcutaneous injections of the nonspecific ß-adrenergic agonist isoprenaline (10 mg/kg/d), to induce left ventricular diastolic dysfunction, or sterile water for 14 days. On Day 14, pulmonary arterial pressures and wedge pressures were measured, echocardiography performed, and the ratio of mitral valve flow velocity (E) to septal lengthening velocity (e’) calculated. Calves were euthanized on Day 15 and lung lesions semi-quantitatively scored. Results: Mean PAWP was 12 ± 1 mm Hg in calves that received isoprenaline and 7 ± 1 mm Hg in controls (P = 0.01). Calves that received isoprenaline tended to have greater relative wall thickness than control calves (P = 0.15) and greater E/e’ ratios (P = 0.16), suggestive of concentric hypertrophy and diastolic dysfunction, respectively. Calves that received isoprenaline also tended to have a left ventricle and interventricular septum that was 29 ± 10 g heavier than control calves (P = 0.10) when controlling for body mass. Hyaline membranes, the hallmark feature of diffuse alveolar damage, were evident in lung sections from all calves that received isoprenaline but none of the controls. Conclusions: Consistent with prior pathological and physiological studies of feedlot cattle, this study provides preliminary evidence that cattle presenting with clinical signs and pathology consistent with early stage acute interstitial pneumonia could be attributable to hydrostatic edema associated with left ventricular failure.",
"keywords": [
"acute respiratory distress syndrome",
"pneumonia",
"congestive heart failure",
"hypertension",
"left ventricle"
],
"content": "Introduction\n\nFeedlot cattle are susceptible to two diseases of the cardiopulmonary system as they approach slaughter weight: congestive heart failure1 and acute interstitial pneumonia (AIP)2. In human medicine, and for the purpose of our study, acute interstitial pneumonia was defined as an acute respiratory distress syndrome (ARDS) of unknown etiology that is not primarily attributable to hydrostatic edema due to congestive left ventricular failure, pulmonary venous hypertension, or fluid overload3. In veterinary medicine, a consensus definition of ARDS has been proposed4. Diagnosing feedlot cattle using the proposed criteria, however, is not feasible largely because the diagnostic tools for ruling out hydrostatic edema are not available; consequently, it has been said that definitive diagnosis of AIP in cattle requires histopathologic evaluation of lung tissue obtained postmortem5. The histological counterpart of ARDS is typically diffuse alveolar damage (DAD), defined by the presence of hyaline membranes6.\n\nIf an animal dies in the acute, exudate phase of AIP, then hyaline membranes and hemorrhage may be the only lesions observed5. This is problematic because these lesions are not pathognomonic for ARDS and may be attributable to hydrostatic edema. Furthermore, there are two notable findings of clinically healthy feedlot cattle that suggest they may be at risk of congestive left heart failure, and therefore pulmonary hydrostatic edema, during the finishing phase: increased pulmonary arterial wedge pressures (PAWP) and pulmonary venous hypertrophy7. This leads us to speculate whether cattle presenting with clinical signs and pathology consistent with early stage AIP could, at least in some instances, be attributable to hydrostatic edema associated with left ventricular failure. The goal of this study was to determine if increased PAWP due to left ventricular dysfunction in a Holstein calf model could lead to pulmonary lesions consistent with the early, exudative phase of AIP in feedlot cattle.\n\n\nMethods\n\nSix, day-old male, intact clinically healthy Holstein dairy calves were collected from a farm in West Texas and individually housed under normoxic conditions (altitude: 975 m). At 7-days of age (Day 1 of the study), calves (n = 3) were given daily subcutaneous injections of the nonspecific ß-adrenergic agonist isoprenaline (10 mg/kg/d) (henceforth, ISO calves) or an equivalent volume of sterile water (controls, n = 3) for 14 days. On Day 14, pulmonary arterial pressures and wedge pressures were measured and echocardiography performed. Calves were euthanized on Day 15. The heart and lungs were weighed, and lung sections histologically evaluated.\n\nInstitutional Animal Care and Use Committee approval was granted prior to initiation of the study (Protocol 17013-02). Efforts were made to ameliorate animal suffering by following recommendations for the housing and care of animals provided by the National Research Council8.\n\nDay-old male, intact, male Holstein calves were obtained from one commercial dairy in West Texas. Six calves were included in this preliminary study as, to our knowledge, no similar studies had been performed in this species. Calves were fed 4 to 5 L of colostrum within 12 hours of birth. They were given 3 L of non-medicated milk replacer (22% crude protein, 15% crude fat) twice per day throughout the study (Purina® Herd Maker®, Gray Summit, MO, USA) and had ad libitum access to water. From 1 week of age (Day 1) calves had ad libitum access to a calf starter (Purina®, Ampli-Calf® Starter 22, Gray Summit, MO, USA) with 22% crude protein (dry matter basis).\n\nAll calves were individually housed in pens with dimensions 1.8 m by 2.3 m (Agri-Plastics, Grassie, ON, Canada). Four calves were housed on a raised slatted floor inside temperature-controlled chambers (temperature 17 ± 3°C). Calves were housed according to body mass so that the heaviest calves at the start of the study (one control and one experimental calf) were housed in one chamber (Calves 4 and 5) and the lightest calves (Calves 1 and 2) in another chamber. One control (Calf 3) and one experimental calf (Calf 6), were housed in shaded outdoor pens with straw bedding on a sloped concrete floor. The pens were moved to new locations every 3 days and the inside of the pens cleaned with disinfectant (Virkon S, DuPont, Wilmington, DE, USA). Soiled straw was removed once daily, and all straw was replaced every 3 days.\n\nTo our knowledge, isoprenaline (DL-Isoproterenol hydrochloride, 98%, Alfa Aesar, Ward Hill, MA, USA) has not been used in large animal models of heart failure with preserved ejection fraction (HFpEF). The dosage used in this study (10 mg/kg sid) was double the dosage used in a study of equivalent duration in a mouse model9. The isoprenaline was dissolved in sterile water (50 mg/mL) at room temperature prior to subcutaneous injection in the neck. Alternating sides of the neck were used. Controls were injected subcutaneously in the neck with an equivalent volume of sterile water (0.2 mL/kg). Treatments were given between 8:30 am and 9:30 am after all calves had been fed milk replacer. Injections were given whilst calves were manually restrained within their pen.\n\nPulmonary arterial pressure (PAP) testing was performed on Day 14. Calves were restrained in a calf chute and a halter used to hold the calf’s head to one side so that the right jugular grove was exposed. The neck was clipped and cleaned with chlorhexidine solution. A 7-French peel-away introducer (IS-07AS, Vascor Medical Corporation, Tarpon Springs, FL, USA) was placed in the jugular vein prior to inserted of a 110 cm, 7 French, polyurethane, modified J-tip wedge pressure catheter (172-110P, Vascor Medical Corporation, Tarpon Springs, FL, USA). A pressure transducer (TranStar DPT, Smiths Medical ASD, Inc., Dublin, OH, USA) was interposed between the catheter to the data acquisition system (IX-TA-220, iWorx Systems, Inc., Dover, NH, USA). The pressure waveforms were recorded and analyzed offline (LabScribe3, iWorx Systems, Inc., Dover, NH, USA). Pressures were analyzed at the end of expiration. The position of the catheter tip was determined by monitoring the change in the pressure waveform as the catheter tip was advanced through the right atrium, right ventricle, and finally into the pulmonary artery.\n\nEchocardiography was performed on Day 14. Calves remained standing throughout the procedure. Fractional shortening and ejection fraction (cubed or Teichholz method10) were obtained from right parasternal long-axis views of the left ventricle obtained between intercostal spaces 3 to 6. Relative wall thickness was calculated as two-times the left ventricular free wall diastolic thickness divided by the left ventricular internal diastolic diameter. Early diastolic (e’) septal lengthening velocities and early mitral flow (E) velocities were obtained from a left parasternal apical four-chamber view. The E/e’ ratio is a measure of left ventricular filling pressure and, consequently, a diagnostic measure of diastolic dysfunction11.\n\nCalves were euthanized with intravenous pentobarbital sodium (85 mg/kg) on Day 15 of the study and exsanguinated. Lung lobes were individually weighed. The atria were separated from the ventricles at the atrioventricular junction. The right ventricular free wall (RV) was separated from the left ventricle and septum (LVS). The RV and LVS were individually weighed.\n\nThe left diaphragmatic lobe was perfused with formalin (10%, neutral buffered) at 15 to 20 cm H2O for approximately 5 minutes. After 5 days of formalin fixation, lung sections were collected midway along the dorsal aspect of the lobe for histology. Paraffinized sections (4 µm) were stained with hematoxylin and eosin and Masson’s trichrome. Lung sections were semi-quantitatively scored based on severity (0 = no lesion; 1 = mild; 2 = moderate; 3 = severe). The investigator was blinded to the calves’ identities and treatment group. The pulmonary parenchymal lesions scored included interstitial edema, intra-alveolar edema, hemorrhage and hemosiderin, inflammatory infiltrates, type II hyperplasia, and interstitial fibrosis. Pulmonary arterioles (< 500 μm) and veins were scored for medial hypertrophy and adventitial fibrosis. Veins were distinguishable from arteries as they have spiral bundles of muscle that give them a beaded appearance.\n\nStatistical analyses were performed using commercial software (Stata version 12.1, College Station, TX). Calf 1 was excluded from statistical analyses because a sub-endocardial lesion was detected postmortem that likely affected his cardiac function and, consequently, pulmonary vascular pathophysiology. Between group differences were evaluated using Student’s t-test with equal variances. Student’s t-test is a suitable statistical method for small sample sizes (n ≤ 5) even if group sizes are unequal as long as the effect size is expected to be large12. Linear regression was used to determine if there was a significant difference between groups in the mass of the right ventricle, left ventricle and interventricular septum, and lung, while controlling for body mass.\n\n\nResults\n\nCalves that received ISO had a greater mean PAWP but a lower mean PAP than controls. Mean PAWP was 12 ± 1 mm Hg in the ISO group and 7 ± 1 mm Hg in the control group (P = 0.01). Mean PAP was 23 ± 2 mm Hg in the ISO group and 43.5 ± 8 mm Hg in the control group (P = 0.05). The control group had a greater heart rate (104 ± 9 bpm) than the ISO group (74 ± 4 bpm; P = 0.03). Results are presented in Table 1.\n\n*Left ventricular endocardial lesion identified post-mortem\n\nThere was no statistical difference in ejection fraction or fractional shortening between the ISO and control groups indicating that left ventricular systolic function was preserved. Ejection fraction was 66 ± 6% in the control calves and 54 ± 6% in the ISO group (P = 0.29). Fractional shortening was 36 ± 4% in the control calves and 29 ± 4% in the ISO group (P = 0.27). ISO calves tended to have greater relative wall thickness than control calves (P = 0.15) and greater E/e’ ratios (P = 0.16) which is suggestive of concentric hypertrophy and diastolic dysfunction, respectively. Results are presented in Table 2.\n\n* Left ventricular endocardial lesion identified post-mortem\n\nThere was no difference in body mass at the end of the study (P = 0.51). The mean body mass was 46.6 kg ± 3.3 kg for the control calves and 42.8 ± 3.4 kg for the ISO group. Calf 1 had endocardial fibrosis extending from the atrio-ventricular boundary to the dorsal aspect of the papillary muscle and approximately 0.5 cm into the myocardium (Supplementary file). Calf 5 had a 1 cm abscess in the right diaphragmatic lobe and Calf 6 had acute fibrinous pneumonia affecting the ventral 20% of the right cranio-ventral lung lobe (Supplementary file). The lungs of Calf 2 appeared hyper-inflated (Figure 1) and Calf 6 had a mottled icteric liver with rounded margins.\n\nMasson’s trichrome. Scale bar = 0.25 mm.\n\nThe ISO group tended to have a left ventricle and interventricular septum that was 29 ± 10 g heavier than control calves (P = 0.10) when controlling for body mass. There was no difference between groups in the mass of the lung (P = 0.81) or right ventricle (P = 0.36) when controlling for body mass. Results are presented in Table 3.\n\n* Left ventricular endocardial lesion identified post-mortem\n\nIn general, tissue areas with lesions consistent with DAD were multifocal and often located adjacent to a lobule with a normal healthy appearance. Occasionally, a gradual change from healthy to DAD was observed within the same lobule. All ISO calves had mild or moderate interstitial and intra-alveolar edema, mild or moderate hemorrhage, and type II hyperplasia. Two control calves (Calves 3 and 5) showed mild interstitial edema in some areas. Interstitial inflammatory infiltrates were evident in two calves, particularly Calf 1. No calves showed evidence of interstitial fibrosis. Calves 4 and 6 showed moderate epithelisation of alveoli. Calf 6 also had severe emphysema and honeycomb parenchymal remodeling (Figure 1). Results are presented in Table 4.\n\n*Left ventricular endocardial lesion identified post-mortem\n\nMild or moderate medial hypertrophy and adventitial fibrosis of pulmonary arterioles was evident in all calves. ISO calves had more substantial medial hypertrophy of the pulmonary veins than controls. Pulmonary veins were not evident in Calf 6. Adventitial fibrosis was not observed except in Calf 4. Results are presented in Table 5.\n\n\nDiscussion\n\nThe findings of this study indicate that left ventricular dysfunction could feasibly contribute to the development of diffuse alveolar damage, the pathologic correlate of acute interstitial pneumonia, an ARDS of feedlot cattle of unknown etiology. Two weeks of daily injections with isoprenaline led to the development of heart failure with preserved ejection fraction (HFpEF); left ventricular systolic function was preserved but diastolic function was reduced. Given that subclinical HFpEF in a Holstein model has many of the pathologic features and physiologic measurements consistent with ARDS13, this preliminary study indicates that the diagnosis of AIP in a feedlot animal can only be considered as tentative if left ventricular dysfunction has not been ruled out as a primary cause.\n\nThe PAWP of the ISO calves were significantly greater than controls but substantially less than the PAWP of 26 mm Hg previously reported in feedlot cattle at the end of the feeding period at the altitude of 1,220 m7. Pulmonary arterial wedge pressures are typically less than 15 mm Hg in non-feedlot cattle and other mammals14; therefore, the DAD seen in the ISO calves in this study is likely attributable to the acute doubling of PAWP over a 2-week period. Feedlot cattle may be able to tolerate much greater PAWP if pressures were to rise insidiously throughout the feeding period. There are a variety of adaptations, in addition to pulmonary arterial and venous remodeling15, that may occur in response to rising pulmonary vascular pressures: first, the lymphatic system is recruitable – it is able to increase the rate of lung-water clearance by over 10-fold.16,17; and second, alveolar fibrosis may occur to reduce alveolar-capillary membrane permeability to water18. The time period over which pulmonary vascular pressure rise is, therefore, likely to be a key determinant of the pulmonary adaptability.\n\nIt is also feasible that isoprenaline induced DAD through forward hemodynamic effects. Increased cardiac contractility and pulse pressure may have had deleterious downstream effects on the pulmonary vasculature19,20 paving the way for increased fluid flux into the pulmonary interstitium. Whether attributable to backward or forward hemodynamic effects, an increase in pulmonary capillary pressure will lead to mechanical injury of the alveolar-capillary membrane leading to increased capillary permeability and impaired gas exchange21. Although acute damage can be reversed, long term blood-gas barrier disruption leads to lung fibrosis, inflammation, impaired alveolar fluid clearance, and muscularization of pulmonary vessels21,22.\n\nInterestingly, the two calves housed in shaded outdoor pens on straw bedding had the greatest pulmonary arterial pressures within their respective treatment groups. In a prior study, calves housed on straw that developed bloody scours had histologic evidence of DAD and greater pulmonary arterial pressures than calves housed on slatted flooring that did not develop scours23. It was speculated that intestinal disease may contribute to the development of pulmonary disease in cattle23. Calves housed on straw bedding in our study may have been in the incubation phase of intestinal disease.\n\nEven though there were only 6 calves in this study, the goal of the study, to determine if left ventricular dysfunction could lead to lesions consistent with the early, exudative phase of AIP, was met. For ethical reasons, decompensated heart failure was not induced; therefore, it is not possible to say whether overt left ventricular failure clinically manifests as an ARDS-like event. Human medical reports suggest, however, that it can present as an ARDS which is why the diagnosis of ARDS requires that acute dyspnea attributable to hydrostatic edema must first be ruled out3. Large-scale prospective cohort studies of feedlot cattle are necessary to determine if pulmonary arterial pressures and PAWP are positively associated with risk of respiratory diseases, such as AIP. We speculate that elevated pulmonary vascular hydrostatic pressures may act synergistically with mediators of acute alveolar damage, such as pathogens or airborne irritants, to promote the development and progression of respiratory disease.\n\n\nData availability\n\nAll raw data underlying this study is available from Harvard Dataverse under a CC0 Public Domain Dedication\n\nImages of the heart and lungs are available at http://dx.doi.org/10.7910/DVN/HD1GEI24\n\nPulmonary histology is available at http://dx.doi.org/10.7910/DVN/UR9MAC25\n\nHepatic histology images are available at http://dx.doi.org/10.7910/DVN/ZBIVAG26\n\nPulmonary vascular pressure recordings are available at http://dx.doi.org/10.7910/DVN/SKBMWZ27",
"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\nNeary JM, Booker CW, Wildman BK, et al.: Right-Sided Congestive Heart Failure in North American Feedlot Cattle. J Vet Intern Med. 2016; 30(1): 326–334. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLoneragan GH, Gould DH, Mason GL, et al.: Involvement of microbial respiratory pathogens in acute interstitial pneumonia in feedlot cattle. Am J Vet Res. 2001; 62(10): 1519–1524. PubMed Abstract | Publisher Full Text\n\nARDS Definition Task Force, Ranieri VM, Rubenfeld GD, et al.: Acute respiratory distress syndrome: the Berlin Definition. JAMA. 2012; 307(23): 2526–2533. PubMed Abstract | Publisher Full Text\n\nWilkins PA, Otto CM, Baumgardner JE, et al.: Acute lung injury and acute respiratory distress syndromes in veterinary medicine: consensus definitions: The Dorothy Russell Havemeyer Working Group on ALI and ARDS in Veterinary Medicine. J Vet Emerg Crit Care. 2007; 17(4): 333–339. Publisher Full Text\n\nWoolums AR: Feedlot Acute Interstitial Pneumonia. Vet Clin North Am Food Anim Pract. 2015; 31(3): 381–9, vi. PubMed Abstract | Publisher Full Text\n\nSweeney RM, McAuley DF: Acute respiratory distress syndrome. Lancet. 2016; 388(10058): 2416–2430. PubMed Abstract | Publisher Full Text\n\nNeary JM: Epidemiological, physiological and genetic risk factors associated with congestive heart failure and mean pulmonary arterial pressure in cattle. Doctoral dissertation, Department of Clinical Sciences, Colorado State University, 2014. Reference Source\n\nInstitute for Laboratory Animal Research: Guide for the Care and Use of Laboratory Animals. 8th Ed. 2011. PubMed Abstract\n\nMa X, Song Y, Chen C, et al.: Distinct actions of intermittent and sustained β-adrenoceptor stimulation on cardiac remodeling. Sci China Life Sci. 2011; 54(6): 493–501. PubMed Abstract | Publisher Full Text\n\nTeichholz LE, Kreulen T, Herman MV, et al.: Problems in echocardiographic volume determinations: echocardiographic-angiographic correlations in the presence of absence of asynergy. Am J Cardiol. 1976; 37(1): 7–11. PubMed Abstract | Publisher Full Text\n\nPaulus WJ, Tschöpe C, Sanderson JE, et al.: How to diagnose diastolic heart failure: a consensus statement on the diagnosis of heart failure with normal left ventricular ejection fraction by the Heart Failure and Echocardiography Associations of the European Society of Cardiology. Eur Heart J. 2007; 28(20): 2539–2550. PubMed Abstract | Publisher Full Text\n\nde Winter JC: Using the Student ’s t-test with extremely small sample sizes. Pr Assessment, Res Evalutaion. 2013; 18(10): 1–12. Reference Source\n\nMatute-Bello G, Downey G, Moore BB, et al.: An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol. 2011; 44(5): 725–738. PubMed Abstract | Publisher Full Text\n\nWill DH, Alexander AF, Reeves JT, et al.: High altitude-induced pulmonary hypertension in normal cattle. Circ Res. 1962; 10(2): 172–177. PubMed Abstract | Publisher Full Text\n\nReid LM: The pulmonary circulation: remodeling in growth and disease. The 1978 J. Burns Amberson lecture. Am Rev Respir Dis. 1979; 119(4): 531–546. PubMed Abstract\n\nUhley HN, Leeds SE, Sampson JJ, et al.: Role of pulmonary lymphatics in chronic pulmonary edema. Circ Res. 1962; 11(6): 966–70, Accessed June 23, 2017. PubMed Abstract | Publisher Full Text\n\nDatar SA, Johnson EG, Oishi PE, et al.: Altered lymphatics in an ovine model of congenital heart disease with increased pulmonary blood flow. Am J Physiol Lung Cell Mol Physiol. 2012; 302(6): L530–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTownsley MI, Fu Z, Mathieu-Costello O, et al.: Pulmonary microvascular permeability. Responses to high vascular pressure after induction of pacing-induced heart failure in dogs. Circ Res. 1995; 77(2): 317–325, Accessed June 23, 2017. PubMed Abstract | Publisher Full Text\n\nLi M, Scott DE, Shandas R, et al.: High pulsatility flow induces adhesion molecule and cytokine mRNA expression in distal pulmonary artery endothelial cells. Ann Biomed Eng. 2009; 37(6): 1082–1092. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi M, Stenmark KR, Shandas R, et al.: Effects of pathological flow on pulmonary artery endothelial production of vasoactive mediators and growth factors. J Vasc Res. 2009; 46(6): 561–571. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuazzi M: Alveolar-capillary membrane dysfunction in heart failure: evidence of a pathophysiologic role. Chest. 2003; 124(3): 1090–1102, Accessed August 17, 2017. PubMed Abstract | Publisher Full Text\n\nChen Y, Guo H, Xu D, et al.: Left ventricular failure produces profound lung remodeling and pulmonary hypertension in mice: heart failure causes severe lung disease. Hypertension. 2012; 59(6): 1170–1178. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGonyeau K, Subbiah S, Klein D, et al.: Development of pulmonary arterial hypertension and diffuse alveolar damage in 2-month old Holstein dairy calves following an acute episode of bloody scours [version 1; referees: 1 approved, 1 approved with reservations]. F1000Res. 2017; 6: 1826. Publisher Full Text\n\nNeary J: Heart and lungs of Holstein calves. Harvard Dataverse, V1. 2018. Data Source\n\nNeary J: Pulmonary histology of Holstein calves. Harvard Dataverse, V1. 2018. Data Source\n\nNeary J: Hepatic histology of Holstein calves. Harvard Dataverse, V1. 2018. Data Source\n\nNeary J: Cardiopulmonary pressures in Holstein calves. Harvard Dataverse, V1. 2018. Data Source"
}
|
[
{
"id": "37016",
"date": "23 Oct 2018",
"name": "Amelia Woolums",
"expertise": [
"Reviewer Expertise Lung disease of cattle",
"including lung infection",
"and immunity to lung disease caused by viruses or bacteria"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n\"If applicable, is the statistical analysis and its interpretation appropriate?\"\n\nFor a small pilot study, the approach to statistical analysis is acceptable. However, in some cases the statistical analysis may have provided results that are misleading. For example: in the Results section, the authors state that there was no difference in body mass between calves in the two groups. How was this tested? If by the Student’s t-test: per the Methods section, the t-test is only appropriate for small samples of unequal size if the effect is expected to be large. But the difference in body mass between the groups was not large, so it’s not convincing that the results of this analysis are accurate. Body mass of the calves is not a particularly important outcome of this study, but if differences are going to be reported, a different statistical test should be used.\n\nSimilarly, while the ejection fraction between the two groups was not significantly different, the distribution of values for this outcome in the calves in the two groups looks like EF would likely have been significantly lower in treated calves if more animals had been included. That is, the study likely lacked power to identify a true difference in ejection fraction. Thus this model may not actually induce left heart failure with preserved ejection fraction, as the authors contend.\n\n\"Are the conclusions drawn adequately supported by the results?\"\n\nGenerally yes, with the caveat that the conclusions are based on a very small number of animals. The data depicted in Table 4 support the authors’ contention that experimentally-induced left heart dysfunction was associated with lung lesions typical of early acute interstitial pneumonia: hyaline membranes and type II pneumocyte hyperplasia.\n\nSpecific comments:\n\nMethods Postmortem exam and histology\n\nWhat were the qualifications of the investigator who identified and scored the histologic lesions? Was that person a board certified veterinary pathologist?\n\nResults Do the investigators have any idea why the ISO treated calves had lower heart rates than the control calves?\n\nDiscussion The writers note: “…this preliminary study indicates that the diagnosis of AIP in a feedlot animal can only be considered as tentative if left ventricular dysfunction has not been ruled out...”\nStrictly speaking, AIP (acute interstitial pneumonia) is a pathologic diagnosis, which can result from a variety of possible primary insults. This study does suggest that acute left heart dysfunction can be associated with the lung lesion consistent with early AIP (hyaline membranes and type II pneumocyte hyperplasia). A histologic diagnosis of AIP can be made based on evidence of diffuse alveolar damage; this work suggests that any cause of acute left heart failure could be one of several possible causes of AIP.\n\nThis is in contrast to the clinical definition of ARDS which, as the authors note in the introduction, currently requires rule out of respiratory failure solely due to heart failure. However, it is usually not possible in the feedlot to make the measurements necessary to confirm ARDS and rule out acute left heart failure, based on current definitions. This research does support the concept that a feedlot steer or heifer with acute severe dyspnea may be suffering from a lung lesion resulting solely from acute congestive left heart failure, as well as a lung lesion resulting from infection, pneumotoxicity, or other possible causes of AIP.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "37946",
"date": "28 Nov 2018",
"name": "Min Li",
"expertise": [
"Reviewer Expertise Cattle model of hypoxia induced pulmonary hypertension",
"including pro-inflammatory phenotype and metabolic reprogramming of pulmonary vascular cells"
],
"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 small pilot study authors tried to determine if increased pulmonary arterial wedge pressure (PAWP) in a Holstein calf could lead to diffuse alveolar damage (DAD), which provide histologic evidence to diagnose acute interstitial pneumonia (AIP). The results showed that left ventricular dysfunction can associate with, or be one of the cause that a feedlot steer has the sign of AIP (hyaline membranes and type II pneumocyte hyperplasia).\n\"If applicable, is the statistical analysis and its interpretation appropriate?\"\nDue to the small number of animals used in the study, a qualified statistician is suggested to verify the statistical analyses.\n\"Are the conclusions drawn adequately supported by the results?\"\nMore histology images (figure 1) with clear labels of different type of lesions and pathology findings will help to support the findings shown in Table 4 and 5.\n\nSpecific comments:\n\n1. Methods Postmortem exam and histology\n\nWhether a board certified veterinary pathologist identified and scored the histologic lesions?\n\nResults More histology images (Figure 1) with clear labels of different type of lesions and pathology findings will help to support the findings shown in Table 4 and 5.\n\n2. In the introduction or discussion part, I would like authors to refine the novelty of this study. Increased PAWP due to left ventricular dysfunction can lead to hydrostatic edema, diffuse alveolar damage, all of which are pathologically correlate with acute interstitial pneumonia. This concept is acknowledged in the cattle field and the results from this study confirmed this concept and suggest that in feedlot, ARDS can not be diagnosed without first rule out LV dysfunction.\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": []
}
] | 1
|
https://f1000research.com/articles/7-374
|
https://f1000research.com/articles/7-60/v1
|
16 Jan 18
|
{
"type": "Case Report",
"title": "Case Report: Acute amyopathic dermatomyositis presenting with isolated facial edema",
"authors": [
"Efthymia Pappa",
"Marina Gkeka",
"Asimina Protogerou",
"Leonidas Marinos",
"Chariclia Loupa",
"Constantinos Christopoulos",
"Marina Gkeka",
"Asimina Protogerou",
"Leonidas Marinos",
"Chariclia Loupa",
"Constantinos Christopoulos"
],
"abstract": "A 45-year-old Asian man presented with acute-onset periorbital and facial edema associated with pyrexia. Muscle weakness was absent. Initial laboratory investigations showed an inflammatory reaction, while screening for infections was negative. Serum muscle enzyme levels were normal. He was hospitalized and treated empirically with antibiotics and corticosteroids, pending the result of facial skin and muscle biopsy. He showed a good clinical and laboratory response but an attempt to discontinue corticosteroids led to a prompt relapse of facial edema and pyrexia, associated with rising laboratory indices of inflammation. Biopsy findings were typical of dermatomyositis. Reintroduction of corticosteroid treatment resulted in complete clinical and laboratory remission. Facial edema as the sole clinical manifestation of dermatomyositis is extremely rare. There have been no previous reports of isolated facial edema in the setting of acute amyopathic dermatomyositis. A high level of suspicion is required to make the diagnosis in the absence of myopathy and the hallmark cutaneous manifestations of the disease (heliotrope rash, Gottron papules).",
"keywords": [
"dermatomyositis",
"amyopathic dermatomyositis",
"inflammatory myopathy",
"edema"
],
"content": "Introduction\n\nA patient presenting with isolated periorbital and facial edema may pose diagnostic challenges. The differential diagnosis includes allergic reactions, soft tissue infections, intracranial venous thrombosis, auto-immune (dermatomyositis (DM), lupus erythematosus) and auto-inflammatory (periodic syndrome) disorders, and trichinosis. In this setting, it may be difficult to make an accurate clinical diagnosis, and the patient is often treated empirically, pending the results of specific laboratory investigations. We present a case where skin and muscle biopsy established the previously unsuspected diagnosis of DM in a patient presenting acutely with periorbital and facial edema without myopathy. To our knowledge, there have been no previous reports of isolated facial edema in the setting of acute amyopathic dermatomyositis.\n\n\nCase report\n\nA 45-year-old Asian man, unskilled worker, who had lived in Greece for the last three years, presented to the Emergency Department with a three day history of painful facial swelling, pyrexia and vomiting. His medical history was notable only for type 2 diabetes mellitus treated with metformin and gliclazide. He denied recent travel abroad.\n\nOn examination there was bilateral, sensitive to palpation, edema involving the eyelids, nose, malar areas, forehead and anterior part of the scalp, associated with painful cervical lymphadenopathy (Figure 1A). The rest of the physical examination, including cardiovascular, respiratory, neuromuscular systems and abdomen, was unremarkable - in particular there were no muscle weakness or arthritis, and no skin eruptions on the trunk or extremities. Urgent laboratory investigations showed a picture of inflammation, with neutrophil leukocytosis (17,800/μL, normal 2,500–8,000), increased C-reactive protein levels (323 mg/L, normal <3.5) and elevated erythrocyte sedimentation rate (80 mm/1h, normal <15). Biochemical profile revealed hyperglycemia (glucose 385 mg/dL, normal 70–100) and mild compensated acidosis (serum bicarbonate 14.5 mmol/L, normal 22–29), but was otherwise unremarkable. Notably, serum muscle enzyme levels (creatine phosphokinase, aspartate aminotransferase, lactic dehydrogenase) were normal. Urinalysis and chest radiogram were normal. Abdominal ultrasound examination revealed cholelithiasis without signs of cholecystitis. The patient was admitted to hospital and, in the absence of a diagnosis, was treated empirically with intravenous ampicillin-sulbactam (3 g q.i.d.), clindamycin (600 mg t.i.d,) dimethindene (4 mg b.i.d. for 3 days, then o.d. for 7 days) ranitidine (50 mg b.i.d.), and methylprednisolone (40 mg t.i.d.). Soluble insulin was administered subcutaneously as required, based on capillary glucose measurements t.i.d.. Blood cultures and screening for infections, including HIV and hepatitis were negative. Computed tomography and magnetic resonance imaging (MRI) of the head showed marked subcutaneous tissue edema of face and cranial vault, while MRI venography was normal.\n\nA: Periorbital edema at presentation. The edema was evolving rapidly and the patient was unable to open his eyes. B: Subsidence of edema was followed by extensive skin desquamation. Erythema is due to corticosteroid therapy. C,D: Development of marked alopecia. Photographs B and D were taken on hospital day 26.\n\nA skin and muscle biopsy was obtained from an affected area of the face. The facial edema and cervical lymphadenopathy remitted gradually over the following days in parallel with falling levels of inflammation markers. An attempt to discontinue corticosteroid therapy on hospital day 9 was quickly followed by relapse of facial edema, pyrexia and cervical lymphadenopathy, associated with a new rise of inflammation markers. Discontinuation of antibiotics and reintroduction of methylprednisolone (40 mg I.V. t.i.d, switching to 16 mg p.o. t.i.d after five days) on hospital day 14 led to a new clinical and laboratory remission within five days.\n\nAdditional investigations showed an elevated antinuclear antibody titer (1:320, normal <1:80), while anti-dsDNA, RNP, Sm, SSA(Ro), SSB(La), Jo1, Mi2 autoantibodies and rheumatoid factor were negative. Trichinella spiralis IgG antibodies were also negative. C1-inhibitor levels were normal, while serum C3 levels were low (26.9 mg/dl, normal 88–135) and C4 levels were within normal range. Serum aldolase levels were normal. The skin and muscle biopsy findings were typical of DM (Figure 2).\n\nA, B: Myositis in the form of a mainly lymphocytic cellular infiltrate of striated muscle fibers with subsequent destruction and regeneration. (Hematoxylin and eosin x250, x400.) C: Leukocytoclastic vasculitis (fibrinoid necrosis and nuclear dust) in septal vessels of the subcutaneous tissue. (Hematoxylin and eosin x400.) D: Telangiectatic vessels in the reticular dermis, edema and a mild lymphocytic infiltrate. (Hematoxylin and eosin x200.)\n\nBy day 33, all laboratory abnormalities had returned to normal. Edema of face and scalp had completely subsided, leaving extensive desquamation and alopecia of involved areas (Figure 1B–D). The patient was discharged taking methylprednisolone 32 mg daily, with arrangements for rheumatology outpatient follow-up. He declined further investigations to exclude an underlying malignancy.\n\n\nDiscussion\n\nIn its typical form, DM presents with the unique combination of inflammatory myopathy and characteristic cutaneous lesions (heliotrope periorbital edema, Gottron papules and sign, shawl and V-sign, holster sign). Internal organs (lungs, heart, and gastrointestinal tract) may become involved, contributing to the significant morbidity and mortality of patients with DM who are not diagnosed and treated at an early stage. The importance of timely diagnosis is enhanced by the fact that approximately 15% of adult patients with newly diagnosed DM either have a preexisting malignancy or will develop one within a few years following diagnosis1,2 When clinical or laboratory manifestations of myopathy (proximal muscle weakness or elevated muscle enzyme levels in the serum) are absent, as was the case with our patient, DM may elude diagnosis. This can occur in the early stages of the illness, when muscle involvement may not yet be evident (“pre-myopathic DM”). The term “amyopathic DM” (synonym: dermatomyositis-sine-myositis) is usually reserved for patients with the hallmark cutaneous manifestations or histological findings of DM who have normal serum muscle enzyme levels and no muscle weakness for at least 6 months after the initial diagnosis (10–20% of all cases of DM, may be higher in Asian populations)3. A limitation of the present report is the lack of long-term outcome information because the patient was lost to follow-up.\n\nAlthough cutaneous manifestations of DM commonly involve the face, isolated facial edema as the sole clinical sign of disease is an extremely rare occurrence. Such an atypical presentation can lead to significant delays in diagnosis. Hall et al. reported a case where DM was diagnosed only when the patient was hospitalized for dysphagia, six months after his initial presentation with severe periorbital edema4. Subcutaneous edema, usually localized or regional, is being increasingly recognized as a manifestation of DM and appears to be associated with more aggressive disease5,6. The presumed pathophysiological mechanism involves increased permeability of capillaries caused by complement-mediated microvascular endothelial damage. In DM, putative antibodies directed against antigens of capillary endothelium activate the classical complement pathway, resulting in deposition of membrane attack complex on capillary endothelial cells early in the inflammatory process7. This is in keeping with the low serum complement C3 levels present in our case. Other, non-immunological mechanisms might be contributing to increased capillary permeability: Levels of vascular endothelial growth factor (VEGF), which induces hyperpermeability by a direct action on endothelial cells, have been found to be increased in muscle tissue and plasma of patients with early-phase DM8,9.\n\nIn conclusion, DM should be included in the differential diagnosis of patients presenting acutely with isolated facial edema of unknown cause, even when other clinical and laboratory manifestations of the disease are absent. An aggressive diagnostic approach with skin and muscle biopsy may enable early diagnosis and treatment of this potentially fatal disease.\n\n\nConsent\n\nWritten informed consent for publication of his clinical details and clinical images was obtained from the patient.",
"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\nWe thank Dr. Spilios Papanikolaou, General surgeon at “Amalia Fleming” General Hospital, for performing the skin and muscle biopsy.\n\n\nReferences\n\nDalakas MC, Hohlfeld R: Polymyositis and dermatomyositis. Lancet. 2003; 362(9388): 971–82. PubMed Abstract | Publisher Full Text\n\nMarie I: Morbidity and Mortality in Adult Polymyositis and Dermatomyositis. Curr Rheumatol Rep. 2012; 14(3): 275–85. PubMed Abstract | Publisher Full Text\n\nGerami P, Schope JM, McDonald L, et al.: A systematic review of adult-onset clinically amyopathic dermatomyositis (dermatomyositis siné myositis): a missing link within the spectrum of the idiopathic inflammatory myopathies. J Am Acad Dermatol. 2006; 54(4): 597–613. PubMed Abstract | Publisher Full Text\n\nHall VC, Keeling JH, Davis MD: Periorbital edema as the presenting sign of dermatomyositis. Int J Dermatol. 2003; 42(6): 466–7. PubMed Abstract | Publisher Full Text\n\nMilisenda JC, Doti PI, Prieto-González S, et al.: Dermatomyositis presenting with severe subcutaneous edema: five additional cases and review of the literature. Semin Arthritis Rheum. 2014; 44(2): 228–33. PubMed Abstract | Publisher Full Text\n\nTu J, McLean-Tooke A, Junckerstorff R: Increasing recognition of dermatomyositis with subcutaneous edema - is this a poorer prognostic marker? Dermatol Online J. 2014; 20(1): 21244. PubMed Abstract\n\nChen M, Daha MR, Kallenberg CG: The complement system in systemic autoimmune disease. J Autoimmun. 2010; 34(3): J276–86. PubMed Abstract | Publisher Full Text\n\nGrundtman C, Tham E, Ulfgren AK, et al.: Vascular endothelial growth factor is highly expressed in muscle tissue of patients with polymyositis and patients with dermatomyositis. Arthritis Rheum. 2008; 58(10): 3224–38. PubMed Abstract | Publisher Full Text\n\nHippenstiel S, Krüll M, Ikemann A, et al.: VEGF induces hyperpermeability by a direct action on endothelial cells. Am J Physiol Lung Cell Mol Physiol. 1998; 274(5 Pt 1): L678–84. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "30305",
"date": "15 Feb 2018",
"name": "Lyubomir A. Dourmishev",
"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 case report presents the diversity of clinical variants in autoimmune inflammatory myopathies. Clinical case is present briefly, but the information is sufficient.\nMy critical remarks are:\nIt is not clear how the authors clinically assess the muscle weakness;\n\nAuthors state creatine phosphokinase, aspartate aminotransferase and lactic dehydrogenase are in normal ranges. What about alanine aminotransferase and aldolase?\n\nI would suppose the term clinical amyopathic dermatomyositis, since muscle biopsy shows definite features of myositis and thus is the key evidence for diagnosis.\n\nIt is not indicated the place of muscle biopsy.\n\nI would recommend discussing the orbital myositis as a possible differential diagnosis.\n\nInvestigation of anti-MDA5 and may have value in this case.\n\nIn conclusion article is interesting and after some corrections will be useful for variety of medical specialists.\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": "3540",
"date": "26 Mar 2018",
"name": "Efthymia Pappa",
"role": "Author Response",
"response": "We thank Professor Dourmishev for his constructive comments, which we took into account in revising the manuscript. Our detailed response to his remarks is as follows: Muscle weakness was assessed by clinical examination. No abnormalities were detected on repeated clinical assessment of muscle tone, power and tendon reflexes. A relevant passage has been included in the revised text. Serum alanine aminotransferase levels were also normal. This has been stated in the revised text. Aldolase levels were normal – this is stated in the original text (paragraph 4, line 5 of case report section). We have used the more accurate term “Clinically amyopathic dermatomyositis” as suggested by the reviewer. We have indicated the site of biopsy (right malar area) in the revised text. Orbital myositis has been included in the differential diagnosis as suggested. We only had access to Jo1 and Mi2 autoantibodies at that time, and therefore no anti-MDA5 or other autoantibodies of potential value in DM could be looked for. We note, however, the low sensitivity of such antibodies. Only 6.9% of DM patients in a recent USA study had positive anti-MDA5, and most of these had overt myopathy (JC Hall et al. Arthritis Care Res. (Hoboken) 2013; 65: 1307-15)."
}
]
},
{
"id": "31365",
"date": "05 Mar 2018",
"name": "Albert Selva-O'Callaghan",
"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 report the case of a patient with periorbital and facial oedema in whom muscle biopsy allowed the diagnosis of myositis (probably DM). The skin biopsy in this case is not specific, absence of mucine and leukocytoclastic vasculitis are not typical of DM. This reviewer, nevertheless, agree that the most probably diagnosis is DM. In this case it would be appropriate to define it as a Clinically Amyopathic DM.\nDetermination of other specific autoantibodies would have been of help: anti-TIF1gamma, anti-PL12, anti-PL7, anti-SRP, anti-MDA5, or anti-NXP2.\n\nIn opinion of the reviewer, this presentation is not so rare.\nPlease comment or modify all these points.\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? Yes",
"responses": [
{
"c_id": "3539",
"date": "26 Mar 2018",
"name": "Efthymia Pappa",
"role": "Author Response",
"response": "We thank Dr. Selva-O’Callaghan for critically reviewing our manuscript. Our response is as follows: We have used the term “clinically amyopathic dermatomyositis” in the revised version, as suggested by both reviewers. We have also clarified that the skin biopsy findings were compatible with but not diagnostic of DM. Among the autoantibodies specific for myositis, we only had access to anti-Mi2 and anti-Jo1. Anti-Mi2 tends to be positive in classical DM with typical skin manifestations and high muscle enzyme levels, both of which were absent in our case. Positive anti-Mi2 was reported in 38.8% of DM patients from the Indian subcontinent (P Srivastava et al. Rheumatol Int 2016, DOI 10.1007/s00296-016-3494-3). In the same ethnic group, Jo1 was detected in 5.4% of DM patients. Although the face is frequently the only area affected in dermatomyositis, particularly in the early stages, we could find no published cases of adult DM presenting with isolated facial edema without rash or muscle enzyme abnormalities."
}
]
}
] | 1
|
https://f1000research.com/articles/7-60
|
https://f1000research.com/articles/7-366/v1
|
23 Mar 18
|
{
"type": "Research Note",
"title": "First record of Doleschallia tongana (Lepidoptera: Nymphalidae) for Guam Island",
"authors": [
"Jake Manuel",
"W. John Tennent",
"Donald W. Buden",
"Aubrey Moore",
"Jake Manuel",
"W. John Tennent",
"Donald W. Buden"
],
"abstract": "A single specimen of the butterfly,Doleshallia tongana Hopkins 1927, was collected on Guam Island on October 23, 2017 (13.430478°N, 144.800419°E). This is a new species record for Guam and Micronesia, indicating a geographical range expansion for D. tongana.",
"keywords": [
"Doleschallia tongana",
"Pacific orange leafwing",
"Guam",
"Micronesia",
"range expansion",
"invasive species",
"new country record"
],
"content": "Introduction\n\nOn October 23, 2017, a butterfly was taken from the underside of a leaf of soursop, Annona muricata, by a student (JM) assembling an insect collection as a requirement for the General Entomology course at the University of Guam. The collection site was the University of Guam campus in Mangilao, Guam (13.430478° N, 144.800419° E).\n\nThe specimen was pinned, images were made (Figure 1), documented in iNaturalist1 and deposited in the University of Guam insect collection (Accession code: iNat8515898).\n\nThis specimen does not match any of the descriptions in Butterflies of Micronesia2, the standard reference for Guam’s butterflies.\n\n\nIdentification\n\nDigital images of the specimen were sent to DB and JT for identification. On 7 November 2017, DB tentatively identified the specimen as a species in the genus Doleschallia, and indicated it possibly belonging to the bisaltide complex. On 24 February, 2018 JT determined the butterfly as Doleschallia tongana Hopkins, 1927, based on images and comparison with the extensive collections of the Natural History Museum, London.\n\nIn common with other species in the “bisaltide species-group, D. tongana is individually variable.\n\nThe convex outer margin of the forewing; the general appearance of the specimen; and geography all suggest D. tongana (tongana Hopkins, 1927, is a name to replace drusias Fabricius, 1781, the type locality for which is Tonga). Some minor ‘unusual’ features include the fact that tongana usually has a sub-apical ‘half-moon’ series of 4–5 spots on the forewing, lacking in this specimen, which only has two, but this lies within the wide individual variation of the species. Considering a distribution of Papua New Guinea (including the Bismarcks), the Solomon Islands, Fiji, Samoa, Tonga and New Caledonia, we are confident of associating this specimen with D. tongana. No doubt further material will confirm this association in due course. The species-group is in need of some revision3. The GBIF Backbone Taxonomy lists the accepted name for this taxon as Doleschallia bisaltide subsp. tongana Hopkins, 19274. However, the taxon record is tagged as a \"name parent mismatch\" issue.\n\nD. tongana is listed in the iNaturalist database5 and has been assigned the vernacular name ’Pacific orange leafwing’.\n\n\nGeographical distribution\n\nD. tongana, as it is currently understood, occurs throughout much of New Guinea, including the island groups in the east (see above).\n\nOccurrence of D. tongana in Samoa is a relatively recently recorded range expansion. It was first detected on Tutuila Island in American Samoa in 19976. Cook and Vargo 20006 state that \"The inclusion of Samoa in this species’ range by Parsons, 19987 appears to be based on a misreading of Hopkins (1927).\"\n\n\nDescription of caterpillar\n\nCook and Vargo 20006 provide a description of a last instar D. tongana caterpillar:\n\n“Just prior to pupation, the caterpillar measured ca. 50 mm in length. It possessed a black ground color with light speckling dorsally and prominent cream colored stripes running longitudinally, located dorso-laterally and ventro-laterally. Each body segment had seven prominent black spines, with numerous smaller secondary spines. The base of each primary spine was pale metallic blue. From a distance, the most prominent features of the caterpillar are the black ground color with metallic blue spots, and the pair of light parallel stripes running longitudinally on each side.“\n\nOnly a few larval host plants have been recorded for D.tongana (Table 1).\n\n\nDiscussion\n\nAn informal survey has been initiated on Guam to search for more specimens of D. tongana and to record host plants.\n\nThis insect has the potential to do economic damage because it has been reported to feed on breadfruit, Artocarpus altilis8.\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\niNaturalist observation: Doleschallia tongana . 2017. Accessed: 2018-02-28. Reference Source\n\nSchreiner IH, Nafus DM: Butterflies of Micronesia. University of Guam, 1997. Accessed: 2018-02-28. Reference Source\n\nTennent WJ: A checklist of the butterflies of Melanesia, Micronesia, Polynesia and some adjacent areas. Zootaxa. 2006; 1178: 1–209. Reference Source\n\nGBIF: Taxon - Doleschallia bisaltide tongana Hopkins, 1927. 2018. Accessed: 2018-02-28. Reference Source\n\niNaturalist taxon: Doleschallia tongana . 2018. Accessed: 2018-02-28. Reference Source\n\nCook RP, Vargo D: Range extension of Doleschallia tongana (Nymphalidae) to the Samoan archipelago, with notes on its life history and ecology. J Lep Soc. 2000; 54(1): 33–35. Reference Source\n\nParsons M: The butterflies of Papua New Guinea: their systematics and biology. Academic Press, 1998. Reference Source\n\nRobinson GS: Macrolepidoptera of Fiji and Rotuma, a taxonomic and biogeographical study. Classey, 1975.\n\nHolloway JD, Peters JV: The butterflies of New Caledonia and the Loyalty Islands. J Nat Hist. 1976; 10(3): 273–318. Publisher Full Text"
}
|
[
{
"id": "32407",
"date": "28 Mar 2018",
"name": "Richard S. Zack",
"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 well-written report and discussion of a new species of butterfly to Guam. The record is a significant range extension for the species. Introductions to islands such as Guam are of concern because of potential economic and environmental effects and reports such as this are important to document. It will be interesting to see if the species has established or if this was a one-time occurrence.\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": "33973",
"date": "23 May 2018",
"name": "Niklas Wahlberg",
"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 simple report on a range extension of a butterfly species. What makes it interesting is that the range extension is to the island of Guam, which is relatively isolated. It seems this species is on a rampage through the South Pacific, colonizing new isolated islands! The only reference I missed was the relatively recent book by Patrick and Patrick (2012) \"Butterflies of the South Pacific\". I unfortunately do not have access to my copy at the moment to see what it says about the species. Perhaps good to look at that?\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/7-366
|
https://f1000research.com/articles/7-365/v1
|
23 Mar 18
|
{
"type": "Research Article",
"title": "Timely referral saves the lives of mothers and newborns: Midwifery led continuum of care in marginalized teagarden communities – A qualitative case study in Bangladesh",
"authors": [
"Animesh Biswas",
"Rondi Anderson",
"Sathyanarayanan Doraiswamy",
"Abu Sayeed Md. Abdullah",
"Nabila Purno",
"Fazlur Rahman",
"Abul Halim",
"Rondi Anderson",
"Sathyanarayanan Doraiswamy",
"Abu Sayeed Md. Abdullah",
"Nabila Purno",
"Fazlur Rahman",
"Abul Halim"
],
"abstract": "Background: Prompt and efficient identification, referral of pregnancy related complications and emergencies are key factors to the reduction of maternal and newborn morbidity and mortality. As a response to this critical need, a midwifery led continuum of reproductive health care was introduced in five teagardens in the Sylhet division, Bangladesh during 2016. Within this intervention, professional midwives provided reproductive healthcare to pregnant teagarden women in the community. This study evaluates the effect of the referral of pregnancy related complications. Methods: A qualitative case study design by reviewing records retrospectively was used to explore the effect of deploying midwives on referrals of pregnancy related complications from the selected teagardens to the referral health facilities in Moulvibazar district of the Sylhet division during 2016. In depth analyses was also performed on 15 randomly selected cases to understand the facts behind the referral. Results: Out of a total population of 450 pregnant women identified by the midwives, 72 complicated mothers were referred from the five teagardens to the facilities. 76.4% of mothers were referred to conduct delivery at facilities, and 31.1% of them were referred with the complication of prolonged labour. Other major complications were pre-eclampsia (17.8%), retention of the placenta with post-partum hemorrhage (11.1%) and premature rupture of the membrane (8.9%). About 60% of complicated mothers were referred to the primary health care centre, and among them 14% of mothers were delivered by caesarean section. 94% deliveries resulted in livebirths and only 6% were stillbirths. Conclusions: This study reveals that early detection of pregnancy complications by skilled professionals and timely referral to a facility is beneficial in saving the majority of baby’s as well as mother’s lives in resource-poor teagardens with a considerable access barrier to health facilities.",
"keywords": [
"Midwives led continuum of care",
"marginalised teagarden communities",
"mothers and newborns",
"referral",
"Bangladesh"
],
"content": "Introduction\n\nGlobally 830 maternal deaths occur every day, 99% of which occur in developing countries1,2. According to the World Health Organization, roughly 303,000 maternal deaths are caused as a result of pregnancy and childbirth related complications3,4. Globally, about 3.7 million neonatal deaths occurred within the first 28 days, with 75% in the first week of life5. Only 19 out of 186 countries have achieved the Millennium Development Goal-5, related to reduction in maternal mortality6; unfortunately, Bangladesh is not one of them. Estimations suggest that about 87% of maternal deaths occurred in South Asian and Sub-Saharan African regions7. According to the Demographic and Health Survey, neonatal mortality rates range from 28 to 54 per 1000 live births in Bangladesh, India and Pakistan8. In 2010 the Bangladesh Maternal Mortality and Health Care Survey (BMMS) claimed that the lifetime risk of maternal death is 1 in 500 due to pregnancy and delivery related complication, and two third of these deaths occurred in the postpartum period. A study in Bangladesh found that 38% of the maternal deaths occurred by haemorrhage, which is the most common cause, 20% occurred by eclampsia, and 8.1% occurred by sepsis9. Another study in the teagarden area of Bangladesh revealed that maternal death in teagarden areas is higher due to lack of knowledge on maternal complication. Ignorance, traditional myths, family restriction on seeking better care, and dependency on traditional birth attendants and village doctors also influence these maternal deaths in teagarden communities10.\n\nReferral is the process of coordinated movement of health care seeker to reach a high-level care within a small window of time11. The goal of timely referral is to minimize or prevent the delay for transportation (called second delay), and ensure pre-hospital care while transporting a patient to the referral facility12,13. In 2014, Directorate General of Health Services (DGHS) reported that out of 120 maternal deaths 47 deaths occurred in the teagarden area of Moulvibazar district of Bangladesh. Estimations suggest that about 46.4% of maternal deaths occurred at home, and 7.1% while the women were on route towards a facility; this indicated the delay occurred as a result of delay in decision making of which facility to take the mothers for management, and arranging transport to go to the facility 14. Another study stated that 22.2% of maternal deaths occurred with more than 6 hours delay in decision-making and 12.9% of deaths occurred with 1–2 hours transportation delay9. In light of this, it can be assumed that ensuring emergency obstetric care services, and quick referral during the perinatal period can help reduce maternal deaths. To safeguard the reproductive age (15–49 years) of a woman, continuous care from family and community, along with support in getting easy access to referral healthcare facilities, is needed15. Transportation support, timeliness of referral, and inter-facility transfer are major contributing factors found to reduce the rate of maternal deaths12,16. A social autopsy study of maternal deaths found that very few mothers sought facility based care during complications, and that ensuring timely referral through transportation saved the lives of many of them14. It is recommended that five Emergency Obstetric and Newborn Care (EmONC) services, including four basic EmONC (BEmONC) and one Comprehensive EmONC (CEmONC), should be available and geographically distributed for each 500,000 individuals of a population17. The component of care (consisting of antenatal care, identification of high risk mothers, safe delivery conduction by skilled birth attendant, timely referral of complicated mothers and postnatal care including essential newborn care.) with high quality services can be ensured by the good referral system at all levels, both in facilities as well as in communities by the trained health care providers9. A shifting process is developed after the identification of high-risk pregnancies from a risk based approach to provide skilled care during delivery, and emergency obstetric care when complications occur18,19. This approach is not adequate to reduce maternal and neonatal mortality as the capacity is limited at the primary level of care, and is difficult to access in the referral facilities remaining in most of the low-income countries20. Professionally, a referral transport system must be managed for providing some basic intervention to the patient before reaching the referral facility21.\n\nAn intervention named “Bagan Mayer Jonno” has been implemented in the selected teagardens in the Moulvibazar district. The project ran through counseling and courtyard meetings of pregnant mothers, as well as an advocacy meeting with their guardians regarding quick referral of complicated mother. This project also supported the communities in detecting high-risk mothers by the active participation of volunteer and professional midwives. It also managed the provision of transportation and assistance of volunteers to ensure a quick and safe referral procedure. The present qualitative study describes the referral system using the midwifery led service delivery in five selected teagardens of Moulvibazar district in Bangladesh.\n\n\nMethods\n\nQualitative method was used to collect information in this study. The referral records of 2016 in selected five teagardens were reviewed retrospectively and qualitative information of selected 15 referral cases were collected though in-depth interviews at the community.\n\nThe average distance between a teagarden and Upazila Health Complex (UHC) varies between 12- 20 kilometers. Approximately, a population of 25,000 people with around 300 pregnant mothers at any point in time live in these gardens.\n\nAs part of the intervention, community volunteers called ‘Bagan Sebika’ were placed in the community, and professional midwives were situated in teagarden health facilities. Bagan Sebika (Paid Volunteer) perform community based activities including home based counseling, courtyard meetings, and advocacy meetings with the pregnant mothers and family members. Bagan Sebika also facilitated the mothers recieving antenatal care (ANC) at the facility. They also accompanying the referred mothers to the referral centre. Midwives’ role at teagarden facility includes ANC, counseling, delivery, referral, and postnatal care (PNC). Midwives also conduct delivery, referral, and PNC at a community level. Midwives also supervised the activities of Bagan Sebika at the community level.\n\nA total of 25 Bagan Sebikas worked in the five selected teagardens. They were assigned to conduct regular home visits to households and met to pregnant mothers. The Bagan Sebikas raised awareness on various issues such as birth preparedness, pregnancy complications, danger signs, and the importance of referral. If the Bagan Sebika identified any complicated or high-risk pregnancy case, they immediately communicated with the professional midwife over mobile phone. Professional midwives are usually experienced in identifying high-risk pregnancies through ANC checkups and previous medical history of the patient. Based on severity of the complication, the professional midwife along with the Bagan Sebika motivate family members of the high-risk pregnant mother to quickly refer to the higher referral centers including the UHC, district hospital and Teagarden central hospital. This counseling assists family members in being aware of the situation and the risk involved, provides them with information on where to seek care, and motivates them to make quick decisions. The Bagan Sebika also assists family members to organize transport, and assist them throughout the referral process. All these steps combined together helps decrease instances of delay in decision making [first delay] and transportation delay or second delay in the target population. In cases of severe complications, the midwives themselves might also help in organizing transportation.\n\nThe present study. The present study was conducted by a facility-based retrospective record review of all referral cases occurring at the referral hospital from the selected five teagardens from January to December 2016. According to 2016 records, a total of 72 high risk pregnant mothers were referred from these five teagardens to the referral centres (Upazila health complex, district hospital and teagarden central hospital). Each teagarden has both permanent workers (registered) and causal workers (unregistered). The teagarden authority provides referral support for the registered mothers (workers), whereas, for the unregistered mothers, the referral support is very low or absent.\n\nThe professional midwives used a structured tool to document the referral history and treatment at the teagarden facilities and did follow up all referral mothers until outcome at the referral facility though Bagan Sebika. [Table 1].\n\nTo conduct the retrospective record review of referral centers, a structured tool was developed by the research team. The tool contained data on mother’s particulars, current pregnancy history, antenatal care, complications, treatment history, referral details, preparedness of the facilities to manage emergency obstetric complications and delivery outcome. This review was carried out by the professional midwives working in the teagarden facilities. The record review included socio-demographics of the mother, medical condition of the referred mother, causes of referral, and view of the feedback of the referred mother and their family members [Table 2].\n\nData collection. A total of 72 referral case data were entered into SPSS software (version 24.0). After entry, all data was checked for missing data and consistency. Once checking was complete, the data was cleaned, and all analysis was done using software SPSS. For case scenario description, a total of 20% of cases (n=15) were purposively selected from the five teagardens (three cases from each garden). Midwives went to the household and organized a meeting for each of the cases. The Midwife invited the family members, relative and neighbours to the meeting to gather on responses from the family and community, as well as understand the referral linkage and service delivery in the facility. The Bagan Sebika in the community organized the meeting based on suitable date and time given by the community. Descriptive statistics were computed for all variables of interest. Frequencies were established to examine the demography of referred mothers, condition of mothers during referral, and documented causes of referral. The project support and remarkable findings of the cases were analyzed through review of the case stories collected from the teagarden facilities. Themes were identified after reading and re-reading of the case stories22,23 and finally thematic analysis was performed.\n\nThis study under “Bagan Mayer Jonno” intervention has been approved by the national ethical review committee of CIPRB (memo- CIPRB/ERC/2016/010). Verbal and written consent were received from each of the referred mothers before collecting the information for the study.\n\n\nResults\n\nFrom the review of records from 2016, Bagan Sebika identified the complicated mothers and immediately informed the project midwife. Then the project midwife decided whether the case needed to be referred. The project midwife also identified mothers as high risk during their routine ANC for referral. A total number of 72 complicated pregnancies (16%) were identified from a total of 450 pregnant mothers. These complicated mothers were identified at different stages during their antenatal visit, or during delivery, or immediately after delivery. Mothers informed the Bagan Sebika if any complication arose. Bagan Sebika also identified complicated mothers during their regular household visit. Then Bagan Sebika immediately informed to the project midwife. Professional midwives ensured immediate referral to the higher center after consultation, and coordinated with garden midwives, doctors and Bagan authorities. Unregistered workers in all cases directly referred to the Upazila or District facility, whereas registered workers were taken immediately to the garden’s existing referral system. In about 85% of cases, the transportation support was provided for referral of the complicated mothers, and of them in 75% of cases the Bagan Sebika (Volunteer) participated during referral of the mothers [Figure 1].\n\nThe referred mothers were mostly young. About 44% of mothers referred were in the age group 17–20 years, whereas 18% and 38% of mothers were from the age group of 21–25 years and 26–35 years, respectively. About 16.7% of referred mothers were housewives and the remaining were from other professions. Highest percentage (51.4%) of referral was among the unregistered teagarden workers (mothers), whereas only over 11% was registered teagarden workers. [Table 3].\n\n39%, 54% and 7% of referred mothers were identified as 1st gravida, 2nd to 3rd gravida and 4th gravida. Most of the mothers referred were in the labour stage (76%), whereas 12.5% were referred during the pregnancy period, and 11.1% after the delivery conduction [Table 3].\n\nWith project support, about 60% mothers were referred to Upazila Health Complex and 28% referred to Sadar district hospital. Only 13% of registered mothers or dependent workers of the teagardens were referred to teagarden referral center [Figure 2]. The time range at which most of the mothers (about 42%) were referred was between 10 a.m. to 2 p.m., where usually doctors, nurses and midwives are available in the government facilities. The remaining referrals occurred at times when only nurses and midwives are available in the facilities. But about 28% and 30% of mothers were referred within the period of 6 a.m. to before 10 a.m., and after 2 p.m. to 8:30 p.m. which is the vital period when doctors or service providers may not be found at government facilities [Table 3].\n\nAbout 14% of referred mothers needed Caesarian section for complications and 86% were normal vaginal delivery conducted by a nurse or midwife in the referral center. 94% of mothers delivered livebirths and 6% delivered stillbirths (2) and intrauterine deaths (2) at referral facilities with the assistance of skilled health care providers [Table 3].\n\nMost frequent causes for referral were due to prolonged labour (31%) and after that pre-eclampsia (about 18%). Moreover, another cause of referral found were retained placenta with post-partum haemorrhage, premature rupture of membrane, severe anaemia, breech presentation, twin pregnancy and others (~11%, ~9%, ~7%, ~7%, ~4% and ~13% respectively) [Figure 3].\n\nThe delay includes first (decision), second (transportation) and third (treatment) delays, which started from the complication arising, up to receiving treatment. In about 46% of cases family members needed more than 4 hours to make a decision as whether to seek care at a facility or not. Whereas about 60% cases reached from teagarden dispensary to the referral center (UHC) within one hour, and 74% cases women received treatment within one hour after arriving at the facility. Midwifery counseling as well as transportation support from the project influenced much in reducing the community delays mainly first and second delay [Figure 4].\n\n(A) First (decision) delay- delay indecision to seek care. (B) Second (transportation) delay- delay in reaching to the facility, Third (treatment) delay- delay in receiving treatment.\n\nA total number of 15 cases were selected randomly out of 72 cases for in-depth analysis and case scenario description. These description includes the socio-demography of the referred mothers, condition of the mothers for referral, responses of the family members and society, referral linkage and services delivery at referral centre [Table 4].\n\n\nDiscussions\n\nThe study revealed that among the referred mothers around 51% were unregistered workers who referred with the support of the project Bagan Mayer Jonno as they were not entitled to get any referral support from teagarden authorities. About 76% of mothers were referred during the period of delivery and 31% referred with the complication of prolonged labour. Most of the mothers (about 60%) were referred to the Upazila Health Complex and after referral about 14% mothers delivered by Caesarian-section at the facilities. A study conducted in rural Tanzania showed that about 28% of pregnant women were referred from primary level of care to tertiary level to ensure their better pregnancy outcome.\n\nThe same study also concluded that the most common referral complications found were multiparity (35%), young age of mother (30%), obstetric complications mostly due to prior history of caesarean section (12%), and previous existed prenatal risks like high blood pressure, severe anaemia etc. (12%)20. On the other hand, our study found that 31% of mothers referred with prolonged labour, 18% with pre-eclampsia, 11% with post-partum haemorrhage (PPH) due to retained placenta, 9% with premature rupture of membrane (PROM), 18% with severe anaemia, breech presentation & twin pregnancy, and remaining 13% with other complications.\n\nProper transportation with cost support along with a good communication technology is the prime concerns in establishing an effective referral17. Our study is also consistent with the findings that almost all referral occurs with transportation support, along with extra assistance from a midwife or volunteer, ensure the lives of many vulnerable mothers. The counseling of the midwife about the severe condition of the mother, as well as its dreadful consequences, and assistance of volunteers during referral motivated the family to quickly make their decision on referral.\n\nOur study showed that about 50% of referred mothers received treatment within 6 hours of referral and 10.6% within 2 hours. Addressing of second delay, or transportation delay, has a significant role in reducing maternal mortalities. Many studies showed that referral transportation should be available within 30 minutes of worsening condition of a mother, so that the complicated mother can be taken to a referral center as early as possible to initiate her treatment24. A mechanism needs to be established for the proper utilization of easily accessible functional transport services, which could be either from government, or from a private referral transport services24,25. Our study found that availability of transport support and assistance of volunteers from that teagarden enhanced quick referral, which consequently reduced the first and second delay.\n\nThis study found that quality ANC support by a midwife from respective gardens not only helped to identify high-risk mothers, but also further assisted the family to make a decision and prepare to delivery at a facility. Projections show that the Government of Bangladesh (GoB) already started midwifery education in all nursing institutes from 2012 and the GoB have the mandate to continue this midwifery led service delivery system until 2021, with the vision to serve hard to reach communities of the country. Another study revealed that to ensure basic and life-saving intervention to the patient, consistent support of a skilled staff should be available until the patient reaches referral facilities. However, several studies stated that it is difficult to pre-determine complication occurrence during pregnancy or childbirth17,24. The government mandate to continue the midwifery led service delivery until 2021, it is therefore necessary to regularly review the referral indicators and counsel on complication readiness, as well as birth planning by a health attendant to improve compliance on maternal referral20.\n\n\nConclusions\n\nEarly detection of complicated mothers and quick transfer to the referral center can ensure the survival of many mothers and neonates. The GoB has plans to scale up the unique midwife led service delivery (both basic and emergency health care services) system to support high-risk mothers of under privileged communities including the teagardens. The teagarden board, owners of the teagardens and local government, including policy makers of every level, must come forward to work together in finding out the best possible way to support the mothers of teagarden. At the community level, professional midwives play a key role in timely referral of a complicated mother to the facility. An integrated approach based on existing government health care delivery system with support from garden health facilities for timely referral of complicated mothers can be beneficial in reducing maternal and neonatal mortality in Bangladesh which in turn will be effective in reaching sustainable developmental goal on time.\n\n\nData availability\n\nData is stored at the CIPRB. Due to sensitivity of the data (contains identifying information), permission is required from the Ethical Review Committee (ERC) of CIPRB, Dhaka, Bangladesh for sharing data with a third party. Data can be requested from the CIPRB, who will contact the Ethical 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 corresponding author in order to request the data though email at info@ciprb.org.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe Bagan Mayer Jonno intervention is financially supported by the United Nations Population Fund (UNFPA), Bangladesh, funding code: Regular resources (RR-FPA90).\n\n\nReferences\n\nWorld Health Organization (WHO): Global Strategy for Women’s, Children’s and Adolescents’ Health, 2016–2030. New York: United Nations; 2015. Reference Source\n\nWorld Health Organization (WHO): Maternal Mortality. Fact Sheet. Media Centre; 2016. Reference Source\n\nWorld Health Organization (WHO): True magnitude of stillbirths and maternal and neonatal deaths underreported. Media Centre, 2016. Reference Source\n\nWorld Health Organization (WHO): Why do so many women still die in pregnancy or childbirth? 2015. Reference Source\n\nLawn JE, Cousens S, Zupan J, et al.: 4 million neonatal deaths: when? Where? Why? Lancet. 2005; 365(9462): 891–900. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization (WHO), United Nations Children's Fund (UNICEF), United Nations Population Fund (UNFPA), et al.: Trends in maternal mortality: 1990 to 2015. 2015. Reference Source\n\nWorld Health Organization (WHO): Knowledge Summary: Women’s & Children’s Health. Progress towards MDGs 4 and 5. Reference Source\n\nOestergaard MZ, Inoue M, Yoshida S, et al.: Neonatal mortality levels for 193 countries in 2009 with trends since 1990: A systematic analysis of progress, projections, and priorities. PLoS Med. 2011; 8(8): e1001080. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHalim A, Utz B, Biswas A, et al.: Cause of and contributing factors to maternal deaths; a cross-sectional study using verbal autopsy in four districts in Bangladesh. BJOG. 2014; 121 Suppl 4: 86–94. PubMed Abstract | Publisher Full Text\n\nBiswas A, Dalal K, Abdullah AS, et al.: Maternal complications in a geographically challenging and hard to reach district of Bangladesh: a qualitative study [version 1; referees: 2 approved]. F1000Res. 2016; 5: 2417. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGiovine A, Ostrowski C: Technical report on Improving Transportation and Referral for Maternal Health: Knowledge Gaps and Recommendations. Wilson Cent. 2010. Reference Source\n\nRaj SS, Manthri S, Sahoo PK: Emergency Referral Transport for Maternal Complication: Lessons from the Community Based Maternal Death Audits in Unnao District, Uttar Pradesh, India. Int J Health Policy Manag. 2015; 4(2): 99–106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPotluri P: Emergency Services in India: Counting on betterment. Asian Hosp Healthc Manag. 2009. Reference Source\n\nBiswas A, Halim MA, Dalal K, et al.: Exploration of social factors associated to maternal deaths due to haemorrhage and convulsions: Analysis of 28 social autopsies in rural Bangladesh. BMC Health Serv Res. 2016; 16(1): 659. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlva S, Wang W, Koblinsky M: The Continuum of Care for Maternal and Newborn Health in South Asia: Determining the Gap and its Implications. Washington DC, 2011; 1–6. Reference Source\n\nSingh S, Doyle P, Campbell OM, et al.: Transport of pregnant women and obstetric emergencies in India: an analysis of the ‘108’ ambulance service system data. BMC Pregnancy Childbirth. 2016; 16(1): 318. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization (WHO), United Nations Population Fund (UNFPA), Children’s Rights and Emergency Relief Organization (UNICEF), et al.: Monitoring emergency obstetric care: a handbook. Geneva WHO, 2009. Reference Source\n\nCampbell OM, Graham WJ, Lancet Maternal Survival Series steering group: Strategies for reducing maternal mortality: getting on with what works. Lancet. 2006; 368(9543): 1284–99. PubMed Abstract | Publisher Full Text\n\nRonsmans C, Graham WJ, Lancet Maternal Survival Series steering group: Maternal mortality: who, when, where, and why. Lancet. 2006; 368(9542): 1189–200. PubMed Abstract | Publisher Full Text\n\nPembe AB, Carlstedt A, Urassa DP, et al.: Effectiveness of maternal referral system in a rural setting: a case study from Rufiji district, Tanzania. BMC Health Serv Res. 2010; 10(1): 326. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMurray SF, Pearson SC: Maternity referral systems in developing countries: current knowledge and future research needs. Soc Sci Med. 2006; 62(9): 2205–15. PubMed Abstract | Publisher Full Text\n\nIrving S: Interviewing as Qualitative Research - A Guide for Researchers in Education and the Social Sciences. Teach Coll Columbia Univ USA, 2006. Reference Source\n\nBoyatzis RE: Transforming qualitative information: Thematic analysis and code development. Sage; 1998. Reference Source\n\nMavalankar DV, Rosenfield A: Maternal mortality in resource-poor settings: policy barriers to care. Am J Public Health. 2005; 95(2): 200–203. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdsul N, Kar M: Study of rogi kalyan samitis in strengthening health systems under national rural health mission, district pune, maharashtra. Indian J Community Med. 2013; 38(4): 223–228. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "32403",
"date": "28 Mar 2018",
"name": "Helen Elsey",
"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 valuable paper providing in-depth information on pregnancy related complications and emergencies in the context of Bangladesh. It draws on the global and Bangladesh literature and situates the study well within the existing evidence.\nThe main concern with the paper the presentation and description of the methods. The study is described as a 'qualitative case study'. The authors have completed a review of record of women with complications during pregnancy and delivery and present the quantitative findings of this review. They have then randomly selected 15 cases for in-depth interviews. This sounds more like a mixed methods study. Greater clarity on the qualitative interviews is required i.e. how were these conducted, by whom, where, was their an interview guide, did the women consent? The decision to randomly select women needs to be justified; with a mixed methods design, the authors could have purposively selected women from their case notes to explore particular issues in the interviews.\nGreater clarification on how the interviews were analysed: were they audio-recorded and transcribed? how did they come up with the 5 headings in the table - are these the emerging themes from the qualitative analysis? While Table 4 is interesting and gives a good insight into the cases, the paper would be greatly strengthened if this descriptive presentation could be synthesises and reported in the results. This synthesis of the key issues emerging from the interviews should also be included in the abstract. The quotations provided in Table 4 come from various people, not just the women e.g. TBAs, nurse, panchayat etc. Does this mean these individuals were interviewed? if so details of the methods used for these qualitative interviews also need to be given.\nFurther details on the socio-economic situation of the tea gardens would help readers understand the context.\nAcronyms and Bangladeshi-specific words should be spelt out for an international audience.\nThis has the potential to be an interesting and valuable paper, but greater clarity on the qualitative methods and analysis is required before being indexed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "32399",
"date": "03 Apr 2018",
"name": "Edwin Roland van Teijlingen",
"expertise": [
"Reviewer Expertise Maternity care",
"South Asia",
"sociology of health & illness",
"qualitative research"
],
"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\nTimely referral saves the lives of mothers and newborns: Midwifery led continuum of care in marginalized teagarden communities – A qualitative case study in Bangladesh\n\nThis is an interesting article on the workings of midwifery, especially referral by midwives, in a district in Bangladesh. The case study approach is appropriate but the description of the Methods is slightly odd. This is a typical case study based on secondary analysis. But the authors do not mention ‘secondary analysis’, let alone give a reference to a methods paper /textbook on the topic. Most record studies are retrospective, i.e. researchers using the record data after it was written. “A qualitative case study design by reviewing records retrospectively...” Also it is possible that authors used a Content Analysis (Krippendorff 2004) rather than a ‘general thematic analysis’?\nThe Discussion needs a section on the Strengths & Limitations of this particular way of using Secondary Analysis in a Case-Study Approach. Any maternity record has incomplete data, unclear recordings, etc. None of this mentioned in the text.\n\nAbstract The expression “76.4% of mothers were referred to conduct delivery at facilities,” is not quite right ‘women perhaps don’t conduct deliveries, you can say women deliver, or women give birth I read the Abstract and wondered why randomly selected in the sentence: “In depth analyses was also performed on 15 randomly selected cases to understand the facts behind the referral.” I would have expected purposively selected case, namely ones that highlight particular aspects of the case the authors would want to highlight/stress. But when I came to page 4 the authors state that the 15 cases are purposively selected. BUT on page 7 of 15 the authors repeat the Abstract “A total number of 15 cases were selected randomly out of 72” This needs to corrected.\n\nIntroduction Perhaps the reader needs a little bit more information about the state of midwifery in the country. In Bangladesh the three-years diploma curriculum following global ICM standards was introduced in 2010 (Bogren et al. 2015). It introduced a six-months post-basic advanced midwifery programme for graduate nurses. So what was the training of the midwives in this study? Where they post 2010 qualified or where some midwives trained prior to this date?\n\nGrammar, style & typos The authors use a mixture of American and British English. I would have preferred British English. They mix words like ‘labour’ (=British English) and in the Abstract ‘hemorrhage’ (=US English) and in the main text on page 3 ‘haemorrhage’(=British English) In the title (and elsewhere in article) I would use a hyphen in ‘ … newborns: Midwifery led continuum of …’ with a hyphen, i.e.: ‘Timely … and newborns: Midwifery-led continuum of …’ Similarly in the Abstract and throughout I would have expected a hyphen in: “pregnancy related complications…” to read: “pregnancy-related complications…” Also page 3 “few mothers sought facility based care during” should be “…sought facility-based care …”. It is perhaps ugly to start a sentence with a number, there are two cases in the Abstract “the facilities. 76.4% of..” AND “section. 94% deliveries…” In Abstract I think plural is needed in the sentence “majority of baby’s as well as mother’s lives” to read: “majority of babies’ as well as mothers’ lives….”\n\nIn the Abstract I think in the word ‘only’ in the sentence “ .. and only 6% were stillbirths…” is a judgement by the authors perhaps not shared by the women/families who had a still birth. Remove the word ‘only’! Page 3 of 15 there are word glued together, e.g. “maternaldeaths”\n\nYou could argue that all mothers are complicated. In the Abstract you should state something like“72 mothers with pregnancy-related complications” instead of “72 complicated mothers” Remove capital in “The Midwife…” page 4 of 15.\n\nReferences: Krippendorff, K. (2004) Content Analysis. An Introduction to its Methodology, London: SAGE Bogren, M.U., Wigert, H., Edgren, L., Berg, M (2015) Towards a midwifery profession in Bangladesh – a systems approach for a complex world, BMC Pregnancy & Childbirth 15:325\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?\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": []
},
{
"id": "32400",
"date": "05 Apr 2018",
"name": "Munia Islam",
"expertise": [
"Reviewer Expertise Qualitative Research in the area of maternal",
"neonatal and child health",
"nutrition and family planning"
],
"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 very well articulated and the findings are supporting the discussion and conclusion very strongly. Additionally, it gives deep insight and shares positive experience of a community based referral intervention in the area of maternal health to a pro-poor, marginalized and isolated community. However, minor language editing will make the article more scientific, lucid, authentic and reader friendly. For example, the author could use the word ‘explore’ instead of ‘evaluate’ in the last line of the introduction part in the abstract. Methodology in abstract part needs reorganizing and rephrasing where qualitative method is described. The introduction of the methodology section could commence like this-\no\n\n“This case study is designed as a mixed-methods retrospective assessment to explore the………” The body of the method section in the abstract can be described in this way, o\n\n“In-depth interviews and retrospective document were carried out to …… Thematic analysis was performed to analyze the qualitative data.”\nThe Bangla phrase, “Bagan Mayer Jonno” should be mentioned in English “garden for mothers”) for the non-Bengali speakers e.g. Method section in the main article needs to be elaborated and re-organized to make it reader friendly and self- explanatory. This section needs revision to maintain cohesion and coherence.\n\nOther than midwife is there any provision of additional service providers in the center inside the tea garden? Do midwives conduct home delivery? How do they maintain referral record? How is it documented? What does mean by the term ‘professional midwife’? Data processing procedure is described under ‘data collection’ sub-heading. It should be renamed as ‘data analysis’ or ‘data processing’. This section needs to be revised to address the cohesion and coherence also. Using the term, ‘case study’ instead of ‘case story’ will shape it more scientific. Need elaboration in the data analysis of qualitative methods. Need to mention, especially what types of qualitative method are used here. Sometimes, ‘case story’ and sometimes ‘IDI’s were mentioned, but the reader may feel difficulty to understand. Case scenario description’ part may go to the method section. Otherwise, you need to rephrase the sub-heading such as ‘findings from the case studies’ or rewrite the body of the paragraph in line with the previous sub-heading, so that it could be understood that you are describing result, not the process or method. The study reveals many opportunities to reduce the maternal mortality and morbidity of tea garden mothers. However, does this study uncover any challenges or obstacles that need further attention? Additionally, one of the recommendations should be ‘the scale-up of this intervention as a model for other marginalized communities in remote areas, who are experiencing ‘poor’ maternal health services.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-365
|
https://f1000research.com/articles/7-239/v1
|
28 Feb 18
|
{
"type": "Case Report",
"title": "Case Report: III° atrioventricular block due to fulminant myocarditis managed with non-invasive transcutaneous pacing",
"authors": [
"Kiran Devkota",
"Ya Hong Wang",
"Meng Yi Liu",
"Yan Li",
"You Wei Zhang",
"Kiran Devkota",
"Ya Hong Wang",
"Meng Yi Liu",
"Yan Li"
],
"abstract": "Fulminant myocarditis is a life-threatening clinical condition. It is the inflammation of myocardium leading to acute heart failure, cardiogenic shock and cardiac arrhythmias. Incidence of fulminant myocarditis is low and mortality is high. Most grievous complications of fulminant myocarditis is mainly cardiac arrhythmias; if there is delay on active management of the patient, it may be fatal. Here, we describe a case of III° atrioventricular block due to fulminant myocarditis that was managed with non-invasive transcutaneous cardiac pacing in the absence of ECMO. The non-invasive transcutaneous pacemaker is a safe, effective and convenient device to revert arrhythmias.",
"keywords": [
"III° A-V block",
"Fulminant Myocarditis",
"non-invasive transcutaneous cardiac pacing",
"ECMO"
],
"content": "Abbreviations\n\nA-V block- Atrioventricular block; ECG- Electrocardiogram; ECMO- Extracorporeal Membrane Oxygenation; LVEF- Left ventricular ejection fraction\n\n\nIntroduction\n\nFulminant myocarditis is a life-threatening clinical condition. It is inflammation of myocardium leading to acute heart failure and cardiac conduction abnormalities with rapid deterioration. There are about 10–38% cases of fulminant myocarditis among all cases of acute myocarditis1. Causes of fulminant myocarditis may be of viral, bacterial or non-infectious origin1–3. Diagnosis of fulminant myocarditis is very difficult because of non-specific symptoms and diagnostic tools. There may be signs of acute heart failure, cardiogenic shock, or life-threatening cardiac arrhythmias. Here we present a case of fulminant myocarditis presenting with different clinical features and IIIº A-V block, which was successfully managed with non-invasive transcutaneous pacing.\n\n\nCase report\n\nA 3 ½ year old female child having a productive cough and 5–6 episodes of the passage of loose stool for 2 days was taken to local hospital after she had sudden convulsion for about 2 minutes. At the hospital, she again had convulsion for 1–2 minutes and her heart rate dropped to 30 bpm. She was given atropine, dexamethasone, dextrose and per rectal choral hydrate at the local hospital (doses not known) and immediately referred to Renmin Hospital. She had no significant past medical history, drug sensitivity or allergies. On arrival she was conscious but lethargic and dyspnoeic. Both pupils were round, 4mm and reactive to light. Her vitals were T36.4 °C, HR 62 bpm, RR 65/min, BP 90/41mmHg and SPO2 80%. Table 1 shows the results of routine laboratory measurements. Her lips were cyanosed and she had pale and cold extremities. Neck and throat examination was normal. Chest examination showed b/l crackles and irregular heart rate with no murmurs. The abdomen was soft and non-tender. Electrocardiogram (ECG) showed- III°atrio-ventricular (A-V) block; left anterior fascicular block, ST-T changes (Figure 1). She had cardiac arrest three times in the emergency department at 15–20 minutes interval. She was resuscitated with chest compression along with Isoproterenol and adrenaline. Subsequently, her heart rate was maintained in between 70–90 beats/min. Provisional diagnosis was acute fulminant myocarditis with bronchial pneumonia.\n\n(A) Electrocardiogram (ECG) at emergency showing- III°atrio-ventricular block; left anterior fascicular block, ST-T changes; (B) ECG recording during transcutaneous pacing; (C) ECG at the time of discharge, which is normal.\n\nThe patient was admitted to hospital after explaining the disease condition and prognosis to parents. She was on continuous oxygen, dopamine, diazepam, Immunoglobulin, ceftriaxone, IV fluids on a maintenance dose and nebulization with Ipratropium bromide (250mcg/ nebulization) along with a high dose of vitamin C, Coenzyme Q 10 and fructose diphosphate and mannitol (for brain edema). However, she again had a cardiac arrest. In addition, ECG showed sinus P wave and no QRS with heart rate dropped from 70 to 20bpm. The patient was resuscitated with chest compression, atropine, and adrenaline. Isoproterenol was started at 1.5mcg/kg/min and increased up to 2mcg/kg/min. Subsequently, her heart rate was maintained at 60–70 bpm. Her heart rate again decreased to 30bpm when isoproterenol was discontinued. As there was no extracorporeal membrane oxygenation (ECMO) machine in our hospital and transfer was not possible, the patient was prepped for non-invasive transcutaneous cardiac pacing.\n\nCardiac pacing was adjusted to 16 mA and rate 90 bpm. The patient’s heart rate was controlled at 80–100 bpm. Her complexion gradually became reddish, cyanosis gradually improved but she had developed eyelid edema. She had passed urine about 130ml twice in 12 hours. Dopamine was increased to 7mcg/kg/min and she was started on furosemide.\n\nAt first 24 hours after cardiac pacing, the patient was conscious. She had passed urine 4 times about 700ml. But facial puffiness was still present. Her heart rate was maintained at the rate of 110–130bpm and SPO2 was 96% with oxygen. Chest pacing was reduced to 14 mA, and the frequency was changed to 70 bpm. After 48hours of pacing, the heart rate was improved to 100–110bpm with few ventricular premature beats. Then pacing was reduced to 12 mA, frequency changed to 60bpm. The pacing current and frequency were gradually slowed down and discontinued. Then, sinus rhythm was established with the heart rate of 100–110bpm with ECG monitoring. The heart rate fluctuated at 80–100bpm with frequent ventricular premature beats. Echocardiography showed left ventricular myocardial wall thickening and thickening of endocardium with left ventricular ejection fraction (LVEF 50%), suggestive of endocardial fibroelastosis (EF). Chest radiograph showed increased lung texture and enlarged cardiac shadow (Figure 2). Captopril, hydrochlorothiazide, and spironolactone were started to reduce cardiac remodeling and to protect heart function. Furosemide was continued. Mannitol was stopped after the patient’s MRI scan revealed normal findings.\n\n(A) Patient on non-invasive transcutaneous pacing; (B) Echocardiography after 48 hours of admission showing left ventricular myocardial wall thickening and thickening of endocardium; (C) Chest X ray showing increased lung texture and enlarged cardiac shadow.\n\nThe patient’s HR was in between 80–100 bpm, with blood pressure was increasing gradually. Dopamine was tapered and stopped at 72 hours, after her BP reached 110/78 mmHg. The chest became gradually clear and her heart sounds were also normal. ECG monitoring also showed improvement with decreased numbers of premature beats and gradual change of S-T segments towards normal.\n\nThe patient was discharged on the 28th day after admission after her all routine investigations returned to normal (Table 1). ECG showed sinus rhythm with heart rate 102 bpm. Echocardiography showed normal cardiac chambers, normal wall motion with EF 60%. Her final diagnosis was fulminant myocarditis with III° A-V block and bronchial pneumonia. On discharge, the patient was advised to continue captopril 6.25mg bid, metoprolol succinate 6.25mg bid, prednisone 1mg / kg orally for six months.\n\nAt six month follow up the patient’s echocardiography had returned to normal with LVEF 65%, and prednisone was reduced to 0.5mg/kg orally for 15 days and with a tapering dose for the next 15 days. After one year follow-up, she had no complaints and no significant abnormalities noted on echocardiography.\n\nAll doses of medications can be seen in Table 2.\n\n\nDiscussion\n\nIn children, sometimes myocarditis is self-limiting. However, if it progresses there is the risk of acute cardiac failure, hemodynamic disturbances, and arrhythmias leading to significant morbidity and mortality. Mortality due to myocarditis for infants is more than 75%, whereas for children it is more than 25%1,4–8. There is no any specific clinical course and investigations to diagnose fulminant myocarditis. Initially, they present with flu-like symptoms and later develop sudden onset of cardiac symptoms that rapidly deteriorate2. Neonates may present with fever, poor feeding, and listlessness and sometimes with danger signs like apnea, episodic cyanosis, and diaphoresis. Older children present with respiratory or gastrointestinal symptoms. Among them only a few present with chest pain9. Diagnosis is mainly done on the basis of: Clinical presentation, blood profile, including CBC, electrolytes, creatinine kinase, creatine kinase MB isoenzyme, C-reactive protein, Troponin T, Troponin I, antistreptolysin O titer, polymerase chain reaction to detect viral antigens, autoantibodies marker, liver enzymes, ECG, Echocardiography, ultrasonography and even Cardiac MRI3 which are mostly supportive. If echocardiography shows low LVEF in children with fulminant myocarditis, the prognosis is poor10. Mortality in fulminant myocarditis is mainly due to cardiac arrhythmias among which structural changes, parameters of ventricular dynamics and vascular changes are responsible for the increased incidence11. This can be managed with percutaneous cardiopulmonary support, ECMO, intra-aortic balloon pumping, or the ventricular assisted device12,13. ECMO remains an effective approach in children for the management of acute fulminant myocarditis14. In addition, intravenous immunoglobulin and high dose steroids help to reduce inflammation15. Patients managed with immunoglobulin, steroids or mechanical support for fulminant myocarditis may have higher survival rate compared to those not receiving these therapies12.\n\nIn the present case, we tried to manage initially with Isoproterenol but were unsuccessful. So we applied the non-invasive transcutaneous pacemaker to revert the A-V block. Pads or electrodes detachment, patient non-cooperation and skin-burn due to high voltage electric current are its limitations. In contrast, intraventricular cardiac pacing is time-consuming; much more risky and surgical site wound infection is common. Beland et al, Kelly et al in their articles note that non-invasive transcutaneous pacemaker is the safe, effective and suitable equipment for children16,17.\n\n\nConclusion\n\nAcute fulminant myocarditis is a grievous condition with high morbidity and mortality. No delay should be had on starting immunoglobulin and steroids if suspected. If there is the arrhythmia, the patient should be immediately started on ECMO, percutaneous cardiopulmonary support or ventricular assisted device. If these are not available, then non-invasive transcutaneous cardiac pacing must be started, which is a safe, convenient and cost-effective device to revert arrhythmias caused by myocarditis.\n\n\nConsent\n\nWe have taken written informed consent from the child`s legal guardian (her father) to use and publish his child`s medical case history and any 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\nAcknowledgements\n\nWe wish to thank Dr. Wu Bing, (Resident Cardiology Department II, Renmin Hospital), entire doctor and nursing staffs Department of Pediatrics I, Renmin Hospital and to the child parents for the consent.\n\n\nReferences\n\nLee EY, Lee HL, Kim HT, et al.: Clinical features and short-term outcomes of pediatric acute fulminant myocarditis in a single center. Korean J Pediatr. 2014; 57(11): 489–495. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLobo ML, Taguchi Â, Gaspar HA, et al.: Fulminant myocarditis associated with the H1N1 influenza virus: case report and literature review. Rev Bras Ter Intensiva. 2014; 26(3): 321–326. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVashist S, Singh GK: Acute myocarditis in children: current concepts and management. Curr Treat Options Cardiovasc Med. 2009; 11(5): 383–91. PubMed Abstract | Publisher Full Text\n\nForcada P, Beigelman R, Milei J: Inapparent myocarditis and sudden death in pediatrics. Diagnosis by immunohistochemical staining. Int J Cardiol. 1996; 56(1): 93–7. PubMed Abstract | Publisher Full Text\n\nMaron BJ, Gohman TE, Aeppli D: Prevalence of sudden cardiac death during competitive sports activities in Minnesota high school athletes. J Am Coll Cardiol. 1998; 32(7): 1881–4. PubMed Abstract | Publisher Full Text\n\nNeuspiel DR, Kuller LH: Sudden and unexpected natural death in childhood and adolescence. JAMA. 1985; 254(10): 1321–5. PubMed Abstract | Publisher Full Text\n\nTopaz O, Edwards JE: Pathologic features of sudden death in children, adolescents, and young adults. Chest. 1985; 87(4): 476–82. PubMed Abstract | Publisher Full Text\n\nNiimura I, Maki T: Sudden cardiac death in childhood. Jpn Circ J. 1989; 53(12): 1571–80. PubMed Abstract\n\nLevine MC, Klugman D, Teach SJ: Update on myocarditis in children. Curr Opin Pediatr. 2010; 22(3): 278–83. PubMed Abstract | Publisher Full Text\n\nPei L, Yang N, Yang YH, et al.: Clinical features and prognostic factors in children with fulminant myocarditis. Zhongguo Dang Dai Er Ke Za Zhi. 2015; 17(11): 1232–6. PubMed Abstract\n\nKlein RM, Vester EG, Brehm MU, et al.: [Inflammation of the myocardium as an arrhythmia trigger]. Z Kardiol. 2000; 89 Suppl 3: 24–35. PubMed Abstract\n\nSaji T, Matsuura H, Hasegawa K, et al.: Comparison of the Clinical Presentation, Treatment, and Outcome of Fulminant and Acute Myocarditis in Children. Circ J. 2012; 76(5): 1222–1228. PubMed Abstract | Publisher Full Text\n\nTaoka M, Shiono M, Hata M, et al.: Child with fulminant myocarditis survived by ECMO Support--report of a child case. Ann Thorac Cardiovasc Surg. 2007; 13(1): 60–4. PubMed Abstract\n\nNing B, Zhang C, Lin R, et al.: Local experience with extracorporeal membrane oxygenation in children with acute fulminant myocarditis. PLoS One. 2013; 8(12): e82258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTucker CE, Fernandez MJ, Morrison CC: Successful Treatment of Fulminant Myocarditis. J Clin Case Rep. 2013; 3: 256. Publisher Full Text\n\nBéland MJ, Hesslein PS, Finlay CD, et al.: Noninvasive transcutaneous cardiac pacing in children. Pacing Clin Electrophysiol. 1987; 10(6): 1262–1270. PubMed Abstract | Publisher Full Text\n\nKelly JS, Royster RL, Angert KC, et al.: Efficacy of noninvasive transcutaneous cardiac pacing patients undergoing cardiac surgery. Anesthesiology. 1989; 70(5): 747–51. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "31290",
"date": "05 Mar 2018",
"name": "Mani Prasad Gautam",
"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 have discussed an interesting case of fulminant myocarditis in a child which have been managed with transcutaneous pacing for the third degree AV block along with other supportive care including intravenous methylprednisolone and immunoglobulin. In overall the article is at satisfactory level in its scientific contents and usefulness to other practitioners. But there is some space to improve its rating. The background information provided here is an overall information, it would have been better if the author has also focused on various arrhythmias associated with fulminant myocarditis as the myocarditis tends to have various refractory arrhythmias including ventricular tachycardia.1 It would have been better if the authors were focused on particular theme, such as on various arrhythmias and their medical management or overall medical management. The case description is somehow lacking smoothness in the flow of information. That could be because lack of cohesion in sentences and writing in the form of clinical vignette can improve its content. Similarly there are a lot of English grammar related errors and rampant use of short form that should be corrected. In addition, it is advised to use scientific words rather than layman terms in academic writings. Taking help from an English language expert might also improve its contents. The case report section should be made little bit short. The discussion section should be more descriptive by discussing clinical features of myocarditis including various arrhythmias and medical management of myocarditis. The use of Vitamin C, Coenzyme Q10, Fructose diphosphate and Mannitol can not be understood from the discussion section which I was expecting there. I hope, the revision after considering above advice will improve the scientific content of the article.\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? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
},
{
"id": "31964",
"date": "14 Mar 2018",
"name": "Sabine Pankuweit",
"expertise": [
"Reviewer Expertise Myocarditis",
"cardiomyopathies",
"heart failure"
],
"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 described a case of III° atrioventricular block due to fulminant myocarditis that was managed with non-invasive transcutaneous cardiac pacing in the absence of ECMO. In this case the non-invasive transcutaneous pacemaker was a safe, effective and convenient device to revert arrhythmias.\nNevertheless, as the authors stated, if there is the arrhythmia, the patient should be immediately started on ECMO, percutaneous cardiopulmonary support or ventricular assisted device. Authors should specify and discuss, what the most important arrythmias with negative prognosis to what extend are.\nAre there any efforts made in this case, to get information on the etiology of the disease (e.g. viral, autoimmune), was there any discussion on an endomyocardial biopsy.\n\nData in children with regard to the prognosis and outcome in fulminant myocarditis 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": []
}
] | 1
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https://f1000research.com/articles/7-239
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https://f1000research.com/articles/7-358/v1
|
22 Mar 18
|
{
"type": "Method Article",
"title": "Model for de novo mutation propagation depending on paternal age at conception and associated neurological disorders",
"authors": [
"Johannes Lawen",
"Ena Wang",
"Ena Wang"
],
"abstract": "This paper presents a computational model for simulating the propagation of de novo mutations and paternal age effects in populations. The model uses data for paternal de novo mutation rates depending on age and demographic data such as age distributions, birth distributions versus age, varying life expectancy, and correlations with fertility. The number of paternal de novo mutations in children increases with the paternal age at conception. This might be of interest considering that the average paternal age has risen significantly in many societies throughout the last century. The model introduced below can superimpose and extrapolate different effects based on demographic dynamics. This includes the assessment of statistically associated neurological disorders in offspring, particularly IQ decay depending on the paternal age and other medical phenotypes which constitute paternal age effects. Yearly paternal mutation rates and correlations with paternal age were used to simulate both, de novo mutation propagation and probabilities for correlating conditions such as IQ decay. The extrapolated effect after several generations of persistently elevated paternal age appears to be drastic. To account for possibly mitigating factors, the paternal age effect has been super-positioned with the Flynn effect in simulated cases. The model automatically generates distributions for varying paternal ages, not just single cases, in convenient 3D distributions. The model simulates each person’s individual reproductive incidents through a particle type approach which is more rigorous than insufficiently adaptive, continuum models based on partial differential equations. The model is not only applicable to humans and yields many valuable conclusions for a wide array of topics including the paternal age effect, correlations with intelligence, evolution, bottlenecks in evolution, as well as the role of de novo mutation.",
"keywords": [
"paternal age",
"de novo mutations",
"IQ",
"demographic models"
],
"content": "Introduction\n\nThe DNA of a child is not exactly a composition of its father’s and mother’s DNA. De novo mutations, base pair changes, do occur during meiosis and throughout the parent’s life, including in male gametes. It has been widely reported (Cannon, 2009; Gauthier & Rouleau, 2012; Kong et al., 2012) that parental age at the time of conception is one factor contributing to a higher mutation rate. The number of de novo mutations rises particularly with the age of the father. Paternal de novo mutations have been found (Kong et al., 2012) to increase by about 2.01 base pairs per year. For example, an age of 45 at conception entails an average 52.5 more de novo mutations in offspring than an age of 20.\n\nKong et al. showed that the father's age is the dominant factor in determining the number of de novo mutations in a child. By sequencing the genomes of 78 parent-child trios, Kong et al. found that the number of mutations increases (Gauthier & Rouleau, 2012; Kong et al., 2012) with the father's age (P = 3.6 × 10-19) at an estimated rate of 2.01 mutations per year (standard error = 0.17). Some of Kong et al.’s rate dimensions imply a first-order relationship whereas others imply rather a zero-order dependency: i.e. respectively reporting an exponential increase with a doubling of paternal mutations every 16.5 years as well as a yearly rate of 2.01 de novo mutations. A first- or zero-order rate are of course two different propositions and, short off alleging a governing influence of a decay in DNA repair and replication, the formation of de novo mutations is proportional to the size of the genome, thus, implying rather zero order kinetics. Either way, for the reported comparison of two extrema of the age at conception the difference in order only affects the assessment of intermediate de novo mutation levels.\n\nThe paternal age effect describes the sensitivity of individual health to the average increase in de novo mutations in offspring due to elevated paternal age: e.g. 30 years of elevated paternal age resulting in ~60 more base pair changes. As per Kong et al.’s reported standard error, the yearly de novo mutation rate has been considered as quasi linear and can thus be considered as of constant logarithmic order of magnitude throughout a man’s life.\n\nRegardless of the age at conception, de novo mutations present a constant limit to the conservation of DNA and a constant degrading effect on an organism’s health, statistically detectable within one generation and even more significant when considered for historic time scales.\n\nIncreasing paternal age has been linked (Cannon, 2009) to a statistically detectable mental decline, an average IQ decline of six points for children of fathers aged 50 (at conception) compared to younger fathers, aged 20, as reported by Cannon. The correlation between de novo mutations and paternal age effect, with respect to the average IQ, might have been considered unexpected due to the logarithmic orders of magnitude separating the number of de novo mutations and the size of the entire human genome, as well as the small fraction of protein coding exon-DNA. A direct link to this statistical effect is nevertheless not only plausible, considering the influence of mutations in intron-DNA on the level of gene expression, but might also be negligent to discard considering the drastic outcome that this might entail for persistent trends of elevated paternal age. This paper presents a model that simulates the paternal age effect on IQ for societies and super-positions it with other effects: e.g. the Flynn effect, which is an increase in IQ points (Flynn, 1987) per generation, that has been found particularly during the 20th century’s development of parenting and education. It has been disputed whether this is a temporal effect as the increases have been most prominent in data from well within the 20th century. Since then the average paternal age has continued to increase. The simulation presented in this paper super-positioned the Flynn effect with rising paternal age and yielded a good agreement with the recently observed halt or decrease of the Flynn effect (Sundet et al., 2004).\n\nThe relevance of paternal age, that is, the relevance of a limited number of de novo mutations, for neurological and psychiatric disorders becomes consistent when considering not only rare mutations in protein coding DNA but also much more frequent genetic alteration in introns, which impacts protein expression, as evidenced by findings that confirmed the long suspected significant role of non-protein coding DNA in protein expression (Le Hir et al., 2003; Nott et al., 2003). Hence, increased random errors in DNA, due to increased paternal age, might plausibly yield statistical decreases in cognitive abilities.\n\nThe developed model can aid to assess these and:\n\n\n\n1. couple de novo mutation accumulation simulations with correlations for IQ decline or probabilities for other health impacts statistically related to paternal age,\n\n2. be useful for both, investigating genetic alteration depending on potentially relevant factors under investigation, such as environmental influences and demographic patterns in terms of reproduction,\n\n3. assess dynamic distributions of neurological and psychiatric disorders (Gauthier & Rouleau, 2012) along multiple generations,\n\n4. simulate the health of organisms and its interconnected dynamic properties during evolution and bottlenecks in evolution,\n\nexplain uncertainty in demographic models which heretofore didn’t consider dependencies between these organism properties and reproductive patterns.\n\n\nMethodology – modeling\n\nA prerequisite to embarking some of the considerations mentioned above, is to recognize that long term developments of properties such as the average IQ belong to the vital interests of societies. Henceforth, even if cautioned that not all influences are yet quantified or even possible to quantify, potential decay factors, their impact potential and long-term impact should be anticipated and extrapolated, particularly assessing the order of magnitude of the potential change in the population’s IQ distribution based on contemporary reproductive trends (here elevated paternal age across multiple generations).\n\nThe developed model simulates reproduction, mutations and IQ individually for each person in the simulated population. The initial age distribution as well as reproductive dynamics can be supplied to the model via demographic data. Reproduction occurs at a particular age with varying probabilities, depending on demographic input data. It is also possible to set a gender bias in the sex ratio of newborns. A for-loop checks every month whether the demographic data suggest reproduction and the associated probability. This has been embedded with a weighted random number ρ and normal distributed random number ρn . Children are born every month based on the set sex ratio and with added de novo mutations according to the paternal age. The IQ is then set randomized as per a normal distribution normed to the parental IQ, plus Flynn effect, minus paternal age effect as per its correlation with the accumulated de novo mutations. Simulated individuals die as per supplied demographic input data. Matrix M contains columns denoting age, gender, life expectancy, de novo mutations, and IQ. Reproduction occurs based on the relation rl + ρ > 1 with l = Mi1 where r is a vector containing the reproductive probability depending on age. If wanted, correlations between intelligence and fertility can be taken into account by involving the IQ in this relation.\n\nThe IQ is denoted in M for a newborn s and calculated with IQ¯=IQ¯min+ΔIQ¯amax−Mi,1/12Δa and Ms,5=Mi,5(1+F)(ρnσ+IQ¯).\n\nIQ is the relative average IQ, Δa is the fertile age interval, σ the spread of the normal distribution, and F is the assumed IQ increase as per the Flynn effect.\n\n\n\nThe simulated cases shown in Figure 1 and Figure 2 below have the specifications: 1.) A population of 20,000; 2.) 300 years; 3.) Average number of children is 2; 4.) No gender bias concerning delivery of baby boys and girls is assumed but could be accounted for by setting a different value for the sex ratio b; 5.) De novo mutation accumulation has been assumed to obey published data (Gauthier & Rouleau, 2012; Kong et al., 2012).\n\nThe start of the decade is varied from 20 to 50. The optimistic assumption of a continuous Flynn effect has been super-positioned.\n\nThe start of the decade is varied from 20 to 50. The optimistic assumption of a continuous Flynn effect has been super positioned which yields very high IQs for young paternal ages after 300 y.\n\nLife expectancy is of little relevance for the simulated results, considering that in most cases death occurs after the fertile age period.\n\nA nonlinear de novo mutation accumulation rate, as plausibility due to changing cell division and DNA repair protein activity throughout life, can be used as already provided for by keeping y as vector. This can easily be incorporated with linear ratios (splines) between each data point yielding varying rates depending on age. The reproduction probability distribution can be set as per demographic data from any population of interest and represented as vector r. This particle type model illustrates that different effects can be conveniently superimposed. Furthermore, it allows to assess the order of magnitude of the problem as shown in the simulated cases where conception occurs in different paternal age intervals, starting with an interval from 20 to 30 and ending with an interval from 50 to 60. That means, 30 different reproductive patterns in terms of paternal age are simulated for comparison, the youngest assuming reproduction to occur between 20 and 30 and the oldest assuming paternal age at conception to be between 50 and 60.\n\n\nResults and discussion\n\nCurrently the model is intended to assess the order of magnitude of the impact of elevated paternal age after multiple generations - not to do forecasting for average properties - since several input distributions, such as for reproduction, age, life expectancy and gender ratios, are set to constant values. The model simulates each individual and thus requires much more computational capacity than models relying on partial differential equations which average every constituent into a continuum. Therefore, if populations >1 million, that is, whole societies or populations, are to be simulated, then supercomputers might support practicable computational times. Several conclusions can be gleaned from the simulation illustrated in Figure 1 to Figure 4 and listed in Table 1.\n\nObviously, limits in the conservation of DNA are usually outpaced by evolution, mating patterns or might be masked by improved social, environmental, parenting, etc. conditions – particularly the later are difficult to model.\n\nAnd heretofore, a constant decay of any health indicator has not been observed. Therefore, there might be a set of mechanisms that effect reproductive patterns and compensate for the about 2 yearly paternal random de novo mutations in inherited DNA. It is not possible for us to identify (e.g. possibly mating choice based on facial symmetry, intelligence, etc.) or quantitatively assess these mechanisms as rigorous as the underlying de novo mutations that they might be mitigating.\n\nNevertheless, considering the constant pace of de novo mutations and detectable associated statistical impact, these mechanisms might entail a similar imminent and substantial response in reproductive patterns. Interestingly, therefore, possibly spelling another uncertainty for demographic modeling.\n\nThe model also illustrates the ill-posed fate and predictability for small populations. If the simulated population is small, e.g. 2,000, then individual fate will cause an uneven surface or curve as in Figure 3 and Figure 4 below. Figure 1 and Figure 2 above were produced from simulations for 20,000 individuals and look more even. Bigger populations should be simulated, e.g. a few hundred thousand, to even out individual fate and further smooth the surface.\n\n\nConclusions\n\nDe novo mutations are accumulated all the time regardless of age at about 2 base pair changes per year, also in children conceived at young age. Mitigating mating choices and reproductive patterns are merely given less opportunity to outpace de novo mutations in case of elevated paternal age. Yet the impact of de novo mutations and the correlation between continuous paternal accumulation of de novo mutations and mild IQ decay has not been discovered before comparing different paternal age groups. Therefore, the effect appears to have been masked by mitigating factors which constitute an uncertainty in modeling. Hence, assessing the paternal age effect due to accumulating de novo mutations in offspring across multiple generations is prone to uncertainty short off an understanding of complex mating dynamics. Nevertheless, simulated cases illustrate the drastic outcomes of persistent elevated paternal age over multiple generations at already one logarithmic order. The model results illustrate highly interconnected patterns in organism health and reproduction in evolution scenarios (not only for humans). The model, if run for small populations, indicates ill-posed, susceptible to random influences, behavior during evolutionary bottlenecks. This statistical particle model shows for the same initial conditions substantially varying outcomes, depending on the set of random numbers for small populations, i.e. isolated communities or evolutionary bottlenecks. Investigated demographic patterns should be empirically supplied to the model, such as: Correlations of intelligence with fertility, correlations of other health indicators with fertility, correlations of de novo mutation levels in newborns with later fertility. To validate paternal age effect correlations, it is recommendable to include data from bioinformatics, which detail affected genes’ functionality, rather than just macroscopic demographic distributions. The effects of continuously elevated paternal age in society can be assessed, particularly with respect to the order of magnitude of the impacts, by super-positioning in simulations impacting and mitigating factors for investigated countries/regions. For example, the simulation returned that, even under the optimistic assumption of a never-ending Flynn effect, if super-positioned with the paternal age effect, then an average paternal age above 40 would lead to the long-term decline of the average IQ in society.\n\n\nSoftware and data availability\n\nThe entirety of the model code is documented in the Methodology section. The code for plotting figures is documented in the Appendix. Input data such as population size and de novo mutation accrual rate are also detailed in the Methodology section. The model can be run on MATLAB 9 or Octave 4.2.\n\n\nAppendix\n\n",
"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\nArslan RC, Willführ KP, Frans EM, et al.: Older fathers' children have lower evolutionary fitness across four centuries and in four populations. Proc Biol Sci. 2017; 284(1862): 1–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCannon M: Contrasting effects of maternal and paternal age on offspring intelligence: the clock ticks for men too. PLoS Med. 2009; 6(3): e42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlynn J: Massive IQ gains in 14 nations – what IQ tests really measure. Psychol Bull. 1987; 101(2): 171–191. Publisher Full Text\n\nGauthier J, Rouleau GA: De novo mutations in neurological and psychiatric disorders: effects, diagnosis and prevention. Genome Med. 2012; 4(9): 71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKong A, Frigge ML, Masson G, et al.: Rate of de novo mutations and the importance of father's age to disease risk. Nature. 2012; 488(7412): 471–475. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLe Hir H, Nott A, Moore M: How introns influence and enhance eukaryotic gene expression. Trends Biochem Sci. 2003; 28(4): 215–20. PubMed Abstract | Publisher Full Text\n\nNott A, Meislin SH, Moore MJ: A quantitative analysis of intron effects on mammalian gene expression. RNA. 2003; 9(5): 607–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSundet JM, Barlaug DG, Torjussen TM: The end of the Flynn effect? A study of secular trends in mean intelligence test scores of Norwegian conscripts during half a century. Intelligence. 2004; 32(4): 349–362. Publisher Full Text"
}
|
[
{
"id": "33258",
"date": "14 May 2018",
"name": "Ruben C. Arslan",
"expertise": [
"Reviewer Expertise paternal age",
"intelligence",
"demographics"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is an interesting attempt to simulate paternal age distributions and associated population level outcomes of interest, such as the number of de novo mutations and cognitive ability (IQ), over generations. The paternal age distributions themselves are only of instrumental interest, the core aim of the model is to shed light on the population patterns in the outcomes under certain assumptions regarding the links between the outcomes and paternal age.\nThe simulations include effectively three steps, each increasing the level of complexity. The first step is to simulate the paternal age distributions. The second step involves linking paternal age to the number of de novo mutations. The third step calls for linking paternal age, or the number of de novo mutations, or both to the social outcome of interest, which in this paper is IQ.\nEach of the three steps involves decisions. The first step of simulating paternal age is in principle straightforward as only demographic input is needed. The authors appear to assume that paternal age has increased throughout the last century. This however runs counter to existing research. For example, Arslan et al. (2017) document that average paternal age has increased in Sweden since 1970, but decreased from 1930 to 1970 and is today still lower than in 1880. It is easily forgotten that although ages at first birth rise, people also have fewer children on average, stopping earlier. Based on the simulations, the authors find that average paternal age over 40 years of age would lead to population level declines in IQ, even if the so-called Flynn effect would continue. This indeed is a result of the simulation. However, an average paternal age of 40 years is currently far beyond observable levels, and it seems to be bold speculation what might happen once such levels were reached.\nThe second step links paternal age to the number of de novo mutations. Existing research that is cited in the paper (Kong et al., 2012) provides numbers that can be used for this, although newer, larger studies should also be considered (Ségurel, Wyman, & Przeworski, 2014; Wong et al., 2016). However, the authors do not cite work on evidence for contemporary evolutionary selection against genetic variants linked to education and presumably intelligence (Kong et al., 2017; Marioni et al., 2016).\nThe third step is perhaps the most challenging one. The authors assume a model that links IQ to the number of de novo mutations, without explicit link to paternal age. Implicitly, via a simulated link between parental age and de novo mutations, the model delivers a monotonic negative link between IQ and parental age. This, however, does not correspond to what is observed in the data (Arslan, Penke, Johnson, Iacono, & Mc Gue, 2014; Carslake, Tynelius, van den Berg, Davey Smith, & Rasmussen, 2017; Myrskylä, Silventoinen, Tynelius, & Rasmussen, 2013). Nor is the simulation code sufficiently clearly documented for us to understand how large a relationship with paternal age is actually assumed, and whether any non-genetic variation in IQ is allowed.\nResearch has predicted negative effects of de novo mutations on intelligence, because of theoretical accounts that the interindividual variation in intelligence is upheld by mutation-selection balance (Arslan, 2017; Penke, Denissen, & Miller, 2007) and empirical results linking de novo mutations to developmental disabilities (Deciphering Developmental Disorders Study, 2017) and rare genetic variants to intelligence (Hill et al., 2017). Although the authors cite Arslan et al. (2017), they do not implement in their simulations that higher paternal age is still linked to lower offspring fertility in all populations we studied. By modelling one force, mutation, but not the potentially countervailing one, selection, as shown for paternal age by Arslan et al. (2017) and for education by Kong et al. (2012) and Marioni et al. (2016), they can obtain the result that intelligence decreases dramatically. Further, although the authors seem to be aware of intelligence-mortality and intelligence-fertility associations, they do not incorporate empirical estimates (Batty, Kivimaki, & Deary, 2010; Kolk & Barclay, 2017) into their model.\nOn a more technical side, the model either has some mistakes or is insufficiently documented. For example, it does not produce IQ values with a standard deviation of 15, as would be implicitly expected. Dead people seem to become female (this seems to be irrelevant to the results), and there is an oddity about age being linked to sex for unexplained reasons. Explicit and descriptive variable names, more comments, and making the simulated data available as a clearly labelled dataset would help with these problems.\nTo summarise, we think demographic predictions such as these are important but require more forethought and modelling complexity than provided here, especially when extrapolated hundreds of years into the future. Previous attempts (Woodley of Menie & Fernandes, 2016; Woodley of Menie, Sarraf, & Fernandes, 2018) have in our opinion (Arslan, 2017; Arslan et al., 2018) also fallen short of addressing this complexity. While we think the current attempt should not be used to announce a future health and intelligence crisis, a revised and refined model could be useful. It should be calibrated with empirical parameters found in the literature and tested according to its ability to predict past time series of the intelligence distribution from parameters such as paternal age which both have been historically recorded in Scandinavian countries.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\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? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No",
"responses": []
},
{
"id": "360290",
"date": "31 Jan 2025",
"name": "Seong Soo A An",
"expertise": [
"Reviewer Expertise Genetics",
"biomarkers and therapeutics in neurodegenerative diseases"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors presented an interesting idea on the effects of paternal ages for neurological diseases.\nEven though authors presented their computational model of mining the significances, authors did not consider the many pathways of the genesis of the de novo mutations, especially in neurological diseases.\nAuthors must consider why neurological diseases? Because there are too many de novo mutations are being reported in cancer patients.\nAmong the neurological diseases, authors need to select a type of neurological diseases. For example, more women come down with Alzheimer's disease, when more men develop Parkinson's disease and similar for ALS.\nIn addition, author must discuss and differentiate the epigenetic factors verse mechanisms of de novo mutations.\nLastly, there are somatic mutations are being reported in patients with epilepsy than other neurological diseases.\n\nIs the rationale for developing the new method (or application) clearly explained? No\n\nIs the description of the method technically sound? No\n\nAre sufficient details provided to allow replication of the method development and its use by others? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-358
|
https://f1000research.com/articles/7-356/v1
|
22 Mar 18
|
{
"type": "Clinical Practice Article",
"title": "Variable phenotypic presentations of renal involvement in Fabry disease: a case series",
"authors": [
"Sarah McCloskey",
"Paul Brennan",
"John A Sayer",
"Sarah McCloskey",
"Paul Brennan"
],
"abstract": "Fabry disease is an X-linked genetic deficiency in the alpha-galactosidase enzyme resulting in intracellular accumulation of glycosphingolipids and multisystem organ dysfunction. Typically 50% of males and 20% of affected females have renal involvement, ranging from proteinuria or reduced renal function, renal parapelvic cysts and progressive renal disease ultimately requiring transplantation or dialysis. The phenotypic presentation of Fabry disease is incredibly varied and will even vary between family members with the same confirmed genetic mutation. In a cohort of patients affected by Fabry disease in the North East of England we examine the different phenotypic presentations of eight index cases (6 male, 2 female) with predominantly renal disease and the renal manifestations within their family members. The mean age of presentation was 40 years of age (range 23-59 years). Various multisystem manifestations were observed including cardiac, neurological, cerebrovascular and skin involvement. Two of the male index patients reached end stage renal disease (ESRD) requiring renal replacement therapy. Two female index patients had phenotypes limited to hypertension and proteinuria at presentation and the remaining patients had either stable or progressive chronic kidney disease at the time of diagnosis. We demonstrate the need for a high index of suspicion in order to consider Fabry disease as a diagnosis and the importance of cascade genetic screening to identify affected family members so that treatment can be initiated in a timely fashion.",
"keywords": [
"Fabry",
"chronic kidney disease",
"proteinuria",
"parapelvic cysts",
"GLA",
"mutation"
],
"content": "Introduction\n\nFabry disease (also called Anderson-Fabry disease) is an X-linked genetic deficiency in the alpha-galactosidase enzyme resulting in an intracellular accumulation of globotriaosylceramide (Gb3) and related glycosphingolipids causing organ dysfunction1. Classic manifestations include acroparesthesia (burning/tingling/numbness at extremities), angiokeratoma (typically over the bathing suit area but can be focal or diffuse) and corneal lipid accumulation known as cornea verticillata or Fleischer vortex dystrophy (seen on slit-lamp examination)2. The condition can also result in progressive renal impairment, gastrointestinal symptoms, heart disease and neurological involvement, including early stroke.\n\nSymptoms of Fabry disease can present in early childhood with overt organ dysfunction usually apparent by the second or third decade, however the phenotype is often variable even within single families3. Furthermore, female heterozygotes often have a degree of alpha-galactosidase activity present resulting in later onset of milder symptoms. Similarly, male patients with atypical variants due to preserved low levels of alpha-galactosidase may present later, typically in the third or fourth decade2.\n\nIt is partly this wide variation in clinical presentation that often leads to a delay in diagnosis. Fabry disease remains a rare disease and a high index of clinical suspicion is required to make a new diagnosis. However, with the advent of enzymatic and genetic screening more patients are being identified through a process of cascade screening allowing earlier initiation of treatment. Each index case diagnosed will typically lead to 3–4 cases on cascade screening4. Study data suggests that enzyme replacement therapy (ERT) may slow progression of renal dysfunction if instituted early however it is of less benefit when patients already have significantly reduced renal function and proteinuria5–7.\n\nAs many as 50% males and 20% of females with Fabry disease have renal manifestations2. Proteinuria and reduced estimated glomerular filtration rate (eGFR) may be present in early childhood8. If a renal biopsy is undertaken the pathognomonic histological renal lesion on electron microscopy is that of myelin-like inclusions known as “zebra bodies”. These can be seen in the lysosomes of cells predominantly within the glomerular and distal tubule due to Gb3 deposition. As the kidney disease progresses podocytes and endothelial cells become hypertrophic with foamy appearing vacuoles. Glomerular mesangial thickening then occurs leading to focal and then global glomerulosclerosis which can be detected under light microscopy9. Urinary epithelial cells may also have a vacuolated appearance containing a number of glycosphingolipids droplets and are then known as oval fat bodies.\n\nA number of small studies and population data have demonstrated an increased prevalence of parapelvic and renal sinus cysts in patients with Fabry disease10. The prevalence of cysts increases with age not unlike the prevalence of simple cysts within the general population. The exact aetiology of parapelvic cysts in Fabry disease is unknown. However the specific finding of renal sinus or parapelvic cysts on imaging in a young adult with renal impairment should raise the possibility of the disease as a diagnosis11. Parapelvic cysts resemble renal cortical cysts in morphology but plunge into the renal sinus from the adjacent parenchyma. If large enough they may compress the pelvicalyceal system causing hydronephrosis but due to their hypoechogenicity they may actually be mistaken for hydronephrosis on ultrasound imaging12.\n\nPreferential involvement of the distal tubules relative to other segments results in a reduced ability to concentrate urine, meaning polyuria may be the first manifestation of renal involvement13. However, it is usually the development of proteinuria or renal impairment that initiates referral to renal services / nephrology departments. Proteinuria and renal impairment progress with advancing age and result in end stage renal disease (ESRD) in almost all male patients and a significant proportion of female patients, if untreated. ESRD is the leading cause of death in male patients with untreated Fabry disease14. Heavy proteinuria is more common in adult males and is associated with a more rapid deterioration in renal function15. Although proteinuria is strongly associated with disease progression, it is not always overt in advanced renal disease.\n\nTreatment of Fabry disease consists of providing patients with recombinant human alpha-galactosidase. Two formulations currently exist and appear to be equally efficacious however both have a significant treatment cost. Some clinicians choose to treat classically presenting males (with low or absent alpha-galactosidase levels) as soon as the diagnosis is established in order to prevent disease progression. Due to the limited evidence of benefits of ERT in patients with established renal impairment, the European Best Practice Group do not advocate treatment in patients who already have established renal impairment (eGFR<60mL/min/1.73m2 or proteinuria>1g/day) unless they have non-renal symptoms that require treatment. A new chaperone therapy called migalastat is able to bind and stabilise certain mutant enzymes, allowing their proper trafficking to the lysosomes where they are able to function. Migalastat offers an alternative treatment option where ERT has lost efficacy, for example in cases of antibody formation to ERT. It also has the advantage of being an orally administered drug and has recently been recommended by NICE (see NICE recommendation for Migalastat). However, the longer-term benefits of this drug are not known. A recent 6 month placebo controlled study of this drug provided disappointing results with no difference in response between drug and placebo16, whilst an 18 month randomised study comparing ERT and open label migalastat produced more encouraging results17.\n\nAs with other forms of proteinuric chronic kidney disease (CKD), hypertension should be treated to a target of below 130/80 mmHg. It is reasonable to treat with an Angiotensin-Converting Enzyme inhibitor or Angiotensin Receptor Blockers as anti-proteinuric agents18. Complications of CKD such as anaemia and renal bone disease should be managed in the same way as for other forms of CKD and ultimately patients should be offered dialysis or transplantation if required.\n\nPatients with Fabry disease have an increased mortality on dialysis when compared to other (non-diabetic) causes of ESRD and the condition is associated with a reduced quality of life on dialysis compared to other patient groups19. Transplantation is therefore recommended as first line treatment for patients with ESRD due to Fabry nephropathy. Post transplantation examination of kidney allograft tissue can show Fabry inclusion bodies however they do not appear to cause graft dysfunction20. Dialysis patients with Fabry disease and transplant recipients may still derive benefit from ERT for extra-renal manifestations such as neurological and cardiac disease.\n\nWithin the North East of England there is a sizeable cohort of patients with Fabry disease. Here we review the varying phenotypic presentations of eight patients with overt renal disease and the renal manifestations of Fabry disease within their families (Table 1). These summaries hopefully help to emphasise the presenting features and patterns of disease progression where the proband has renal features. The importance of a full family history cannot be understated (Figure 1), as well as looking for evidence of male to male transmission of disease, which would exclude X-linked inherited disease\n\nCKD, chronic kidney disease; ESRD, end stage renal disease; HCM, hypertrophic cardiomyopathy; *3 cases reported EGL Genetic Diagnostics (Eurofins Clinical Diagnostics)\n\nMales are represented by squares, females by circles. Affected males are coloured black. Carrier and affected females are marked with a dot. The proband for each family is marked with an arrow.\n\n\nFamily A\n\n37 year old male was referred to nephrology following the detection of skin lesions that were identified as angiokeratoma. He was noted to have reduced serum alpha-galactosidase levels. At the time his blood pressure and renal function were documented as normal (serum creatinine within reference range) with no evidence of proteinuria. Within 3 years his kidney function deteriorated and he unfortunately progressed to ESRD, requiring haemodialysis. He was also was noted to have a mild hypertrophic cardiomyopathy.\n\nSeparately, his older brother, aged 47 years, was also noted to have skin lesions, having previously had an ischaemic stroke at the age of 46. A diagnosis of Fabry disease was confirmed by reduced serum alpha galactosidase levels. He also was documented as having normal renal function (serum creatinine within reference range) and left ventricular hypertrophy (LVH). He was treated with ERT. Over the last decade there has been only a small decline in his renal function (eGFR 67 to 52 mL/min/1.73m2) with no evidence of proteinuria.\n\nMutation analysis in the proband and his brother confirmed a pathogenic GLA mutation p.Thr412Serfs and cascade screening has allowed the offspring of the two brothers to be genetically tested. A daughter and a grandson were identified as inheriting this pathogenic allele but are currently asymptomatic. Interestingly, the brothers had a maternal aunt who was noted to have undergone renal transplantation for an unknown cause of ESRD. There was no other family history of renal disease.\n\n\nFamily B\n\nA 39 year old lady with no family history of Fabry disease presented to Renal Services with hypertension, proteinuria but preserved renal function. A renal biopsy demonstrated multiple myelin inclusion bodies, typical of Fabry disease. Genetic tests confirmed a diagnosis of Fabry disease(GLA c.999-1G>C) and allowed testing of her son and her sister, who were mutation negative. She commenced ERT approximately 1 year after presentation, and responded with a reduction in proteinuria. Her renal function has remained normal throughout (eGFR>90 mL/min/1.73m2).\n\n\nFamily C\n\nA 38 year old man presented with proteinuria (Urine Protein Creatinine Ratio (UPCR) 56 µmol/mmol creatinine), preserved renal function (estimated Glomerular Fitration Rate (eGFR) 79 mL/min/1.73m2) and a finding of possible renal cysts on ultrasound. He was also found to have a dilated left ventricle and aortic root on echocardiography. MR imaging of his kidneys demonstrated that his renal cysts were parapelvic in origin (Figure 2), pointing to a diagnosis of Fabry disease. He underwent subsequent genetic testing which confirmed a GLA splicing mutation and commenced on ERT within a year of his presentation. He has no contactable family members. His renal impairment progressed despite treatment, reaching ESRD 10 years later at which point he received a pre-emptive live donor transplant.\n\nMRI scan of proband from family C demonstrating bilateral parapelvic cysts.\n\n\nFamily D\n\nA 50 year old male was diagnosed with Fabry disease following presentation to neurology services with gait disturbance and thigh pain. Brain MRI revealed a previous ischaemic stroke. At the time of diagnosis he had evidence of CKD (eGFR 82 mL/min/1.73m2) and proteinuria with macroscopically normal kidneys on ultrasound. He was also noted to have hypertrophic cardiomyopathy. He was commenced on enzyme replacement therapy. His renal function has declined since diagnosis 9 years ago and eGFR is now 43 mL/min/1.73m2 with significant proteinuria (UPCR 108 µmol/mmol creatinine).\n\nA year later his brother was referred to Renal Services aged 43 years with progressive renal dysfunction (estimated Glomerular Filtration Rate (eGFR) 57 mL/min/1.73m2) and proteinuria. He had been made aware that his brother had a diagnosis of Fabry disease and his diagnosis was confirmed by low serum levels of alpha-galactosidase and genetic testing (missense mutation in GLA p.Ile117Ser). He was established on ERT but his renal function deteriorated over the last 10 years to an eGFR of 23 mL/min/1.73m2. He is currently being worked up for a pre-emptive renal transplant. He is otherwise asymptomatic.\n\nCascade screening in this family revealed that some of the brothers’ relatives were also affected, including a male maternal cousin who had presented at 20 years of age with progressive CKD resulting in ESRD. His originally documented cause of ESRD was of unknown aetiology. He subsequently underwent genetic testing which confirmed the same GLA mutation (p.Ile117Ser) as his cousins.\n\nThis large family had a number of other relatives who have presented to Renal Services or have been diagnosed as a result of cascade screening, including a 32 year old male cousin (also on the maternal side) who presented late with ESRD. He had been made aware of the diagnosis of Fabry disease within the family and his diagnosis was confirmed on renal biopsy and by genetic testing. He is now established on haemodialysis and receives ERT. His 39 year old brother was also diagnosed with Fabry disease the following year and was commenced on ERT. He reported classic neurological symptoms of acroparathesia and gastrointestinal upset and had normal renal function and only microalbuminuria at presentation. Both brothers have young daughters who have mild neurological symptoms that may be attributed to Fabry disease however they have normal renal function and have not yet undergone genetic testing.\n\n\nFamily E\n\nA 59 year old man presented with severe hypertension that was refractory to anti-hypertensive therapy and he was referred to Renal Services for consideration of atypical causes. It came to light that his older brother had presented to Renal Services within the same centre 35 years before, aged 25 with severe hypertension and CKD. The older brother had rapidly progressed to ESRD within 3 years and was maintained on haemodialysis until he received a renal transplant aged 30 years. A review of the histological specimens taken at the time of a bilateral nephrectomy aged 34 (performed for severe hypertension) confirmed the presence of myelin inclusion bodies (zebra bodies) on electron microscopy.\n\nA younger brother had undergone investigations over the years for a variety of symptoms and was also noted to have LVH, normal renal function but had documented microalbuminuria. The 3 brothers were found to have low levels of alpha-galactosidase activity and genetic analysis confirmed that all three siblings shared the identical p.Arg301Gln missense mutation. This degree of phenotypic variation within a family with the same genetic mutation is unusual and could potentially be explained by the role of environmental factors or additional modifying genes influencing disease manifestations27. A number of relatives have been identified through cascade screening but are currently asymptomatic or have no evidence of renal impairment.\n\n\nFamily F\n\nA 45 year old female presented with proteinuria and progressive CKD but then developed cardiomyopathy. She had initially undergone a renal biopsy which reported findings consistent with focal segmental glomerular sclerosis (FSGS) however on review, the electron microscopy was found to have evidence of podocyte vacuolation in keeping with a diagnosis of Fabry disease. Mutation analysis confirmed a pathogenic GLA mutation p.Arg227*. A number of her relatives have been diagnosed with the same genetic mutation following cascade screening however they are currently asymptomatic with no evidence of renal involvement.\n\n\nFamily G\n\nA 23 year old male presented to renal services with haematuria, proteinuria and progressive CKD. He had classical features of Fabry disease including angiokeratoma and LVH. Genetic testing confirmed a p.Arg112Cys GLA mutation. Enzyme replacement therapy was commenced. Unfortunately, he had a progressive decline in renal function resulting in ESRD at the age of 33 years of age. He was treated with haemodialysis and renal transplantation. He was troubled with significant acroparasthesia. He died aged 41 years. Family screening confirmed that his mother and sister were carriers of the p.Arg112Cys variant. His mother lived to 77 years of age and died of congestive cardiac failure. His sister, aged 51 years has an absence of haematuria and proteinuria, with preserved renal function.\n\n\nFamily H\n\nA 32 year old male was referred to renal services with a finding of elevated serum creatinine and reduced eGFR (45mL/min/1.73m2) and was also noted to have angiokeratoma. He underwent a renal biopsy which had electron microscopy findings consistent with a diagnosis of Fabry disease. He suffered with mild neuropathic pain but had no evidence of cardiac involvement. Mutational analysis of GLA revealed a nonsense mutation c.679C>T, p.Arg227*.\n\nHis brother was seen the following year aged 22 and was noted to have slightly reduced renal function (eGFR 79mL/min/1.73m2) with no evidence of microalbuminuria but with evidence of left ventricular diastolic dysfunction on echocardiogram. Both brothers have been commenced on ERT and six other family members have been identified through cascade genetic screening.\n\n\nDiscussion\n\nInherited renal disease is the fifth most common cause of ESRD28. It is vital that a detailed family history of renal and extra-renal phenotypes is taken for any new patient presenting to renal services, whether the presentation is with hypertension, proteinuria or CKD. Only by doing this will the practitioner be able to piece together the often subtle clues that the patient may have an inherited disorder. If the family pedigree suggests or is compatible with X-linked inheritance (i.e. no evidence of male to male transmission) then Fabry disease should always be considered. Similarly the diagnosis should be considered in male patients with renal impairment and a finding of parapelvic cysts on renal ultrasound11 as in the case of Family C. The European Best practice Group also recommend screening of any males under the age of 50 with unexplained CKD and to consider screening in females of any age19.\n\nMany of the manifestations of Fabry disease are non-specific and can occur in other systemic disorders such as diabetes and hypertension. As a result of this and a highly variable phenotype there is often a significant lag time between presentation to services and a diagnosis of Fabry disease. Females and atypically presenting males may only have hypertension or mild phenotypes and it may be difficult to recognize a pattern of inherited disease. Family D demonstrates how it may be helpful to revisit the family history of a patient with ESRD of unknown cause and consider Fabry disease as a potential diagnosis.\n\nRenal biopsy, or as in the case of the patient in Family E, examination of renal tissue from nephrectomy, can often be helpful making the diagnosis. A small number of patients are identified co-incidentally through renal biopsy for investigation of CKD or proteinuria without any significant family history and when the diagnosis has not previously been considered. Screening for levels of alpha-galactosidase in at risk populations provides a non-invasive method of identifying patients. Similarly cascade genetic testing rapidly identifies affected family members in the absence of symptoms or clinical manifestations; potentially allowing treatment to be commenced earlier and prevention of disease progression.\n\n\nConclusion\n\nThe pedigrees and disease spectrum we have described demonstrate the significant variation in phenotype, even amongst family members who have been confirmed to have the same genetic mutation. This along with variation in disease phenotype, including the existence of atypical variants of Fabry Disease, means that there is often a significant delay before patients receive a diagnosis and are therefore able to start ERT and other protective treatments. The importance of a high index of clinical suspicion of Fabry disease in patients with unexplained CKD and the determination of a full family history cannot be stressed enough. With the advent of both dry blood spot alpha-galactosidase testing and molecular genetic screening the speed and ability to detect affected family members has been significantly improved. This allows a precise diagnosis to be made and for patients to be commenced on ERT early in their disease course with the hope of preventing worsening of symptoms and organ damage.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patients and/or relatives of the patients.\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 work was funded by Northern Counties Kidney Research Fund (award to JAS).\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 thank the patients and family members involved in this study.\n\n\nReferences\n\nMacDermot KD, Holmes A, Miners AH: Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 60 obligate carrier females. J Med Genet. 2001; 38(11): 769–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGermain DP: Fabry disease. Orphanet J Rare Dis. 2010; 5: 30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRigoldi M, Concolino D, Morrone A, et al.: Intrafamilial phenotypic variability in four families with Anderson-Fabry disease. Clin Genet. 2014; 86(3): 258–63. 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J Med Genet. 2017; 54(4): 288–96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJain G, Warnock DG: Blood pressure, proteinuria and nephropathy in Fabry disease. Nephron Clin Pract. 2011; 118(1): c43–8. PubMed Abstract | Publisher Full Text\n\nTsakiris D, Simpson HK, Jones EH, et al.: Report on management of renale failure in Europe, XXVI, 1995. Rare diseases in renal replacement therapy in the ERA-EDTA Registry. Nephrol Dial Transplant. 1996; 11 Suppl 7: 4–20. PubMed Abstract\n\nSessa A, Meroni M, Battini G, et al.: Chronic renal failure, dialysis, and renal transplantation in Anderson-Fabry disease. Semin Nephrol. 2004; 24(5): 532–6. PubMed Abstract | Publisher Full Text\n\nGal A: Molecular Genetics of Fabry Disease and Genotype–Phenotype Correlation. In: Elstein D, Altarescu G, Beck M, Fabry Disease. Dordrecht: Springer Netherlands; 2010; 3–19. Publisher Full Text\n\nShabbeer J, Yasuda M, Luca E, et al.: Fabry disease: 45 novel mutations in the alpha-galactosidase A gene causing the classical phenotype. Mol Genet Metab. 2002; 76(1): 23–30. PubMed Abstract | Publisher Full Text\n\nIshii S, Sakuraba H, Suzuki Y: Point mutations in the upstream region of the alpha-galactosidase A gene exon 6 in an atypical variant of Fabry disease. Hum Genet. 1992; 89(1): 29–32. PubMed Abstract | Publisher Full Text\n\nDavies JP, Winchester BG, Malcolm S: Mutation analysis in patients with the typical form of Anderson-Fabry disease. Hum Mol Genet. 1993; 2(7): 1051–3. PubMed Abstract | Publisher Full Text\n\nYasuda M, Shabbeer J, Benson SD, et al.: Fabry disease: characterization of alpha-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele. Hum Mutat. 2003; 22(6): 486–92. PubMed Abstract | Publisher Full Text\n\nRichfield L, Bruce R, Baker R, et al.: Phenotypical expression of mutation R227X exon 5 of the GLA gene (nucleotide change p.Arg227X c.608C > T) identified in a large UK kindred spanning eight generations in a well-documented family pedigree. Acta Paediatr. 2006; 95: 127–8. Reference Source\n\nBrady M, Montgomery E, Brennan P, et al.: Diagnosing Fabry disease--delays and difficulties within discordant siblings. QJM. 2015; 108(7): 585–90. PubMed Abstract | Publisher Full Text\n\nDevuyst O, Knoers NV, Remuzzi G, et al.: Rare inherited kidney diseases: challenges, opportunities, and perspectives. Lancet. 2014; 383(9931): 1844–59. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "32417",
"date": "26 Apr 2018",
"name": "Shabbir Moochhala",
"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 describes families in whom a diagnosis of Fabry was made via renal services. This is a valuable piece of work and is a useful contribution to the renal literature. The descriptions are careful and succinct, and are complete with mutations analysis data and pedigree charts. Thus they illustrate the authors' main points which are of high clinical suspicion and attention to family histories in general renal clinics. There are also examples of rare presentations given, e.g. presentation with hypertension in a female patient and confirmed on biopsy.\nSome specific observations:\nIntroduction:\n\nPara 4: There is a differential diagnosis associated with zebra bodies. It may be worth noting this (e.g. antimalarials, amiodarone) as they can give false positives.\n\nPara 8: Tahir, J Am Soc Nephrol 2007 Sep;18(9):2609-17 provides evidence that ACE/ARB use in addition to ERT is essential to reduce proteinuria, and this is regarded as standard practice i.e. it is more than \"reasonable\" as stated in para 8.\nPara 9: Ref 19 was an observational study performed in untreated Fabry disease (i.e. pre-ERT era) so this should be stated.\n\nCases:\nFamily A, brother 2: Was he also treated for LVH? If so, an ACE or ARB may have augmented the response from ERT.\nFamily E: Were other investigations performed to exclude other more common causes of hypertensive renal disease in the index patient? Such severe hypertension as an initial presentation of Fabry is regarded as very rare. Another diagnosis may have co-existed.\nFamily F: It would be unusual to find no lamellated bodies anywhere on an initial electron microscopic report of a renal biopsy from a patient with Fabry.\nDiscussion:\nScreening for alpha gal in at risk populations is only reliable in males, not the whole population.\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": "32323",
"date": "27 Apr 2018",
"name": "A. Neil Turner",
"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 well-written account from a renal unit gives a clear detailed account of the range of presentations of Fabry Disease in 8 local patients with mainly renal manifestations of this rare lysosomal storage disorder. An excellent summary of the condition is followed by details of index patients and relatives.\nThe condition is increasingly recognised, but only some nephrologists are not fully aware of the range of presentations and manifestations. It is important as its very expensive treatments need careful consideration, though heavy proteinuria or other evidence of significant renal consequences would usually considered to be a treatment indication. The unexplained association with parapelvic cysts is nicely illustrated.\nThere is no doubt that there are unrecognised patients still; whether they meet treatment criteria is important, and not addressed. But that's OK.\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/7-356
|
https://f1000research.com/articles/6-2051/v1
|
27 Nov 17
|
{
"type": "Research Article",
"title": "Funding knowledgebases: Towards a sustainable funding model for the UniProt use case",
"authors": [
"Chiara Gabella",
"Christine Durinx",
"Ron Appel",
"Christine Durinx",
"Ron Appel"
],
"abstract": "Millions of life scientists across the world rely on bioinformatics data resources for their research projects. Data resources can be very expensive, especially those with a high added value as the expert-curated knowledgebases. Despite the increasing need for such highly accurate and reliable sources of scientific information, most of them do not have secured funding over the near future and often depend on short-term grants that are much shorter than their planning horizon. Additionally, they are often evaluated as research projects rather than as research infrastructure components. In this work, twelve funding models for data resources are described and applied on the case study of the Universal Protein Resource (UniProt), a key resource for protein sequences and functional information knowledge. We show that most of the models present inconsistencies with open access or equity policies, and that while some models do not allow to cover the total costs, they could potentially be used as a complementary income source. We propose the Infrastructure Model as a sustainable and equitable model for all core data resources in the life sciences. With this model, funding agencies would set aside a fixed percentage of their research grant volumes, which would subsequently be redistributed to core data resources according to well-defined selection criteria. This model, compatible with the principles of open science, is in agreement with several international initiatives such as the Human Frontiers Science Program Organisation (HFSPO) and the OECD Global Science Forum (GSF) project. Here, we have estimated that less than 1% of the total amount dedicated to research grants in the life sciences would be sufficient to cover the costs of the core data resources worldwide, including both knowledgebases and deposition databases.",
"keywords": [
"Bioinformatics",
"Data Resources",
"Knowledgebases",
"Funding",
"Long-Term Sustainability",
"Open Science"
],
"content": "1 Introduction\n\nKnowledgebases are organized and dynamic collections of information about a particular subject where data from multiple sources are not only archived, but also reviewed, distilled and manually annotated by experts. These digital infrastructures are essential to the effective functioning of scientific research and for the whole life science community: they serve as encyclopaedias, concentrating high quality knowledge collected from many different sources. In life sciences, knowledgebases are in general manually curated by experts, i.e. highly qualified scientists —called biocurators —who manually select, review and annotate the information on a particular subject. As a result, knowledgebases are collections of continuously updated data, providing a highly reliable source of scientific knowledge, with the data being validated and enhanced. There is a substantial difference between a repository and a knowledgebase. Both represent the computationally tractable accumulation of (pieces of) information and knowledge processed in such a way that the data is easily readable, understandable and exported. However, knowledgebases, which are a synthesis of knowledge that is built on many datasets, do not rely on data deposition by the users, as the information in general requires to be carefully selected and processed by experts. Knowledgebases allow research to be faster and more efficient as they:\n\npromote knowledge transfer to different sectors (e.g. between industry and academics),\n\npromote the re-use of the data, with new analysis/methodologies and comparisons,\n\nreduce the need to recreate or regenerate duplicate data,\n\nspeed up research through easy access to integrated data, leading to considerable time and efficiency gains for researchers,\n\nmake data available for teaching,\n\ngenerate scientific input and motivation for new research, by allowing scientists to apply computational methods to analyse new data in light of prior knowledge.\n\nIn life sciences, manual expert curation plays a fundamental role in the creation of high quality knowledgebases. One example is represented by the Universal Protein Resource (UniProt), a key resource for protein sequences and functional information1. Expert curation constitutes a core activity in the development and maintenance of the UniProt Knowledgebase (UniProtKB), which is composed of UniProtKB/Swiss-Prot - the reviewed section containing expert curated records with information extracted from the literature and curator-evaluated computational analysis, and UniProtKB/TrEMBL - the unreviewed section with automatically annotated records. Despite the clear and increasing necessity for such high quality knowledgebases, the question of their sustainability in the long term is frequently raised, due to the current lack of an appropriate funding model.\n\nManual curation is acknowledged to be highly accurate2,3, but criticism is often raised about the necessity for such a time- (and cost-) consuming activity as opposed to the use of programs for automated or semi-automated information extraction (Information-Extraction programs —IE programs). In reality, current IE programs are not able to extract the large amount of information or compare data with the same accuracy as professional curators do, but they can be extremely useful for identifying mentions of single entities in the scientific publications, using for instance name-entity recognition tools2. Consequently, manual curation cannot be fully replaced by the existing Artificial Intelligence (AI) technology. Text-mining is, however, often used as a first-line method for data extraction and identification of relevant literature.\n\nThe cost of professional curation is surprisingly low compared to the cost of open access journals’ publication charges, or to the cost of performing the related research. The Swiss National Science Foundation (SNSF) allows to claim CHF 30001 (€2790) for costs of Open Access (OA) publication from agreed research funding. The Open Access Co-ordination Group in the UK estimates average fees at £15862 (€1863). Per year, the UniProtKB/Swiss-Prot curators read and/or evaluate between 50,000 and 70,000 papers, of which they fully curate approximately 8,000 publications. This means that in one year they read, evaluate and capture the output of research associated with OA publication costs of €100 to €200 million, significantly more than the budget of UniProt as a whole (∼ €15 million per year). Similarly, each publication that is read and/or evaluated, is the result of a research project grant with a typical value of ∼ $ 450,0003 (∼ €400,000). The cost of integrating the output (of the 8,000 publications) in UniProtKB, corresponds roughly to less than 0.1% of the cost to generate the research associated. Coherently with this analysis, a recent paper demonstrated that the costs of curation are quite modest on a per-article basis, and represent a fraction of the cost of the original research: the cost of biocuration of articles for the EcoCyc database is estimated at $ 219 (€193) per article over a 5-year period, corresponding to 6—15% of the cost of open-access publication fees for publishing biomedical articles, and to 0.088% of the cost of the overall research project associated4. Additionally, a recent analysis on UniProt showed that expert annotation is sustainable given that a large part of the literature is redundant and/or not relevant for the curation5. Thus, curation costs are affordable in an absolute sense and represent a small fraction of the cost of the overall associated research projects that generated the experimental data.\n\nUniProt is an open access knowledgebase: its curated data are “digital online, free of charge, and free of most copyright and licensing restrictions”, i.e. without price barriers (subscriptions, licensing or pay-per-view fees) and permission barriers such as copyright and licensing restrictions6. But open access is not to be confused with cost-free: making the data available involves significant labour, service and technology cost. The European Commission policy on open access data is very clear:\n\n“The vision underlying the Commission’s strategy on open data and knowledge circulation is that information already paid for by the public purse should not be paid for again each time it is accessed or used, and that it should benefit European companies and citizens to the full. This means making publicly-funded scientific information available online, at no extra cost, to European researchers and citizens via sustainable e-infrastructures, also ensuring long-term access to avoid losing scientific information of unique value.”4\n\nThe scientific and political community is generally in favour of open access: data repositories/archives and knowledgebases mostly contain data produced through research work funded by public grants, and in principle, the information already paid for by the public purse should not be paid for again each time it is accessed or used. This is often the case for research articles, which are one of the primary media of knowledge dissemination. In many cases, papers in peer-reviewed journals are only available for a fee or, by open access charges. Moreover, data produced in research is not necessarily a finished product suitable for immediate usage or storage in data resources. So, dedicated funding is necessary to curate and structure the data so that they can be accessible and usable by the scientific community. This is not “paying again for already paid for information”. This is additional funding necessary to make the information accessible in a usable manner, so as to avoid additional, larger costs. Manually curated knowledgebases face the problem that they are insufficiently and unsustainably funded by public funds. As an example, the UniProtKB/Swiss-Prot efforts in Switzerland are exclusively funded by Swiss and US funds, while the resource is being used by researchers all over the world. The search for a sustainable funding model that ensures the maintenance and the future development of such resources remains thus a critical challenge. At the beginning of this millennium, a survey on existing databases reported that more than two-thirds (68%) of 153 considered databases had uncertain near futures (living expectation for 1–5 years of funding). Fifteen years later, only 24% of them were still alive (or rebranded) and 76% were no longer maintained, showing that a viable, sustainable framework for long-term data stewardship is sorely and urgently needed7.\n\nUntil now, public knowledgebases have mainly been funded through institutional funding, user fees and/or research grants8. The latter are grants intended specifically for research projects rather than infrastructures or databases. Funding through research grants is not an effective model on the long term, as it presents major limitations. Firstly, grants are competitive and they reward innovation: curated databases end up competing with innovative research projects (that, ironically, most often could not even be carried out without these databases). In addition, in order to obtain these grants that are focusing on innovation, databases and knowledgebases are typically pushed to adding new features, thus increasing the cost further. Secondly, grants are cyclic, with rounds of 3–5 years, with review criteria that are often not appropriate and applicable to infrastructures as they are conceived for research projects. Funds are thus not stable in the long term: often grants may not be renewed, or the funding for the renewed grant could be dramatically decreased. Alternatively, institutional funding could in principle guarantee the long term sustainability of the research infrastructures as contracts are often negotiated and fixed over several years, allowing data centres to plan in advance and build the infrastructures. At the same time, the weakness of such model lies indeed in its inflexibility, which may not always allow to keep the pace with the growing data volume and improving techniques. Also, data access charges through subscription or user fees remain incompatible with the rising principles of open access. For these reasons, it is very common to see data resources combining the longer-term, but rather inflexible, institutional funding, with more flexible shorter-term research grants. It is important to mention that many data resources depend on research grants particularly in their early stages, as they are often the result of a research study. Yet, while this funding model often allows to identify the need for certain resources in the scientific communities, it is not intended to sustain them on the long term, inconsistently with the long living scope of such resources.\n\n\n2 Overview of existing funding models\n\nIn this paper, twelve funding model have been identified and are described here below. They include existing funding models for data resources and facilities, as well as possible scenarios that are currently considered by various international initiatives. The list is not exhaustive, but introduces the major funding sources in the various sectors of research, with a particular focus on the life sciences. Some funding models that are currently not specifically supporting data resources are also included, as they could be implemented for data stewardship and preservation.\n\nThe twelve models can be grouped in three main categories in function of the revenue origins (Figure 1). Most of the models rely on funds coming from national budgets and allocated to research and/or infrastructure, redistributed among the applicants, institutions or services according to various rules and conditions. A second category embraces all the models dependent on user fees. Finally, there are models counting on voluntary donations and participations, or third party funding. On top of these, models exist that are a mixture of these categories, as they benefit together from national funding and commercial fees or investments, or they take advantage of funding from government bodies, industries and voluntary donation.\n\nThe 12 considered models are represented depending on the origin of the revenues.\n\n1. National funding: governmental agencies fund the infrastructure directly, through non-cyclical funding programmes. For research infrastructures, funds derive directly from the domestic R&D budgets. Often, users are charged for some subscriptions or special services. Examples are:\n\nNational archives, libraries such as the National Library of Medicine at the National Institutes of Health (NIH), statistical agencies;\n\nNASA archives, State archives;\n\nPublic universities.\n\n2. Infrastructure model9: funding agencies pay directly for data resources as a necessary part of the research infrastructure, through a percentage of the research funding that is specifically set aside. The grants themselves are only allocated to research projects. A percentage of each grant is then retained and assigned to a budget for data stewardship, and subsequently redistributed among the relevant infrastructures, including knowledgebases. This model is similar to the National model (model 1), but in this case funding agencies are not necessarily national (they can also be private, thus with different budget constraints). The funding agencies contribute financially in proportion to the grant volume that they allocate to research. This model is not implemented yet as a funding model for life sciences knowledgebases.\n\n3. Institutional support: universities or institutions have their own repository/data bank that is maintained through the “internal” institutional funds. Grants can be cyclic or long-term, and usage may be restricted to the institution’s members or be open to the worldwide community.\n\nIt is often used to support specialist resources, such as CAZY —the Carbohydrate-Active enZYmes Database, funded through the French National Center for Scientific Research (CNRS) and the Aix-Marseille University\n\nUniProt is also partly institutionally funded through the SIB Swiss Institute of Bioinformatics (with governmental funding) and the European Bioinformatics Institute (EMBL-EBI) (with member states funding).\n\n4. Research project grants: competitive cyclic research or dedicated resource grants from national funding agencies such as the NIH, the National Science Foundation (NSF) or the Swiss National Science Foundation (SNSF). They request a submission by the applicant every 3–5 years. Access is free for the user. This category includes also a few existing grants specifically conceived for databases and resources5. Most of the databases and knowledgebases in the life sciences are supported by these type of grants, including:\n\nFlyBase —database for Drosophila genetics and molecular biology: grants from the National Human Genome Research Institute (NHGRI) at the NIH. Support is also provided by the British Medical Research Council, the Indiana Genomics Initiative, and the NSF;\n\nZFIN —Zebrafish Model Organism Database: NHGRI and small amounts from NSF;\n\nMGI —Mouse Genome Informatics: NIH grants;\n\nRGD —Rat Genome Database: NIH grant;\n\nTAIR —The Arabidopsis Information Resource, from 1999 to 2013: NSF grant;\n\nPeptideAtlas —database of re-analysed Mass Spectrometry peptides identification: grants from the European Commission and three institutes of the NIH;\n\nPDB —Protein Data Bank, the 3D structure database: grants from seven federal sponsors through the ww-PDB organization of four international partners.\n\n5. Content licensing/industrial support model10: requires commercial users to pay a fee for access to the data and for-profit reuse, whereas data are free for non-commercial users.\n\nBetween 1998 and 2004, for-profit users were paying an annual fee for access to Swiss-Prot (now part of UniProt), whereas academic researchers had free access. Swiss-Prot returned to an all-user-free access model in 2004 after the SIB Swiss Institute of Bioinformatics, the European Bioinformatics Institute (EMBL-EBI), and the Protein Information Resource (PIR) formed the UniProt consortium and obtained a grant from the NIH. For more details on this case study, see Section 3.\n\n6. User subscription fees: users are charged on a time base (e.g. every month or year) or on download sizes, and they have access to the entire database. At the end of the validity, the subscription must be renewed to continue the access.\n\nMany scientific journals, including prestigious ones, such as Nature, Science or Cell;\n\nKEGG, the Kyoto Encyclopedia of Genes and Genomes: a pathway database;\n\nTAIR, since 2013: curators formed a non-profit company (Phoenix Bioinformatics) and relied on tiered subscription revenues (national, institutional or individual subscriptions)11. So far this model has been described as successful in maintaining the database’s quality and user base12.\n\n7. Value-added/asymmetrical pricing model (freemium service)8: a basic data set within the database is freely available to anyone. Individual scientists or companies that are willing and able to pay a higher fee can buy additional levels of service, better data access or additional tools and resources.\n\nTRANSFAC13 —knowledgebase of eukaryotic transcription factors and their regulated genes: has a free public version dated 2005, while the professional version, that is susceptible to subscription to provide full access, is regularly updated and presents more advanced tools and an easy-to-use interface;\n\nPlatforms such as LinkedIn, Dropbox, . . .\n\n8. Infrastructural razor & blades14: an attractive, inexpensive or free initial offer (“razor”) encourages continuing future purchases of follow-up items or services (“blades”).\n\nApplied to the public sector information environment, this model sees datasets stored for free on cloud computing platforms and accessible by everyone via APIs (“razor”). Re-users are charged only for the computing power that they employ on-demand (“blades”). Application of this model is limited to contexts and domains in which the computational costs to access the datasets are significant;\n\nGENEINVESTIGATOR —search engine for gene expression: 7-days free access to the professional edition and permanent free access to the Basic edition for academics.\n\n9. Public-private consortium7: is a mixture of funding from government bodies and industries. The funders mandate the research subjects and supporting companies do not receive priority access to data.\n\nThe SGC —Structural Genomic Consortium: it consists of three academic laboratories in Oxford, Toronto and Stockholm and is funded by a consortium of 13 public and private bodies including GlaxoSmithKline, Genome Canada, Merck, Novartis, the Swedish Foundation for Strategic Research and the Wellcome Trust. The three laboratories solve protein structures chosen by the funders. All solved structures are deposited in a data bank, but supporting companies do not benefit of priority access.\n\n10. Online advertising and corporate sponsorship: corporate sponsorship is part advertising and part dealmaking —the corporation pays to support a database that provides value to its potential customers.\n\nGeneCards —database of human genes that provides genomic, proteomic, transcriptomic, genetic and functional information on all known and predicted human genes: it is free for academic non-profit institutions; other users need a commercial license. Advertisings appear on the website as banner ads. However, the income from advertising does not allow GeneCards to be self-sustainable: other funds come from academic grants and database licences’ royalties.\n\n11. Open source volunteering (or wiki approach)15: replacing part of data curation by community participation can be attractive as it has a low cost. It depends, however, on drawing contributions from busy users. In addition, contributions tend to be sporadic, leaving many gaps. Hence it can only replace (a small) part of curation and therefore still requires funding for curation, software engineers, storage space, and operating costs.\n\nGeneWiki —informal collection of pages on human genes and proteins;\n\nWikiProteins —web-based, interactive and semantically supported workspace based on Wiki pages of biomedical concepts16;\n\nTOPSAN —a collaborative annotation environment for structural genomics17.\n\n12. Donations: philanthropic funding such as grants and donations can generate income. They partly depend on the impact on and awareness of the (user) population.\n\nHuman Protein Atlas —funded by the Knut & Alice Wallenberg Foundation;\n\nHuman Cell Atlas —funded by the Chan Zuckerberg Initiative;\n\nWikipedia —funded by small voluntary donations from thousands of users.\n\n13. Mixed models: most of the knowledgebases rely on diversified multiple funding streams. This approach has the obvious advantage of increasing resilience if one of the sources disappears. Some example of databases or knowledgebases supported by a mixed model are:\n\nUniProtKB: Swiss government through the SIB Swiss Institute of Bioinformatics (4-year grant), NIH (4-year grant) and EMBL-EBI;\n\nPRIDE —PRoteomics IDEntification database, part of ProteomeXchange: 25% EMBL-EBI, 50% Wellcome Trust (5-year grant), and 25% UK Biotechnology and Biological Sciences Research Council (BBSRC - research infrastructure grant);\n\nOMIM —Online Mendelian Inheritance in Man, a catalogue of human genes and genetic disorders, with a particular focus on the gene-phenotype relationship: NIH and, also, very recently through donations;\n\nInterPro —database for protein sequence analysis and classification: EMBL-EBI, BBSRC and Wellcome Trust;\n\nEnsembl —genome database and browser for the retrieval of genomic information: Wellcome Trust, NIH, EU FP7 and EMBL-EBI;\n\nEurope PMC —on-line database of free access biomedical and life sciences research literature: managed and developed by the EMBL-EBI on behalf of an alliance of 26 research funders, led by the Wellcome Trust.\n\nThe models are summarized for comparison in Table 1. Each model is described in terms of its compatibility with open access policies and its equity among the potential users and institutions, i.e. whether or not wealthier institutions or certain users are particularly favoured. Also, the forecasted stability of the models over time and the key dependency of each funding stream are indicated. Associated factors such as national/international economic situation dependency (which are obviously relevant within each model described) has been indicated only when representing the main dependency. The dependency of the funding is crucial to describe the vulnerability of the models and also needs to be taken into account when setting up a mixed model. The best funding model would combine models that are dependent on different factors.\n\nThe aspects that favour open access, equity of users and stability over time are highlighted in bold.\n\n\n3 Funding situation of the UniProt knowledgebase, past and present\n\nFor the purpose of this work, the UniProt knowledgebase is used as a case study. UniProt contains a reviewed collection of high-quality annotated and non-redundant protein sequences, and brings together experimental results, computed features and scientific conclusions. At present, UniProt is developed and maintained by the UniProt consortium, a collaboration between the SIB Swiss Institute of Bioinformatics, the European Bioinformatics Institute (EMBL-EBI), and the Protein Information Resource (PIR). The UniProt knowledgebase (UniProtKB) is the central resource that combines UniProtKB/Swiss-Prot and UniProtKB/TrEMBL. UniProtKB/Swiss-Prot contains sequences that have been curated by expert biocurators, while UniProtKB/TrEMBL provides sequences that have been annotated by automatic annotation systems. UniProt also includes the UniProt Reference Clusters (UniRef), a database of clustered sets of sequences from the UniProtKB, and the UniProt Archive (UniParc) that provides a complete set of known sequences, including historical obsolete sequences.\n\nThe UniProt knowledgebase is an interesting case study because it passed through various funding models, as well described in the literature7,18,19. It started under the name of Swiss-Prot, a research project at the University of Geneva in 1986. At that time it was funded through a Swiss National Science Foundation (SNSF) research grant, which lasted until 1996, when the knowledgebase suffered a funding crisis20,21. After negotiations with the Swiss Government, an agreement was reached with the creation of an institutional framework for the knowledgebase: the SIB Swiss Institute of Bioinformatics, born on 30 March 1998 as a non-profit foundation that could fund 50% of the knowledgebase. Simultaneously to SIB, the company Geneva Bioinformatics (GeneBio) S.A. was established as the exclusive commercial representative of SIB, to compensate the other 50% of the costs. GeneBio was selling licenses to commercial users, the fee depending on the number of users in the company, while academics users had free access. The royalties greatly exceeded the portion of the budget provided by the Swiss Federal Government. The Swiss-Prot group grew rapidly to the size of 80 people in 2004, while the database more than quadrupled in content in 6 years. In 2002, the funding model of Swiss-Prot changed and returned freely accessible to all the users. SIB and EMBL-EBI joined with PIR to form the UniProt consortium and applied for a NIH grant. Today the UniProt consortium has three main funders: the Swiss government (from 1996 and through SIB from 1998), recently with a 4-year grant from 2017 to 2020 accounting for about 38.7% of the total costs, an NIH grant ending in April 2018 (∼ 32.5%), funds from the European Molecular Biology Laboratory (EMBL, ∼ 25.4%) and other funding of different sources (∼ 3.4%). Swiss-Prot has also been supported by some EU funding, which ended in 2009. Now, despite the fact that about 28% of its users are from Europe, only a little portion of the curated part of the UniProtKB is currently supported by European funding.\n\nThe yearly total income of UniProt is in the order of $ 17 million (∼ €15 million), of which more than 90% is going to the staff salaries. This income hardly allows the curation of the current relevant literature5. However, it does not allow any expansion that is required by the fast-growing need of literature biocuration.\n\n\n4 Application of the models to the UniProt case\n\nIn this section, the models presented in Section 2 are applied to the case study of the UniProt knowledgebase. For each model, the conditions to obtain an income equivalent to the UniProt annual effective costs, rounded up to €20 million, are analysed.\n\nWhen possible, the analysis of the feasibility of these models is extended to a theoretical global cost of the ensemble of the bioinformatics major core data resources for life science research (i.e. repositories and knowledgebases), estimated to about €190 million. By extension and to simplify the reading, this amount will be referred to as the budget for the “total core data resources”. This value has been assessed from an estimated cost of the ELIXIR candidate core data resources (∼ €70 million6, per 435 million inhabitants for the ELIXIR’s Member States), extrapolated to a virtual geographical area that includes Europe, USA and Japan (respectively of 743 million, 320 million and 128 million inhabitants, for a total of 1.19 billion inhabitants). Other studies22,23 reported different values for other groups of resources and / or infrastructure, which may be in contrast with the data presented here. Yet, it is important to emphasize that the amounts presented in this work are rough estimations and should not be taken as financial reference data: their main purpose is to illustrate how the funding models could be applied to a real case study.\n\nAll estimations are based on available data (number of users, data download, ads, etc.). The revenue values are not intended to be an exact calculation, but should be used as an indicator of the income potential for the different models. Usage data of UniProt have been obtained through Google Analytics, with adjustments for the user population as in 23,24. This triangulation leads to an estimation of unique users per month at 83,000 units.\n\nWhenever possible, data are presented in the original currency from which they are derived, transformed in euros for ease of understanding (US$ 1 = €0.88, CHF 1 = €0.93, currency rates as at July 2017). To simplify the reading, amounts are approximated and each model is considered as a single model (i.e. no mixed model). The depth of the analysis of each model depends in general on its applicability to the UniProt case study. Some models that are theoretically applicable, but dependent on several variable parameters, are also not presented in greater detail as they would require a separate business analysis that is out of the scope of the present study. Also note that the analyses are performed under the hypothesis that the choice of the model does not influence the parameters of the same model (i.e. no feedback loop).\n\n1. National funding. In this model, the countries having the highest access rates of the UniProt website would pay an amount to the UniProt consortium, proportional to the usage (2016 data) or to the national wealth (OECD data7). Four parameters have been separately taken into account for this analysis: the UniProt usage rate, the Gross Domestic Product (GDP), the Net National Income (NNI) and the R&D domestic spending. Table 2 gives an overview of the costs for the top-10 user countries, both for the UniProt case and for the total core data resources. For the first parameter, the costs respectively for UniProt and for the total core data resources are distributed among the user countries according to their usage rate. A very small percentage of the national budget (0.00025-0.00035‰) or the R&D spending (0.014‰) would allow the sustainability of UniProt. Similarly, supposing the same geographical usage distribution for total core data resources infrastructure as for UniProt, it is possible to estimate that 0.0024-0.003‰ of the total budget or 0.13‰ of the total R&D spending could sustain the total core data resources.\n\nPotential amounts from the top-10 UniProt user countries to sustain UniProt (orange columns) and the total core data resources (blue columns). Costs per country as a function of (1) usage, (2) Gross Domestic Product (GDP), (3) Net National Income (NNI) and (4) R&D domestic spending.\n\nThis model guarantees secure funds for knowledgebases, and is stable over time. It is compatible with the criteria of open access and equity for users and institutions, and coherent with the idea that the countries with the highest number of users contribute to the maintenance of the infrastructures from which they are benefitting. A contribution based on the R&D spending might be preferred to the GDP as these two do not always correlate. For a country with a large population such as India, where the expenditures in research represent only the 0.85% of the GDP (compared to 3.2% for Japan, for example), the contribution might be seen as unfairly large for the government.\n\nAt the international level, some recommendations that are consistent with this model have been recently put forward. The European Commission’s High Level Expert Group on the European Open Science Cloud (HLEG - EOSC) proposed that about 5% of the total research expenditure should be spent on properly managing and ‘stewarding’ data in an integrated fashion. The implementation of this model requires, however, that the different governments or national funding agencies agree to contribute with a fixed percentage of their R&D budgets, which in general cover other research domains other than the life sciences. This model can therefore not be put into place in a short timeframe, and governance costs may represent a considerable fraction of the funds obtained.\n\n2. Infrastructure model. The cost of data stewardship would be covered directly by the funding agencies that fund field-related research projects. This model can be implemented as a sort of revised version of National model, model 1: funding bodies (not only governmental, but also private agencies) allocate a fixed percentage of their life science grants to a budget that is subsequently distributed to the infrastructures, knowledgebases included, according to well-defined selection criteria. To estimate the percentage needed to sustain UniProt and, more generally the total core data resources, the budgets reserved to the life sciences from five theoretically selected funding agencies, have been considered (the Swiss National Science Foundation8, the National Institutes of Health (NIH)9, the Wellcome Trust10, the Japan Science and Technology Agency11, and the European Commission12. The total yearly budget assigned to the life sciences by these five agencies adds up to ∼ €21 billion. A very small fraction of this budget, in the order of 0.1%, would then be sufficient to sustain UniProt. Only 1% of the total amount dedicated by these five funding bodies to grants in the life sciences would suffice to cover the cost of the total core data resources (0.9% of these budgets corresponds to approximately €190 million). Extending this model to other major funding agencies would increase the income and reduce the percentage needed from each agency.\n\nThis approach is very attractive in terms of equity and potential of income. It requires the major funding agencies to collaborate at an international level, and represents a significant evolution in the way how research infrastructure is funded. It also necessitates that countries that are currently not funding life science databases, or in a small proportion compared to their usage, start contributing. In general, funding agencies’ revenues can come from different sources, not only from the national budget (as in model 1); therefore, the participation of a certain funding agency to this model would likely depend on the availability of its own local budget, while the identification of the knowledgebases to which the budgets are allocated would require a selection process based on well-defined indicators (as it is currently done for grant assignments) and a lead agency or an institution that would take care of the funding distribution process. See Section 5 for an in-depth discussion about this model.\n\n3/4. Institutional support + research project grants. These two models are equivalent to the current funding scheme of UniProt, with 63% of the budget covered by institutional support (from Switzerland through SIB and from the EU through the EMBL-EBI), and 32% by NIH funding, granted for four years until 30 April 2018.\n\n5. Content licensing. UniProt is used by many life science companies to carry out business, research and to generate profit. The potential income of a commercial paywall is thus estimated, by assuming that all the life science for-profit companies would subscribe a licence to UniProt. A (non-exhaustive) list of the life science companies of 30 major countries in the world, irrespective of their size, was extracted13 together with a classification of all manufacturing companies, in terms of their size14. By assuming that the relative proportions of small, medium and large companies in the life sciences sector are similar to the proportions in the whole manufacturing area, the distribution of the life science companies in terms of their size was estimated. In this case, even low licence prices would generate an income of about €20 million, e.g.:\n\n€500 to small companies (0–9 employees)\n\n€1,000 to companies of 10–19 employees\n\n€3,000 to companies of 20–49 employees\n\n€5,000 to companies of 50–249 employees\n\n€10,000 to companies of 250+ employees.\n\nThe licence prices were intentionally underestimated in order to balance with the overestimation of the number of subscribing companies (>15,000).\n\nWhen this model was applied to Swiss-Prot in 1998, the licence fees ranged from €2,500 for small companies (typically start-ups) to €90,000 for the largest companies. At that time, the necessary annual budget for Swiss-Prot was ∼ €8 million. When calculating the revenues using these licence fees, a subscription by 1/10 of the total companies calculated above, would allow for a sustainable model for the knowledgebase. The implementation of this model requires some extra administrative costs, such as the creation of an adequate platform for the payment of the licences (probably on the order of few FTEs) and the costs for negotiation with the companies. This model has a large potential of income and allows maintaining a free access to the knowledgebase for academic users, while not for commercial users. It is though not compatible with the principles of open access and could hamper licensing and reuse of data by other resources that might see their access limited or blocked.\n\n6. User subscription fees. As estimated, UniProt has a traffic of about 83,000 unique users/month and average monthly data download from the FTP site of 30 TB. Charging the single user with a subscription fee of €20/month would allow an income sufficient to sustain the resource. Similarly, charging the user according to data download a fee of €0,055/MB download, would cover the yearly budget of €20 million. While these amounts are comparable to many subscriptions for software or applications, this model remains inconsistent in terms of equity and open science. Moreover, the implementation of this model would also require the setup of a platform for the payments, or the adoption of an existing one, with some additional (but probably negligible) costs. As the previous model, it is not compatible with the principles of open access.\n\n7/8. Freemium service / Razor & blades. The potential income for UniProt through these two models is difficult to estimate. Their implementation would imply that a selected part of the information (or old releases) was available for free and additional features (or the latest releases) were dependent on the payment of a fee. The data within UniProt would therefore have to be split into “free\" and “not-free-but-worth-paying-for\", in terms of data selection or old/new releases, which would require a more in-depth analysis and a careful selection of the type of data to charge and release. This model is currently used by some scientific journals such as the Proceedings of the National Academy of Sciences (PNAS): access to the complete PNAS Online is limited to paying subscribers and to members; without a subscription, all content older than 6 months is accessible at no cost. Similarly, TAIR adopted the same policy: up-to-date curated data are available to subscribers and one year later they become freely available for anyone to download. Interestingly, from an early analysis, the group reported that the introduction of a paywall did not decrease the use of the database11. These models, however, are not compatible with the principles of open access and they also require an infrastructure comparable to the subscription model to support the fee services. Moreover, they cannot guarantee the long term survival if all the resources will have to rely on subscription fees: a paywall for accessing each resource will heavily charge the user that will inevitably choose to dismiss some of them.\n\n9. Public-private consortium. A biotechnology/pharma company consortium financing UniProt is an option that would allow academic users a free access, with the budget of the resource being supported by a consortium of companies that make use of the knowledgebase. Yet, it is hard to estimate how many and which companies would be willing (or able) to participate, among which the cost (or part of it) would be distributed. A similar model to cover part of the costs could also in principle see field-specific companies funding the part of UniProt aligned with their interests, but without a privileged access to the data. In this way, the resource would remain open access for the users, although funded by private companies. However, the history of Swiss-Prot has shown that commercial users prefer to pay a (compulsory) licence subscription because a voluntary contribution is not easily defendable in the annual budget. The recent experience of the TAIR knowledgebase also shows that support from companies as a voluntary participation lags behind mandatory fees11.\n\n10. Advertising. This model could in principle be applied to UniProt in many different manners. One possibility is to have banner ads of related companies on the web page sides proposing pharmaceutical products, lab tools, antibodies, reagents, etc. This option has the inconvenience that the advertisements may damage the high quality image of the database, in addition to being intrusive and annoying for the users. A second possibility may be to add links to company websites that are selling products related to the proteins findable through the UniProt search tool. Also, the addition of some sponsor services, as for instance the inclusion of a comparative table of products, can be of added value to the knowledgebase. Another possibility is to collect users’ data (e-mails, searches, locations . . . ) and to exchange - provided permission from the users is obtained - the information with advertisers or partners. This is a model adopted by services as Google, Facebook and Apple, and by scientific journals such as Nature and Science. This model raises criticisms in terms of privacy and high scientific quality of the database. Moreover, the setup of an advertising platform is associated with additional costs and staff to support the structure. An advertising model requires a high volume of visitors to provide a sustainable income. To generate $ 50,000 (€44,000) per year in advertising revenues, a website needs approximately 2 million page visits per year10 The UniProt traffic of about 56 million page views per year could generate a maximum of €1.3 million, less than 1/15 of the annual budget. This model can therefore not be the unique funding source for UniProt, but rather a complementary stream to other models, as compatible with the principle of open access.\n\n11. Wiki approach. The model does not generate a revenue, but describes a way how data could be annotated, i.e. through voluntary participation of the community. The added value of UniProt lies in the high quality of the annotated data, curated by professional expert biocurators. Public contributions cannot maintain this high level of accuracy of the data. There exist many examples of resources that adopted a “Wiki-based” approach, in genomics (GeneWiki, WikiGenes), in proteomics (WikiProteins, TOPSAN), as well as for RNA annotation (Rfam, miRBase). However, these systems still encounter many obstacles such as usability, authorship recognition, and information reliability25. In addition, many of these Wiki resources are based on the integration of already processed data collected from existing knowledgebases such as UniProt. New approaches have been presented to increase reliability and usability, such as mechanisms to track authorship and to encourage community participation25, but many issues remain to be addressed. For example, a Wiki approach still requires funds for the basic infrastructure (servers, technical staff, . . . ). It could therefore be implemented, combined with other more robust models, as a solution to reduce the total cost of the knowledgebase. Studies have evaluated the multiple attempts to take advantage of the significant experience of the life science community (passionate scientists, students, retired researchers, . . . ) through some sort of crowd-sourcing. Yet, crowd-sourced curation appears to have a very low participation rate. In general, the more complex a database is, the more likely professional curation is to be favoured over crowd-sourced curation22,26. Therefore, this approach is definitely not applicable to the case of UniProt: high quality data thanks to professional expert curation are at the heart of this resource, and quality could not be guaranteed through a crowd-sourced curation.\n\n12. Donations. Many online databases and journals rely on donations from people around the world. The most famous is Wikipedia, the free collaborative collection of knowledge. Even though Wikipedia’s content comes from active users on a voluntary base (i.e. at cost zero), the site has running costs that are covered primarily by individual donations, in addition to other funding sources that allow to sustain specific projects. In 2016, the Wikimedia Foundation received $77.2 million (€72.5 million) from 5.4 million users (∼ 1% per year of all users, with an average donation of about $15 ∼ €13)15. By applying a similar model to the UniProt case, the same fraction of users could potentially contribute with similar donations to the knowledgebase. However, donations would contribute to less than 1% of Uniprot’s budget (∼ €115,000). Also, this model is highly unpredictable, as donations depend on individuals, the awareness of the funders and a strong involvement in the cause. Moreover, some extra costs are to be included, as such a model requires setting up a fundraising infrastructure (people, campaign, department, . . . ). Yet, it is worth considering this model as a complementary model.\n\n\n5 Discussion and proposal for a longterm sustainable funding model for knowledgebases\n\nAs described above, most life science knowledgebases are currently heavily dependent on grants and paid subscriptions: these funding models present many limitations that are described in Section 2. The ideal funding model for UniProt, with possible extensions to the total core data resources, should respond to the following criteria:\n\nTo guarantee open access and equal opportunity\n\nTo generate revenues that are sufficient to fully cover the costs and that are stable over time\n\nTo derive from transparent sources\n\nTo combine different revenue streams, in order to reduce the risk of lacking income if one of the sources is discontinued; the different revenue streams have to depend on different external factors or different entities (see Table 1) to further increase resilience.\n\nTable 3 summarizes the pros and cons for each model, with a focus on UniProt, and an estimation of the time frame necessary for its implementation. A complex model would obviously require a longer period of time to be put in place and accepted by the community.\n\nA model relying on access fees (model 5, User subscription fees, as well as model 6, Content licensing) would likely guarantee the sustainability of UniProt, at least as long as the resource remains useful and has an impact for the community. If academics could have a privileged free-of-charge access, commercial entities would contribute financially to the maintenance of the knowledgebase through a subscription fee. The introduction of a paywall, even for a part of the users, would probably possible impact data reuse, access and submission. One of the principal concerns is that a paywall may prevent researchers from linking to data in other databases. And of course, scientists would need to use their grant money to pay for subscriptions. One option to recoup the usage costs could be a “virtual coins model”, in which the costs for using the resources is included directly in the grant applications and a virtual budget is assigned specifically for that purpose. In this way, the resource is maintained as long as it is sufficiently used and the researchers receive pre-paid credits to access the infrastructure. In theory, the difference with a subscription fees model is that the user doesn’t subtract part of his research budget to pay the infrastructure, as the amount that s/he needs to pay is foreseen in the project estimates. The closest scenario is perhaps the BD2K Cloud Model16, though it relates to data storage and deposition databases. It hardly applies to resources such as knowledgebases with manual curation as it might be very hard for the scientists to estimate in advance the amount of usage that they will need for a project. Moreover, this implementation is not compatible with the principles of open access. Yet, in its recent experience of the application of a subscription model, TAIR claims to minimize these drawbacks by balancing subscriber-only privileges and the publication of special releases which can be downloaded and reused by other data resources. TAIR is actually also supported by a grant from the SLOAN Foundation, which allowed the transition to the subscription-based funding model11.\n\nThe table summarizes the potential of income of each model and the complexity of the implementation. Refer to Section 5 for the calculations.\n\nAnother possibility is the Consortium Model, in which major biotechnology/pharma companies contribute to the budget. Currently, there exist some joint programmes, such as the Innovative Medicines Initiative (IMI17), a partnership between the European Commission and the European Federation of Pharmaceutical Industries and Associations, that could in principle also support infrastructure and data resources. However, implementing this model for more than one database (for example for all ELIXIR Core Data Resources) may hinder the negotiations with the commercial partners. Alternatively, the idea of a separate consortium for each database is likely prohibitive.\n\nOn the basis of these observations, in this work, the Infrastructure Model is proposed as a sustainable model for all the life science knowledgebases, since it is compliant with the criteria we put forward above. Its process is illustrated in Figure 2. The funding agencies distribute research grants only to research projects (but not to databases), in function of their field/topic. A percentage of each grant is retained and assigned to a budget for data stewardship and subsequently redistributed among the relevant infrastructures, including Data Management Plans providers, deposition databases and knowledge-bases.\n\nOn the left, the current model, in which databases compete cyclically for grants against research or resource projects. On the right the Infrastructure Model, in which the funding agencies distribute research grants only to research projects. A percentage of each grant is retained and assigned to a budget for data stewardship, and subsequently redistributed among the relevant infrastructures, including Data Management Plans providers, deposition databases and knowledgebases.\n\nIn addition, whereas UniProt’s current funding is almost entirely coming from the USA, Switzerland and the EMBL member states, this model has the advantage of distributing the cost over the countries according to the composition of the science community and thus allows a considerable diversification of the revenue streams. Importantly, at a larger scale, with less than 1 percent of the life science budget of five major funding agencies in Europe, Switzerland, Great-Britain, Japan and the USA, this model would be able to fund €190 million to cover the costs of the total core data resources. Should such a model be implemented at an even wider international level, it will involve funding agencies from other countries, thus increasing the income and further diversifying the streams. The Infrastructure model has also the advantage to scale with the amount of data that is generated. The implementation of such a model requires however the appointment of a super partes lead agency or an institution that takes care of the funding distribution process and the selection criteria. This function could be played by ELIXIR, as the European reference for the life science bioinformatics resources, or by another non-profit organization. This model could also be combined with others, such as the advertising model or some donations or institutional support.\n\nThe Infrastructure model as presented could in principle be valid in the case of a consortium of funding agencies with similar volume of grants investments. However, if there is a large discrepancy among the parties, this model turns out to be unfair, as the contribution of each agency to the total budget is directly proportional to its research spending. As a consequence, “large” funders will end up in paying the largest fraction of the figure and the model will not be fair. In this situation, a variation to the model can be conceived: funding agencies are classified by size in terms of their research spending, as “small (S)”, “medium (M)” and “large (L)” funders and contribute to the total cost with a fixed percentage, depending on their category. In this way, costs will be redistributed more evenly among the funders and spread across the entire research community. A third possibility is to setup a fixed “entry fee” from each agency, that would guarantee a minimal income. The rest of the costs are distributed among the three categories of funders, again depending on their size. Figure 3 presents a comparison of the three variations of the Infrastructure Model, with the representations of the distribution of the UniProt cost among the 5 funding agencies considered for this study (NIH, EU, Wellcome Trust, SNSF, JST, see Section 4. In Case (i), each funding agency contributes with the 0.1% of its life science budget to the total cost. As the difference in research spending of the funders is so massive (the investment of the NIH into the life sciences corresponds to more than 6500% of the investment of the SNSF), the NIH ends up in paying more than 3/4 of the total cost. In Case (ii) the five funding agencies are classified depending on their life science spending and the total cost is shared among the categories: NIH as “large”, contributing for 49% of the cost, EU as “medium”, contributing for 30% of the cost and Wellcome Trust, SNSF and JST as “small”, contributing for 7% of the cost each. As clearly visible in the figure, this model has the advantage of redistributing the costs among such different funders. Case (iii) represents the extension of Case (ii), in which a fixed 2% entry fee is required from each funder (irrespective of the size) and the rest of the cost is covered by a contribution depending on the classification (S - M - L). This last variation may be perceived as the fairest, as it allows a redistribution of the costs and ensures a minimal income. The entry fee should be then set at a level that would not discourage the small funders from participating. Worldwide, similar initiatives have already been started. At the European level, the already mentioned commission High Level Expert Group on the European Open Science Cloud (HLEG-EOSC) was created in September 2015 to provide strategic advice to the European Commission on the European Open Science Cloud initiative as part of the Digital Single Market. In its discussion on the financing of research infrastructures, including e-infrastructures (e.g. ESFRI, e-IRG and Horizon 2020-related groups), the group proposed that well-budgeted data stewardship plans should be made mandatory to all research proposals and speculated that on average about 5% of research expenditure should be spent on properly managing and stewarding data. The analysis carried out in this work demonstrated that less than half of this value could actually be sufficient to maintain the core data resources.\n\nCase (i) is the classic model, in which the cost is covered by 0.1% of life science budget of each agency. In Case (ii), the five funding agencies are classified depending on their life science spending and total cost is shared among the categories with different percentages, but constant inside each group. In Case (iii) a fixed 2% entry fee is required from each funder (irrespective of the size) and the rest of the cost is covered by a contribution depending on the classification (S - M - L), as in Case (ii).\n\nIn parallel, the USA’s NIH has launched a virtual space called Commons, a shared computing resource and a repository for data and informatics tools. In 2015, the NIH started a pilot study to test the efficacy of the Commons Cloud Credits Business Model that is designed to provide unified access to a selected choice of compute resources. In this pilot project, the researchers obtain cloud credits as part of their project grant, i.e. dollar-denominated vouchers that can be used with the cloud provider of the investigator’s choice. The cloud provider has to be Commons-compatible by meeting a set of NIH standards for capacity and capabilities. This approach is supposed to provide the researchers with a cost-effective way of accessing cloud computing resources22. However, cloud providers still rely exclusively on NIH funding.\n\nBoth these initiatives concern mainly digital research infrastructures, such as archives, storage and data stewardship, while discussions on curated databases are still in their infancy. The Human Frontiers Science Program Organisation (HFSPO) with the Global Life Science Data Resource Working Group, as well as the ELIXIR Long Term Sustainability Working Group and the OECD Global Science Forum project (GSF) are all working on the issue of sustainable business models for data repositories and curated databases. The HFSPO has recently proposed that an international coalition should be set up to support the core data resources in the life sciences. The coalition would first define indicators to establish the core data resources eligible for international support, develop models that provide free global access, and help assess the fraction (an estimation of 1.5/2% has been proposed) of total research funding for such resources27,28. A similar project carried out by the OECD GSF is exploring the complexity of the problems connected to the future support of the data resources in the life sciences. Discussions are based on a strong consensus that core data resources for the life sciences should be supported through coordinated international efforts that better ensure long-term sustainability and appropriately align funding allowing for access at no charge.\n\nThe model presented in this work is in line with these considerations: its approach encourages equity, internationality and economic dependability, but it necessitates major changes to the way funds are distributed. It thus requires negotiating with the funding agencies at an international level, with probably less effort than with all the user country governments, and the introduction of a suitable structure to support this model in the life science community.\n\n\nData availability\n\nAll data required to reproduce the analysis presented in this study are included in the manuscript.\n\n\nNotes\n\n1http://www.snf.ch/SiteCollectionDocuments/Dossiers/dos_OA_regelung_auf_einen_blick_e.pdf\n\n2http://www.universitiesuk.ac.uk/policy-and-analysis/reports/Documents/2015/monitoring-the-transition-to-open-access.pdf\n\n3https://report.nih.gov/nihdatabook/charts/Default.aspx?chartId=155&catId=2, averaged on the last 10 years\n\n4Communication of the Commission ‘ICT infrastructures for e-Science’ of 5.3.2009, COM(2009) 108 final\n\n5https://grants.nih.gov/grants/funding/ac_search_results.htm\n\n6Corresponding to an estimated cost of the 26 candidate core data resources (5 archives, 15 knowledgebases and 6 declared as being both archive and knowledgebase, for the equivalent of 320 FTEs) submitted to ELIXIR on 1 December 2016.\n\n7https://data.oecd.org\n\n8http://p3.snf.ch/Default.aspx?id=AR2015\n\n9https://www.report.nih.gov/award/index.cfm\n\n10https://wellcome.ac.uk/funding/managing-grant/grant-funding-data-2015-2016\n\n11http://www.jst.go.jp/EN/JST_Brochure.pdf\n\n12http://ec.europa.eu/research/horizon2020/pdf/press/fact_sheet_on_horizon2020_budget.pdf, page 4\n\n13http://www.biotechgate.com/gate/v3/statistics.php\n\n14https://data.oecd.org/entrepreneur/enterprises-by-business-size.htm#indicator-chart\n\n15https://wikimediafoundation.org/wiki/2015-2016_Fundraising_Report\n\n16https://commonfund.nih.gov/bd2k/cloudcredits\n\n17https://www.imi.europa.eu/",
"appendix": "Competing interests\n\n\n\nUniProt is partially funded by the SIB, the Swiss Node of ELIXIR.\n\n\nGrant information\n\nThis work was done in the context of an ELIXIR Implementation Study linked to the ELIXIR Data platform and is funded by the ELIXIR Hub.\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 Amos Bairoch, Alex Bateman, Maïa Berman, Niklas Blomberg, Lydie Bougueleret, Alan James Bridge, Robert Kiley, Lydie Nso Nso, Sylvain Poux, Nicole Redaschi, Andy Smith, Heinz Stockinger, Daniel Teixeira and Ioannis Xenarios for the fruitful discussions and valuable suggestions.\n\n\nReferences\n\nWu CH, Apweiler R, Bairoch A, et al.: The Universal Protein Resource (UniProt): an expanding universe of protein information. Nucleic Acids Res. 2006; 34(Database issue): D187–D191. 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Publisher Full Text\n\nMatys V, Kel-Margoulis OV, Fricke E, et al.: TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Res. 2006; 34(Database issue): D108–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFerro E, Osella M: Eight business model archetypes for psi re-use. In Open Data on the Web Workshop. Google Campus, Shoreditch, London. 2013. Reference Source\n\nSalzberg SL: Genome re-annotation: a wiki solution? Genome Biol. 2007; 8(1): 102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMons B, Ashburner M, Chichester C, et al.: Calling on a million minds for community annotation in WikiProteins. Genome Biol. 2008; 9(5): R89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeekes D, Krishna SS, Bakolitsa C, et al.: TOPSAN: a collaborative annotation environment for structural genomics. BMC Bioinformatics. 2010; 11: 426. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBairoch A: Serendipity in bioinformatics, the tribulations of a Swiss bioinformatician through exciting times! Bioinformatics. 2000; 16(1): 48–64. PubMed Abstract | Publisher Full Text\n\nBairoch A, Boeckmann B, Ferro S, et al.: Swiss-Prot: juggling between evolution and stability. Brief Bioinform. 2004; 5(1): 39–55. PubMed Abstract | Publisher Full Text\n\nButler D: Bidding heats up for protein database. Nature. 1996; 381(6580): 266. PubMed Abstract | Publisher Full Text\n\nWilliams N: Unique protein database imperiled. Science. 1996; 272(5264): 946. PubMed Abstract | Publisher Full Text\n\nBourne PE, Lorsch JR, Green ED: Perspective: Sustaining the big-data ecosystem. Nature. 2015; 527(7576): S16–S17. PubMed Abstract | Publisher Full Text\n\nBeagrie N, Houghton J: The value and impact of the european bioinformatics institute. 2016. Reference Source\n\nFomitchev MI: How google analytics and conventional cookie tracking techniques overestimate unique visitors. In Proceedings of the 19th International Conference on World Wide Web. WWW ’10, New York, NY, USA, ACM. 2010; 1093–1094. Publisher Full Text\n\nChen IM, Markowitz VM, Palaniappan K, et al.: Supporting community annotation and user collaboration in the integrated microbial genomes (img) system. BMC Genomics. 2016; 17: 307. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarp PD: Crowd-sourcing and author submission as alternatives to professional curation. Database (Oxford). 2016; 2016: pii: baw149. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnderson WP, Group Global Life Science Data Resources Working: Data management: A global coalition to sustain core data. Nature. 2017; 543(7644): 179. PubMed Abstract | Publisher Full Text\n\nAnderson W, Apweiler R, Bateman A, et al.: Towards coordinated international support of core data resources for the life sciences. bioRxiv. preprint 110825 first posted online 2017, 2017. Publisher Full Text"
}
|
[
{
"id": "28423",
"date": "30 Nov 2017",
"name": "Helen M. Berman",
"expertise": [
"Reviewer Expertise Structural bioinformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper analyzes of sustainability models for knowledgebases using Uniprot as an example. It makes some important points and is definitely worthy of indexing. However, there are aspects of the presentation that could be improved. Here are some suggestions.\nIntroduction\nA simple statement about the differences between repositories and knowledgebases is required. Sustainability is a problem for all data resources not just knowledgebases. It would be better to make generalizations in the introduction and wait until section 3 to discuss UniProt in particular. After the statement about the sustainability framework there should be some discussion of other similar studies. These should include the Michigan study, the HFSP study, and the commentaries by Lorsch et al. among others. Then I suggest that there be a short statement about what this paper will cover. The comments in last paragraph of this section belongs in the appropriate parts of section 2. In particular, the difficulty of sustaining resources from research funding sources is a key issue facing many new and existing resources.\n\nOverview\nThis section is very clear. I think that some comments from the last paragraph of the introduction could be incorporated in the appropriate model descriptions. All of the data resources need references.\n\nFunding situation of Uniprot\nThe history of UniProt funding exemplifies the problems in the current sustainability models. Table 1 is very useful and compares well with the Michigan study. Although that study focused on domain repositories in all of science, the conclusions are similar. I do not understand the references given in the Infrastructure model section. Can any estimate be made of about further gains in automating information extraction that can be anticipated from improvements in machine learning tools and techniques? In other words, is there scope for significant future reduction in manual biocuration. In the concluding section, it would be useful to elaborate further on the how the availability of subsidized cyber infrastructure and services alone would impact the long term UniProt sustainability.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3517",
"date": "22 Mar 2018",
"name": "Chiara Gabella",
"role": "Author Response",
"response": "We want to thank the authors of the report for their interesting suggestions. We have implemented the suggestions which have improved the text presentation and the flow of the paper. We are grateful to the authors for their comments. In particular, we have: Enhanced the difference between repositories and knowledgebases in the introduction; Made the introduction as general as possible, without mention to the UniProt case Added statement about sustainability as problem for all data resources Moved the manual curation description to the appropriate paragraph and added considerations about the future of manual curation, which have also been discussed with Prof. Berman in the F1000 blog https://blog.f1000.com/2018/02/07/how-best-to-fund-knowledgebases/ Added references to all the data resources presented in the text Made more specific references to the previous comparable studies that are mentioned in the discussion section."
}
]
},
{
"id": "28421",
"date": "27 Dec 2017",
"name": "Eva Huala",
"expertise": [
"Reviewer Expertise biocuration",
"data resource sustainability"
],
"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\nCore data resources for the global biological research community are essential for the progress of science. Financial support for such resources is not guaranteed even given high usage and a clear need from the community. The authors of this paper describe 12 different ways to fund a core bioinformatics data resource like UniProt and advocate the adoption of the ‘infrastructure model’ for such a purpose.\n\nGeneral Comments:\n\n1. The authors discuss a very important issue and raise excellent points on the value and necessity of curation and how paying for curation is not ‘paying again for already paid for information’. They also make the valid point that while many highly used resources are used globally, they are financially supported by only a fraction of their users. An equitable distribution of the financial burden of data resource maintenance and improvement is a desirable goal.\n\n2. We think it would be useful to reexamine the criterion that open access equals free access. The paper defines open-access as “digital online, free of charge, and free of most copyright and licensing restrictions”. We propose that charging a modest fee that most of the community can afford in combination with mechanisms to enable access for those who can't afford it does not prevent access of the resource by those who need it.\n\nComments on the Overview of existing funding models:\n\n1) Models 7 and 8 are not clearly distinguished, these should probably be combined.\n2) Model 10, 'online advertising and corporate sponsorship', appears to us to be a mixed model.\n3) Some discussion of the 'pay to submit' model would be helpful, it is not clear why this model was not included. Example is the Dryad Digital Repository (http://datadryad.org/).\n4) The Cambridge Crystallographic Data Centre (CCDC, https://www.ccdc.cam.ac.uk/) is an important example of the freemium model that should be included.\n\n5) Model 11, Open source volunteering, doesn’t belong in income generating models. Even though this is stated later in the manuscript, for clarity it should be removed from this discussion.\n\nComments on the Recommendation of the Infrastructure Model\n\nThe proposed advantages of the infrastructure model strongly depend on the details of its implementation. In particular, the mechanism of allocation across existing and new resources will be challenging to design in a way that is: a) fair across countries and research disciplines, b) provides sufficient support to resources across a spectrum of resources ranging from those requiring intensive curation and therefore higher cost to resources with a less intensive approach and associated lower costs, c) provides a way to reevaluate the distribution periodically and shift resources where they are most needed, d) preserves some incentive for resources to maintain high quality and serve their users well, e) in spite of the possibility for shifting resources, still enables long term planning and is stable enough to ensure long term sustainability. In practice, this model will likely necessitate some sort of periodic evaluation of each existing or new resource to determine which will be funded and at what level; in other words, a mechanism that is very like a grant process. Careful planning would be required to avoid the acknowledged drawbacks of the existing grant funding paradigm. We think a discussion of some of these issues would improve the paper and enable a fairer comparison against existing funding mechanisms where the implementation details have been extensively worked out and the drawbacks are therefore more clear.\nA few points of clarification on TAIR, the resource with which we, the authors of these comments, are associated.\n\n1. The paper classifies TAIR (as currently funded) as an example of the “User Subscription Fees’ model. We think it should be considered a Mixed Model as its revenue derives in part from the National model (China and Switzerland country-level subscriptions), in part from the User Subscription Fees model (academic institutional, individual, and corporate subscribers), and in part from the Foundation model (Sloan Foundation grant). There are also elements of the Freemium model as non-subscribers have some free page views before encountering a monthly limit.\n\n2. The paper states, “TAIR is actually also supported by a grant from the SLOAN Foundation, which allowed the transition to the subscription-based funding model.” TAIR had already transitioned to subscription funding before the Sloan grant was received and funding from the Sloan Foundation was “to enhance the technology behind TAIR’s subscription funding model” and “[develop] a next-generation, flexible and customizable technology platform capable of serving other databases and research resources wishing to shift to user-based funding.”\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": "3516",
"date": "22 Mar 2018",
"name": "Chiara Gabella",
"role": "Author Response",
"response": "We thank the reviewers for the positive feedback and the constructive comments. We have addressed the major concerns raised in the report. We have corrected in the text the TAIR description and classification and added as an example of mixed model. We are grateful for those clarifications on TAIR. We have maintained the definition of open access equals free of charge, but we acknowledge that very modest fees could not prevent access to the resource by those who need it. We have added a comment on open access in the discussion section. Models 7 & 8 look indeed similar (see comment to Dr Blake): the analysis is merged for the two as they entail similar limitations. The difference relies on the period of time during which the “free” version is available. While for model 7 there is no limit in time, model 8 sets a time frame for free usage (which implies that if a user do not pay, he does not access the resource). We preferred to retain the distinction between the two, while we agree that they could in principle be merged. Model 10 could indeed be seen as a type of mixed model. At a deeper analysis, most of the models are mixed models or can easily be combined to form mixed models (see comment to Dr Blake). We preferred to maintain this selection and generate mixed models from the 12 described. Of course, other classifications according to different criteria could have been done. The “pay-to-submit” model was intentionally excluded as it is mainly a funding model for repositories. Knowledgebases, who do not rely on user data deposition, could not be funded through this model – unless merged with others-. The CCDC has been included, thank you for this precious suggestion Model 11 does not generate income. We preferred to retain it in the description as it has been recently considered as a possible sustaining model for some types of resources. As stated in the discussion, we agree that it cannot be a solution for curated databases."
}
]
},
{
"id": "28422",
"date": "02 Jan 2018",
"name": "Judith A. Blake",
"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 and interesting approach to considering the funding of expert biological infrastructure funding. Using UniProt as the test case, 12 models of funding and sustainability are investigated and discussed. Funding of infrastructure in life sciences is an incredibly important and pressing problem as increased levels of data generation and computational analysis accessing highly structured and integrated digital data are flooding the research environment.\nThe paper, far from being a generalized overview or editorial, dives deeply into an investigation of alternative funding models. It will be very useful to the user and to the funding communities to have this outline of different approaches. While I see overlap between a few of the funding mechanisms, the more important impact of the paper is that careful thought has gone into considering a range of mechanisms.\n\nWhile adequate linkages to data resources are presented, I think it would be a useful addition to add a table of sources and the particular result from that resource that is included in the discussion. Authors state all data to reproduce are included in the study, but actually the data are extracted from external reports. That said, all data for the evaluation presented in Figure 3 are available in the study. This is a minor quibble.\n\nThe challenge for important, comprehensive, and extensively used resources such as UniProt, the Model Organism Databases, and others is that while they are essential infrastructure for advancement of scientific investigations, no one agency or organization wants over responsibility. The effort to globally fund digital infrastructure will require cooperation and consideration of many parties. This paper reports on the issues and possible solutions\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": "3515",
"date": "22 Mar 2018",
"name": "Chiara Gabella",
"role": "Author Response",
"response": "We thank Dr Blake for the very positive review. We very much appreciate that the article is not perceived as a “generalized overview or editorial”, rather a call to action. We are very grateful to Dr Blake for pointing that out.We acknowledge that some of the models might look similar (e.g. model 7 and model 8) and in fact we have joint the analysis of those two models. As it is mentioned in the text, the list is likely not exhaustive: it is rather a subjective selection of the most used funding sources for data resources. It is also worth saying again that to simplify the reading, the study is conducted as if data resources were relying on one unique funding stream. In reality, most of the resources depend on mixed models and different funding sources. Considerations should therefore be weakened and merged, by taking into account many various parameters, in order to represent the current situation. This type of analysis falls out of the scope of this work."
}
]
}
] | 1
|
https://f1000research.com/articles/6-2051
|
https://f1000research.com/articles/6-2028/v1
|
20 Nov 17
|
{
"type": "Research Article",
"title": "Evaluation of Trichoderma spp., Pseudomonas fluorescence and Bacillus subtilis for biological control of Ralstonia wilt of tomato",
"authors": [
"Shiva Yendyo",
"Ramesh G.C.",
"Binayak Raj Pandey",
"Shiva Yendyo",
"Ramesh G.C."
],
"abstract": "Background: Ralstonia solanacearum is the major bacterial disease in tomato, which invades the roots of diverse plant hosts and colonizes xylem vessels causing wilt, especially in tropical, subtropical and warm-temperate regions. R. solanacearum produces several toxins helping it to invade the plant’s natural defense mechanism. Native isolates of Trichoderma spp., Pseudomonas fluorescence and Bacillus subtilis can be used as biocontrol agents to control the bacterial wilt and combined application of these beneficial microbes can give better results. Methods: Bacterial wilt infection in the field was identified by field experts and the infected plant part was used to isolate Ralstonia solanacearum in CPG media and was positively identified. Subsequently, the efficacy of the biocontrol agents was tested and documented using agar well diffusion technique and digital microscopy. 2ml of the microbial concentrate (109 cfu/ml) was mixed in one liter of water and was applied in the plant root at the rate of 100 ml per plant as a treatment method. Results: It was observed that the isolated Trichoderma spp. AA2 and Pseudomonas fluorescence PFS were most potent in inhibiting the growth of R. solanacearum, showing ZOI 20.67 mm and 22.33 mm, respectively. Digital microscopy showed distinct inhibitory effect on the growth and survival of R. solanacearum. The results from the field data indicated that Trichoderma spp. and Pseudomonas fluorescence alone were able to prevent 92% and 96% of the infection and combination of both were more effective, preventing 97% of infection. Chemical control methods prevented 94% of infection. Conclusions: Results showed the bio-efficacy of the native isolates. The various level of antagonistic effect against R. solanacearum shown by all 13 isolates, including the results shown by native isolates in the field, manifested the promising potential of the biocontrol agents, like Trichoderma and Pseudomonas against controlling the bacterial wilt infection.",
"keywords": [
"Trichoderma",
"Pseudomonas fluorescence",
"Bacillus subtilis",
"Ralstonia solanacearum",
"biocontrol agent",
"bio-efficacy",
"bacterial wilt",
"tomato"
],
"content": "Author endorsement\n\nDr. Sushil Thapa 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. Dr. Sushil Thapa declares he has no competing interests. Affiliation: Texas A&M AgriLife Research, Amarillo, TX 79106, USA.\n\n\nIntroduction\n\nBacterial wilt caused by Ralstonia solanacearum (formerly called Pseudomonas solanacearum) is a major bacterial disease for tomatoes. It invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing bacterial wilt disease1–3. It is a devastating plant disease most commonly found in tropical, subtropical and warm-temperate regions4,5. R solanacearum produces several known virulence factors, including extracellular polysaccharide (EPS), and a combination of plant cell wall-degrading enzymes, such as endoglucanase (EG) and polygalacturonase (PG). Mutants lacking EPS and EG shows reduced virulence6–8. The major virulence factors for this pathogen are plant cell wall-degrading polygalacturonases (PGs)9. Various biocontrol agents are used to control the bacterial wilt caused by R. solanacearum. Trichoderma harzianum10,11, Trichoderma viride12, Trichoderma asperellum13, Trichoderma virens14, Pseudomonas fluorescence15,16 and Bacillus subtilis17 are used as biocontrol agents to control bacterial wilt. Combination treatment methods using two or more of these agents are more effective in managing the disease than treatment using a single biocontrol agent10,18,19. Chemical bactericides and fungicides induce resistance in pathogens during long term use, which ultimately makes the pathogen tolerant to these chemical applications20–23. Hence, there should be a focus on the use of biological methods to control plant disease.\n\nThis study focuses on evaluating the efficacy of different native isolates of Trichoderma species, B. subtilis and P. fluorescence against bacterial wilt disease caused by the pathogen R. solanacearum in the tomato plant. The study hypothesizes that the native isolates of Trichoderma spp., B. subtilis and P. fluorescence can be used as bioantagonistic agents to control bacterial wilt (R. solanacearum) of tomato. This study tries to reflect the bioantagonistic effects by the microscopic examinations, agar well diffusion technique and by applying the concentrate of biocontrol agents in tomato plots, to calculate their efficiency by comparing them with chemical methods of treatment.\n\n\nMethods\n\nThe sample collection and field trials were done at Agro Narayani Farm, Sukranagar, Chitwan District, Nepal (Latitude: 27.582016 and Longitude: 84.272259) where the bacterial wilt infection was recorded in the previous harvest. The tests in the field were conducted from 11 March 2017 to 8 July 2017 for 120 days in new transplants. At the time of transplant, compost fertilizer at the rate 2.94 kg/m2, urea at the rate of 23.59 gm/m2, potash at the rate of 29.49 gm/m2, DAP 19.66 gm/m2, borax at the rate 1.97 gm/m2 and zinc at the rate 1.97 gm/m2 were applied. At 45 days and 90 days of transplant NPK 20:20:20 was applied at the rate 9.83/m2. Spacing of the plant was 50 cm in double row system of 50 × 50 cm. The plot size was 50 m2 and total of 8 plots were used having 100 plants per plot. Weather data were also collected from online resources as reference.\n\nPhysical symptoms, such as wilting of young leaves, discolored tissue at the dissected part of the stem base, and white, slimy ooze when the dissected part of the plant was kept in the glass of water, were used for the identification of infected plants24.\n\nBacterial wilt infection in tomato plants (Variety: Manisha) grown in Chitwan, Nepal, was positively identified by S. Yendyo of Kishan Call Center (KCC). Six whole plants were brought to the Quality Control laboratory of Agricare Nepal Pvt. Ltd. in a sterile bag for the isolation of the pathogen.\n\nR. solanacearum was isolated from dissected sections of the infected tomato plants on Casamino acid-Peptone-Glucose (CPG) Agar (casein hydrolysate 1 g/l, peptone 10 g/l, glucose 5g/l, agar 15g/l). Briefly, the stems of the infected plant were washed three times with autoclaved distilled water and then blot dried. After drying the stem were washed with 80% ethanol solution, then 1% sodium hypochlorite (NaOCl) solution was applied for 2 minutes. Final washing was done with autoclaved distilled water three times. The xylem of 2–3 cm from the stem was dissected and the sap was rubbed in CPG medium and inoculated for 48h at 28°C. Identification was done on the basis of the morphology of the colony on CPG medium, Gram staining and microscopic examination25,26.\n\nA total of 13 strains were used in this study (see Table 1). Six strains of Trichoderma spp., two strains of Pseudomonas fluorescence and one strain of Bacillus subtilis were isolated from soils and root soils collected from different sites in Nepal. ATCC 13525 strain of P. fluorescence from Microbiologics, St. Cloud, MN 56303, France was used as the reference for isolated Pseudomonas species. Trichoderma harzianum, Trichoderma virens and Trichoderma asperellum strains were provided by Tamil Nadu Agricultural University (TNAU), Coimbatore, Andhra Pradesh, India, and were used as reference species for isolated Trichoderma spp.\n\nThis includes the short code given to the isolates for identification in this study.\n\nTrichoderma spp. were isolated by serial dilution method using TSM agar plate (K2HPO4: 0.9 g/l, MgSO4: 0.2 g/l, KCl: 0.15 g/l, NH4Cl: 1.05 g/l, Glucose 3 g/l, Rose Bengal: 0.15 g/l, agar 20 g/l, streptomycin: 100 mg/l, tetracycline: 50 mg/l)27. 1 gm of each soil sample were suspended in 9 ml of sterile distilled water and vortexed (Accumax India, New Delhi-110058, India) for 5 min. The soil suspension was then serially diluted to 10-3 and 10-4. Pour plate technique was used by mixing 1 ml of the diluted soil suspension in 3 TSM agar plates for each sample and incubated at 25°C for 5 days. The strains were purified on TSM agar plates using sub-culture technique.\n\nPseudomonas fluorescence were isolated by serial dilution method using King’s B agar (Peptone: 20g/l, K2HPO4: 1.5 g/l, MgSO4: 1.5 g/l, glycerol: 10 ml/l, agar: 20g/l)28. 1 gm of each soil sample were suspended in 9 ml of sterile distilled water and vortexed (Accumax India, New Delhi-110058, India) for 5 min. The soil suspension was then serially diluted to 10-6. Pour plate technique was used by mixing 1 ml of the diluted soil suspension in 3 King’s B agar plates for each sample and incubated at 27°C for 48 h. The strains were purified on King’s B agar plates using sub-culture technique.\n\nBacillus subtilis was isolated by serial dilution method using Nutrient Agar (Peptic digest of animal tissue: 5 g/l, NaCl: 5 g/l, beef extract: 1.5 g/l, yeast extract: 1.5 g/l, agar: 20g/l)29. 1 gm of each soil sample were suspended in 9 ml of sterile distilled water and vortexed (Accumax India, New Delhi-110058, India) for 5 min. The soil suspension was then serially diluted to 10-6. Pour plate technique was used by mixing 1 ml of the diluted soil suspension in 3 NA plates for each sample and incubated at 27°C for 48 h. The strains were purified on NA agar plates by using sub-culture technique.\n\nAll thirteen isolates were screened against R. solanacearum by agar well diffusion technique30. R. solanacearum on CPG agar plates were transferred to nutrient broth and shaken in a rotary shaker (Talboys, Henry Troemner, LLC, USA) at 100 rpm at 27°C for 24 h. Similarly, the TSM, King’ B and NB were prepared for all Trichoderma spp., P. fluorescence and B. subtilis, respectively, and incubated for 7 days, 48 h and 48h, respectively. After incubation of the antagonists, 5 ml of broth suspension were centrifuged at 5000 rpm for 5 min and the supernatant was stored at 4°C for further procedure. Then, R. solanacearum suspension of 108 cfu/ml was prepared as per McFarland 0.5 turbidity method31 and was swabbed on NA plates. Holes of 5 mm were punched into the agar plate and 40 µl of the supernatant prepared were added separately and the plates were incubated at 27°C for 48 h. Inhibition of R. solanacearum growth was assessed by measuring the radius in mm of the zone of inhibition (ZOI) after incubation.\n\nFor microscopic visualization of the inhibition, CPG agar plates were prepared to provide the most favorable growth to R. solanacearum, and the respective 5 mm mycelial discs of Trichoderma species were added in the center of the plate after cotton swabbing from the CPG broth of R. solanacearum. For B. subtilis and P. fluorescence, the line was streaked parallel to the streak of R. solanacearum in two different CPG agar plates using dual culture technique. After 72 hr of incubation, live microscopic examination on the culture plate was done using a digital microscope (Olympus CX-43, Tokyo, Japan). Images were captured to visualize the interaction of the individual strains of biocontrol agents with R. solanacearum.\n\nThere is common practice in Nepal of keeping bio-based products in 5–10% (w/v) sucrose solution for 2–4 h before application to the plant. Hence, to evaluate the effect of 5% (w/v) sucrose solution on the cell number (growth) of the biocontrol agents, 1 ml of concentrate containing 1 × 109 cells ml-1 was kept in 5% sucrose solution, made using autoclaved distilled water. Cell count was taken using a Hemocytometer (Reichert, Buffallo, NY, USA) with trypan blue at 1 h, 2 h, 3 h and 24 h to observe the effect on microbial population.\n\nFor the field study, the concentrates containing 109 cells/ml of the respective biocontrol agent were used. The densities of the cells were determined using Hemocytometer (Reichert, Buffallo, NY, USA)32. The TSM broth of all six isolated native Trichoderma species viz. AA2, AG3, AKD, A5, A9 and A10 were mixed in equal proportion to prepare 1 liter of concentrate containing 109 cells/ml. Similarly, two native P. fluorescence species viz. PFB and PFS were mixed in an equal proportion to prepare 1 liter of concentrate containing 109 cells/ml. Concentrate of native B. subtilis viz. BS containing 109 cells/ml was used to analyze the effect of B. subtilis as a possible biocontrol agent.\n\nBefore the application in the tomato plots at Agro Narayani Farm, the prepared concentrate of biocontrol agents were taken to the field and were further diluted at the rate of 2ml/l of tap water containing 5% (w/v) sucrose. After 2 h of incubation in 5% sucrose water, the diluted solutions were applied in the root of tomato plants at the rate of 100 ml per plant. The processes of applications were repeated every 7 days for 8 weeks (total of 8 applications) by preparing fresh dilutions in 5% sucrose solutions 2 h prior to applications. Effects after the 8 weeks of continuous application were measured in the field by identifying the number of plants that underwent recovery after treatment. 6 plots were treated with the biocontrol agent and 2 plots were used as controls. Chemical treatment was done in one plot (positive control plot) using the combination of Agricin (9% Streptomycin Sulphate and 1% Tetracycline Hydrochloride) at the rate of 100 ml of 0.1% (w/v) solution per plant from Agricare Nepal Pvt. Ltd., Nepal and Bavistin (50% carbendazim) at the rate of 0.2 % (w/v) solution per plant from Crystal Crop Protection Pvt. Ltd., India. For negative control no treatment methods were selected in one plot.\n\nThe treatment plots were designed such that the effects of the individual biocontrol agent and effects of combination treatment can be studied (Table 2).\n\nOne plot comprised of 100 tomato plants and 8 plots in total were studied; area of 50 m2 per plot.\n\nStatistical analysis of the data was done using IBM SPSS Statistics (ver 23) and figures and data were made through Microsoft Excel 2007 and Microsoft Word 2007.\n\n\nResults\n\nWeather data of Chitwan District for temperature, humidity and rainfall were collected from www.worldweatheronline.com from March 2017 to July 2017 (see Table 3).\n\nSource: www.worldweatheronline.com\n\nThe bacterial wilt outbreak was reported in the late May, 2017, when the temperature and humidity level increased. Higher temperature and moisture favors the growth of R. solanacearum33\n\nFrom the field examination of the tomato plants, observation revealed that the leaves were flaccid, adventitious roots started to appear on the stem and ooze appeared after dipping the stem in water. Also, field experts from KCC confirmed the presence of bacterial wilt infection, due to their years of experience in plant disease diagnosis.\n\nInfected plant saps from six xylems showed similar bacterial colonies on CPG medium. All the colonies were similar to avirulent type, as the appearance was white or cream-colored, irregularly-round, fluidal, and opaque on CPG medium34,35. Gram staining and observation using a microscope showed that the bacteria were gram negative, rod shaped and non-spore forming, which further confirmed that the bacteria was R. solanacearum.\n\nAll 13 strains tested showed antagonistic effect against R. solanacearum, with inhibition zone radii ranging from 13 to 21.33 mm (Table 4). P. fluorescence PFS isolated from Parthenium rhizoplane soil was most potent compared to other P. fluorescence strains. Trichoderma virens ATV and Trichoderma harzianum ATH provided by TNAU were the least and most potent species. Among six natively isolated Trichoderma sp., AA2 isolated from Parthenium rhizoplane soil was most potent and AKD and A9 were least potent. However, the activities of native Trichoderma spp. were satisfactory in the term of inhibition zone shown. Bacillus subtilis BS isolated from Bhaktapur top soil did not show satisfactory inhibition activity.\n\nAll the data are generated using three replications. Values are means (±SE) zone of inhibition (ZOI) in mm against R. solanacearum (n=3, P <0.05). S30 denotes streptomycin sulphate used at the dilution of 30 mcg. 5 mm diameter of punch hole is included in the data. Code is in reference to Table 1.\n\nThe effect of biocontrol agents against R. solanacearum was analyzed at a microscopic level in dark phase using image analyzer (Olympus CX-43, Tokyo, Japan). Figure 1–Figure 3 show the distinct inhibitory effect on the growth and survival of R. solanacearum caused by different biocontrol agents. From the figures it can be seen that the most of the pathogenic cells (R. solanacearum) were either killed or growth was retarded or limited in or towards the region of growth of antagonists as compared to region away from the growth of antagonists.\n\nA1–J1 shows strains AA2, AG3, AKD, A5, A9, A10, ATA, ATH and ATV, respectively (see Table 1), growing on R. solanacearum, which was cotton swabbed onto CPG agar plates. A2–J2 represents growth of R. solanacearum 4 cm away from the growth of Trichoderma spp., viz., A2, AG3, AKD, A5, A9, A10, ATA, ATH and ATV, respectively, on the plate. Negative control (i.e., blank plate without any swabbing) and positive controls (i.e. R. solanacearum without Trichoderma species) are also included. The images reveal that the population of R. solanacearum is significantly less and most of the cells are dead in the region of growth of samples treated with Trichoderma spp., compared to the region 4 cm far from the growth of Trichoderma spp.\n\nPFA, PFB and PFS represent P. fluorescence (PF) species (see Table 1), and RS represents R. solanacearum. Both PF and RS were streaked near to each other to see the interaction between the two species. PF tended to grow on the side of RS, whereas RS tended to restrict the growth towards the PF species. RS positive control (without PF streak nearby) tended to spread, which confirms the spreading pattern of RS.\n\nBS represents Bacillus subtilis species (see Table 1), and RS represents Ralstonia solanacearum. Both BS and RS were streaked near to each other to see the interaction between two species.BS has completely overgrown the RS streak on the CPG agar plate, which suggests that there has been an interaction between RS and BS.\n\nThe effect of sucrose (5% w/v) in the concentrate mixture of individual biocontrol agents was analyzed (Figure 4), which showed that there was profound increase in the number of cells of biocontrol agents after 2h of incubation compared with the initial population. The cell count between 2h and 3 h of incubation was not significant as compared with 2 h and 24 h of incubation. Thus, 2 h of incubation in sucrose solution can be considered as optimal time, as lengthier time can result in growth of contaminant in the solution whilst using tap water in the field.\n\nAn increase in a number of cells of biocontrol agents was seen over time when these agents were kept in 5 % (w/v) sucrose solution (n=3).\n\nBefore applications, 2ml of respective biocontrol agents of (109 cells/ml) were incubated in 5% (w/v) of sucrose solution and incubated for 2 hr. The prepared dilution was thus applied at the rate of 100 ml per plant in the root region every week. 8 applications were done over 8 weeks. 8 plots (100 plant/plot) were selected, out of which one plot was used as positive control/chemical treatment plot (Agricin+Bavistin) and one plot as negative control/zero treatment plot (no treatment given).\n\nThe results are displayed in Table 5 and show that the application inhibited the bacterial wilt infection (R. solanacearum) in tomato plants by and the highest rate of plant recovered was 97% from treatment using antagonists, which was comparable with the plant recovery of 94% using the chemical treatment (Agricin and Bavistin). Only 37% of plants were recovered in the plot where no treatment methods were applied. Field Images (Figure 5) show a clear visualization of growth of plants and severity of infection in treated and untreated plot.\n\nThere was a significant recovery of plants using a mixture of Trichoderma species and Pseudomonas fluorescence, and the recovery rate was higher than that of chemical treatment. Bacillus subtilis did not show significant recovery rate.\n\nField images clearly reveal that the treatment with biocontrol agents has helped to eliminate the bacterial wilt disease in the field after 8 weeks of application. (A) shows the growth and vigor of plants treated with biocontrol agents; (B) shows growth and severity of infection occurred in untreated plot.\n\n\nDiscussion\n\nThis research covers the results of the effectiveness of native biocontrol agents in both laboratory and field settings, providing a complete portfolio from research in the lab to application in the field. This research also provides the application strategies of biocontrol agents at the field level from years of experience of expert field technicians. Also, from literature reviews10–17, it has been shown that these biocontrol agents can be used to control various other bacterial and fungal diseases, such as Fusarium wilt. Hence, application of these biocontrol agents can also help to prevent other diseases in various crops.\n\nThe present results, using microscopy, showed that different species of Trichoderma, Pseudomonas fluorescence and Bacillus subtilis clearly hinders the growth of Ralstonia solanacearum, which causes bacterial wilt in tomato plants. Trichoderma spp. secret different compounds against bacteria and also produce various secondary metabolites that promote plant growth and yield36–38. P. fluorescence produces various compounds that suppress the growth of R. solanacearum and also induces systemic resistance in the plant39–41. B. subtilis is well known to induce systemic resistance in plants by secreting various kinds of lipopeptides and secondary metabolites, and this agent also improves plant growth42–44. Analysis of data from research, field trials and scientific journal reviews suggests that the application of B. subtilis may not immediately show results, but continuous application of this strain in the agricultural field will slowly induce resistance of plants against pathogenic diseases42.\n\nPre-application of biocontrol agents can successfully prevent the disease attack45, induce systemic disease resistance in plants and increase the yield from secondary metabolites secreted by the beneficial bacteria in the biocontrol agent. Also, the farmers can sell their products by considering them as IPM (Integrated Pest Management) product or as an organic product, giving better monetary value for the farmers.\n\nFrom the results, 2 h incubation of the biocontrol agent in 5% (w/v) sucrose solution was judged as suitable practice being carried out in Nepal for application of biocontrol agents, as it was seen that number of cells of biocontrol agents were increased during the incubation period. In addition, the water they use for drip irrigation generally is unsterilized and comes from an underground source, which may promote the growth of contaminants if kept for longer periods.\n\n\nConclusions\n\nIn the present study, Trichoderma spp. and P. fluorescence seem to be the best biocontrol agents in controlling bacterial wilt induced by R. solanacearum. The zone of inhibition shown by the various antagonists reveals that native isolates were successful in inhibiting the growth of R. solanacearum. The digital microscopy also supports the antagonistic effects of the native isolates. Also, combination therapy using both Trichoderma spp. and P. fluorescence seems to be more effective than treatment using each individual biocontrol agent. A 97% control rate was achieved using combination treatment in the field.\n\nAlso during field application, mixing with 5% (w/v) of sucrose solution and keeping it for 2 h seems to be an effective strategy in better management of bacterial wilt, as it increased the population of the microbes as the biocontrol agent before the actual application. The application strategies of biocontrol agents with the rate of 100 ml per plant per week successfully recovered the plants from the attack of the pathogen. However, the application rate and amount of biocontrol agents can be varied according to disease severity. Also, the application of the multiple numbers of biocontrol agents can be performed to achieve better results. Trichoderma spp. and Pseudomonas fluorescence provide better results in controlling bacterial wilt in tomato. Bacillus subtilis did not perform well in the immediate control of disease. Data from Table 5 reveals that biocontrol agents can be used as the sole method to control bacterial wilt, and the use of chemical methods can be avoided in the field.\n\nHence, native isolates of Trichoderma spp. and Pseudomonas fluorescence can be used as biocontrol agents to control the bacterial wilt and combined application of these beneficial microbes as bioantagonist can give better results in controlling bacterial wilt infection by R. solanacearum.\n\n\nData availability\n\nOSF: Raw values of zone of inhibition by antagonist against Ralstonia solanacearum. ZOI is shown in mm and the data were used for statistical analysis. http://doi.org/10.17605/OSF.IO/9TQCE46\n\nOSF: Raw values of the 5% sucrose treatment. The average of these values in cells/ml was taken to create the data in the manuscript. http://doi.org/10.17605/OSF.IO/Q8FVU47\n\nFigshare: Images of the plates showing the zone of inhibition by different antagonists against Ralstonia solanacearum. Clear zone of inhibition obtained by agar well diffusion technique indicates the bioefficacy of the selected bioantagonists against R. solanacearum. https://doi.org/10.6084/m9.figshare.5562058.v248\n\nFigshare: Raw digital images (400X) of microscopic analysis representing the interactions of different antagonist against Ralstonia solanacearum. Collection of raw images obtained from digital microscopy in both dark field and bright field microscopy at 400 X zoom. https://doi.org/10.6084/m9.figshare.5561968.v149\n\nFigshare: Field Images of plot design showing pictures of before the treatment and effects after the treatment. There is significant decrease in the occurrence of disease for the treated plots whereas the bacterial wilt has severely affected in the untreated plot. https://doi.org/10.6084/m9.figshare.5562373.v250\n\nData are available under the terms of the Creative Commons Attribution 4.0 license (CC-BY 4.0).",
"appendix": "Author contributions\n\n\n\nSY was actively involved in the generating the data from the field where as RGC was actively involved in generating data from the laboratory. BRP was actively involved in the generating data from both the field study and the laboratory as well as writing the manuscript. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\nAuthor endorsement: Dr. Sushil Thapa 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. Dr. Sushil Thapa declares he has no competing interests. Affiliation: Texas A&M AgriLife Research, Amarillo, TX 79106, USA.\n\n\nGrant information\n\nThis research was funded as a part of public private partnership activities of Agricare Nepal Pvt. Ltd with Winrock International Nepal for USAID’s KISAN Project, the Presidential Feed the Future Initiative to develop bio-products locally in Nepal. The grant number was AID-367-C-13-00004 with sub-awardee DUNS number 557770037.\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 first like to thank Ms. Mona Sharma, Public Private Partnership Manager, Winrock International, Nepal and USAID for providing moral and financial support to conduct this research. We like to thank Prof. Dr. Sevugapperumal Nakkeeran, Department of Plant Pathology, Tamil Nadu Agricultural University (TNAU), Coimbatore, India for his supportive ideas in the research. We would also like to thank Ms. Santoshi Sharma, Ms. Shushilata Sapkota and Ms. Sweta Shrestha, interns at Agricare Nepal Pvt. Ltd., Nepal, Mr. Sarkal Jyakhwo, Field Technician at Kishan Call Center, Nepal and Mr. Aashish Khanal, Production Officer at Agricare Nepal Pvt. Ltd., Nepal for their supportive efforts during the project. Lastly, we would like to thank Mr. Deepak Gurung, the tomato farmer in Sukranagar, Chitwan, Nepal for reporting the problem of bacterial wilt and providing his field for the plot design and the study.\n\n\nReferences\n\nTans-Kersten J, Huang H, Allen C: Ralstonia solanacearum needs motility for invasive virulence on tomato. J Bacteriol. 2001; 183(12): 3597–3605. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPradhanang PM, Momol MT, Olson SM, et al.: Effects of plant essential oils on Ralstonia solanacearum population density and bacterial wilt incidence in tomato. Plant Dis. 2003; 87(4): 423–427. 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Publisher Full Text\n\nAliye N, Fininsa C, Hiskias Y: Evaluation of rhizosphere bacterial antagonists for their potential to bioprotect potato (Solanum tuberosum) against bacterial wilt (Ralstonia solanacearum). Biol Control. 2008; 47(3): 282–288. Publisher Full Text\n\nJi X, Lu G, Gai Y, et al.: Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain. FEMS Microbiol Ecol. 2008; 65(3): 565–573. PubMed Abstract | Publisher Full Text\n\nLiu HX, Li SM, Luo YM, et al.: Biological control of Ralstonia wilt, Phytophthora blight, Meloidogyne root-knot on bell pepper by the combination of Bacillus subtilis AR12, Bacillus subtilis SM21 and Chryseobacterium sp. R89. Eur J Plant Pathol. 2014; 139(1): 107–116. Publisher Full Text\n\nLarkin RP, Fravel DR: Efficacy of various fungal and bacterial biocontrol organisms for control of Fusarium wilt of tomato. Plant Dis. 1998; 82(9): 1022–1028. 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FEMS Microbiol Lett. 1999; 173(1): 155–162. PubMed Abstract | Publisher Full Text\n\nCarls RA, Hanson RS: Isolation and characterization of tricarboxylic acid cycle mutants of Bacillus subtilis. J Bacteriol. 1971; 106(3): 848–855. PubMed Abstract | Free Full Text\n\nBoukaew S, Chuenchit S, Petcharat V: Evaluation of Streptomyces spp. for biological control of Sclerotium root and stem rot and Ralstonia wilt of chili pepper. BioControl. 2011; 56(3): 365–374. Publisher Full Text\n\nDoughari JH: Antimicrobial activity of Tamarindus indica Linn. Trop J Pharm Res. 2006; 5(2): 597–603. Publisher Full Text\n\nLouis KS, Siegel AC: Cell viability analysis using trypan blue: manual and automated methods. Methods Mol Biol. 2011; 740: 7–12. PubMed Abstract | Publisher Full Text\n\nAlvarez B, Biosca EG, López MM: On the life of Ralstonia solanacearum, a destructive bacterial plant pathogen. Current research, technology and education topics in applied microbiology and microbial biotechnology. 2010; 1: 267–279. Reference Source\n\nWilliamson L, Nakaho K, Hudelson B, et al.: Ralstonia solanacearum race 3, biovar 2 strains isolated from geranium are pathogenic on potato. Plant Dis. 2002; 86(9): 987–991. Publisher Full Text\n\nKumar A, Prameela TP, Suseelabhai R: A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences. J Biosci. 2013; 38(2): 267–278. PubMed Abstract | Publisher Full Text\n\nTapwal A, Singh U, Singh G, et al.: In vitro antagonism of Trichoderma viride against five phytopathogens. Pest Technol. 2011; 5(1): 59–62. Reference Source\n\nMukherjee PK, Horwitz BA, Herrera-Estrella A, et al.: Trichoderma research in the genome era. Annu Rev Phytopathol. 2013; 51: 105–129. PubMed Abstract | Publisher Full Text\n\nRuano-Rosa D, Cazorla FM, Bonilla N, et al.: Biological control of avocado white root rot with combined applications of Trichoderma spp. and rhizobacteria. Eur J Plant Pathol. 2014; 138(4): 751–762. Publisher Full Text\n\nLi H, Li H, Bai Y, et al.: The use of Pseudomonas fluorescens P13 to control sclerotinia stem rot (Sclerotinia sclerotiorum) of oilseed rape. J Microbiol. 2011; 49(6): 884–889. PubMed Abstract | Publisher Full Text\n\nPark KH, Lee CY, Son HJ: Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth-promoting activities. Lett Appl Microbiol. 2009; 49(2): 222–228. PubMed Abstract | Publisher Full Text\n\nAlsohim AS, Taylor TB, Barrett GA, et al.: The biosurfactant viscosin produced by Pseudomonas fluorescens SBW25 aids spreading motility and plant growth promotion. Environ Microbiol. 2014; 16(7): 2267–2281. PubMed Abstract | Publisher Full Text\n\nOngena M, Jourdan E, Adam A, et al.: Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environ Microbiol. 2007; 9(4): 1084–1090. PubMed Abstract | Publisher Full Text\n\nKloepper JW, Ryu CM, Zhang S: Induced Systemic Resistance and Promotion of Plant Growth by Bacillus spp. Phytopathology. 2004; 94(11): 1259–1266. PubMed Abstract | Publisher Full Text\n\nBernal G, Illanes A, Ciampi L: Isolation and partial purification of a metabolite from a mutant strain of Bacillus sp. with antibiotic activity against plant pathogenic agents. Electron J Biotechnol. 2002; 5(1): 7–8. Publisher Full Text\n\nIppolito A, Nigro F: Impact of preharvest application of biological control agents on postharvest diseases of fresh fruits and vegetables. Crop protection. 2000; 19(8–10): 715–723. Publisher Full Text\n\nPandey BR, Yendyo S, GC R: Raw values of zone of inhibition by antagonist against Ralstonia solanacearum. 2017. Data Source\n\nPandey BR, Yendyo S, GC R: Raw values of the 5 % sucrose treatment. 2017. Data Source\n\nPandey BR, Yendyo S, GC R: Images of the plates showing the zone of inhibition by different antagonists against Ralstonia solanacearum. figshare. 2017. Data Source\n\nPandey BR, Yendyo S, GC R: Raw digital images (400X) of microscopic analysis representing the interactions of different antagonist against Ralstonia solanacearum. figshare. 2017. Data Source\n\nPandey BR, Yendyo S, GC R: Field Images of plot design showing pictures of before the treatment and effects after the treatment. figshare. 2017. Data Source"
}
|
[
{
"id": "30061",
"date": "05 Feb 2018",
"name": "Teresa A. Coutinho",
"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, particularly the introduction and discussion is poorly written. Each paragraph is an entity on its own and should contain one topic, with a introductory, body and concluding sentence. Many of the paragraphs contain too many topics.\n\nPlease correct the spelling of Pseudomonas fluorescens throughout the manuscript.\n\nIn the abstract the authors mention that R. solanacearum produces several toxins – toxins are not the major virulence factor in this pathogen – why this sentence was included in the abstract and no where else in the manuscript, is difficult to undertand.\n\nThe use phenotypic methods to identify bacteria is no longer sufficient. None of the biocontrol agents or even the causal agent of the wilt symptoms observed where appropriately identified in this study. Based on the latest publications - two Ralstonia species can cause bacterial wilt - R. solanacearum and R. pseudosolanacearum. So which one were they working with? How where the references strains of the biocontrol agents used? Not clear - again their identity needs to be verified by sequencing at the very least (of 16S rRNA).\n\nIt is unclear how the inoculum concentrations of the bacterial control agents were determined.\n\nIn the experimental plot the authors mention that bacterial wilt was present the previous year. Although I would agree that the pathogen would be present the following year, how did they insure that all areas of the experimental plots were contaminated with the pathogen? Was there 100% loss the previous year?\n\nThe use of sucrose in the preparation of the biocontrol agents is concerning. Although the authors noted that it is a common practice in Nepal, the reasons why they would do this need to be provided.\n\nThe Figures 1-3 are not very useful. I am not exactly sure what the authors are trying to show. Also, in Figure 1 how do your know the bacterial cells are dead?\n\nThere are no error bars on Figure 4.\n\nIn Table 4 what do the authors mean by variance.\n\nThe discussion and conclusions should be reworked into a comprehensive section. Currently the conclusions are longer than the discussion which is not correct.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "3404",
"date": "21 Feb 2018",
"name": "Binayak Raj Pandey",
"role": "Author Response",
"response": "Dear Dr. Teresa A. Coutinho,Thank you for your creative comments. Your comments have helped us to add few points to make the manuscript more clear. I have tried to address most of the issues in version 2 of the manuscript.1. Regarding the issue- The manuscript, particularly the introduction and discussion is poorly written. Each paragraph is an entity on its own and should contain one topic, with an introductory, body and concluding the sentence. Many of the paragraphs contain too many topics.-The introduction and discussion section has been re-written. English language has already been reviewed and corrected by native speakers.2. Regarding the issue- Please correct the spelling of Pseudomonas fluorescens throughout the manuscript.-The spelling of Pseudomonas fluorescens throughout the manuscript has been corrected. We found some authors using previous spelling as cited in Reference 11 of the manuscript.3. Regarding the issue- In the abstract, the authors mention that R. solanacearum produces several toxins – toxins are not the major virulence factor in this pathogen – why this sentence was included in the abstract and no where else in the manuscript, is difficult to understand-In the abstract, the statement that R. solanacearum produces several toxins has been replaced by virulence factors. Thanks for the indication.4. Regarding the issue- The use phenotypic methods to identify bacteria is no longer sufficient. None of the biocontrol agents or even the causal agent of the wilt symptoms observed were appropriately identified in this study. Based on the latest publications - two Ralstonia species can cause bacterial wilt - R. solanacearum and R. pseudosolanacearum. So which one were they working with? How where the references strains of the biocontrol agents used? Not clear - again their identity needs to be verified by sequencing at the very least (of 16S rRNA). -R. solanacearum has been replaced by Ralstonia spp. to address the fact that the bacterial wilt was present but which species of Ralstonia has caused it needs genetic services to identify. I agree that 16S rRNA is a recently evolved tool and Microbiologist are using Phenotypic methods to identify the microbes from decades. Microbiological tool is still a valid method to identify the microbes to genus level and is still being used as a tool in hospitals, pathology labs and research centers to do so where human life is involved and 16S rRNA is used to identify to the species level. Nepal being a low-income country don't have any sequencing service in any of government or private organization till the date. Hence, the issue with species-level identification has been added in the \"Discussion\" section of the manuscript and throughout the manuscript, we have mentioned only the genus level of the microbes except Pseudomonas fluorescens as they both can be distinguished easily by morphological and microscopic examination and fluorescence that they emit when subjected to UV light. As far as Bacillus subtilis is concerned I have mentioned the GeneBank Accession Number for it where I am the first author. -The wilt symptoms are clearly mentioned and cited properly in the \"Isolation and identification of Ralstonia\" subheading of \"Methods section\" in the manuscript.- I have now clearly mentioned that How where the references strains of the biocontrol agents used? in \"Isolation of antagonists\" sub-heading of \"Methods\" section.5. Regarding the issue- It is unclear how the inoculum concentrations of the bacterial control agents were determined. -It has clearly been mentioned in \"Evaluating the effects of biocontrol agents in the field\" subheading in \"Method Section\" that a Hemocytometer was used. I don't think we need to mention the protocol of using Hemocytometer to count the cells as it is a very simple procedure adopted even in the diploma curriculums and re-writing such simple protocols will make the manuscript a mesh and will make the readers diverted from the main contents in the manuscript. 6. Regarding the issue- In the experimental plot the authors mention that bacterial wilt was present the previous year. Although I would agree that the pathogen would be present the following year, how did they insure that all areas of the experimental plots were contaminated with the pathogen? Was there 100% loss the previous year?-We did not mention the word \"previous year\" in any section of the manuscript rather we mentioned \"Previous Harvest\". In Nepal Harvest can be done 2 times in a year due to pleasant climatic conditions. I have addressed the issue by mentioning that the last harvest was done 3 month before the test started and there was heavy loss in the crop due to wilt infection. We have seen the case that even after 2 years there has been re-occurrence of the wilt in the field, its just 3 months, I think we can buy the fact that the pathogens were present in the field also we tested the soil for presence of pathogen and i have added the same in the manuscript. No, the loss was not 100 % as it has clearly been indicated in Table 5 that with the Negative control (w/o any treatment) there was 63 % loss in the plot. I agree that we should have injected bacterial wilt in the field but it went against our ethics to deliberately destroy the crop of the poor farmer. Hence unlike any other research paper where only field data is taken into account to evaluate the efficiency by injecting the pathogen into the field we thought to evaluate the efficacy in the lab and then comparing the validity of the data from the field. We in the research paper have clearly indicated that the evaluation in the field was done in a natural environment giving all the necessary fertilizers as needed without adultering the pathogen population in the soil where the previous infection occurred. The distance between the treated and untreated plots were merely 0.5 meter in length hence it seems impossible that the pathogens cannot effect the nearby plot. If you seen the pictures from data availability section of the manuscript then you can clearly note that the plot where Bacillus subtilis was given as treatment method was effected by the wilt (16 % infection) whereas the infection was 8 % in the plot that was merely 0.5 m from the BS treatment plot. The Negative Control plots (w/o Any Treatment) was also merely 0.5 m from all chemically treated plot but the infection rate was 63 % for Negative Control Plots and 6 % for chemically treated plot. Is that even possible if the pathogen was not present in the soil ? 7. Regarding the issue- The use of sucrose in the preparation of the biocontrol agents is concerning. Although the authors noted that it is a common practice in Nepal, the reasons why they would do this need to be provided. -We have now clearly mentioned and restated in a single paragraph in \"Discussion\" Section. We just wanted to share our experience that if followed for commercial formulations of Biocontrol Agents gives a better result than just directly spreading it in the field. The other farmers & scientists in another country can adopt the same practice and share their outputs of the trials. The practice that has been mentioned in the paper merely concern the output that paper is trying to give.8. Regarding the issue- The Figures 1-3 are not very useful. I am not exactly sure what the authors are trying to show. Also, in Figure 1 how do your know the bacterial cells are dead?- We have now clarified in detail in \"Result\" section that how the bacterial cells were assumed to be dead and what we are trying to show.9. Regarding the issue- There are no error bars in Figure 4.-We have changed the style of the graph and also added the error bars. The detail data can be found in the \"Data Availability\" Section.10. Regarding the issue- In Table 4 what do the authors mean by variance.- We have removed the variance column, we thought that the variance data could be useful to some statisticians. The detail data can be found in the \"Data Availability\" Section.11. Regarding the issue- The discussion and conclusions should be reworked into a comprehensive section. Currently, the conclusions are longer than the discussion which is not correct.-We have reworked both the conclusion and discussion section and made discussion longer than the conclusion.There are a lot of manuscripts being published on microbes without 16S rRNA sequencing results and we should consider the fact that in some cases 16S rRNA sequencing alone is not sufficient to identify the species. We will try to identify the species in future manuscripts and if the species are novel then we will be happy to publish the results of that too.Hope I have addressed the major issues in this manuscripts and I assure you that the research from the beginning has been extensively monitored by the professionals from USAID and the manuscript has been reviewed by agricultural scientists from Nepal and USA as mentioned in the manuscript itself at the beginning. I think you can easily understand that it's a challenging work when the biocontrol agents come and the interactions are complex with the pathogens as you have already worked with Ralstonia and we have also cited your work in Reference 25 of our manuscript. Hence this manuscript is just a preliminary work to give light on how the interaction can be and such research in Nepal can promote the use of Biocontrol Agents in Nepal and we are continuously thriving to establish the use of these beneficial microbes by working with USAID, Winrock International and FAO in Nepal. The results that we are observing from the fields are astonishing and hope we will soon publish more articles on biocontrol agents where genetic works are done. With Regards,Binayak Raj Pandey"
}
]
},
{
"id": "28139",
"date": "09 Feb 2018",
"name": "Dhurva Prasad Gauchan",
"expertise": [
"Reviewer Expertise Plant and agriculture biotechnology",
"genetic diversity and plant tissue culture",
"organic farming"
],
"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 contributes data to the biological control of Bacterial wilt of tomato using microbial consortium which can be turned into commercial product in Nepal. The work is well planned, the methods seem adequate, the results are in a non-negligible amount and discussed on the basis of a convenient literature set. But, the English used in the paper needs to be corrected. If author/s are not expert, they can take help from English expert to rewrite the Sentences. I believe that the manuscript is worthy of being indexed and that it could be accepted after a minor revision, for which I give now some comments, with the aim of helping the authors in obtaining a definitely acceptable version.\n\nTitle:\n\nChange the title Evaluation of Trichoderma spp. and Pseudomonas fluorescence for biological control of Bacterial wilt of tomato.\n\nAs Bacillus results are not satisfactory the author supposed to concentrate on only one bacterial and fungal species.\n\nAbstract:\nBackground: Ralstonia solanacearum is the major bacterial disease in tomato, which invades the roots of diverse plant hosts and colonizes xylem vessels causing wilt, especially in tropical, subtropical and warm-temperate regions.\n\nRewrite the sentence: Ralstonia solanacearum is the major bacterial disease (Ralstonia solanacearum is the not the disease, it is causal organism)\n\nResults: Incorporate the findings of Bacillus subtilis (Remove if findings are not up to the mark)\n\nConclusion: Rewrite the conclusion as it gives confusing information.\n\nIntroduction:\nThis study focuses on evaluating the efficacy of different native isolates of Trichoderma species, B. subtilis and P. fluorescence against bacterial wilt disease caused by the pathogen R. solanacearum in the tomato plant. The study hypothesizes that the native isolates of Trichoderma spp., B. subtilis and P. fluorescence can be used as bioantagonistic agents to control bacterial wilt (R. solanacearum) of tomato. This study tries to reflect the bioantagonistic effects by the microscopic examinations, agar well diffusion technique and by applying the concentrate of biocontrol agents in tomato plots, to calculate their efficiency by comparing them with chemical methods of treatment.\n\nRewrite and give stress to firm objectives, sentences are poorly written need more accurate writing.\n\nMethods: Write the Latitude: 27.582016 and Longitude: 84.272259 in scientific manner. Trial design is not described (Field Experimentation: Experimental Design). Weather data is not needed. Table: 1 Write full scientific name of plants with authority. Follow strictly ICBN system wherever plant name is used.\n\nBriefly, the stems of the infected plant were washed three times with autoclaved distilled water and then blot dried. Omit Briefly\n\nTable: 1 Scientific names of source are not uniform. Write complete scientific name of plants with authority. What is the scientific reason to collect samples all from tropical region except Bacillus which is collected from mid hill regions of Nepal. Justify.\n\nIn this section: Effect of sucrose on the population of biocontrol agents: quote supportive references for this methods. Choose appropriate statistical tools to evaluate your generated data. Used statistical tools are insufficient.\n\nResults:\nOnly 4 months weather data is not justifiable until it is compared for two consecutive years. Better skip this data which does not show relevance with your work.\n\nBacillus subtilis BS isolated from Bhaktapur top soil did not show satisfactory inhibition activity. When does Bacillus give not satisfactory results then better omit Bacillus content from the paper.\n\nTable 4. Inhibition zone made by the isolates used as biocontrol agents against Ralstonia solana cearum using agar well diffusion technique.\n\nIt is very nice if some supportive pictures are added in this section.\n\nDiscussion:\nThe present results, using microscopy, showed that different species of Trichoderma , Pseudomonas fluorescence and Bacillus su btilis clearly hinders the growth of Ralstonia solanacearum , which causes bacterial wilt in tomato plants. AND Results: Bacillus subtilis BS isolated from Bhaktapur top soil did not show satisfactory inhibition activity.\n\nBoth statements are contradictory. Justify Make a common consensus and write obtained results clearly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "3435",
"date": "21 Feb 2018",
"name": "Binayak Raj Pandey",
"role": "Author Response",
"response": "Dear Dr. Dhurba Gauchan Sir, The comments were extremely helpful to realize few shortcomings of the manuscript. We have briefly detailed and corrected few abnormalities in Abstract, Introduction, Methods, Results and Conclusion sections of the manuscript. Your suggestions has definitely taken the manuscript to next level. Bacillus subtilis did not show satisfactory results and we have added few points to make it clear that the results were not satisfactory. We have added the clarification of why the Bacillus subtilis was taken from Bhaktapur. The other research articles and our observations (not mentioned in this research) suggest that Bacillus subtilis can induce systemic resistance in a plant with long-term use. Also, the scientific names of the plants were added with authority in version 2. From next research onwards we will try to incorporate the ideas and suggestions from this manuscript and make also record the weather data for two consecutive years. It's a challenge to work with Biocontrol agents in Nepal as very few genetic tools are available with no option for sequencing and we have to depend on the 3rd world for such services which possess the risk of native potent strains being claimed by others. Hope this lack will end soon in Nepal. Only few scientist are working in the field of Biocontrol agents and organic farming in Nepal and we are honored that you who fall among those scientist have reviewed our article. Regards, Binayak Raj Pandey"
}
]
}
] | 1
|
https://f1000research.com/articles/6-2028
|
https://f1000research.com/articles/7-348/v1
|
21 Mar 18
|
{
"type": "Opinion Article",
"title": "Unintended consequences of the potential phase-out of gamma irradiation",
"authors": [
"Jacquelyn W Chou",
"Michelle Skornicki",
"Joshua T Cohen",
"Michelle Skornicki",
"Joshua T Cohen"
],
"abstract": "The radioisotope cobalt-60 (Co-60) is important for commercial, medical, and agricultural applications. Its widespread use has meant that Co-60 can be found in less secured facilities, leading to the fear that unauthorized persons could obtain and use it to produce a “dirty bomb”. This potential security concern has led to government calls for phasing-out Co-60 and other radiation sources, despite ongoing safety and security regulations for handling, transport and use of radioactive sealed sources.\nThis paper explores potential implications of phasing out radioisotopic technologies, including unintended safety and cost consequences for healthcare and food in the US and globally.\nThe use of Co-60 for healthcare and agricultural applications is well-documented. Co-60 is used to sterilize single-use medical devices, tissue allografts, and a range of consumer products. Co-60 is used in Gamma Knife treatment of brain tumors in over 70,000 patients annually. Co-60 is also used to preserve food and kill insects and pathogens that cause food-borne illness.\nCo-60 is effective, reliable, and predictable. Limitations of alternative sterilization technologies include complex equipment, toxicities, incompatibilities with plastic, and physical hazards. Alternative ionizing radiation sources for wide-reaching applications, including e-beam and x-ray radiation, have advantages and drawbacks related to commercial scale capacity, penetrability, complexity and reliability.\nIdentifying acceptable alternatives would require time, costs and lengthy regulatory review. FDA testing requirements and other hurdles would delay replacement of existing technologies and slow medical innovation, even delaying access to life-saving therapies. A phase-out would raise manufacturing costs, and reduce supply-chain efficiencies, potentially increasing consumer prices, and reducing supply.\nThese consequences are poorly understood and merit additional research. Given Co-60’s importance across medical and non-medical fields, restrictions on Co-60 warrant careful consideration and evaluation before adoption.",
"keywords": [
"Sterilization",
"Co-60",
"gamma irradiation"
],
"content": "Introduction\n\nNaturally occurring cobalt is a stable element. One of its synthetic isotopes, Cobalt-60 (Co-60), has an extra neutron in its nucleus that makes it unstable. As it breaks down, Co-60 emits high energy, “ionizing” radiation that can break molecular bonds. Co-60 plays an important role in a wide variety of commercial, medical, agricultural and research applications because it is a “radioisotope” and hence generates steady, predictable ionizing radiation.\n\nOne of the most useful applications of radioisotopes in general and Co-60 in particular is sterilization. The list of single-use medical devices sterilized using Co-60 is lengthy, including surgical instruments, gloves, gowns, dressings, masks, catheters, laparoscopic equipment, implants, probes, and other objects that enter sterile tissue or the vascular system. Another major healthcare use of Co-60 is the sterilization of tissue allografts, including bone, skin, amniotic membrane and soft tissues used to treat severe burns, non-healing ulcers, and to facilitate organ transplants1,2. (https://www.aatb.org/?q=about-us) Co-60 is also used to sterilize consumer products such as bottle teats for premature babies, medical bandages and a variety of personal health and hygiene products, and raw materials for cosmetics3,4. (https://www.nei.org/Knowledge-Center/Other-Nuclear-Energy-Applications/Consumer-Products)\n\nA second critical application of Co-60 is cancer treatment. Gamma Knife technology, developed in 1968, uses gamma radiation to target small brain tumors. By precisely targeting high doses of ionizing radiation generated by Co-60, Gamma Knife therapy can treat small brain tumors while mitigating damage to surrounding normal, healthy tissue. Gamma Knife therapy is used in over 70,000 patients annually5,6. (http://gammaknife.com/what-is-gamma-knife/)\n\nCo-60 has also been used since the 1920s to preserve food. (https://www.fda.gov/food/ingredientspackaginglabeling/irradiatedfoodpackaging/ucm081050.htm.) By killing microorganisms, insects, and pathogens that can cause food-borne illness, such as salmonella and escherichia coli (E. coli), ionizing radiation extends food shelf life and improves food safety7,8. (https://www.fda.gov/food/ingredientspackaginglabeling/irradiatedfoodpackaging/ucm081050.htm)\n\nBecause they produce radiation, radioisotopes have raised security concerns. The fear is that loss of radioisotopes – due to accident, oversight, or sabotage – could result in their acquisition by unauthorized persons or terrorists who could then produce a radiological dispersal device (“dirty bomb”)9. Motivated by these concerns, some members of Congress proposed legislation in the 2015 Appropriations Bill that would have phased out the use of radioisotopes in the United States, including Co-60. (http://www.nrc.gov/reading-rm/doc-collections/cfr/part020/) Though that legislation did not pass, the suggestion that use of Co-60 and other radioisotopes should be phased-out has persisted. In 2015, the Committee on Homeland and National Security created the Interagency Working Group on Alternatives to High-Activity Radioactive Sources (GARS) to develop best practices to transition to non-radioisotopic technologies.\n\nThis paper explores the merits and potential downside of shifting away from radioisotopic technologies. We describe unintended safety and cost consequences of a Co-60 phase-out for healthcare and food applications.\n\nThe widespread commercial use of Co-60 means it is often housed in less well-guarded facilities, such as hospitals, that are unlike heavily guarded nuclear facilities9. Experience in other countries has demonstrated the potential for mishaps. For example, in 2013, thieves in Mexico stole a truck transporting Co-60. The thieves ultimately abandoned the Co-60, as the material itself did not appear to be the target, and the authorities recovered it10. Also in 2013, online fashion retailer Asos recalled a batch of metal-studded belts contaminated with Co-60 likely introduced in scrap metal from India or another Asian country11. In both of these cases, it was incidental exposure to Co-60 that posed the greatest public health risk, rather than the potential for a terrorist act. To date, these types of Co-60 mishaps have not been reported in the United States, perhaps because of this country’s stricter regulations. There have been no deaths from exposure to radiation or any history of contaminated groundwater at irradiators in the US. The US Nuclear Regulatory Commission (NRC) has reviewed cases of radiation incidents and has developed strict requirements designed to reduce future risk of incidents (10 CFR (Code of Federal Regulations) Part 36, implemented in 1993.) (https://www.nrc.gov/reading-rm/doc-collections/fact-sheets/commercial-irradiators.html)\n\nOngoing regulatory changes and additional safety and security requirements have been implemented over the years to further increase and assure maintenance of safe and secure handling, transport and use of high activity radioactive sealed sources. The US NRC promulgated 10 CFR Part 37 in March 2013 with adoption required by US NRC licensees by March 2014 and Agreement State licensees by March 2016. (https://www.nrc.gov/reading-rm/doc-collections/cfr/part037/) Part 37, “Physical Protection of Category 1 and Category 2 Quantities of Radioactive Material”, provides specific detail of control in the following areas:\n\ni) Background investigation and access control program of people and facilities (criminal history checks and related other elements of personnel involved in handling, transport, and use of this material; access authorization to areas where such material is housed; personnel access authorization and control where material is used; and protection of information)\n\nii) Physical protection requirements during use (comprehensive security program for facilities and people; security zones; monitoring/detection/assessment; maintenance, testing; security program review; reporting of events)\n\niii) Physical protection in transit, including preplanning and coordination of transit; physical protection during transit; and personnel/vehicle controls\n\niv) Record keeping, and\n\nv) Enforcement\n\nCo-60 has three main advantages. First, the gamma radiation it produces is versatile. Gamma radiation can deeply penetrate a wide range of low- and high-density materials1,12,13. Deep penetration is important for sterilizing medical supplies and to reach pathogens deep within the food matrix14. Because gamma radiation does not require high temperature, it can be used on temperature-sensitive items, products can be irradiated in bulk, and sterilization can take place after final packaging15,16.\n\nSecond, Co-60 is reliable. It has a simple and predictable decay pattern and a relatively long half-life (5.27 years). Treatment with Co-60 is precise and reproducible;15,17–19 (http://www.world-nuclear.org/information-library/non-power-nuclear-applications/radioisotopes-research/radioisotopes-in-medicine.aspx, http://www.world-nuclear.org/information-library/non-power-nuclear-applications/overview/the-many-uses-of-nuclear-technology.aspx) therefore, instrument calibration standards rely on Co-60 as a “gold standard” yardstick20. Co-60 gamma irradiators are simple to use and control1,3,12, and because the Co-60 itself generates the radiation, it is energy-efficient15. These properties minimize operational maintenance requirements15.\n\nFinally, Co-60 has favorable physical characteristics that make it ill-suited to manipulation that could pose security risks. It cannot start a fission chain reaction, it is non-flammable, and it cannot poison a water supply because it is insoluble. Moreover, because Co-60 is not readily dispersible, it does not emit neutrons or leave residues, or cause other surrounding materials to become radioactive15,16.\n\nAlternative sterilization technologies include chemical treatment, non-ionizing radiation, and other ionizing radiation sources (Table 1). Chemical treatments pose a series of challenges.1,21. Ethylene oxide (EO) requires complex equipment, is toxic, and flammable, and poses an explosive hazard. EO can be inconsistent because its use depends on multiple variables—including temperature, time, pressure, vacuum, and concentration—to address differences in the target material’s physical characteristics (e.g., density and porosity), packaging, and humidity3,12. Peracetic acid-ethanol, although rapid22 and compatible with a wide variety of materials, significantly reduces biomechanical strength, decreases remodeling activity in ligament grafts1 and does not reduce infection risk22. Heat treatment damages many materials. For example, steam autoclaving damages plastics which comprise many single-use items. Although evidence is limited and it can only treat heat-resistant materials22, microwave treatment has been shown to effectively sterilize some bone allografts1.\n\nAlternative ionizing radiation sources have both advantages and drawbacks. Like gamma radiation, e-beam can sterilize health care products on a commercial scale1 and is currently used in many large facilities. E-beam delivers radiation rapidly and can be scaled3. However, because the radiation is machine-generated, rather than a material by-product (as is the case with Co-60-generated gamma radiation), the equipment is complex and costly to install and operate. Nor is e-beam radiation as predictable or uniform as Co-60 gamma radiation1. Finally, e-beam radiation does not penetrate materials as well as gamma radiation. X-ray radiation achieves penetration comparable to that achieved by gamma rays3. However, similar to equipment used to generate e-beam, x-ray generating equipment is complex, expensive and less reliable than Co-60, with very few such sterilization units operating globally3,13,17,23. (http://www.world-nuclear.org/information-library/non-power-nuclear-applications/overview/the-many-uses-of-nuclear-technology.aspx, http://www.iaea.org/inis/collection/NCLCollectionStore/_Public/29/057/29057259.pdf)\n\n\nImpacts – Medical – United States\n\nBecause ionizing radiation has desirable properties and radioisotopes reliably generate this type of radiation, limiting the use of radioisotopes could have implications for medical care, including single-use medical supplies, allografts, and therapeutic technologies. These impacts extend beyond the US population since the US supplies a significant amount of sterile medical devices globally.\n\nCurrent estimates indicate that the sterilization industry is divided between EO (50%), gamma (40.5%), e-beam (4.5%) and other (including x-ray) (5%), and there are 200 healthcare facilities worldwide with commercial gamma sterilization capability23. Co-60 is the most widely used form of radiation for sterilizing single-use medical products16, as heat sterilization is often damaging, and alternative methods might not achieve sufficient penetration17,23. (http://www.world-nuclear.org/information-library/non-power-nuclear-applications/overview/the-many-uses-of-nuclear-technology.aspx) Single-use medical products were used in over 52 million surgical procedures in the US in 2011, with 45% of single-use products sterilized using Co-6023,24.\n\nCurtailing radioisotope sterilization would make availability of some single-use products uncertain, hence potentially jeopardizing millions of surgical procedures yearly. At the very least, products for which the effectiveness of alternative sterilization technologies has not been established would have to undergo significant and both time and cost intensive testing25,26. In the case of products for which adequate sterilization proved infeasible, single-use medical products could become unavailable going forward. Predicting the impact of this disruption on health is difficult. We do not know how many technologies would be affected, for how long, or the adequacy of substitute or redesigned products. However, with the possibility that millions of surgeries could be affected, it is clear that curtailing use of radioisotope materials for single-use medical supply sterilization could be highly disruptive.\n\nMore than 2 million allografts each year support more than 1 million annual tissue transplants2. Used in reconstructive surgery for musculoskeletal injuries, allografts avoid the major complications associated with use of autogenic materials1. In addition, allograft skin and amniotic membrane have unique properties that make them irreplaceable and indispensable in the treatment of serious burn injuries1. The risk of transmitting infectious disease from donor to recipient necessitates sterilization.\n\nSterilization alternatives for tissue allografts (ethylene oxide, peracetic acid-ethanol, thermo-disinfection), microwave, electron beam) lack the same demonstrated effectiveness as gamma radiation1,21. Studies have identified insufficient penetration as a key limitation of alternative sterilization technologies1. Although some evidence indicates that microwave sterilization is effective for bone allografts28, evidence supporting microwave sterilization in general, and its use for other tissues is limited compared to the evidence for gamma radiation.\n\nEven if more extensive evidence were available, a phase-out of radioisotope sterilization could trigger FDA regulatory testing requirements21 and delay replacement of existing sterilization technologies, potentially affecting the supply of tissue allografts.\n\nGamma Knife is a stereotactic radiosurgery technique that relies on Co-606. Approximately 70,000 Gamma Knife surgeries take place worldwide each year, with nearly 1 million surgeries having been conducted from 1991–2013. (https://gammaknife.com/downloads/Facts%20in%20short_1028438.01.pdf) While alternatives exist, the Gamma Knife technique, described in Table 2, is the most established, well-researched and validated form of radiosurgery. (http://nyulangone.org/locations/center-for-advanced-radiosurgery/gamma-knife-radiosurgery)\n\nGamma Knife is specifically indicated for brain surgeries. Radiosurgery using Co-60 is non-invasive and accurate to 0.15mm. Because it can target small areas, Gamma Knife can be used more extensively than competing technologies that deliver larger tissue doses of radiation because they cannot be as finely focused29,30. The more precise targeting achieved by Gamma Knife also causes less damage to healthy tissue, speeding recovery and minimizing side effects31. In a prospective cohort study in which physicians assigned and treated patients with either gamma knife radiosurgery or whole brain radiotherapy (follow-up of 1200 days, or 3.3 years), the mortality rate was lower for Gamma Knife patients (74.4% vs. 97.1%), and the median survival time greater (9.5 months for Gamma Knife versus 8.3 months for whole brain radiotherapy patients)32. With approximately 70,000 gamma knife surgeries each year6 and 1.2 added months (9.5-8.3) gained per surgery, eliminating Gamma Knife could potentially cost 7,000 life-years annually. Curtailing Co-60, which the Gamma Knife depends on, would eliminate the only demonstrated treatment for certain tumor types.\n\nA ban on radioisotope sterilization technologies could also slow medical innovation. Modifications to sterilization modalities could involve costly redesign and require additional validation for the sterilization process, the sterilization product itself, and its packaging23. First, altering the sterilization method constitutes a major change to new drug applications (NDA) and abbreviated new drug applications (ANDA), requiring FDA approval prior to distribution of drug products26. The added effort could divert resources away from development of new technologies. Alternative sterilization methods may require new 510(k) or premarket applications (PMA). For 510(k) applications, the FDA determines whether the device is at least as safe and effective as a legally marketed device (“substantial equivalence”)5,8. Although the process is supposed to take no more than 90 days, one sterilization company noted that the full FDA clearance process lasts 9 months. (http://www.revoxsterilization.com/sites/default/files/Revox_OsteoArticle.pdf) The FDA believes that novel sterilization technologies “carry a substantial risk of inadequate sterility assurance if not conducted properly”. (https://www.fda.gov/downloads/MedicalDevices/.../ucm109897.pdf) Therefore, recent guidance indicates that in the context of a novel sterilization process, FDA intends to inspect manufacturing facilities before clearing a 510(k). (https://www.fda.gov/downloads/MedicalDevices/.../ucm109897.pdf) PMA applications or supplements require up to 180 days of review time, depending on the product’s regulatory classification, design, and other required changes5.\n\nSecond, because these reviews will divert FDA’s resources, FDA’s approval of other new technologies will likely slow. The FDA already has a backlog of products awaiting approval. Further slowing the overburdened approval system could delay patient access to life-saving therapies33. (https://www.cato.org/publications/commentary/fda-can-be-dangerous-health)\n\nThird, a Co-60 phase-out will raise manufacturing costs. It costs on average $31 million to bring a low-to-moderate 510(k) product from concept to market, with approximately three-quarters of the cost related to FDA-dependent or related activities. Costs for PMAs are $94 million, with $75 million related to the FDA process34. By raising costs, a Co-60 phase-out could disincentivize future innovation.\n\nFinally, Co-60 sterilization facilities are often located near medical device manufacturers or distribution hubs. Phasing out of these facilities would reduce the attendant supply chain efficiencies, potentially increasing consumer prices, delaying product availability, and reducing supply.\n\n\nImpacts – Non-Medical – United States\n\nBecause of the reliability and predictability of Co-60 for sterilization, its use extends beyond medical products. A phase-out has implications for both the food supply and the multitude of consumer goods that use Co-60 for sterilization.\n\nThe US food supply relies heavily on gamma radiation. Co-60 is used for food preservation, shelf-life extension, and reduction of food-borne illness for domestic and international food products, including microbial disinfection of spices. (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm279485.htm) Most spices sold in the US are grown overseas in developing countries, where pollution and water issues can contaminate food shipped to this country. (http://www.washingtonpost.com/wp-dyn/content/article/2010/03/13/AR2010031301111.html) Irradiation is also used as a quarantine treatment for fresh horticultural commodities, and as a substitute for fumigants in Asian countries and the US7. (https://uw-food-irradiation.engr.wisc.edu/Process.html)\n\nPhasing-out Co-60 will likely affect US and global food supply chains as alternatives are established. Alternatives to Co-60 have characteristics that limit their ability to treat all food products requiring irradiation. Because gamma irradiation can completely penetrate a product, it can deactivate both surface pathogens and those found within the food matrix (see Table 3)14. With almost a quarter of the world’s food irradiation units located in the US35, switching to alternatives to Co-60 irradiation may cause some disruption to the global food supply system. There is potential for impacts to food prices, supply, and access, depending on how quickly the global food irradiation system would be able to respond to a US phase-out.\n\nLike food products, consumer goods prices will likely increase in response to a Co-60 phase-out, as decommissioning current Co-60 facilities will be costly. In February 1999, a decommissioning project commenced for a gamma irradiation facility at the Brookhaven National Laboratory (BNL), located on Long Island, New York36. The decommissioning process was involved, taking over a year to conduct three main phases: 1) preparation of the facility; 2) packaging and shipment of irradiation sources for disposal; 3) disposal/discharge of pool water (used for cooling) and dismantling. Ultimately, all 24,000 curies of cobalt-60 were removed.\n\nBuilding replacement facilities is also resource-intensive, with food irradiation facilities estimated to cost between $3-5 million, (https://uw-food-irradiation.engr.wisc.edu/Process.html) and the equipment alone required for an electron accelerator for medical device sterilization estimated to cost between $1 and $2 million37. There is little publicly available information on the full spectrum of cost for Co-60 facilities, including facilities large enough for medical sterilization, but conservatively, they likely cost several million dollars. Consumer products, medical products, and food products that involve Co-60 for sterilization would all be affected by the cost of decommissioning and construction of new facilities for replacement sterilization.\n\n\nWorldwide impacts\n\nRestrictions on Co-60 in the US may shift its use to other countries where weaker regulations may have additional economic and security consequences38. At a recent IAEA conference on Safety of Radiation Sources and the Security of Radioactive Materials, held in 2000, countries submitted reports describing their use of radiation38. Though the regulatory and enforcement status of these countries has likely improved since 2000, the conference proceedings identified several countries with serious regulatory limitations. Angola had begun to use radiation sources, including Co-60, but lacked appropriate infrastructure to control their sources, relying on technical assistance from IAEA and other Member States. Bangladesh was facing financial and administrative hurdles to train and motivate personnel, and to create necessary infrastructure and facilities to achieve safety standards compatible with IAEA International safety standards. To the extent that a national-security motivated phase-out of Co-60 applications in the US shifts Co-60 use to other countries, such changes could aggravate security risks.\n\nA Co-60 phase-out in the US may also increase the cost (and reduce quality) of medical devices and therapies elsewhere. Fifty-percent of the world’s sterile single use medical devices come from the US. (http://documentslide.com/documents/1-a-profile-of-the-radiation-source-sector-committee-on-radiation-source-use.html) Depending on the implementation details of a phase-out and transition, there could be an initial decrease in supply of single-use medical devices from the US, as well as a price increase. This outcome could affect the safety and efficacy of healthcare in other countries, particularly those with more limited resources.\n\n\nDiscussion\n\nThe effectiveness, reliability, and predictability of Co-60 have made it a primary source of gamma irradiation for a wide variety of medical and non-medical applications in the US. Its widespread use, though a strength, has also meant that Co-60 can be found in less secured facilities. This potential security concern has led to calls to phase out Co-60 and other radiation sources.\n\nThese concerns should be considered in the context of trade-offs that Co-60 restrictions in the US would impose. These trade-offs include unintended consequences for both medical care and consumer access to products in the US and worldwide. The use of Co-60 for Gamma Knife surgery, sterilization of tissue allografts, and sterilization of single-use medical devices is highly effective and well-documented. Although there are alternative sterilization technologies, they all have limitations. Similarly, Co-60 is used across a range of food products, helping to maintain the quality and safety of food supplies in the US and abroad. Even if acceptable alternative technologies are identified, identifying those alternatives would take time and necessitate costly and lengthy regulatory review.\n\nJust in the US, a phase-out of Co-60 would impose direct monetary costs, time costs, and limitations to access. However, a US phase-out could also shift gamma irradiation processing offshore, particularly for food processing. The establishment of additional Co-60 facilities in countries that may lack rigorous safety and security regulations on par with the US could exacerbate security concerns.\n\nThese consequences are not well understood and merit additional research. First, a systematic risk assessment and a cost-benefit analysis of a Co-60 phase-out should be undertaken. The potential trade-offs described in this paper should be quantified and weighed against risk and potential cost of security failure scenarios. An evaluation of efficacy, implementation timeline, and cost for Co-60 alternatives should be included.\n\nSecond, a comprehensive assessment of the risk of all radioactive isotopes should be undertaken, along with an evaluation of additional regulatory steps that could shore up security without a complete ban on use of these radiation sources. US regulations for transport, storage, and security already provide a measure of safety. However, given the recent calls for a complete phase-out, it appears more can be done to further improve security.\n\nLastly, how a phase-out of Co-60 in the US might influence the shift of Co-60 facilities to locations abroad, and how the spread of Co-60 use might influence the threat posed should be carefully evaluated. A follow-up meeting to the year 2000 Safety of Radiation Sources and the Security of Radioactive Materials conference could help to assess the regulatory progress made in each country.\n\nNational security concerns are always important, but they can be difficult to assess, particularly when it comes to preventing an event that has yet to occur. However, it is possible to assess the current use of Co-60 in the US and the impact of a potential phase-out. Given its importance across medical and non-medical fields, restrictions on Co-60 merit careful consideration and evaluation before their adoption.",
"appendix": "Author contributions\n\n\n\nStudy concept and design were contributed by Chou and Cohen. Data was collected primarily by Skornicki and Chou, with assistance from Cohen, and interpreted by all authors. The manuscript was drafted by Skornicki and Chou, and revised by all authors.\n\n\nCompeting interests\n\n\n\nSkornicki and Chou are employed by Precision Health Economics. Cohen serves as a consultant to Precision Health Economics. The authors have no competing interests to disclose.\n\n\nGrant information\n\nSterigenics provided financial support for this research to Precision Health Economics.\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\nResearch assistance and support during manuscript outline preparation were provided by Caroline Huber, Kabirraaj Toor, and Lara Yoon. The authors thank Paul Gray of Nordion Inc. (Retired), Nancy Glick of MSL Group, and Kathy Hoffman of Sterigenics, for their helpful feedback.\n\n\nReferences\n\nSingh R, Singh D, Singh A: Radiation sterilization of tissue allografts: A review. World J Radiol. 2016; 8(4): 355–69. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmerican Association of Tissue Banks: About Us. 2016; [cited 2016 October 28]. Reference Source\n\nTrends in Radiation Sterilization of Health Care Products. International Atomic Energy Agency. 2008. Reference Source\n\nNuclear Energy Institute: Consumer Products. Reference Source\n\nUS Food and Drug Administration: Premarket Notification PMA. 2017.\n\nElekta: What is Gama Knife? 2016; [cited 2016 October 27]. Reference Source\n\nMorehouse KM, Komolprasert V: Irradiation of Food and Packaging: An Overview. 2004. Reference Source\n\nUS Food and Drug Administration: Premarket Notification 510(k). 2017. Reference Source\n\nCapala J, Goetsch SJ, Orton CG: Point/Counterpoint. Medical use of all high activity sources should be eliminated for security concerns. Med Phys. 2015; 42(12): 6773–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartinez G, Partlow J: Stolen cobalt-60 found in Mexico; thieves may be doomed. The Washington Post. 2013. Reference Source\n\nNeville S: Asos pulls belts in radioactive scare. In The Guardian. 2013.\n\nSilindir M, Ozer Y: Sterilization Methods and the Comparison of E-Beam Sterilization with Gamma Radiation Sterilization. FABAD J Pharm Sci. 2009; 34: 43–53. Reference Source\n\nInteragency Working Group on Alternatives to High Activity Radioactive Sources (GARS): Transitioning from high-activity radioactive sources to non-radioisotopic (alternative) technologies. A best practices guide for federal agencies. Washington, D.C, 2016; 20502. Reference Source\n\nDiCaprio E, Phantkankum N, Culbertson D, et al.: Inactivation of human norovirus and Tulane virus in simple media and fresh whole strawberries by ionizing radiation. Int J Food Microbiol. 2016; 232: 43–51. PubMed Abstract | Publisher Full Text\n\nWorld Institute for Nuclear Security: Considerations for the Adoption of Alternative Technologies to Replace Radioactive Sources. 2016. Reference Source\n\nIndustry Experts: Sterilization Technologies: A Global Market Overview. 2016. Reference Source\n\nWorld Nuclear Association: The Many Uses of Nuclear Technology. 2014; [cited 2016 October 28]. Reference Source\n\nKomolprasert V: Packaging for Foods Treated by Ionizing Radiation. 2007. Publisher Full Text\n\nWorld Nuclear Association:Radioisotopes in Medicine. 2016. Reference Source\n\nInteragency Working Group on Alternatives to High-Activity Radioactive Sources, Subcommittee on Nuclear Defense Research and Development, Committee on Homeland and National Security of the National Science and Technology Council: Transitioning from High-Activity Radioactive Sources to Non-Radioisotopic (Alternative) Technologies: A Best Practices Guide for Federal Agencies. 2016. Reference Source\n\nSchmidt T, Grabau D, Grotewohl JH, et al.: Does sterilization with fractionated electron beam irradiation prevent ACL tendon allograft from tissue damage? Knee Surg Sports Traumatol Arthrosc. 2017; 25(2): 584–594. PubMed Abstract | Publisher Full Text\n\nRutala WA, Weber DJ, Health care Infection Control Practices Advisory Committee (HICPAC): Guideline for Disinfection and Sterilization in Healthcare Facilities. Centers for Disease Control and Prevention. 2008. Reference Source\n\nGamma Industry Processing Alliance (GIPA): A Comparison of Gamma, E-beam, X-ray and Ethylene Oxide Technologies for the Sterilization of Medical Devices and Other Products. 2017. Reference Source\n\nWorld Overview, Surgical Procedures (000s), in Pharmaceutical Fact Book. 2012.\n\nShekelle PG, Wachter RM, Pronovost PJ, et al.: Making health care safer II: an updated critical analysis of the evidence for patient safety practices. Evid Rep Technol Assess (Full Rep). Rockville MD. 2013; (211): 1–945. PubMed Abstract | Free Full Text\n\nUS Food and Drug Administration: Guidance for Industry: Changes to an Approved NDA or ANDA. US Department of Health and Human Services: Washington, DC. 2004. Reference Source\n\nFölsch C, Kellotat A, Rickert M, et al.: Effect of thermodisinfection on mechanic parameters of cancellous bone. Cell Tissue Bank. 2016; 17(3): 427–37. PubMed Abstract | Publisher Full Text\n\nSingh R, Singh D: Sterilization of bone allografts by microwave and gamma radiation. Int J Radiat Biol. 2012; 88(9): 661–666. PubMed Abstract | Publisher Full Text\n\nKubicek GJ, Turtz A, Xue J, et al.: Stereotactic Radiosurgery for Poor Performance Status Patients. Int J Radiat Oncol Biol Phys. 2016; 95(3): 956–959. PubMed Abstract | Publisher Full Text\n\nWatanabe Y, Lee R, Dusenbery KE, et al.: Cumulative Dose to Brain After Multisession Gamma Knife Stereotactic Radiosurgery for Treatment of Multiple Metastatic Tumors. Int J Radiat Oncol Biol Phys. 2016; 96(2): E83. Publisher Full Text\n\nPhan J, Yang JN, Ghia AJ, et al.: Dosimetric Comparison of Gamma Knife Extend to Linear Accelerator Base Volumetric Modulated Arc Therapy Plans for Fractionated Stereotactic Radiosurgery. Int J Radiat Oncol Biol Phys. 2015; 93(3): E615–E616. Publisher Full Text\n\nLee WY, Cho DY, Lee HC, et al.: Outcomes and cost-effectiveness of gamma knife radiosurgery and whole brain radiotherapy for multiple metastatic brain tumors. J Clin Neurosci. 2009; 16(5): 630–4. PubMed Abstract | Publisher Full Text\n\nSilverman E: FDA still struggling with backlog of generic drug applications. March 2, 2016, Dec 2, 2016. Reference Source\n\nMassdevice: Medical device makers spend millions to meet FDA rules, study finds. Medcitizens. 2010. Reference Source\n\nInternational Atomic Energy Agency: Directory of Gamma Processing Facilities in Member States. Vienna, Austria, 2004. Reference Source\n\nBowerman BS, Sullivan PT: Decommissioning the Brookhaven National Laboratory Building 830 Gamma Irradiation Facility. 2001. Publisher Full Text\n\nPharmaceutical & Medical Packaging News: When in-house sterilization makes sense. 1999. Reference Source\n\nInternational Atomic Energy Agency: National Regulatory Authorities with Competence in the Safety of Radiation Sources and the Security of Radioactive Materials. International Conference of National Regulatory Authorities with Competence in the Safety of Radiation Sources and the Security of Radioactive Materials. Buenos Aires, Argentina. 2000. Reference Source"
}
|
[
{
"id": "32270",
"date": "17 Apr 2018",
"name": "Steven J. Goetsch",
"expertise": [
"Reviewer Expertise I have performed Gamma Knife radiosurgery for the last 23 years."
],
"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\nJacqueline Chou and others have done an excellent job of evaluating the consequences to the United States from a ban of Co-60 gamma emitting radioisotopes in medicine and industry. The problem is actually far larger than that. Several more gamma emitting isotopes are also in widespread use. Gamma radiation from Co-60 is highly penetrating which makes it ideal for some applications and unsuitable for others. It is comparable in energy to a 4MV linear accelerator.\nMany medical devices contain Cesium-137. This isotope has a much longer half-life than Co-60 (30 years vs. 5.26 years) and emits gamma radiation with much lower energy, making it easier to shield. It is the isotope of choice for blood irradiators. Much of the US blood supply is irradiated before infusing into human patients, particularly those who are immune-compromised.\nAnother extremely prevalent isotope is Ir-192.This isotope of iridium emits a multiplicity of gamma rays with about half the average energy of Cs-137 and one fourth the mean energy of Co-60 gamma radiation. Ir-192 is most commonly used in industrial radiography. There are literally thousands of such devices used in almost every major construction site in the country involving the use of structural steel. There is no other practical way to verify the competence of welding at a construction site which is vital to the safety of the bridges and buildings under construction.\nAnother important field which makes extensive use of radioisotope sources is the oil and gas drilling industry. Well logging sources (primarily Cs-137 and Americium-241 mixed with beryllium) are inserted into a well and reflected radiation is analyzed to determine the geologic structure of the layer being penetrated.\n\nEach of these applications is important and very difficult to replace with radiation producing electronic devices.\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": "34941",
"date": "24 Jul 2018",
"name": "L. John Schreiner",
"expertise": [
"Reviewer Expertise Medical physics and radiation therapy."
],
"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 presented quite a complete and compelling analysis of some unintentional and, perhaps, overlooked consequences for the USA and the world if use of the radioisotope cobalt-60 (Co‑60) is severely limited because of security motivated restrictions. They provide the motivation for controlling access to Co-60 sources in their review and analysis. And they lay out some consequences and risks through a good survey of the value of Co-60 in commerce, medicine and agriculture and the potential impact of restrictions. They reason that the restriction of Co-60 needs to be more carefully analysed, particularly in sterilization and medical applications, since other irradiation technologies and approaches are not yet fully developed for all these applications. They note that the ability to configure the radioactive Co-60 sources in purpose-built devices has provided robust solutions which may not be as easily, or economically, achieved with more technologically complex x-ray or electron irradiators. And associated difficulties of increased complexity and decreased reliability may delay the transition to alternate approaches not yet in place. These are important points that have not been analysed broadly in the literature previously, and to bring them forward here presents a timely and important addition to the literature.\n\nMuch of their discussion is limited to select applications in which Co-60 based irradiators have had a lead role in the United States and high income countries (HICs). And the analysis of these applications is quite good but there are two points I would suggest the authors consider in future work regarding the impact of the elimination of Co-60 treatment units in radiation therapy.\n\n1)\n\nWhile the description in the paper on the implications of Gamma-knife restriction does give some context to the importance of Co-60 based brain cancer treatment, it has missed recent technological and clinical developments. The ability to treat small lesions and targets with high energy external beam radiation treatment (EBRT) has been extended to x-ray linear accelerator based units; this includes specialised devices and techniques such as small field robotic x-ray units (the CyberKnife, Accuray, Sunnyvale, CA) and advanced EBRT units with volumetric modulated arc radiotherapy (VMAT) capabilities that are now available in cancer clinics. That is not to say that the Gamma-knife inventory currently deployed is not a critical resource. It is just to note that alternative devices are already available for this clinical application, although a transition to these devices would have considerable challenges in funding and clinical implementation.\n\n2)\n\nA further aspect of radioisotope restriction that could benefit from additional analysis is the impact of the removal of the Co-60 treatment units in low and middle income countries (LMICs) throughout the world. One can argue that the development of high photon energy Co-60 based devices (with effectively ~1.25 MeV mono-energetic beams) in the 1950s inaugurated high energy EBRT. As noted by the authors, Co-60 radiation therapy devices have been essentially replaced in much of HICs (aside from select devices such as the Gamma-Knife, the recent ViewRay MRIdian Co-60 Co-60/MRI unit, and some other specialised units). But over 2000 conventional Co-60 EBRT units are still in use in LMICs throughout the world, in large part because of the simple and robust design of these units and the low power and water supply infrastructure requirements for the devices (which often limit the feasibility of linear accelerator based x‑ray EBRT units in certain locales). This is an important inventory already in place for cancer treatment, particularly in LMICs; the removal of these devices because of Co-60 security concerns may have critical negative effects. The World Health Organization has reported that LMICs annually account for over 60% of the world’s new cancer case, yet despite being home to 85% of the world’s population, they have less than 35% of the world’s radiotherapy facilities.\n\nThe increased burden of cancer and the associated requirement for an increased distribution of EBRT radiation units in LMIC’S has also been highlighted by the International Atomic Energy Agency and the Global Task Force on Radiotherapy for Cancer Control. The restricted availability of Co‑60 devices potentially introduces a critical impediment to the important increased deployment of EBRT units as governments and international health agencies work to deal with this deficit. While research groups and radiotherapy vendors are working on developing simpler x-ray units for locales with limited infrastructure, it seems there will be a period over which we will rely on the simple Co-60 devices already deployed as well as additional installations (perhaps with new devices specially designed for improved performance and enhanced security of sources) to meet the cancer burden. And, as the authors discuss, the very countries requiring Co-60 radiotherapy deployment may be the same countries where Co-60 security is a challenge. Innovative solutions to ensure availability of needed Co-60 units with effective security control requires attention and the authors may be well set to undertake the analysis and discussion informing such solutions\n\nI have also added some suggested references that discuss some points on Co-60 EBRT in LMICs.\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": []
}
] | 1
|
https://f1000research.com/articles/7-348
|
https://f1000research.com/articles/7-346/v1
|
21 Mar 18
|
{
"type": "Brief Report",
"title": "Association of delivery procedure with APGAR scores among neonates born to healthy Pakistani mothers: a pilot study",
"authors": [
"Muhammad Ali Khalid",
"Rida Ghani",
"Muhammad Fahad Khalid",
"Muhammad Saad Malik",
"Ahmed Waqas",
"Rida Ghani",
"Muhammad Fahad Khalid",
"Muhammad Saad Malik",
"Ahmed Waqas"
],
"abstract": "Background: The present study explores the factors associated with poor APGAR scores among singletons born to healthy Pakistani mothers. Methods: This cross-sectional study was conducted at a Tara Urea Medical Center, Iskandarabad Colony, district Mianwali, Pakistan from April 1 to August 30, 2017. Data was collected using a preformed proforma by a gynecologist and pediatrician during the birth procedure. The questionnaire comprised of two sections including neonatal and maternal characteristics. All data were analyzed in SPSS v.20. Results: Regression analysis revealed that vaginal deliveries were associated with higher APGAR scores at five minutes than those delivered by cesarean section. However, maternal age and BMI and weight of the baby did not yield significant association with APGAR scores at five minutes. APGAR scores assessed at one minute were significantly associated with weight of the neonate. Conclusion: APGAR scores of the neonates at birth are significantly associated with birth procedures. Therefore, birth procedure should be selected and managed effectively to reduce the risk of low APGAR scores.",
"keywords": [
"APGAR",
"neonate",
"factors",
"predictors"
],
"content": "Introduction\n\nIn recent years, much has been published regarding the morbidity and mortality among newborns. However, most research studies are limited to the paradigm of Western countries. A limited contribution in terms of large scale prevalence studies, and culture and resource sensitive interventions have been made from the developing and third world countries. This is a major public health concern because the majority of newborn deaths occur in poorly resourced developing world. According to the World Health Organization (WHO), 2.6 million neonates died in 2016, accounting for a staggering 7,000 newborn deaths daily, with 1 million dying on the first day of life. Therefore, it is of paramount importance to assess the health status of the neonate during the birth procedure using validated methods such as the APGAR scoring system.\n\nThe APGAR score (AS) is validated, reliable and one of the most widely used clinical tools for evaluation of health status of the newborn immediately after birth1. The APGAR scoring system was originally developed by Virginia Apgar (1953) based on five characteristics including heart rate of the newborn, respiratory effort, muscle tone, irritability, and color1. Thus, APGAR scores provide a convenient method to recognize life threatening situations for the newborn and their subsequent response to resuscitation techniques2. A low APGAR score is also a significant predictor of neonatal mortality, long term neurological development, IQ scores, motor impairment, cerebral palsy, mental retardation, epilepsy and poor academic performance3,4.\n\nPrevious literature has shown that APGAR scores of neonates is dependent on a number of factors including maternal age, body mass index, education level, parity, previous miscarriages, emotional well-being, use of nicotine and antidepressants, gestational age, medical comorbidities, and multiple pregnancies5. Method of delivery of the newborn has also been demonstrated as a significant predictor of APGAR scores among the newborns.\n\nHowever, the evidence regarding the association between mode of delivery and APGAR scores is controversial. For instance, Kilsztajn and colleagues reported that compared to vaginal deliveries, neonates born through c-section had higher APGAR scores6. However, they found that this association was spurious and driven by confounding with indication. A few studies also stratify the risk of low APGAR scores according to the urgency of birth procedure. For instance, Lai et al., reported that only emergency cesarean and instrumental vaginal deliveries are associated with low to moderate APGAR scores2. Similar results have been corroborated by Johnson et al. and Prior et al. in their studies exploring predictive factors of neonatal outcomes7,8. This variation in APGAR scores among neonates born through different birth procedures is likely due to oxidative stress experienced by them, however, studies exploring this hypothesis have also yielded mixed results9–11.\n\nIn addition to delivery procedures, a number of other factors have been recognized as independent predictors of APGAR scores among neonates. These include non-cephalic presentation, intramuscular narcotic use, presence of meconium, nulliparity, preterm birth, body mass index, emotional distress, type of pregnancy, and antenatal healthcare2,5.\n\nDue to paucity of data, the present study explores the factors associated with poor APGAR scores among singletons born to healthy Pakistani mothers.\n\n\nMethods\n\nThis cross-sectional study was conducted at Tara Urea Medical Center, Iskandarabad Colony, district Mianwali and Majid Medical Complex, Lahore, both in Pakistan from April 1 to August 30, 2017. Participants undergoing birth procedures at this center were recruited randomly using an online computer software. Only those women were found eligible who had a term birth, no previous history of comorbid medical disorders and antepartum complications were included. All participants provided written informed consent and they were ensured anonymity and that no individual data would be disclosed. Ethical review was sought and granted by the research committee at the Tara Urea Medical Center, Iskandarabad Colony, district Mianwali, Pakistan (Reference # IRB-001).\n\nData was collected using a preformed proforma by a gynecologist and pediatrician during the birth procedure. The questionnaire (Supplementary File 1) comprised of two sections including neonatal and maternal characteristics. Maternal characteristics included body mass index, age and the birth procedure used for delivering the newborn. Neonatal characteristics included gender, weight, admission to neonatal intensive care unit, complications during delivery, and APGAR scores at 1 and 5 minutes.\n\nUsing Gpower v3.1.7, a minimum sample size of 92 was considered to be appropriate for this study to achieve a power of 80% for an anticipated moderate effect size and five predictors. All data were analyzed using SPSS v.20 (IMB, Chicago, IL). Quantitative variables were presented as mean (SD) and categorical as frequency (%). Normality of quantitative variables was assessed by visualizing histograms. Thereafter, linear regression analysis was run to explore the predictors of APGAR scores at one and five minutes. Both maternal and neonatal characteristics were introduced as independent variables in these models.\n\n\nResults\n\nOut of 100 women undergoing birth procedures, a total of 98 (98%) agreed to participate in the study. There were 49 male (50%) and 49 female (50%) neonates. A majority of the neonates were born though supra-vaginal delivery (67, 68.4%) followed by cesarean section (22, 22.4%) and episiotomy (9, 9.2%). A total of six (6.1%) neonates experienced complications during delivery and four (4.1%) were admitted to neonatal intensive care unit. Mean weight of mothers was 72.51 (6.32) kg, height 160.85 (4.64) cm, age 23.94 (2.81) years and BMI 28.06 (2.25). Mean weight of the neonates was 2.99 (0.25) kg, APGAR score at one minute 6.18 (1.37) and APGAR score at five minutes was 9.67 (0.69).\n\nLinear regression analysis delineating predictors of APGAR scores at one minute explained only 5.3% variation. According to it, maternal age, BMI and type of delivery procedures were not associated with APGAR scores assessed at one minute after delivery. However, it was inversely associated with weight of the neonate.\n\nRegression modelling for APGAR scores assessed at five minutes explained 70.60% of variation in it. According to it, vaginal deliveries and higher maternal BMI were associated with higher APGAR scores at five minutes than those delivered by cesarean section. However, maternal age and weight of the baby did not yield significant associations with APGAR scores at five minutes. Detailed results are presented in Table 1.\n\nBMI= Body mass index\n\nStd. Error= Standard Error\n\n\nDiscussion\n\nIn present study, delivery by cesarean section and low maternal BMI was associated with low APGAR scores assessed at five minutes. Whereas, the APGAR scores at one minute was significantly associated with weight of the neonate.\n\nOur research evidence has several limitations and therefore, should be generalized with caution. The cross-sectional nature of the study design limits inferences regarding causality and temporality. Moreover, a small sample size (n=98) limits the statistical power of this study, therefore, future studies should include greater study samples.\n\nIn conclusion, a newborn’s health status is associated with the birth procedure as well as the birth-weight. These results have several implications for neonatal health in Pakistan on designing culture sensitive policies and educational interventions in antenatal care and preference for birthing procedures among expectant mothers as well as the practicing obstetricians and gynecologists. Expectant mothers should be delivered educational interventions during regular antenatal check-ups, aiming at improving overall health including an optimum weight of the fetus. Moreover, the rising preference of birth by cesarean sections among Pakistani expectant mothers should be discouraged.\n\n\nData availability\n\nDataset 1: APGAR factors 10.5256/f1000research.13784.d19811812\n\nCoding Scheme: Gender: 1= Male, 2= female; Mode of delivery: 1= C-section, 2= supra-vaginal delivery, 3= SVD with episiotomy; Admission to NICU= 1=yes, 2=no; Complications during delivery= 1=yes, 2=no. Mode of delivery binary= 1=C-section, 2=other",
"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 – Copy of study questionnaire\n\nClick here to access the data.\n\n\nReferences\n\nCasey BM, McIntire DD, Leveno KJ: The Continuing Value of the Apgar Score for the Assessment of Newborn Infants. N Engl J Med. 2001; 344(7): 467–71. PubMed Abstract | Publisher Full Text\n\nLai S, Flatley C, Kumar S: Perinatal risk factors for low and moderate five-minute Apgar scores at term. Eur J Obstet Gynecol Reprod Biol. 2017; 210: 251–6. PubMed Abstract | Publisher Full Text\n\nMoore EA, Harris F, Laurens KR, et al.: Birth outcomes and academic achievement in childhood: A population record linkage study. J Early Child Res. 2014; 12(3): 234–50. Publisher Full Text\n\nRazaz N, Boyce WT, Brownell M, et al.: Five-minute Apgar score as a marker for developmental vulnerability at 5 years of age. Arch Dis Child Fetal Neonatal Ed. 2016; 101(2): F114–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlmeida NKO, Pedreira CE, Almeida RMVR: Impact of maternal education level on risk of low Apgar score. Public Health. 2016; 140: 244–9. PubMed Abstract | Publisher Full Text\n\nKilsztajn S, de Souza Lopes E, Nunes do Carmo MS, et al.: [Apgar score associated with mode of delivery in São Paulo State, Brazil]. Cad Saude Publica. 2007; 23(8): 1886–92. PubMed Abstract | Publisher Full Text\n\nJohnson JH, Figueroa R, Carry D, et al.: Immediate maternal and neonatal effects of forceps and vacuum-assisted deliveries. Obstet Gynecol. 2004; 103(3): 513–8. PubMed Abstract | Publisher Full Text\n\nDunn L, Prior T, Greer R, et al.: Gender specific intrapartum and neonatal outcomes for term babies. Eur J Obstet Gynecol Reprod Biol. 2015; 185: 19–22. PubMed Abstract | Publisher Full Text\n\nRaijmakers MT, Roes EM, Steegers EA, et al.: Umbilical glutathione levels are higher after vaginal birth than after cesarean section. J Perinat Med. undefined. degruyter.com [Internet]. 2003; 13(6), [cited 2018 Jan 11]. Publisher Full Text\n\nMehmetoglu I, Kart A, Caglayan O, et al.: Oxidative stress in mothers and their newborns in different types of labour. Turk J Med Sci. 2002; 32: 427–9. Reference Source\n\nFogel I, Pinchuk I, Kupfermine MJ, et al.: Oxidative stress in the fetal circulation does not depend on mode of delivery. Am J Obstet Gynecol. 2005; 193(1): 241–6. PubMed Abstract | Publisher Full Text\n\nKhalid MA, Ghani R, Khalid MF, et al.: Dataset 1 in: Association of delivery procedure with APGAR scores among neonates born to healthy Pakistani mothers: a pilot study. F1000Research. 2018. Data Source"
}
|
[
{
"id": "142234",
"date": "29 Jun 2022",
"name": "Chitkasaem Suwanrath",
"expertise": [
"Reviewer Expertise Maternal fetal medicine"
],
"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 study has too small a sample size to determine predictive factors of Apgar scores. I have raised concerns on many points.\nMethod:\nIn the sample size calculation, the authors stated five predictors, please give details of the predictors\n\nWhich references the author used to calculate sample size (N =92)?\n\nAre there any interventions in this study?\n\nWere cesarean sections on demand allowed in the study hospitals?\n\nRoute of delivery is a categorical variable, not quantitative data. Is it appropriate to use linear regression?\nResults:\nWhat does supra-vaginal delivery mean? I have never heard this term?\n\nRoutes of delivery are classified as vaginal delivery and cesarean section. Episiotomy is in the category of vaginal delivery.\n\nSince the sample size is small. Data distribution is unlikely normal. Data presented as median (min, max) or IQR may be more appropriate.\n\nPlease give details of the indication for cesarean section\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? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-346
|
https://f1000research.com/articles/6-1270/v1
|
28 Jul 17
|
{
"type": "Research Article",
"title": "Consumption of traditional alcoholic beverages in children from a rural village in Northern Peru, 2017",
"authors": [
"Juan M. Ramírez-Ubillus",
"Martín A. Vilela-Estrada",
"Shirley A. Herrera-Arce",
"Estefany Mejía-Morales",
"Christian R. Mejia",
"Juan M. Ramírez-Ubillus",
"Shirley A. Herrera-Arce",
"Estefany Mejía-Morales",
"Christian R. Mejia"
],
"abstract": "Introduction: Alcoholic beverages have a proven impact on neuronal development and other areas of the body, primarily the heart, kidneys and liver, which is why their consumption in children is prohibited. However, there are traditional drinks that may have alcohol content. The aim of this study was to characterize the consumption of traditional alcoholic beverages in children of a rural village in Northern Peru. Methods: This study was an analytical cross-sectional study. Mothers were recruited by census sampling and reported the consumption by their children of two traditional drinks with alcoholic content: Chicha de Jora (Ch) and Clarito (Cl), which are derived from the fermentation of maize. The frequency of consumption, accessibility and perception of consumption risk were described. Results: Data were collected about 300 children, 61% (183) of whom consumed Ch. and 31% (92) of whom consumed Ch and Cl. Regarding drink accessibility, the majority of mothers said that these drinks were cheap (Ch: 69.0% and Cl: 60.7%). Additionally, the vast majority of families sometimes consumed or always consumed such beverages (Ch: 81.3% and CI: 65.7%). One in three mothers perceived Ch and Cl as being nutritious and helping their children grow. 25% of mothers perceived that there was no risk to their children from the consumption of the beverages, whereas >60% said that there could be a risk due to the beverages’ alcohol content. Conclusions: Our study found that traditional beverages containing alcohol are consumed frequently by children in a village in Northern Peru. Mothers provide accessibility to the beverages and perceive the risk the drinks have, yet they continue to provide such drinks to their children putting their health in great danger. We advise that future studies concerning the intervention of these attitudes are performed, for a better future and development of children.",
"keywords": [
"Alcohol",
"Children",
"Health Problems",
"Peru"
],
"content": "Introduction\n\nAlcoholic beverages, which are traditionally derived from the fermentation of sugars and yeast1, currently have a large socio-economic impact. The World Health Organization states that 3.3 million deaths are caused every year worldwide by the harmful use of alcohol2. It is well known that these types of drinks cause a series of physiological problems (renal, digestive, hepatic, etc.)3,4, as well as behavioral problems, which include maladaptation to the family and social environment, and, in extreme situations, could lead to suicide5.\n\nAccording to worldwide data, alcohol use has 5.1% comorbidity (high blood pressure, cirrhosis, renal disease, etc.) in the age group between 20–39 years6. However, some countries, such as Colombia and Argentina, have reported onset at an earlier age7. In Peru, there is almost no information on this subject (information that is provided is mostly provided by local institutions); however reports show that the median age when alcohol consumption begins is 13 years, while in locations where children have greater access to alcoholic beverages, consumption starts at 10 years8.\n\nChicha de Jora (Ch) and Clarito (Cl) are drinks derived from the fermentation of maize that have been consumed since Pre-Hispanic times9 throughout the northern coast of Peru. Consumption is high due to their low production cost, ease of access, and tradition10.These factors can create a problem if such drinks are consumed by children and teenagers. The objective of this study was to characterize the consumption of these traditional alcoholic beverages in children of a rural village in Northern Peru.\n\n\nMethods\n\nA cross-analytical cross-sectional study was carried out between February and May 2017, in which the mothers and/or guardians of the Northern Peruvian settlement of \"La Piedra\", where 308 children under the age of 15 reside, were surveyed. Household visits were completed for the purposes of the study. Thanks to the information provided by the governor, the surveys were carried out in each of the homes of the mothers and/or guardians using census sampling. A sample size was calculated for a descriptive study, for the local population of children, with a statistical power of 99%, a 95% confidence level and a maximum prevalence of 50%. A minimum sample of 300 children was obtained; this was captured non-randomly.\n\nAll mothers residing in the populated center (small town) during the interview were included. Mothers who did not wish to participate in the study, as well as those mothers who responded inadequately to our survey were excluded. After reading through the informed consent and agreeing to participate the mothers were enrolled in the study. Those who did not respond adequately to the survey (unanswered questions and/or incomplete answers) were excluded. Rate of rejection = 2.5%, thus achieving a total of 300 surveys applied, obtained from the interview of 103 mothers or guardians (in some cases the mothers or guardians had more than one child).\n\nFor the present study, a survey was carried out, which was previously validated by a pilot study in a sample of 50 individuals, where a Cronbach's alpha of 0.781 was obtained. The previous pilot study was not published, the results were only for the evaluation of the survey. The survey had minor modifications after the pilot study. These were used to specify the details of consumption, access and even the consequences of the consumption of alcoholic beverages. The final survey had two main sections (Supplementary File 1):\n\nSocio-demographic data: Basic data was provided, such as the child’s age, weight, height and school grade, and in addition the number of household members and household income.\n\nCharacteristics of drinking habits in liquids/beverages: These characteristics were evaluated through closed questions, in which inquiries were made about the daily consumption of different drinks, primarily the consumption of beverages containing alcohol (Ch and Cl). The following information was obtained: The frequency of consumption, the accessibility of the drinks, whether or not they were consumed by the person responding to the survey and by the whole family, and if consumption of the drinks was perceived to be harmful or nutritional for the child’s health. Finally, other exploratory variables were captured, such as the consumption of other types of beverages (gas, pure water, milk, lemonade, Chicha Morada, etc), and a section where the child's socio-academic problems were assessed was included. These exploratory variables are not discussed in the present study.\n\nAll surveys were anonymous and were conducted by a researcher belonging to the study. The approximate duration of the survey was 20 minutes. At all times the assigned researcher was properly trained to be able to solve doubts about any of the questions.\n\nFor the data analysis, a double digitizing system (data processed by two researchers separately, and then checked for errors manually) was performed, for a better control of the data collected. Surveys were entered in the Microsoft Excel program (version 2015), then proceeded to make a first filter for checking the data. Following this, the data were processed in Stata 11.1 (StataCorp LP, College Station, TX, USA).\n\nFor descriptive statistics, we worked with frequencies/percentages for categorical variables, and medians and interquartile ranges for the quantitative variables. The chi-square statistical test was applied for the association of the consumption of the drinks versus the perception that the consumption of the drinks could be bad for children. P<0.05 was considered statistically significant.\n\nPermission and support was provided by local authorities (governor, health center doctor and school director). Since children were the target of this study, all precautions were taken to ensure anonymity and respect for ethical precepts. The study was approved by the Ethics Committee of the San Bartolomé National Hospital, endorsed by the National Health Institute (NIH; approved March 5, 2016; Office No. 422). This committee was chosen since there is no committee that monitors the approval of the NIH where the study was conducted. This committee also approved the pilot study. The ethical standards on human experimentation of the Declaration of Helsinki of 1975 were taken into account. The results will be given to the sanitary authorities of the region, so that they can learn about this reality and put forward strategies of help. The study was carried out under the permission of the mothers/guardians, who gave written informed consent.\n\n\nResults\n\nData were collected about 300 children, 51.3% (154) were girls, and the median age was 9 years (interquartile range: 5–12 years). 15.8% (41) studied at an initial level, 53.5% (139) studied in a primary school and 30.7% (80) studied in secondary school. 61.0% (183) and 30.7% (92) consumed Ch and Cl, respectively (Table 1).\n\nMost of the mothers reported that they consumed Ch (84.7%) and Cl (62.7%) when they were children, and the majority also consume the drinks now (Ch: 74.0% and Cl: 47.7%). Regarding accessibility of the beverages, the majority of mothers said that these drinks were cheap (Ch: 69.0% and Cl: 60.7%), and the vast majority of families sometimes consumed or always consumed such beverages (Ch: 81.3% and Cl: 65.7%) (Table 2).\n\n35% of mothers perceived that Ch is nutritious and helps growth, while 33% and 35% of mothers perceived that Cl is nutritious and helps growth, respectively (Figure 1). 25% of mothers perceived that there was no risk for their child to consume the beverages. However, >60% said that there could be a risk due to the alcohol contained in the drinks (Table 3).\n\nFigure 2 shows that although women perceive consumption of beverages as bad for their children, 46% and 34% still gave their children Ch and Cl, respectively.\n\n\nDiscussion\n\nThe consumption of alcohol in children is still a very important problem, as evidenced in this study, where out of 300 children surveyed, 183 and 92 children consumed Chicha de Jora (Ch) and Clarito (Cl), respectively, every week. These results of consumption are greater than in different studies from different countries. For example, in Brazil only 12.8% consumed some type of alcoholic drink before the age of 1011; in the Province of Buenos Aires, 55.4% of teenagers between the ages of 11 and 14 years consumed alcohol12; while a study in Colombia, with a mean age of 14.4 years, concluded that the pattern of alcohol abuse measured by the CAGE scale was 14.6%13.\n\nThe consumption of these traditional beverages also occurred during the mothers' childhood, with a majority stating that they had consumed both drinks. Many of the mothers expressed that they still consume them. A report of a population study in Chile, of 408 alcoholic respondents, reported that 27.2% lived with children in the house and in 46.3% of cases the drinker was either the father or the mother14. Another report in Angola showed that 56% of mothers of 319 children had regular alcohol habits. Our study showed that this percentage was higher at 84.7% of mothers who consume Ch and 62.7% who consume Cl15. Also in Brazil, Argentina, Colombia, Chile, and Mexico, it was reported that occasional consumption of alcohol is associated with family context, influence of friends, antisocial behavior, and skills and experiences already acquired in childhood, which could be circumstances that encourage the consumption of alcohol in children11–13,16,17.\n\nThe consumption of alcohol in younger populations has risen in recent years, which has the potential to cause harm and create addictive behavior17. In our population, the acquisition of Ch (69.0%) and Cl (60.7%) was considered economical because of their low cost of production; therefore making them more accessible and frequently consumed. One in every three mothers perceived that the Ch and Cl are nutritious and help the growth of their children, and this is a perception that could lead them to giving these drinks to their children. A study from Spain reported that fathers and mothers do not consider their children's alcohol consumption to be a problem18, thus increasing their early intake without restriction. Unfortunately, no studies about the consumption of alcohol or drugs by children and adolescents guided by therapeutic or beneficial purposes from parents has been reported until now.\n\nIn the present study, most mothers knew about the risk of alcohol consumption by children. However, it was observed that the consumption in most of their children remained high. Studies carried out in Spain and Cuba indicate that the family can be a protection, but also a risk factor. In both cases, the maternal figure tends to have a positive influence on the child, which differs from what was found in the present study18,19. We can infer that this is mainly due to a socio-cultural characteristic where the community (and especially the mothers) view the consumption of these traditional alcoholic beverages as normal.\n\nThe study had the limitation of selection bias, since it was completed in a sample that does not represent the total population of Peru. However, this study used census type sampling in a population that had not been previously reported; therefore, these results can be taken as preliminary. Notably, these findings can be used to alert the responsible authorities, so that screening and support measures can be impletmented, so that the families of this village, and other similar locations that present similar conditions of consumption, can receive the necessary support.\n\n\nConclusions\n\nAccording to the present study, it is concluded that children consume a large quantity of traditional alcoholic beverages and their mothers provide accessibility. Even though the mothers perceive the risk that these beverages have, they still provide them to their children.\n\n\nData availability\n\nDataset 1: Raw data from the responses of mothers/guardians concerning their children’s consumption of traditional alcoholic beverages (n=300 children). doi, 10.5256/f1000research.12039.d17015820",
"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: Survey for mothers/guardians relating to the consumption of traditional alcoholic beverages in their children. This survey is provided in Spanish and English.\n\nClick here to access the data.\n\n\nReferences\n\nWorld Health Organization: Glossary of Terms of Alcohol and Drugs. [Internet]. [Quoted June 20, 2017]. 1994. Reference Source\n\nWorld Health Organization: Alcohol. Descriptive Note No. 349. [Internet]. [Quoted June 20, 2017]. 2015. Reference Source\n\nGárate B, Villagra D, Puente E, et al.: Effects of Alcohol on the Nervous System. National Agrarian University la Molina. [Internet]. [Quoted June 20, 2017]. 2013. Reference Source\n\nEstruch R: Effects of alcohol on human physiology. Addictions. 2002; 14(Suppl. 1): 43–61. Reference Source\n\nRodríguez-Míguez E, Mosquera Nogueira J: Measuring the impact of alcohol-related disorders on quality of life through general population preferences. Gac Sanit. 2017; 31(2): 89–94. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: World Situation Report on Alcohol and Health 2014. [Internet] .201 [Quoted June 20, 2017]. Reference Source\n\nde la Espriella Guerrero RA, Rodríguez V, Rincón CJ, et al.: [Alcohol Consumption in the Colombian Population, 2015 National Mental Health Survey]. Rev Colomb Psiquiatr. 2016; 45(Suppl 1): 76–88. PubMed Abstract | Publisher Full Text\n\nNational Commission for the Development and Life without Drugs-DEVIDA. Executive Report: IV National Study on the Prevention and Consumption of Drugs in High School Students 2012. [online]. [Accessed June 20, 2017]. 2013. Reference Source\n\nPalacios MFP: Application of Chicora de Jora in 30 standard recipes. [Thesis to choose the title of Graduate in Gastronomy and Food and Beverage Services]. Cuenca: University of Cuenca; 2010. Reference Source\n\nDe La Cruz CA, Janampa GAC: Heat treatment to stabilize the girl from Jora. [Thesis to choose the title of Chemical Engineer]. Lima: National University of Engineering; 2012.\n\nde Lima Argimon II, Campana A, Estefenon S, et al.: Consumo de alcohol en niños y adolescentes de un municipio en el sur de brasil. Revista Argentina de Clínica Psicológica. Date of reference: 8 / julio / 2017. 2016; XXV: 267–274. Reference Source\n\nDuffy D: Risk Factors and protective factors associated with the consumption of alcohol in children and adolescents. Argentina: Buenos Aires University, Buenos Aires. Salud & Sociedad [online]. 2014; 5(1): 40–52. Reference Source\n\nRueda-Jaimes GE, Ramírez JL, Martínez-Villalba AM, et al.: [Alcohol abuse and Associated Factors in Student Children and Adolescents]. Rev Colomb Psiquiatr. 2012; 41(2): 273–283. PubMed Abstract | Publisher Full Text\n\nGrigoravicius M, Iglesias A, Ponce P, et al.: Family Context and Consumption of Psychoactive Substances in Children between 8 and 12 Years Old. Acta de Investigación Psicológica. 2013; 3(2): 1149–62. Publisher Full Text\n\nMartínez-Cárdenas A, González-Sábado R: Riesgos psicosociales de salud mental en niños de 0-10 años asistidos en el consultorio del Hospital Provincial de Bengo. Angola. Multimed Revista Médica Granma [revista en Internet]. [citado 2017 Jul 8]; [aprox. -91 p.]; 2017; 21(2). Reference Source\n\nMaturana HA: Alcohol and drug consumption in adolescents. Rev Med Clin Condes. Department of Psychiatry. Child Psychiatrist Unit. 2011; 22(1): 98–109. Reference Source\n\nTegoma-Ruiz VM, Cortaza-Ramírez L: Prevalencia del consumo de alcohol en adolescentes de una secundaria de Coatzacoalcos, Veracruz. Enfermería Universitaria. 2016; 13(4): 239–45. Publisher Full Text\n\nMarch Cerdá JC, Prieto Rodriguez MA, Danet A, et al.: [Parental stance towards alcohol consumption in 12- to 17-year-old adolescents from six urban areas in Spain]. Gac Sanit. 2010; 24(1): 53–58. PubMed Abstract | Publisher Full Text\n\nPérez Rosabal E, María Soler Sánchez Y, Pérez Rosabal R, et al.: Factores de riesgo y consumo de alcohol en adolescentes. Multimed. 2016; 20(2): 308–321. Reference Source\n\nRamírez-Ubillus JM, Vilela-Estrada MA, Herrera-Arce SA, et al.: Dataset 1 in: Consumption of traditional alcoholic beverages in children from a rural village in Northern Peru, 2017. F1000Research. 2017. Data Source"
}
|
[
{
"id": "24666",
"date": "31 Jul 2017",
"name": "Dirk W. Lachenmeier",
"expertise": [
"Reviewer Expertise Risk assessment of unrecorded alcohol"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a pilot study into the consumption of traditional alcoholic beverages in rural Peru. The article is interesting and novel as there is clearly a lack of data on unrecorded alcohol from South America. It is also quite disturbing to read that considerable alcohol exposure may occur in children.\nThe following revisions should be considered:\nFor the international reader the beverages Chicha de Jora and Clarito are almost unknown. Can some information about these beverages be provided as background? For example, are they similar to maize beers? What is the typical alcoholic strength of these beverages? Are they commercially and legally sold by some kind of artisanal small-scale industry, or are they illegally sold? Should they be considered as falling into the WHO category of “unrecorded alcohol” (see Rehm et al.1 for definition).\n\nThe conclusion states that the products are providing “great danger” to health. While this may be true, there is not much in the data that would allow for such a conclusion. The study appears to be non-quantitative in nature and the alcoholic strength of the product appears to be unknown. Hence no calculation of daily alcohol exposure can be made, which would allow for a quantitative risk assessment (such as by using the margin of exposure approach2). With the currently available data I would suggest to conclude that there may be a health hazard, but quantitative intake assessment as well as chemical characterization of the beverages would be necessary for risk assessment.\n\nThe introduction of the abstract is written in a rather vague fashion. “... traditional drinks that may have alcohol content”. Are Ch and Cl without alcohol available? This should be clarified.\n\nIntroduction, last paragraph. The reference 9 mentions “Chicora de Jora”. Is this the same as “Chicha de Jora”? Reference 10 mentions “girl from Jora”? Are these translation mistakes? Are there any more reliable peer-reviewed references with background on the beverages than some theses? Can a link to the source of reference 10 be provided?\n\nMethods, characteristics of drinking habits in liquids/beverages: what beverage is “gas”? Clarify that Chicha morada is non alcoholic.\n\nTable 3: check values for line “dangerous”. Is this logical that both are “0”?\n\nDiscussion, first sentence: Please provide percentages to make the values more easy to compare with the data from Brazil and Colombia.\n\nPage 6, first line: please clarify where the consumption has risen (in Peru?)\n\nPage 6, 4th line: “low cost of production”. Please provide some comparison for the low cost. Are the alcoholic beverages cheaper than non-alcoholic alternatives such as milk or fruit juices?\n\nPage 6, 1st paragraph, last line “no studies about consumption of alcohol and drugs by children”. I wonder about this request and why this is seen as unfortunate. I would find it highly unethical to study alcohol and drug consumption in children, and I can predict that we will never see such a study. This sentence should be deleted.\n\nDiscussion, last paragraph: please include the non-quantitative nature as limitation. Considering your comment that “the consumption of these traditional alcoholic beverages” is seen as “normal”, is there at least some information how much of the beverages is consumed by the children?\n\nConclusions: “it is concluded that children consume a large quantity of traditional alcoholic beverages”. This conclusion appears to be not founded in the data. No quantitative measurements were conducted.\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": "3511",
"date": "21 Mar 2018",
"name": "Martín Vilela",
"role": "Author Response",
"response": "Dear reviewer, the changes requested in the comments have been made. On behalf of the Authors, very grateful"
}
]
},
{
"id": "24709",
"date": "25 Aug 2017",
"name": "Paul Anthony Camacho López",
"expertise": [
"Reviewer Expertise Epidemiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is interesting, but the presented analysis only is descriptive, losing the relevance and applicability. I suggest that Authors enlarge the analysis, which should evaluate the potential risk factors. The conclusion is adequate, but only they could describe the consumption of traditional alcoholic beverages. The introduction is not enough to explain the problem compared to the use of alcoholic beverages. In the methods, authors should clarify the patterns of consumption, the frequencies of consumption per week or monthly. The data analysis only centered in the descriptive the mother´s consumption (perceptions), accessibility and perception of risk, but they did not analyze the association or relation with child´s age, weight, height and school grade, household income and number members.\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": [
{
"c_id": "3510",
"date": "21 Mar 2018",
"name": "Martín Vilela",
"role": "Author Response",
"response": "Dear reviewer, the requested changes have been made in the comments, in the same way, we communicate that this is a preliminary study and we take into account in a future study to consider data in associations, as well as associated factors. On behalf of the Authors, very grateful"
}
]
}
] | 1
|
https://f1000research.com/articles/6-1270
|
https://f1000research.com/articles/7-343/v1
|
20 Mar 18
|
{
"type": "Research Article",
"title": "Adolescent girls’ attitudes toward female genital mutilation: a study in seven African countries",
"authors": [
"Koustuv Dalal",
"Zhanna Kalmatayeva",
"Sourav Mandal",
"Gainel Ussatayeva",
"Ming Shinn Lee",
"Animesh Biswas",
"Koustuv Dalal",
"Zhanna Kalmatayeva",
"Sourav Mandal",
"Gainel Ussatayeva",
"Ming Shinn Lee"
],
"abstract": "Background: The study’s aim is to examine adolescent girls’ attitudes toward the continuation or discontinuation of female genital mutilation (FGM) in association with their demographics in seven different countries in Africa. Methods: Data from the women’s survey of the Demographic and Health Surveys (DHS) conducted by the respective ministries (of Health and Family Welfare) in Egypt, Guinea, Kenya, Mali, Niger, Senegal and Sierra Leone were used. Adolescent girls (15–19 years) were included in the current analysis: Egypt (N=636), Guinea (N=1994), Kenya (N= 1767), Mali (N=2791), Niger (N=1835), Senegal (N=3604), Sierra Leone (N=1237). Results: Prevalence of supporting the continuation of FGM among adolescent girls was in Egypt 58%, Guinea 63%, Kenya 16%, Mali 72%, Niger 3%, Senegal 23%, and Sierra Leone 52%. Being Muslim and having low economic status were significantly associated with supporting the continuation of FGM in five of the participating countries. Girls having no education or only primary education in Guinea, Kenya, Mali and Sierra Leone exhibited a higher likelihood of supporting FGM than girls with secondary or higher education. In Egypt, Niger and Senegal there was no association between education and supporting FGM. The girls who stated that they had no exposure to media showed the higher likelihood of supporting FGM in Guinea, Kenya, and Senegal than those with exposure to media. Conclusions: The current study argues that increasing media coverage and education, and reducing poverty are of importance for shifting adolescent girls’ attitudes in favor of discontinuation of FGM.",
"keywords": [
"female genital cutting",
"attitudes",
"Egypt",
"Guinea",
"Kenya",
"Mali",
"Niger",
"Senegal",
"Sierra Leone."
],
"content": "Introduction\n\nFemale genital mutilation (FGM) is a major public health problem in some parts of the world, especially in Africa and the Middle East1–3. It is recognized as a violation of the human rights of girls and women3, and is a violation of the Convention on the Rights of the Child, which has been ratified by all of the countries where FGM is common except Somalia4, as the majority of FGM procedures being carried out on young girls5. FGM has no known health benefits5 and it can cause many health problems immediately and later in life, for example pain, bleeding, infections, and increased risks during childbirth both for the mother and the baby2,6. The number of victims is estimated to between 100 and 140 million1,3,7. Around 3 million girls a year face the risk of FGM3,7. In the seven countries included in this multi-country study the estimated prevalence of FGM among girls and women between 15 and 49 years is high (over 85%) in Egypt, Guinea, Mali, and Sierra Leone. In Kenya and Senegal it is almost 30% and in Niger 2%3. However, the prevalence could vary much within countries by ethnic group.\n\nThe first step toward changing the practice of FGM is to change attitudes toward it, even though this can be difficult and psychologically painful8. Knowing what demographic factors appear to influence attitudes towards FGM can help in deciding on the actions to take to promote changes. It is a question for both governments and the people. Among the governments of the included countries, the prevailing attitude is that FGM should be prevented, and many have signed agreements or passed laws banning it7,9. Even if such laws exist, they may be badly disseminated in some countries due to a lack of central administration7,9. Many governments also turn a blind eye to the practice1 or are accused of being slow to act9. For example, half of Gambian Health Care professionals working in rural areas support the continuation of FGM10.\n\nMore women than men support the practice11,12. Social pressure and tradition are the most compelling factors for the continuation of FGM12,13. There are divergent results on the support for FGM among younger girls. WHO14 emphasizes that support for discontinuation is high among younger women, while Masho and Matthews12 and Sipsma and colleagues15 conclude that younger girls are more supportive of FGM. One reason for this could be their having less experience of circumcision, either their own or that of their daughters15. There are also contradictory results on religious beliefs. Islam was not a significant predictor of a favorable attitude towards FGM in Guinea, although a majority believed that FGM was accepted by their religion11. In western Africa it was a predictor except in Niger and Nigeria15. In Ethiopia being Muslim was a predictor12. One explanation that has been offered is that in Ethiopia religious beliefs are based on transmitted interpretations, not on the original religious texts13. Another divergent predictor of attitudes towards FGM is household wealth. Higher levels of household wealth increased women’s support for discontinuation in Guinea11. In some countries wealth was associated with supporting FGM and in some the opposite15. Media also plays a major role in clarifying doubts and misconceptions about FGM13. Decision-makers, leaders in the community, and religious leaders are important channels for modifying cultural beliefs about FGM16. Women with lack of exposure to mass media supported the continuation of FGM to a higher extent than others12. Higher education however, is the main factor associated with supporting discontinuation of FGM in most of the studies11,12,15–17. Empowerment is also a key factor in the elimination of FGM18, although Afifi19 concludes that only high empowerment combined with high education played a significant role.\n\nA multi-country comparison is a unique opportunity to analyze what demographic factors that are associated with attitudes toward the continuation of FGM. It is especially interesting to focus on young women (girls 15–19 years), a group that will play an important role in future decisions on FGM among young girls, that is, their daughters.\n\nThe aim of this study is to examine adolescent girls’ attitudes toward the continuation of female genital mutilation in association with their demographics in seven different countries in Africa.\n\n\nMethods\n\nThis study was part of the women’s survey of the Demographic and Health Surveys (DHS) conducted by the Ministries of Health and Family Welfare in Egypt, Guinea, Kenya, Mali, Niger, Senegal and Sierra Leone, respectively. Household interviews were performed using the same structured questionnaires (the Women’s Questionnaire) in Egypt DHS 200820, Guinea DHS 201221, Kenya DHS 200822, Mali DHS 200623, Niger DHS 201224, Senegal DHS 2010–1125, Sierra Leone DHS 200826.\n\nInitially, we identified the countries with high prevalence of FGM in WHO reports2,3. We then searched in DHS databases for FGM prevalence in the selected countries. Due to language barriers, we have only selected countries with English databases for FGM. Seven countries were ultimately selected for the current study.\n\nThe sample for the women’s surveys included women of reproductive age (between 15 and 49 years) from both rural and urban areas. The sampling within each country used the sampling procedure probability proportional to population size (PPS) based on the sizes of the state’s/region’s urban and rural populations, which led to nationally representative samples. In each of these seven countries the DHS used almost identical multistage sampling procedures. Primary sampling units (PSUs) were selected from all administrative regions in both rural and urban areas using the probability PPS based on the most recent census of each country. Households were then selected randomly from the PSUs. Finally, based on the selection criteria, respondents were selected from the households. In the current study, seven countries with differing populations (in 2008) are included: Egypt (74.9 million), Guinea (10.3 million), Kenya (38.0 million), Mali (12.7 million), Niger (14.7 million), Senegal (12.7 million), Sierra Leone (5.5 million). In each country, sampling is based on PPS of the population. Therefore, population size has no potential influence on the current study. More details of the sampling procedures and survey methods are available in Egypt DHS 200820, Guinea DHS 201221, Kenya DHS 200822, Mali DHS 200623, Niger DHS 201224, Senegal DHS 2010–1125, Sierra Leone DHS 200826.\n\nAll DHSs are global initiatives to monitor demographic and health issues, including the Millennium Development Goals, in the developing countries. The respective governments, the United States Agency for International Development (USAID), and other international donor agencies finance DHSs. Macro International Incorporated (Calverton, MD) provides the technical support for conducting DHSs. DHSs are well controlled by field experts at national and international level and therefore are rigorously planned, well organized, strictly monitored, reliable, and widely used, especially in the developing countries. Experts have provided training and guidance to the field workers to develop the awareness and skills necessary to facilitate the optimal response from the respondents. Interviewers also received training in handling private responses without putting the respondents or the interviewer at risk. An interviewer’s manual was developed and provided to the field workers. The main questionnaire was initially formulated in English and then translated into local languages as needed, using appropriate scientific methods (translations and back-translations). The current study used the secondary data generated by the above-mentioned DHSs.\n\nIn the current study, female adolescents (15–19 years) were identified in the seven aforementioned sample sets and were included in the current analysis: Egypt (N=636, 4% of total women respondents), Guinea (N=1994, 21.8%), Kenya (N=1767, 20.3%), Mali (N=2791, 21.1%), Niger (N=1835, 17%), Senegal (N=3604, 23%), Sierra Leone (N=1237, 17.1%).\n\nThe main variable of interest for the current study was “Circumcision should continue or be stopped?” The respondents had the options “should continue” and “should not continue.” The current study used this as the dependent variable in all bivariate and multivariate analyses.\n\nPlace of residence: Urban or rural area\n\nReligion: Muslim and non-Muslim. The original questionnaire included other religions as well. However in the current analysis, all other religions than Muslim have been combined into new variable: non-Muslim.\n\nEducation: None, or primary, secondary, or higher education. In the current analysis, secondary and higher education were merged into a new variable “Secondary+.”\n\nExposure to media: The survey questionnaire had asked respondents about reading newspapers or magazines, listening to radio, and watching television. The current study created a new variable “exposure to media” by merging these three variables. Several studies have indicated that media plays an important role in the discontinuation of FGM12,13,16.\n\nEconomic status: This was a composite measure of the cumulative living standard of the households, considering all economic assets (such as radios, televisions, bicycles), materials used for construction of houses, types of water access, and sanitation facilities. Principal components analysis (PCA) was used to estimate individual households on a continuous scale of relative wealth20–26. This was a standardized scale in the normal distribution with a mean of zero and a standard deviation of one. Then, using the standardized scores, wealth quintiles were created: poorest, poorer, middle, richer, and richest.\n\n\nEthical issues\n\nThe current study uses secondary data collected from the DHS Program. The DHS had received ethical permission from the Institutional Review Board of Opinion Research Corporation (ORC) Macro International Inc.\n\nPrevalence was estimated for each country to reflect adolescent girls’ supportive attitudes on continuation of FGM. The proportions and chi-squared tests were used to explore the cross relationships between dependent and independent variables. Multivariate logistic regressions were performed to study the potential associations between justification of female genital mutilation and respondents’ socioeconomic factors and exposure to media. Data were analyzed using IBM SPSS version 20.0.\n\n\nResults\n\nThe sample consisted of 15–19 year-old girls in seven countries in Africa. The mean age of respondents was 17 years in six countries, and 18 years in Egypt. The proportion of respondents residing in rural areas was: in Egypt 77.4%; Guinea 55.6%; Kenya 75.5%; Mali 57.4%; Niger 62.8%; Senegal 57.5%; Sierra Leone 45.8%. Respondents were mainly of the Muslim religion in (Egypt 97.2%; Guinea 88.5%; Mali 88.9%; Senegal 94.8%). Kenya had only 17.7% Muslim respondents, and Sierra Leone had 69% Christian. Figure 1 shows the educational levels of the respondents by country.\n\nPrevalence of FGM among all respondents of reproductive age (15–49 years) was in Egypt 94.4% (15–19 years: 91.5%); Guinea 97.9% (95.7%); Kenya 31.6% (22.7%); Mali 88.7% (88.6%); Niger 4.2% (3.5%); Senegal 40% (40.3%) Sierra Leone 90.9% (70.6%).\n\nIn Figure 2 we have assessed the prevalence of FGM among adolescent girls and their attitudes towards continuing FGM. Prevalence of supporting the continuation of FGM among adolescent girls was in Egypt 58%, Guinea 63%, Kenya 16%, Mali 72%, Niger 3%, Senegal 23%, and Sierra Leone 52%.\n\nIn Niger no significant differences were found between demographic factors and supporting the continuation of FGM (Table 1) (due to the missing cases or the low proportion of support). In all other countries, girls living in rural areas or with poor economic status were more likely to support FGM. Also lower-educated girls and girls who were not exposed to media supported the continuation of FGM to a greater extent, except in Egypt.\n\nP-values of chi-square test.\n\nMali, Niger, Guinea, and Senegal had more than 40% uneducated adolescent girls (15–19 years). Almost two-thirds of respondents (15–19 years) in Kenya and Egypt had primary and secondary education respectively. Figure 1 has presented the education levels for adolescent girls (15–19 years) in seven African countries\n\nAfter adjusting for all other independent variables in this study, using a logistic regression for each country independently, some results are found that are similar between the countries and one that differs between the countries (Table 2). Religion and economic status were significantly associated with supporting the continuation of FGM in five of the participating countries. Non-Muslim participants (Odds Ratios between 0.093 and 0.502, p<0.005) were less likely to support FGM than Muslim participants in all countries except Egypt and Niger. Lower economic status (Odds Ratios between 2.226 and 7.802, p<0.005) gave a greater likelihood of supporting FGM than did the richest group in all countries but Mali. In Guinea, Kenya, Mali and Sierra Leone, having no education (Odds Ratios between 1.937 and 4.657, p<0.005) or only primary education (Odds Ratios between 1.616 and 2.108, p<0.005) resulted in a greater likelihood of supporting FGM than having secondary or higher education. In Egypt, Niger and Senegal there was no association between education and supporting FGM. The girls who stated that they had no media exposure (Odds Ratios between 1.439 and 2.837, p<0.005) showed a greater likelihood of supporting FGM in Guinea, Kenya and Senegal than those with media exposure. In Mali (Odds Ratio=0.486, p<0.005) the result showed the opposite: girls with no media exposure were less likely to support FGM than those with media exposure.\n\n1.0 denotes reference category. Significance level: “*=> p<0.001”; “† => p<0.050”\n\n\nDiscussion\n\nIn the seven countries compared in this paper, support for FGM among adolescent girls (15–19 years of age) ranged from 3% to 71%. The countries where the support for FGM was over 50% were Egypt, Guinea, Mali, and Sierra Leone. In Senegal slightly more than 20%, and in Kenya slightly fewer than 20% of respondents supported the continuation of FGM. In Niger only 3% supported FGM; however there was a large proportion of missing cases, because the FGM prevalence is very low. The current study has indicated that adolescent girls from highly FGM-prevalent countries were more supportive of continuing FGM than those from low prevalence countries. Guinea, Kenya, Senegal, and Sierra Leone have demonstrated that exposure to media (newspapers, magazines, radio or television) has a positive effect for reducing the justification of FGM among adolescent girls. The situation in Mali is the complete opposite. Adolescent girls in Mali who are exposed to media supported the continuation of FGM more than those who are not exposed to media. The Mali study was the oldest (2006) among the seven countries, and this inconsistent finding may indicate that media did not play its expected role of protecting girls from FGM. In a recent UN report, a number of organizations have urged more media coverage to motivate the discontinuation of FGM in the affected countries27. The current study echoes these calls for stronger media coverage towards discontinuation of FGM. An integrative review study has assessed 16 studies of which only five have focused on attitudes towards FGM in the country of origin13. The current study uses nationally representative data from seven countries.\n\nNo education, rural residence, and lack of information ignite attitudes in favor of the continuation of FGM. Previous studies have also signaled the same findings12. Empowerment is often considered an explanatory factor of supporting the discontinuation of FGM18,19. In this study the participants are 15–19 years old, live with their families, and have no socioeconomic empowerment. This could explain why the girls in the current study to some extent accepted family-oriented socioeconomic and cultural norms and supported the continuation of FGM.\n\nEconomic status and education are factors that could change attitudes toward FGM16,27. The current study similarly recommends more education and less poverty as ways to shift attitudes in favor of a discontinuation of FGM.\n\nThis study is cross-sectional in nature which leads to difficulties in assigning causal relationships between respondents’ background factors and favoring FGM. Prevalence of FGM has been determined from respondents’ self-reporting. No clinical examination was performed to verify their reported genital mutilation. Therefore, the reported prevalence could over- or underestimates the actual rate. Though all the seven DHSs have tried to select samples in all strata of the country using probability proportional to population size (PPS), after finalization of the study data, some countries have fewer respondents in the adolescent group (15–19 years). Also, countries like Niger have very low FGM prevalence. Polarization in the distribution of data (such as by religion) may lead to non-significant results. The current study used the same questionnaires for identifying the FGM prevalence and for assessing teenagers’ attitudes towards continuation of FGM using quantitative research. However, we have no answer to why the youngest generation of women supports FGM. Appropriate qualitative studies among adolescent girls exploring the reasons for such support could lead to more effective policy-making to eradicate FGM. Due to the large variations within countries, the importance ascribed to country-specific data in tailoring approaches to effectively reduce and eliminate FGM is in our view well warranted12,15.\n\n\nPolicy implications\n\nTo the best of our knowledge, the current study is the first to examine the attitudes of adolescent girls (15–19 years) regarding the discontinuation of FGM in seven countries. The findings of the current study support the provision and dissemination of adequate education to eliminate FGM, and emphasize the important role of media. The study has considerable implications for policy aiming to eliminate FGM, as higher prevalence indicates a higher supportive attitude among adolescent girls. UN bodies are advocating for the elimination of FGM. However, if teenage girls support FGM, they are likely to continue the practice with their daughters when they become mothers. Previous studies have recommended educational, community, and media intervention1,4,14–16. The findings presented here indicate that the youngest women should be targeted for necessary interventions.\n\n\nData availability\n\nThe DHS Program owns data used in this study. The DHS data for all the seven countries are available for researchers interested in further analyses. Researchers should contact The DHS Program and get permission to use the required data.",
"appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe DHS Program for collecting and providing data for scientific research and publications. Assoc. Professor Susanna Geidne, PhD, Örebro University for her generous contribution and comments.\n\n\nReferences\n\nEke N: Female genital mutilation: What can be done? Lancet. 2000; 356(Suppl): S57. PubMed Abstract | Publisher Full Text\n\nWHO: Eliminating Female genital mutilation: An interagency statement. Geneva: World Health Organization; 2008. Reference Source\n\nWorld Health Organization: Female genital mutilation and other harmful practices: Prevalence of FGM. [cited 2018 Feb10], Accessed 2017-09-18. Reference Source Accessed 2017-09-18\n\nConvention on the Rights of the Child. [cited 2018 February 10]. Reference Source\n\nWorld Health Organization: Female Genital Mutilation. [cited 2018 February 10]. Reference Source\n\nRushwan H: Female genital mutilation: A tragedy for women's reproductive health. Afr J Urol. 2013; 19(3): 130–133. Publisher Full Text\n\nUNFPA-UNICEF joint programme on female genital mutilation/cutting. [cited 2018 February 12]. Reference Source\n\nLien IL, Schultz JH: Internalizing knowledge and changing attitudes to female genital cutting/mutilation. Obstet Gynecol Int. 2013; 2013: 467028. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWakabi W: Africa battles to make female genital mutilation history. Lancet. 2007; 369(9567): 1069–1070. PubMed Abstract | Publisher Full Text\n\nKaplan A, Hechavarria S, Bernal M, et al.: Knowledge, attitudes and practices of female genital mutilation/cutting among health care professionals in The Gambia: a multiethnic study. BMC Public Health. 2013; 13: 851. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGage AJ, Van Rossem R: Attitudes toward the discontinuation of female genital cutting among men and women in Guinea. Int J Gynaecol Obstet. 2006; 92(1): 92–96. PubMed Abstract | Publisher Full Text\n\nMasho SW, Matthews L: Factors determining whether Ethiopian women support continuation of female genital mutilation. Int J Gynaecol Obstet. 2009; 107(3): 232–235. PubMed Abstract | Publisher Full Text\n\nReig Alcaraz M, Siles González J, Solano Ruiz C: Attitudes towards female genital mutilation: an integrative review. Int Nurs Rev. 2014; 61(1): 25–34. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: Female genital mutilation and other harmful practices. [cited 2018 January 30]. Reference Source\n\nSipsma HL, Chen PG, Ofori-Atta A, et al.: Female genital cutting: current practices and beliefs in western Africa. Bull World Health Organ. 2012; 90(2): 120–127F. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDalal K, Lawoko S, Jansson B: Women's attitudes towards discontinuation of female genital mutilation in Egypt. J Inj Violence Res. 2010; 2(1): 41–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlo OA, Gbadebo B: Intergenerational attitude changes regarding female genital cutting in Nigeria. J Womens Health (Larchmt). 2011; 20(11): 1655–1661. PubMed Abstract | Publisher Full Text\n\nIsman E, Ekéus C, Berggren V: Perceptions and experiences of female genital mutilation after immigration to Sweden: an explorative study. Sex Reprod Healthc. 2013; 4(3): 93–98. PubMed Abstract | Publisher Full Text\n\nAfifi M: Women's empowerment and the intention to continue the practice of female genital cutting in Egypt. Arch Iran Med. 2009; 12(2): 154–160. PubMed Abstract\n\nEgypt Demographic and Health Survey 2008. [çited 2017 December 20]. Reference Source\n\nInstitut National de la Statistique Ministère du Plan Conakry Guinée: Guinée Enquête Démographique et de Santé et à Indicateurs Multiples (EDS-MICS) 2012. [cited 2017 December 20]. Reference Source\n\nKenya Demographic and Health Survey 2008–09. [cited 2017 December 20]. Reference Source\n\nCellule de Planification et de Statistique du Ministère de la Santé (CPS/MS), Direction Nationale de la Statistique et de l’Informatique du Ministère de l’Économie, de l’Industrie et du Commerce (DNSI/MEIC), et al.: Mali Enquête Démographique et de Santé (EDSM-IV) 2006. [cited 2017 December 20]. Reference Source\n\nInstitut National de la Statistique (INS), ICF International:Niger Enquête Démographique et de Santé et à Indicateurs Multiples 2012. [cited 2017 December 20]. Reference Source\n\nAgence Nationale de la Statistique et de la Démographie (ANSD) [Senegal], ICF International: Senegal Demographic and Health Survey – Multiple Indicator Cluster Survey (EDS-MICS) 2010–2011. 2012. [cited 2017 December 20]. Reference Source\n\nSierra Leone Demographic and Health Survey 2008. [cited 2017 December 20]. Reference Source\n\nUNFPA: Female Genital Mutilation. [cited 2018 January 20]. Reference Source"
}
|
[
{
"id": "33115",
"date": "17 May 2018",
"name": "Els Leye",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper provides a secondary analysis of attitudes of adolescent girls towards continuation of FGM in association with a number of variables (demographic). The paper is well written, straightforward, and shows some surprising results. I think it is worthwhile publishing, as it sheds light on a particular group of interest when it comes to prevention of FGM, i.e. adolescent girls.\n\nSome minor comments: . - use most recent prevalence data (200 million with FGM), from UNICEF 2016 - typo in 3rd paragraph of introduction, page 3: One reason for this could be they're (not their)... - typo in 4th paragraph of introduction, page 3: remove 'that' in second line.\nPage 4, methods section: Millennium Development Goals have expired; you should update with the Sustainable Development Goals.\nPage 8, last sentence of first paragraph of discussion: I do not really see the relevance.\nPage 8, second column, line 5: typo: should be underestimate.\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": "34761",
"date": "18 Jun 2018",
"name": "Nesrin Varol",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important article that points out the most crucial aspects of the process of abandonment of FGM. Education, financial independence for women, and addressing poverty are some of the key factors to change attitudes and protect girls and women.\nPlease see my other comments:\nPage 1 Abstract\nMethods Please delete “different” in “…seven different countries in Africa.” Countries are different by definition.\n\nResults Please correct grammar here and again in results section. “Prevalence of supporting the continuation……girls was 58% in Egypt, 63% in Guinea, etc…” Or “Prevalence of supporting the continuation…..girls was: 58% Egypt, 63% Guinea, etc…..”\n\nPage 3 Introduction 1. “…..where FGM is common except Somalia4, as the majority of FGM procedures being carried out on young girls5.” Meaning of the end of this sentence is unclear. You mean that Somalia is the only country that hasn’t ratified the Conventions. You also mean that FGM is a violation of human rights etc because it’s being performed on children / young girls. The grammar needs to be changed here. Perhaps write: “It is recognized as a violation of the human rights of girls and women3, and is a violation of the Convention on the Rights of the Child, as the majority of FGM procedures being carried out on young girls5 These have been ratified by all of the countries where FGM is common except Somalia4.\n\n2. “In the seven countries included in this multi-country study the estimated prevalence of FGM among girls and women between 15 and 49 years is high (over 85%) in Egypt, Guinea, Mali, and Sierra Leone.” You write “in the seven countries” but then only write the names of four, i.e Egypt, Guinea, Mali and Sierra Leone.\n3. “More women than men support the practice11,12 Please write which countries this statement refers to or detail the statement a little further.\n\n4. “A multi-country comparison is a unique opportunity to analyze what demographic factors that are associated with attitudes toward the continuation of FGM.” Please delete “that”.\n\n5. “The aim of this study is to examine adolescent girls’ attitudes toward the continuation of female genital mutilation in association with their demographics in seven different countries in Africa. “ Change to FGM. Once you have written an acronym, continue to use it throughout the article. Delete “different” again.\n\nPage 4 Methods “All DHSs are global initiatives to monitor demographic and health issues, including the Millennium Development Goals, in the developing countries.” The MDGs are the older Goals that were used. Also perhaps change from “developing” to “low-income”. So: “….including the past Millennium ….., in low-income countries.”\nResults “Respondents were mainly of the Muslim religion.” The religion is Islam, the people who follow Islam are Muslims. Please change to “….of the religion of Islam.” or “…of the Islamic religion.”\nPage 5 Results 1. “Almost two-thirds of respondents (15–19 years) in Kenya and Egypt had primary and secondary education respectively. “ Please place a comma after “…education, …”.\n2. Figures 1 & 2. “Sierra Leone” is missing the “e” at end of Leone.\n\n3. “Non-Muslim participants (Odds Ratios between 0.093 and 0.502, p<0.005) were less likely to support FGM than Muslim participants in all countries except Egypt and Niger. “ It would be interesting for the reader to know the details of the results in Egypt and Niger. It is an important result and should be nuanced in its presentation. Are Muslims less likely to support FGM than non-Muslims? If so, please state that with the appropriate numbers.\nPage 8 “The situation in Mali is the complete opposite. Adolescent girls in Mali who are exposed to media supported the continuation of FGM more than those who are not exposed to media. The Mali study was the oldest (2006) among the seven countries, and this inconsistent finding may indicate that media did not play its expected role of protecting girls from FGM. ” Yes, this is indeed an interesting finding. It would be good for you to speculate on why this may be the case. Your interpretation is part of a discussion of your findings. Perhaps the reasons may be that the social obligation / pressure is even more entrenched in Mali? Or religious pressure? Or even lower levels of education as compared to the other countries?\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": "35488",
"date": "09 Jul 2018",
"name": "Gregory E Gilbert",
"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\nThank you very much for the opportunity to review this potentially important manuscript. Unfortunately, it suffers from a fatal flaw.\n\nFirst, recommendations. This reviewer strongly recommends English language editing to improve clarity, readability, and flow of the text. The authors are to be congratulated for authoring a coherent manuscript and have done much better than this reviewer could do in (what is presumed to be a language other than their primary language).\n\nThe fatal flaw has to do with study design. A complex survey design was used in at least to survey the Kenyan (and I suspect other) population(s). This two-stage sampling design oversampled urban populations and undersampled those in the North East province. Not taking the sampling design into account when doing the analysis has critical implications and renders and conclusions wrong. This reviewer is unaware of SPSS survey sampling software; however, SAS has several survey procedures (PROC SURVEYMEANS, PROC SURVEYFREQ, PROC SURVEYPHREG), R (which is free) has a package for complex survey analysis (survey), and RTI (SUDAAN) all have software for handling complex surveys designs.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-343
|
https://f1000research.com/articles/7-329/v1
|
15 Mar 18
|
{
"type": "Research Article",
"title": "Evaluating disparities in the U.S. technology transfer ecosystem to improve bench to business translation",
"authors": [
"James Weis",
"Ashvin Bashyam",
"Gregory J. Ekchian",
"Kathryn Paisner",
"Nathan L. Vanderford",
"James Weis",
"Ashvin Bashyam",
"Gregory J. Ekchian",
"Kathryn Paisner"
],
"abstract": "Background: A large number of highly impactful technologies originated from academic research, and the transfer of inventions from academic institutions to private industry is a major driver of economic growth, and a catalyst for further discovery. However, there are significant inefficiencies in academic technology transfer. In this work, we conducted a data-driven assessment of translational activity across United States (U.S.) institutions to better understand how effective universities are in facilitating the transfer of new technologies into the marketplace. From this analysis, we provide recommendations to guide technology transfer policy making at both the university and national level. Methods: Using data from the Association of University Technology Managers U.S. Licensing Activity Survey, we defined a commercialization pipeline that reflects the typical path intellectual property takes; from initial research funding to startup formation and gross income. We use this pipeline to quantify the performance of academic institutions at each step of the process, as well as overall, and identify the top performing institutions via mean reciprocal rank. The corresponding distributions were visualized and disparities quantified using the Gini coefficient. Results: We found significant discrepancies in commercialization activity between institutions; a small number of institutions contribute to the vast majority of total commercialization activity. By examining select top performing institutions, we suggest improvements universities and technology transfer offices could implement to emulate the environment at these high-performing institutions. Conclusion: Significant disparities in technology transfer performance exist in which a select set of institutions produce a majority share of the total technology transfer activity. This disparity points to missed commercialization opportunities, and thus, further investigation into the distribution of technology transfer effectiveness across institutions and studies of policy changes that would improve the effectiveness of the commercialization pipeline is warranted.",
"keywords": [
"Commercialization",
"Technology Transfer",
"Technology Licensing",
"Patents",
"Licenses",
"Startups"
],
"content": "Introduction\n\nThe transfer of inventions from academic institutions to private industry is a major driver of economic growth and human welfare. Broadcom, Google, Akamai, Yahoo, Biogen, Bose, and Genentech represent just a handful of pioneering companies with academic roots (Kenney, 2017). Indeed, many of today’s defining technologies originated in academic labs, including nuclear energy and the internet (Busbin, 1995; Manyika & Roxburgh, 2011; Nelson & Byers, 2015).\n\nTechnology-driven progress demands not only the development of new inventions, but also their dissemination throughout society. Our national capacity to fuel growth and improve human well-being through new technologies depends on our ability to pass these technologies through a commercialization pipeline. This national need for an efficient and effective technology handoff between academia and industry motivated our analysis of the current United States (U.S.) academic technology transfer environment.\n\nLeveraging data from the Association of University Technology Managers (AUTM) U.S. Licensing Activity Survey, we characterized the performance of research organizations across different steps of the technology transfer process. Our findings indicate that the translational abilities of research organizations across the U.S. vary widely, with a small minority of institutions producing the vast majority of technological and economic benefits. To begin addressing this gap, we surveyed initiatives aimed at improving technology transfer and propose remedies for observed disparities in institutional performance.\n\n\nMethods\n\nThe AUTM Licensing Survey solicits responses annually from around 300 institutions, including universities, hospitals and research institutions, to quantify the total technology transfer activity at these institutions. These metrics are derived from a set of core questions that AUTM deems essential for assessing transfer and licensing activity. A detailed description of each metric from the AUTM survey data is given in Supplementary Table 1. We defined the “commercialization pipeline” (Figure 1) by identifying a set of key questions asked in each AUTM survey, and extracting relevant data from the 2010 to 2014 AUTM surveys. We use this commercialization pipeline to measure and compare relative levels of technology transfer activity at different institutions, and at different steps along the pipeline. The distributions of each metric across every surveyed institution are visualized as linear and log histograms, as well as empirical cumulative distributions, in Supplementary Figure 1 and Supplementary Figure 2.\n\nEach step in this pipeline corresponds to a metric in the AUTM survey. We use the health of the pipeline as a proxy for the overall health of the U.S. technology transfer ecosystem.\n\nWe ranked each institution from the AUTM Licensing Survey data by each step in the commercialization pipeline. Any institution ranked in the top 10 (about the top 5%) in at least one stage of the pipeline was included in the our list of top performing institutions. This resulting list of 25 institutions (approximately 12% of all surveyed institutions) was then sorted based on mean reciprocal rank (MRR):\n\n\n\nwhere N = 7 is the number of stages in the pipeline and Ri is the ranking of the institution in step i of the pipeline. We chose this scoring system to identify institutions with consistently high performance across the commercialization pipeline while avoiding heavily penalizing anomalous weak performances in just a single metric.\n\nGiven value xi for institution i and xj for institution j, we calculate G, the Gini Coefficient, such that:\n\n\n\nThe Gini coefficient is a measure of statistical dispersion used to assess inequality in a population. A high Gini coefficient indicates high levels of inequality where, in this case, a few institutions contribute a substantial amount of total translational activity. Conversely, a low Gini coefficient indicates that each institutions contributes an equal share.\n\nVariance estimates (υ) for the Gini coefficient for each step were derived via jackknife resampling (Karagiannis & Kovacevic’, 2000; Yitzhaki, 1991):\n\n\n\nwhere N is the number of observations, G is the Gini coefficient when all observations are considered, and Gi is the Gini coefficient value when the ith observation is removed. The confidence intervals of the log-normal fits were computed to the 95% confidence levels using the Jacobian of the parameter estimates assuming normally distributed residuals. All statistical analysis was performed in MATLAB 2016b (Mathworks, Natick, MA, USA).\n\n\nResults\n\nOur goal was to understand how much each institution contributed to each step of the commercialization pipeline and to determine any notable overall trends in U.S. technology transfer. Histograms (Supplementary Figure 1 and Supplementary Figure 2) of contributions from each institution along the commercialization pipeline reveal highly skewed distributions. The distributions of each metric are generally well approximated by a log-normal fit. Note that the x-axes is on a log scale and therefore the significant skew in the distribution is not immediately apparent. The effectiveness of a log-normal fit decreases towards the end of the commercialization pipeline (Startups and Adjusted Gross Income).\n\nThe majority of institutions contribute a small amount to overall technology transfer regardless of how activity is measured (Figure 2 and Supplementary Figure 3). Specifically, the top 20% of institutions contribute over 60% of total commercialization activity. Importantly, this trend is robust to normalization by research expenditures, which indicate that differences in research funding do not explain the gap in productivity (Supplementary Figure 3). In fact, the top 10% of institutions contribute over 40% of “startups per dollar of research expenditures” and over 70% of “adjusted gross income per dollar of research expenditures”.\n\nA small number of institutions contribute to the majority of commercialization activity.\n\nWe identified the 25 top-performing institutions by sorting all top-performing institutions by the average of their reciprocal ranking at each step in the commercialization pipeline (Table 1). Most organizations that perform well do so across the entire commercialization pipeline, indicating strong and broad technology transfer abilities (e.g. University of California and University of Texas Systems; MIT; and Stanford). On the other hand, some organizations excel in only specific parts of the commercialization pipeline (e.g. University of Washington in Licenses and Options Executed; California Institute of Technology in New Patent Applications; and University of Georgia in Licenses and Options Executed), which reveals focused, less-robust technology transfer capabilities.\n\nBar plots show the mean value over the years under consideration for each institution for each step in our commercialization pipeline.\n\nWe extended this analysis by calculating the Gini coefficient, a measure of statistical dispersion that is often used to quantify income inequality (Gini, 1912). In this analysis, a low Gini coefficient indicates that each institution is contributing roughly equally to U.S. technology commercialization, whereas a high Gini coefficient indicates that a few institutions are producing the majority of the commercialization output.\n\nAs shown in Figure 3 and Supplementary Figure 3, high levels of inequality exist throughout the pipeline. For context, the Gini coefficient of patents issued in the U.S. is above 60%, while the Gini coefficient of all U.S. household income is 48% (U.S. Census Bureau). We believe this indicates that the majority of U.S. research organizations have significant untapped commercialization potential, the full realization of which could lead to new technologies and, overall, improved U.S. productivity.\n\nError bars represent one standard deviation of uncertainty as estimated via jackknife resampling (Karagiannis & Kovacevic’, 2000; Yitzhaki, 1991).\n\nMany of the top performing institutions have invested significant effort and resources in supporting entrepreneurs at each stage of the commercialization pipeline. Top performing institutions have ensured continuity in their support structure to enable the efficient and effective translation and development of both institute-owned and student-created intellectual property. Table 2 highlights active programs at MIT and Harvard, two top performing translational institutions. Our summary of these initiatives span university incubators, student organizations, university venture capital funds and business plan competitions (Table 2).\n\nThe shaded regions denote which areas of the pipeline each program most directly addresses.\n\nThe overview of successful programs (Table 2) provides a blueprint for universities that would like to foster improved technology transfer and innovation. While some of these programs would require a significant undertaking on the part of the university, many can be achieved in a straightforward and lightweight manner via the support of student-led activities and partnership with government and private organizations. Examples of grassroot student groups that have launched many new programs exist at both MIT and Harvard. For instance, the MIT Biotech Group group has partnered with the MIT Alumni Angels of Boston to launch a life sciences-focused track to improve access to capital for early-stage startups. The Harvard Biotechnology Club runs an incubator program to develop and translate academic research. These programs represent student-led efforts that require little to no university expenditure or resources. For larger undertakings, university/corporate collaborations can provide an efficient means to achieve significant progress. A prime example of this is JLABS @ M2D2, the medical device incubator partnership between Johnson & Johnson and the University of Massachusetts Lowell (McCarthy et al., 2013).\n\n\nDiscussion\n\nExpense, time, infrastructure, and the lack of partnerships are among the most common barriers to research commercialization and alleviating these bottlenecks allows more inventions to enter the marketplace (Vanderford et al., 2013). Programs to increase support for inventors at less well performing institutions to file disclosures, pursue patent prosecution, and seek licensing deals could significantly boost translational output. Sharing best practices from the leaders in technology commercialization may help bring more new technologies to market.\n\nOne salient feature of the top-performing institutions is their broad portfolio of commercialization-focused initiatives. Individually, these projects typically target only a few steps on our commercialization pipeline (for example, business plan competitions target the latter stages of the technology transfer process). However, the best performing universities have a large number of these efforts which, in aggregate, fully span the commercialization pipeline. This observation indicates a potential strategy for improvement of those less well served technology transfer pipelines; specifically, the cultivation of commercialization focused initiatives, such as incubators, business plan competitions, innovation prizes, law clinics, and student organizations. The value of these efforts goes beyond their immediate impact. For example, although when taken at face value, a business plan competition may seem to serve only the winning team, its merit truly stems from bringing together students, entrepreneurs, investors, and the media in a constructive setting. The resources required for such projects are small, and, given the disparity in commercialization, potential societal benefits are vast.\n\nA clear barrier to effective commercialization of university technology is the widespread lack of access to experienced, motivated, and well-resourced technology transfer offices (TTO). Many institutions are unable to support a comprehensive TTO, hampering efforts to introduce new technology into industry. The use of consultants can help alleviate some shortcomings, but faces its own barriers to widespread adoption (AUTM Technology Transfer Practice Manual).\n\nAlternatively, a coalition of institutions could create a third-party technology licensing organization whose charter is to serve the technology transfer needs of those institutions. Like a sports agent, this third-party organization would use its expertise to strike technology transfer deals between institutions and licensees, freeing universities to focus on their strengths. Funded directly by the institutions and, in part, by licensing revenue, this organization would have the necessary resources and freedom to hire top-tier technology transfer professionals who can effectively interface between stakeholders in industry and in academia, while negotiating on behalf of the parent institutions. These teams would work to creatively package and license technologies to maximize their utility to society, as well as to assure that the parent institutions receive a fair return on their investment.\n\nOperating outside of the university, this organization would be free to make decisions much more quickly than traditional TTOs. Similarly, its employees would be incentivized to work in the best interest of the parent institutions by ensuring the process is both efficient and maximizes value for all stakeholders. This outsourced model of technology transfer speaks towards the latent need for more efficient, properly incentivized, and more widespread efforts to commercialize academic research and development efforts.\n\n\nConclusion\n\nAs the U.S. economy becomes increasingly driven by technological change, understanding and improving the commercialization pipeline is critically important. The significant disparity in technology transfer performance is evident as the top few institutions produce a very large share of the country’s total technology transfer. We believe this disparity points to missed commercialization opportunities, which we as a society are paying for by missing out on potentially highly impactful innovations.\n\n\nData availability\n\nThe AUTM Licensing Activity Survey data are available on the organization’s website (https://www.autm.net/resources-surveys/research-reports-databases/licensing-surveys/) by fee or institutional subscription/membership. As such, the raw data analyzed for this study cannot be provided in the context of this article. The 2010–2014 survey data used for this study was obtained as part of an institutional membership (University of Kentucky).",
"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 Figure 1. Histograms of each step in the commercialization pipeline shown in Figure 1. Insets show cumulative distributions, with shaded rectangles indicating the number of institutions necessary to reach 80% of total activity.\n\nClick here to access the data.\n\nSupplementary Figure 2. Histograms of each step in the commercialization pipeline shown in Figure 1 with a log x-axis. Note that the x-axes is on a log scale and therefore the significant skew in the distribution is not immediately apparent.\n\nClick here to access the data.\n\nSupplementary Figure 3. Quantized and normalized distribution for each step in the commercialization pipeline. The shaded bars represent the percentage of the total of each category owned by institutions in percentiles indicated.\n\nClick here to access the data.\n\nSupplementary Table 1. Pipeline descriptions.\n\nClick here to access the data.\n\n\nReferences\n\nAssociation of University Technology Managers Technology Transfer Practice Manual. 3rd ed. Reference Source\n\nBusbin JW: The Role of Marketing Research and Decision Systems in the Marketing Process. Journal of Nonprofit & Public Sector Marketing. 1995; 2(2–3): 167–90. Publisher Full Text\n\nGini C: Variabilità E Mutabilità, Contribrito Allo Studio Delle Distribuzioni E Delle Relazioni Statistiche: Fascicolo Ier: Introduzione - Indici Di Variabilità - Indici Di Mutabilità. 1912. Reference Source\n\nKaragiannis E, Kovacevic’ M: A Method to Calculate the Jackknife Variance Estimator For the Gini Coefficient. Oxford Bulletin of Economics and Statistics. 2000; 62(1): 119–22. Publisher Full Text\n\nKenney M: The Diverse Roles of Universities in Regional Innovation Ecosystems: Case Studies from University of California Campuses*. 2017.\n\nMcCarthy SP, Tello S, Hafer N: Medical Device Innovation in Massachusetts- M2D2: Massachusetts Medical Device Development Center. UMass Center for Clinical and Translational Science Seminar Series. October 17 2013. Reference Source\n\nManyika J, Roxburgh C: The Great Transformer: The Impact of the Internet on Economic Growth and Prosperity. McKinsey Global Institute, October 2011. Reference Source\n\nNelson A, Byers T: Organizational Modularity and Intra-University Relationships between Entrepreneurship Education and Technology Transfer. In Advances in the Study of Entrepreneurship, Innovation & Economic Growth. 2015; 275–311. Publisher Full Text\n\nU.S. Census Bureau: Current Population Survey, 1968 to 2015 Annual Social and Economic Supplements. Reference Source\n\nVanderford NL, Weiss LT, Weiss HL: A survey of the barriers associated with academic-based cancer research commercialization. PLoS One. 2013; 8(8): e72268. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYitzhaki S: Calculating Jackknife Variance Estimators for Parameters of the Gini Method. Journal of Business & Economic Statistics: A Publication of the American Statistical Association. 1991; 9(2): 235. Publisher Full Text"
}
|
[
{
"id": "32039",
"date": "19 Mar 2018",
"name": "Karen I. Deak",
"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\nOther than the Materials and Methods dealing with the underpinnings of the statistical methodolgy, which are well beyond me (my failure, not the authors'), the article is clear and easy to understand. It provides an interesting look at the University technology transfer process, and is probably of interest to a wide variety of readers -- both those in the technology transfer field and those who care what federal research dollars can provide.\nThe authors demonstrate that some universities are better than others at commercializing the research that their professors undertake. They provide an examination of whether universities can be good at all steps of the process; or whether it is more effective for a TTO to specialize at one, more high-leverage, point in the process.\n\nThe article is clear, and the conclusions and findings are well-reported. The authors additionally provide a few suggestions of how to improve or make more efficient the technology transfer process. These takeaways will be of interest to readers in TTOs, especially.\nIt would be interesting for a follow-on article for the authors to examine tech transfer efforts at different \"types\" of universities -- with or without a medical school; or at places where there have been blockbuster licensing deals vs. not. But clearly those ideas are well outside the scope of this article.\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": "32556",
"date": "06 Apr 2018",
"name": "Adriana Bankston",
"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\nGeneral comments: This publication highlights an important topic in examining the effectiveness of universities to facilitate the transfer of new technologies into the marketplace. This work also raises awareness to the fact that only a small number of institutions contribute to the vast majority of the total commercialization activity. This is only one of the many disparities found across U.S. institutions, and the work presented here very effectively puts this particular disparity into a broader context.\nTherefore, this type of research has the potential to be utilized as a way to press for change in the system as a whole. Particularly for this field, a change in the system would require advocating for the implementation of successful practices from high-performing institutions across a higher number of U.S. institutions, so that eventually these technologies can become commonplace in all universities. To this end, I believe it is particularly important to consider how we might provide similar types of resources to U.S. institutions with less of an access to these tools or with fewer number of tools at their disposal as compared to the high-performing institutions in the area of technology transfer.\nThe publication itself is very well-written, with the motivation for pursuing this work and its purpose being very clearly defined. The authors have also very succinctly articulated the gap filled by this work and its overall significance to the field, and the results support the conclusions drawn. Overall, I believe this paper is suitable for publication in its current form. I have just a few suggestions for improvement, or ideas that might be useful to consider if expanding or revising this paper in the future. These are mostly broad recommendations related to whether and how the disparities described here could be reduced across universities.\nGeneral considerations: I would like to commend the authors for detailing the steps of the commercialization pipeline early in the publication - this is very helpful for those who might not be very familiar with it. Similarly, the explanations of the data analysis parameters, including the Gini coefficient, are very useful in understanding the conclusions drawn from these studies. One suggestion I would have is to move the information from the Supplementary Table 1 into a main table in the publication, to enable the reader to more easily and quickly refer to these concepts in conjunction with reading Figure 1.\nI also appreciate the fact that the commercialization pipeline allows the authors to answer key questions using relevant data in the AUTM Licensing Survey. Hower, it would be really helpful to also conduct a more in-depth investigation of how each of the universities in the AUTM Licensing Survey measures up in every step of the commercialization pipeline, in order to obtain a view of the national landscape on this issue beyond just the top 25 institutions. While I realize that looking at all 300 universities may be cumbersome and that analyzing the top 25 was a relevant strategy in this case, a more detailed analysis might allow for looking at broader trends that exist across U.S. institutions in the technology transfer area, which could then be utilized to guide future reforms.\nIn terms of the actual reforms, I would also be curious to know more about the types of policies which the authors propose to be implemented in U.S. universities in order to improve the effectiveness of the commercialization pipeline. As part of this question, I recognize the importance mentioning the grassroot student groups who have launched successful programs at MIT and Harvard. Perhaps it would be helpful to suggest how such universities can best share their strategy for success in technology transfer with other universities in the U.S., which could potentially enable the creation of a shared collection of online resources, and/or a network of professionals interested in helping universities improve in this area who would meet regularly to discuss this issue.\nGiven that there are multiple programs described from these two institutions in Table 2, it may be helpful to discuss more about the specifics of these programs, in terms of the similarities and differences (perhaps in a supplementary figure) in order to point out particular elements that would be useful for other universities to utilize and incorporate into a more universal program that could be adopted widely across the U.S. to improve the technology transfer field. Alternatively, are there differences between these programs that could be pointed out in order to determine their suitability for being adopted by other universities as multiple independent programs?\nI also wonder if there are other additional types of actions that could be undertaken by graduate students and postdocs who might have an interest in this area, or by university administrators who could assist in potentially reducing some of these disparities described here. One area that might be worth exploring in this regard is implementing technology transfer programs in all U.S. universities (perhaps these could be designed by a local technology transfer office) geared towards training both researchers and administrators in this area, in order to facilitate the commercialization of the findings produced by researchers in U.S. institutions.\nI believe the idea of a third party organization, which is mentioned, could work very well. However, there could be limitations in terms of what universities (or researchers) are willing to share with an outside group, depending on how the agreement terms are formulated. Therefore, it may be useful to also consider other individuals within universities who could be involved in implementing reforms in the technology transfer process from the inside. I also wonder how to ensure that the third party organization will associate with the institutions that are most in need of this partnership, instead of linking to the already high performing institutions, the latter which could in fact ensure their own success. To this end, these agreements would need to be carefully crafted with the interest of both parties in mind.\nAt the same time, multiple parties should contribute to the implementation of such reforms, including those outside the university. Perhaps it would be useful to add into the current Figure 1 some suggestions at every step along the way, indicating where improvements could be made and by whom (i.e. which stakeholder). This broader analysis might also help with further discussions of the types of changes which should be implemented in universities in order to ensure that all U.S. institutions have the same resources and as the currently high-performing institutions in technology transfer, therefore aiming to reduce some of these disparities and create a more uniform way to analyze this problem across the board. In addition, as multiple groups are likely to be involved in this process, perhaps it would also be useful to expand this discussion by adding in more details on the efforts already undertaken successfully by each of these groups which could help reduced these disparities. For example this could include direct links to such efforts, with clear instructions and concrete actions which other universities could easily adopt and implement locally.\nSpecific suggestions: Overall, the motivation for each part of the study and the actual results are very clearly explained and are insightful. I was particularly intrigued by the findings in Table 1, and the idea that performance of institutions in such these areas can be measured. This is encouraging to see in thinking more broadly about the variables that can be quantified from U.S. institutions. One suggestion is that it may be useful, after each result, or perhaps in the discussion section, to include further explanations of how each of these findings contributes to our broader understanding of academic technology transfer. This might also lead to an obvious list of particular weakness in the commercialization pipeline that must be corrected across institutions.\nGetting more into the data itself, with respect to Table 1, I understand the top performing institutions are ranked as high performing in all of the presented categories. However, there are a few institutions in the list that I would consider high performing academically, but which have been indicated to perform in a variable manner in some of the particular areas examined within the commercialization pipeline. I am wondering whether more discussion on these “gray area” institutions could be added, and how their particular performance in a given area translates to benefiting the economy itself. For example, does high performance in certain variables lead to benefits in the local economy, and is high performance in all of these areas required to benefit the U.S. economy as a whole, or is this a more mixed population?\nAlong these lines, in thinking about the differences in available resources for technology transfer between U.S. universities, I also wonder if it is possible to perform a type of analysis that would take into account this difference and measure performance in a way that is dependent upon the available resources. For example, a particular university may be considered successful overall for their local area given the limited amount of resources, depending on how this analysis is performed and analyzed. However, if we only consider universities that are high performing overall in all categories, it may be more difficult to analyze the individual success of those institutions who only show high performance in certain areas.\nOverall, I believe Table 2 contains a wealth of information that could be further analyzed and discussed to draw additional conclusions from the data presented here, including potentially by examining the entire dataset of 300 institutions, which could add another layer of depth and complexity to these findings.\nThe authors also mentioned that this analysis was performed using 2010-2014 AUTM Licensing Survey data. As this survey data is being reported every year, it may be interesting in the future to expand beyond this timeframe and examine more general trends in the factors presented here over a longer period of time (for example in the last 10 years). That kind of analysis could also provide more insights into the landscape of technology transfer as a whole, and highlight particular recommendations that could be made (or maybe have already been made) to improve the technology transfer system in the U.S. Finally, if available, obtaining and sharing the raw data from the Survey itself may also allow other individuals to perform their own additional analyses of interest.\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": "32093",
"date": "09 Apr 2018",
"name": "Evan Facher",
"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 tackle a very relevant question virtually all technology commercialization organizations at academic institutes have – what can they do to enhance translation of innovations developed at their organizations. In order to assess the current situation and provide some thoughts on approaches to ameliorate the challenges their colleague institutes have, the authors took a well reasoned, data intensive approach to examine a large set of information. The AUTM data is a valuable source of self-reported figures and a large amount of it was assessed to reach the presented conclusions. The design was appropriate and relevant to answer the hypotheses posed in this article. Further, a fairly robust statistical analysis was performed. Given the data is available (for purchased access) to any institute of higher learning, the analyses described in the paper can be readily replicated, and potentially expanded on, by any group that seeks to build on it.\n\nThe conclusions drawn as a result of this assessment are supported by the data and provide multiple areas for practioners of technology transfer to examine further for implementation. I believe the manner in which the authors broke down the commercialization pipeline was sound and is a good model for others to use in assessing their own processes. The only suggestion I would offer is that universities are increasingly focusing on software licensing, as a result, not all translation will be of patented innovations but rather copyrights. As such, there may be additional information that can be garnered for these inventions. One additional area that may be interesting to include for the authors if they chose to eventually expand on the analysis beyond this submission is understanding the disparity of human resources at the AUTM schools to understand how much, if any, this specific infrastructure component is tied to commercialization activity and AUTM metrics.\n\nIn conclusion, I enjoyed the opportunity to review this article and believe it adds to the literature on technology transfer. A significant amount of data was used to identify recommendations that align with opportunities most organizations can adopt.\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": "32326",
"date": "11 Apr 2018",
"name": "Dipanjan Nag",
"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\nReferee #1\nThe authors correctly point out that this is a skewed industry, with the top performing schools in any given category contributing more than their share of returns. (Page 1) The selection of top 10 institutions in the initial analysis and then expanding to top 25 institutions is somewhat arbitrary. (Page 3) This data is coming from institutions of all sizes that vary according to the research dollars as well as the overall size of institutions. For an objective analysis the data needed to be normalized along those lines. (Page 3) Some of the stages described by the authors in Figure 1 are metrics that can be manipulated. For example the number of patents applied for and issued really are not metrics of measuring efficiency or impact of an office. Large number of patents can be applied for and granted, the real measure of performance will be the number of patents that are licensed. (Page 3) The approach of creating a composite metric is a good one, rather than just looking at the revenue number. The Milken study is another one that has utilized this approach and is an objective measure. The difference is that the Milken study normalized the data, as well as provided the weighting of each factor. (Page 3) In most cases the large revenue numbers can be attributed to a single large license or a monetization activity from a university. The weighting of that single license will skew the result enormously. Combined with the lack of data normalization, the results will not be reflective of the true impact to technology commercialization from universities. The weighting factor for each of the \"steps\" and how they overall impact the ranking is unclear. The weighting of the input metrics vs. the output metrics cannot be the same, hence the rankings would change quite radically. That explanation should be very clearly pointed out. (Page 4) The majority of institutions here are based on their size and hence a normalization is needed to avoid picking institutions by size only. Smaller schools often times are more efficient. In looking at Table 1 their analysis shows the 1% and the top 19% percent of the institutions. There are 300 institutions in the survey. Why did they pick 25 to rank? In addition Table 1 shows that the top 20% account for 60% of results. Why didn't they cite the top 60 institutions. It would seem logical to look at the top 20%. (Table 1)\n\nThe sharing of best practices has existed for decades in the very collaborative technology transfer community. The AUTM Leadership Conference is precisely that effort, along with the LES IP100 meeting. The fact is that, it is not easy to solicit inventions from faculty. It takes years if not decades to get them on board with the idea that commercialization is a good thing. (Page 6) These programs are ideal in nurturing an \"ecosystem\" for technology commercializatin. The authors are absolutely right about taking a holistic approach rather than just trying to increase disclosures, patents, startups or revenues. (Page 6) The whole idea of hiring consultants to fix technology transfer does not work. I have personally been a consultant and a technology transfer professional for many years. The notion of fixing tech transfer through consultant is not economically viable. Consultants most of the time do not have the necessary understanding of the university structure and functions. Many aspects of technology transfer will not have economic impact or metrics based impact, but they need to be done nonetheless. Analogy of hiring consultant would be like replacing full time and dedicated servicemen with mercenaries. Having said that, hiring of consultants for focused engagements is highly recommended and successful offices do that often times in areas such as patent prosecution, valuation, licensing comparable, or market analysis. If there are significant examples that the authors can cite to support the replacement of technology transfer offices with consultants, that would cause readers to consider such an approach. Short of that authors should either reconsider adding this to the article or reframing the recommendation. (Page 6) This model has been tried in Japan, where both TTOs and TLOs existed. The model failed miserably. Not only is this model not economically viable, but it creates an atmosphere of trying to create a consortium out of disparate companies like Amazon, Google, Ford and IBM. Some consortiums on a limited scope such as a particular technology focus, e.g. IoT, with a few institutions could work. Again, the authors should cite specific examples that clearly demonstrate the validity of the proposed model otherwise reconsider adding this as a recommendation. (Page 6) The picking of two institutions which are ranked according to the authors at #3 and #19 does seem highly biased analysis. Additionally, the programs that the authors have described are student and faculty programs. Technology transfer performed by universities across the board and the data reported by AUTM do not take into account any student programs. Hence, the impact that authors have contemplated at each stage are possibly not accurate. Suggest the inclusion of universities from different \"ecosystems\" as well as exclusion of student programs from the analysis. (Page 7) From a practical standpoint this model will be at best difficult if not impossible with the interest of the parties diverging because of a simple reason, royalty stacking. Each inventor or assignee wants to have the highest value for their portion of the invention. That is just one factor from an economic standpoint. There are many other intangible factors that would be barriers. Anyone who has setup consortia can attest to these challenges. (Page 7) Overall, the article has significant challenges: 1. Authors are predominantly from the Boston area and are analyzing their own institution and another institution which is in close proximity. There are more than 300 institutions and a number of thriving \"ecosystems\". This is a significant bias and in some ways looks more like a marketing piece as opposed to an objective article. The reason the two institutions are successful could be because of the existence of industry players, local VCs, selective admission of students at two private colleges, and many other factors.\n\n2. Their analysis seems to have consisted solely of sorting the AUTM data, not much analysis (interviews, or other supporting data) about the drivers of those outcomes. Hence, they don’t show any support for their statements about technology transfer offices being under performing.\n3. The recommendations or solutions that the authors are proposing are not supported by any data or examples and are simply anecdotal. If such models have existed and worked then analysis of those models would be helpful.\n\n4. As we all know the Milken study was published with different results than the authors present. They do not reference the Milken study or explain the differences. Other articles which have attempted to perform similar analysis have cited the Milken study. (e.g. Mature Biotech Entrepreneur in 2014)\n5. Technology transfer is indeed a complex interplay of a various factors that are not always easy to understand. The intangible factors such as location, concentration of research areas, public versus private institution, can all have impact that cause significant disparity in the results. Authors need to consider analyzing or at least mentioning some of those factors in this article. (Page 8 - Conclusion)\n\nReferee #2\nThe years 2010-2014 is a small, five-year sample of data, from which broader trends and conclusions are difficult to draw. The industry moves and changes rapidly, so the 2014 data (which is FY2014, or July 2013 – June 2014 for most institutions), is a relatively old data set. Why were only the top 10 in each category of the Commercialization pipeline included in the MRR analysis? The parameters identified in the Commercialization pipeline (Figure 1) to measure the health of a tech transfer office are, in many instances, inadequate or irrelevant. If one is trying to measure the health of a university (not simply its TTO), then one might use research expenditures as a reasonable variable. However, this reviewer fails to see the direct correlation between an institution’s research expenditures and health of the TTO. Furthermore, technology commercialization is a non-linear, dynamic process. The notion that commercialization proceeds from left-to-right (i.e. research expenditure-invention-patent-license-startup-revenue) is incomplete. Similarly, with respect to patent applications and patents issued, such numbers depend heavily on an individual institution’s IP strategy. For example, California Institute of Technology, files provisional patent applications on nearly every invention disclosure that is received. How does such a policy speak to the health of the TTO? Licenses and options are certainly one reasonable metric that can be used to assess the health of a TTO. However, all licenses and options are not created equal. Some universities will license 30 technologies or patents in a single license, and some will license the same 30 technologies or patents in 30 different licenses. Is the second university healthier than the first? The authors have failed to address or account for differences in quality of licenses and options between universities. The quantities by themselves don’t paint an accurate picture. For the Startups metric, the authors should address, again, the quality of the startups coming out of the universities. Some universities create large numbers of startups in a given year, but some of those may not be legitimate, growth-oriented startups with qualified management. Adjusted Gross Income should not be used as a metric to measure the health of TTOs. The TTO has no control over this value. In the Results section on page 4, I find it disappointing that the authors go through so much analysis to demonstrate the truth of the Pareto principle in technology transfer. The fact that 20% of the universities contribute to 60% of “total commercialization activity” is entirely unsurprising. Again, in the Results section on page 4, “Highly performing institutions”, I don’t see that the data were normalized for research expenditures. If so, the analysis needs to be rerun. Furthermore, the authors need to remove the University of California, University of Texas, and University of Maryland systems because those systems report to AUTM statewide. Alternatively, the authors could retrieve any desired data from the individual institutions in those states. I am not clear on the analogy between the Gini coefficient of patents and US household income. How do the authors conclude that a 60% Gini coefficient for patents means that universities have significant untapped commercialization potential? Page 5, “Improving the pipeline”, along with Table 2, reads more like an advertisement for Harvard and MIT, particularly the Biotechnology Clubs of which some of the authors are members. The paper would be strengthened if they identified activities conducted outside of Boston-based universities that are contributing unexpected positive results to the tech transfer ecosystem.\n\nDiscussion section comments\nSharing best practices across institutions is a common and frequent activity in the university tech transfer community. Organizations like AUTM exist for such a reason. Where do the authors propose universities obtain resources to promote additional commercialization activity? Do the authors have any data to support one of their proposed solutions, the outsourcing of technology transfer to consultants?\n\nOverall questions and comments\nSpecifically, outside of Harvard and MIT, what are institutions in the top 25 doing that other institutions can replicate to improve their TTOs? Are the authors aware of similar programs at other universities as those highlighted in Table 2? Have the authors considered non-traditional factors that may contribute to the success of TTOs, such as overall university mission, geographic location, appetite for risk, patience for technology development timelines, etc.?\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? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-329
|
https://f1000research.com/articles/7-324/v1
|
15 Mar 18
|
{
"type": "Research Article",
"title": "Improving paper-based discharge process; a continuous full-cycle quality improvement project in low resource setting",
"authors": [
"Ihab B Abdalrahman",
"Mohammed Elsanousi Huzaifa Mohammed",
"Abdelmohaymin A Abdalla",
"Sulaf Ibrahim Abdelaziz",
"Aboaagla Abdalbagi Ali",
"Dina Nader Mutwakel Osman",
"Ahmed Abdelmoniem Ahmed",
"Hagir Emad Elwasila",
"Romaisa Hashim Mokhtar Mohammed",
"Mohamed Dafaalla",
"Ihab B Abdalrahman",
"Mohammed Elsanousi Huzaifa Mohammed",
"Abdelmohaymin A Abdalla",
"Sulaf Ibrahim Abdelaziz",
"Aboaagla Abdalbagi Ali",
"Dina Nader Mutwakel Osman",
"Ahmed Abdelmoniem Ahmed",
"Hagir Emad Elwasila",
"Romaisa Hashim Mokhtar Mohammed"
],
"abstract": "Background: The moment of hospital discharge is a time for vulnerability for many patients and might jeopardize their safety. We found that the current structure of the discharge card at Soba University Hospital (SUH) does not improve the quality of the discharge summary. This hinders the delivery of valid, relevant and adequate health information and can negatively affect outpatient care.\n\nMethods: We implemented a new discharge card design with structured headings at the Department of Medicine at Soba University Hospital from the beginning of March to April 15th, 2017. This was coupled with educational sessions highlighting the problems that might occur if there were gaps in patient transition from inpatient to outpatient. Results: There was a significant improvement in documentation of the majority (>90%) of the items, including name, age, source of admission treating doctor, diagnosis and medication, but there was a drop in documentation of comorbidities. We also noticed that the new discharge summary format significantly improved the documentation of the majority of the headings (all P values were <0.001), yet, there was a drop in documentation of comorbidities and dates for follow up. Conclusions: Recording of paper-based health records like discharge summaries could be substantially improved by use of well-structured formats and practical training sessions. Improvement is a dynamic process. Some gaps might appear during execution, these need monitoring and continuous improvement to establish sustainability.",
"keywords": [
"discharge summary",
"audit",
"SCAR",
"Soba University Hospital"
],
"content": "Introduction\n\nHospital discharge describes the point at which inpatient hospital care ends, with ongoing care transferred to other primary, community or domestic environments. This transfer process requires documentation of the medical encounter to be sent to the receiving services, such as primary care providers and referral clinics. The discharge process is a complex one and requires coordination of several departments1. The moment of hospital discharge is a real time of vulnerability for many patients. Missing important information or sharing inaccurate information might lead to unintended omission or delay of necessary treatment, or to duplication of work. A major concern is the negative effect on safety and quality of health care. In one study, 19% of patients had adverse events after discharge, 31% of them were preventable adverse events2,3.\n\nHoyer et al. reported in a retrospective study that a delay in completion of the discharge summary was associated with higher rates of readmission4. In some healthcare settings the discharge card is sent directly to the primary care provider or the receiving facilities. In other settings, the discharge summary is handed to the patient following the discharge process, and subsequently most of the information will accompany the patient along with the transfer of medical care. This highlights the importance of effective, highly informative, and patient-centered discharge cards. The card serves as the main tool of communication between healthcare professionals in different healthcare facilities. Many interventions are suggested to ensure this safe transition of care5; including education and training of medical staff, designing universal discharge protocol and medication reconciliation; and involving patients and their relatives in the discharge process6,7. Some facilities use an electronic discharge summary and computer-based innovations to improve the discharge process, which include notifications of pending tests at discharge. This has been challenged by the cost and ability of end-user to utilize8. Some organizations (like the Royal College of Physicians) created discharge cards with top level headings for medical records to help guiding clinicians in the discharge process.9 They are thought to improve doctors' performance and have a pronounced effect in patient outcome9,10.\n\nThe audit team at Soba Center for Audit and Research (SCAR) conducted an audit evaluating the quality and content of discharge cards (11). We found that a significant proportion of the discharge summaries are not written in the structured-predesigned format of the discharge card, which halts the delivery of valid, relevant and adequate health information. We concluded that the current structured format does not improve the quality of the discharge summary11. We implemented a new discharge card design to improve the reporting of discharge summaries. In this report we aimed to assess the impact of the implemented interventions and compared the quality of the old cards to the new ones (old and new discharge cards can be found in Supplementary File 1 and Supplementary File 2 respectively).\n\n\nMethods\n\nWe evaluated the quality of information documented in Soba University hospital discharge cards in two separate audit cycles. The study was conducted at Soba University Hospital (SUH). The first cycle included 146 discharge summaries and was done in April 2014 and the second cycle included 65 summaries and was completed between March 1st and April 15th, 2017. The audit team consisted of residents from the department of internal medicine, medical officers from the Soba Center for Audit Research (SCAR), supervised by consultant physicians. The discharge summaries that were analyzed were written for patients discharged from the Department of Internal Medicine at the Soba University Hospital.\n\nThe first audit cycle showed inadequate documentation of information in the discharge summary11. The audit team reviewed the literature, conducted several meetings and assessed local environmental factors. This resulted in restructuring of the discharge card format with appropriate headings, based on a standard cards designed by NHS-UK, used as a benchmark9. As a result, we applied structured educational sessions and changed the format of the discharge summary as the primary interventions to improve documentation. The sessions were delivered in May 2017 and included demonstration and training on effective documentation in discharge summaries. The sessions were delivered by the audit team and candidates were Soba University hospital trainees (registrars and foundation doctors). After the interventions were applied, we reevaluated the quality of the discharge summary documentation in the second cycle.\n\nThe study was approved by Soba Center for Audit and Research SCAR at Soba University Hospital. Consent was waived by the research committee because the study is a quality improvement project that doesn’t involve human subjects. Data was extracted from the discharge summaries using a structured preformat and formulated as present or absent. Incomplete data that needs seeking information outside the discharge summary were considered absent.\n\nWe used Microsoft excel 2016 for statistical analysis to compare between the two cycles. We calculated the frequencies and percentages of availability of mandatory information that should be available in the discharge summaries initially and after applying the intervention to assess the impact of intervention plan. We used SPSS v22 to calculate the P value by applying one sample t-test.\n\n\nEthical statement\n\nThe audit was approved by SCAR at Soba University Hospital. Consent was waived by the research committee because the study is a quality improvement project that doesn’t involve human subjects.\n\n\nResults\n\nThe results showed that there was universal improvement in all aspects of documentation of the discharge summary (Table 1). Age, date of discharge, drug name, duration, and investigations were documented in more than 90% of summaries. Drug-related data and in-patient management are illustrated in Figure 1 and Figure 2, respectively. Table (2) shows differences in follow-up data documentation between old and new cards. Although there was remarkable improvement in documentation of telephone numbers and action plans, only around half of the summaries contained this information. We also noticed that the new discharge summary format significantly improved the documentation of the majority of the headings (all P values were <0.001), yet, there was a drop in documentation of comorbidities and dates for follow up.\n\n\nDiscussion\n\nThe aim of this study was to maintain patient safety at the time of transfer of care, by improving documentation in the discharge summary card. Previous research that we conducted (11) showed variability in documentation of different headings, with the majority being deficient. The variability of the card documentation made the interpretation of the card more difficult. In fact, the information on the headings, including gender, mode of admission, diagnosis, name of follow up clinician, future plans, drug duration, and drug route are absent in >60% of discharge cards. As a result, we concluded that the quality of the previous discharge summary format was not at an acceptable level.\n\nDocumentation of patient telephone numbers increased to 51.6% with the new discharge cards, compared to 0.7% with the old discharge cards. Although this shows a significant improvement, it does not meet our goals. It affects our ability to follow up those patients and to notify them about important issues. Claassen et al. reported that utilizing telephone-based follow-up procedures can maximize patient data retention in longitudinal studies12.\n\nThe exact date of follow-up was not documented in 35% of the cards. We attribute this result to the fact that some patients were discharged at the end of the day or after hours. It might be difficult for the discharging doctor to set an appointment. This reflects gaps in communication between in-patient team and out-patient departments; making future plans of follow-up not clear at time of discharge. Corrective intervention will be explored in future plans. Previous studies have suggested that adverse events complicate 1 out of every 5 hospital discharges, in the Medicare (a national social insurance program administered by the U.S. federal government) population alone, 19.6% of patients dismissed from the hospital will be readmitted within 30 days and 34.0% of patients will be readmitted within 60 days13. Good discharge planning and good communication tools between the healthcare facilities, healthcare providers and patients are needed to streamline a safe discharge process.\n\nAlthough we were able to induce substantial change by implementing a new well-structured format and effective educational session, maintenance of satisfactory level of documentation may be challenging especially in a dynamic hospital environment. The service at the department of medicine at SUH is led by consultants. Most of the work is done by registrars (residents in postgraduate training) and house staff (interns who just graduated). Central bodies assign registrars and house staff to hospitals; registrars are assigned by the Sudan Medical Specialization Board and interns by the Federal Ministry of Health. Interns usually stay in service for 3 months and registrars for 4–8 months. These are temporary workers, by the time they master the game they move to another facility or service with different structure and processes. This creates uncertainty regarding the reproducibility and transferability of activities and improvements related to working practices and calls for a universal or national implementation. We will continue to educate and monitor, and future plans may consider electronic records. However, electronic health records as the sole mode of communication in hospitals may be costly and impractical, particularly in low resource settings8.\n\n\nConclusions\n\nDocumentation of paper-based health records like discharge summaries could be substantially improved by use of well-structured formats and practical training. These structured formats should be tailored according to the hospital discharge plan. Monitoring, continuous education and proactive solutions are needed to achieve safe care and establish sustainability.\n\nWe recommend that managers and heads of departments at SUH as well as other faculties should be informed about the results of this work, and that deficiencies should be addressed by appropriate interventions. Moreover, we strongly encourage the program to be transferred to other departments, and advocate for other facilities to be designed.\n\n\nData availability\n\nDataset 1: Second cycle data and summary of the first cycle data 10.5256/f1000research.13359.d19541914",
"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: Old style discharge card.\n\nClick here to access the data.\n\nSupplementary File 2: New style discharge card.\n\nClick here to access the data.\n\n\nReferences\n\nAudit Commission: The Way to Go Home: Rehabilitation and Remedial Services for Older People. London Station Off. 2000. Reference Source\n\nForster AJ, Murff HJ, Peterson JF, et al.: The incidence and severity of adverse events affecting patients after discharge from the hospital. Ann Intern Med. 2003; 138(3): 161–167. PubMed Abstract | Publisher Full Text\n\nKripalani S, LeFevre F, Phillips CO, et al.: Deficits in communication and information transfer between hospital-based and primary care physicians: implications for patient safety and continuity of care. JAMA. 2007; 297(8): 831–41. PubMed Abstract | Publisher Full Text\n\nHoyer EH, Odonkor CA, Bhatia SN, et al.: Association between days to complete inpatient discharge summaries with all-payer hospital readmissions in Maryland. J Hosp Med. 2016; 11(6): 393–400. PubMed Abstract | Publisher Full Text\n\nRoyal College of Nursing: Discharge planning: A summary of the Department of Health’s guidance Ready to go? Planning the discharge and the transfer of patients from hospital and intermediate care. Dep Heal. 2010; 16.\n\nWaring J, Marshall F, Bishop S, et al.: An ethnographic study of knowledge sharing across the boundaries between care processes, services and organisations: the contributions to ‘safe’ hospital discharge. Heal Serv Deliv Res. 2014; 2(29): 1–160. PubMed Abstract\n\nLaugaland K, Aase K, Barach P: Interventions to improve patient safety in transitional care--a review of the evidence. Work. 2012; 41(Suppl 1): 2915–24. PubMed Abstract | Publisher Full Text\n\nDalal AK, Poon EG, Karson AS, et al.: Lessons learned from implementation of a computerized application for pending tests at hospital discharge. J Hosp Med. 2011; 6(1): 16–21. PubMed Abstract | Publisher Full Text\n\nRoyal College of Physicians - Health Informatics Unit: RCP Approved ‘Generic Medical Record Keeping Standards’. Clin Med (Northfield. Il). M: 2007; 3–4.\n\nCarpenter I, Ram MB, Croft GP, et al.: Medical records and record-keeping standards. Clin Med (Lond). 2007; 7(4): 328–331. PubMed Abstract | Publisher Full Text\n\nDafaalla MD, Abdalrahman I, Kheir A, et al.: Adequacy of the discharge summary at Soba university hospital: where are we now? Online J Clin Audit. 2014; 6(4): 11. Reference Source\n\nClaassen C, Kurian B, Trivedi MH, et al.: Telephone-based assessments to minimize missing data in longitudinal depression trials: A project IMPACTS study report. Contemp Clin Trials. 2009; 30(1): 13–19. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJencks SF, Williams MV, Coleman EA: Rehospitalizations among Patients in the Medicare Fee-for-Service Program. N Engl J Med. 2009; 360(14): 1418–1428. PubMed Abstract | Publisher Full Text\n\nAbdalrahman IB, Mohammed MEH, Abdalla AA, et al.: Dataset 1 in: Improving paper-based discharge process; a continuous full-cycle quality improvement project in low resource setting. F1000Research. 2018. Data Source"
}
|
[
{
"id": "38605",
"date": "08 Oct 2018",
"name": "Pratap Kumar",
"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\nSummary: The article builds on an important recognition that current paper-based discharge forms do not contain information required for expected quality of post-discharge care and its coordination. The authors have designed an intervention to improve documentation in discharge forms, which includes a re-design of the form, and educational sessions for physicians who fill the forms. While the intention is clear, the study has some significant limitations in its executions and/or reporting:\n1. The theory of change for their implementation of \"a new discharge card design to improve the reporting of discharge summaries\" is not clear - how and why were the changes made (both the restructured form and the educational sessions) expected to improve reporting? 2. Introduction/referencing: The approach for the redesign is not adequately described. 3. Ethical issues: Discharge forms can and often contain identifiable patient information. The authors should describe how the data was collected, stored and analysed while ensuring safety and confidentiality of the data. 4. Methods: How did the old and new forms differ? The forms are central to the study, but the authors do not describe how they differed. 5. Methods: The data appear to be from two samples (old and new discharge cards), but comparisons were analysed using a one sample t-test. This appears wrong. 6. Methods: Details on the intervention (dates and numbers of cards analysed, etc) provided are inadequate. 7. Results: Different parts of the form (demographics & identifiers, drug-related information, in-patient management, follow-up) are presented in separate tables and figures. The reader does not get an overview of how the old and new forms were compared, under which categories, and how what numbers of cards were analysed for each category. Ideally the numbers of discharge summaries analysed and the p-values of the differences should be presented along with the percentages. 8. Discussion: The authors present results from a previous study in the first paragraph, which is confusing. 9. Discussion: The statement \"Although this shows a significant improvement, it does not meet our goals\" refers to a goal that is not described or justified. 10. Discussion: The negative results (decrease in documentation of co-morbidities and date of follow up) are not discussed. These are clearly important information that can affect quality of follow-up.\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? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-324
|
https://f1000research.com/articles/7-317/v1
|
14 Mar 18
|
{
"type": "Study Protocol",
"title": "Repeated sessions of bilateral transcranial direct current stimulation on intractable tinnitus: a study protocol for a double-blind randomized controlled trial",
"authors": [
"Arash Bayat",
"Miguel Mayo",
"Samaneh Rashidi",
"Nader Saki",
"Ali Yadollahpour",
"Arash Bayat",
"Miguel Mayo",
"Samaneh Rashidi",
"Nader Saki"
],
"abstract": "Background: Transcranial Direct Current Stimulation (tDCS) is reportedly a potential treatment option for chronic tinnitus. The main drawbacks of previous studies are short term follow up and focusing on the efficacy of single session tDCS. This study aims to investigate the therapeutic efficacy, adverse effects (AEs) and tolerability of repeated sessions of bilateral tDCS over auditory cortex (AC) on tinnitus symptoms Methods: This will be a double-blinded randomized placebo controlled parallel trial on patients (n=90) with intractable chronic tinnitus (> 2 years) randomly divided into three groups of anodal, cathodal, and sham tDCS. In the sham treatment, after 30 sec the device will be turned OFF without informing the patients. The tDCS protocol consists of 10 sessions (daily 20 min session; 2 mA current for 5 consecutive days per week and 2 consecutive weeks) applied through 35 cm2 electrodes. The primary outcome is tinnitus handicap inventory (THI) which will be assessed pre- and post-intervention and at one month follow-up. The secondary outcomes are tinnitus loudness and distress to be assessed using a visual analogue scale (VAS) pre-intervention, and immediately, one hour, one week, and one month after last stimulation. The AEs and tolerability of patients will be evaluated after each session using a customized questionnaire. Possible interactions between the disease features and treatment response will be evaluated.\n\nDiscussion: To our knowledge this is the first study to investigate the effects of repeated sessions of tDCS on chronic tinnitus symptoms with one month follow-up. In addition, the AEs, and tolerability of patients will be studied. In addition, the possible interactions between the disease specific features including the hearing loss, laterality, type of tinnitus, and treatment response will be evaluated.\n\nTrial registration: The study has been registered as a clinical trial in Iranian Registry of Clinical Trial (IRCT2016110124635N6) on the 01/06/2017.",
"keywords": [
"Transcranial direct current stimulation",
"Repeated sessions",
"bilateral",
"Intractable chronic tinnitus",
"Tolerability",
"adverse effects"
],
"content": "Abbreviations\n\ntDCS: transcranial direct current stimulation; AC: auditory cortex; DLPFC: dorsolateral prefrontal cortex; THI: tinnitus handicap inventory; BDI-II: Beck Depression inventory; BAI: Beck Anxiety Inventory; dBHL: decibels hearing level.\n\n\nBackground\n\nTinnitus is a subjective auditory phantom perception without an external physical sound source1, which exists in different forms including pulsatile or continuous, buzzing, ringing, hissing, tone, or a combination of them2. It is a relatively common disorder with 10–15% prevalence among adults worldwide. Tinnitus is a debilitating condition which severely affects the quality of life. It is accompanied by different comorbidities such as anxiety, depression, and sleep disturbances3.\n\nThere is no definitive treatment for tinnitus and a large proportion of tinnitus become intractable1,3. During the recent years, different non-pharmacological techniques have been developed for treatment of chronic tinnitus including cognitive behavioral therapies, hearing aids, neurofeedback, and noise-masking techniques4–6. Although some of these techniques have shown therapeutic outcomes, the therapeutic efficacies of these techniques are limited and studies to develop more efficient techniques are ongoing.\n\nTranscranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique and recent studies have reported promising therapeutic outcomes in improving cognitive functions in healthy individuals7 as well as symptoms of different neuropsychiatric disorders such as depression8, obsessive compulsive disorder9 auditory-verbal hallucinations10, and chronic pain11. Several studies have investigated the therapeutic efficacy of different tDCS protocols in chronic tinnitus11–15. Results were in general promising, despite some controversial findings. In treating tinnitus with tDCS, there are two main targets as the site of stimulation including the dorsolateral prefrontal cortex (DLPFC)16,17, and the auditory cortex18–20. The neural anomalies, hyperactivity, or maladaptive plasticity of the primary and secondary auditory cortices have been reportedly the main causes of tinnitus loudness and disturbances in non-auditory structures and networks such as anterior cingulate cortex, and particularly DLPFC are the main causes of distress or annoyance experienced in tinnitus2,3,21. The main hypothesis for therapeutic application of tDCS in tinnitus is disturbing ongoing abnormal neural activities responsible for tinnitus. According to this approach, the areas involved in the pathophysiology of tinnitus are targeted by tDCS. The other hypothesis of eliciting therapeutic outcomes from tDCS is inducing plastic changes to alter the maladaptive plasticity of the involved regions. The important point for treatment of tinnitus is that there is a large network consisting of different auditory and non-auditory regions with overlapping functions22–24.\n\nMost of the studies performed to date, have focused on the effects of single tDCS session on tinnitus symptoms, with short term follow ups ranging some hours to some days. To our knowledge, there is no published study investigating the effect of repeated sessions of tDCS over the auditory cortex (AC) on tinnitus symptoms as well as the AEs of and tolerability to the tDCS with one month follow up. This study aims to investigate the effects of repeated sessions of tDCS on tinnitus symptoms with one month follow-up. Our main hypothesis is that repeated sessions will lead to aggregative therapeutic effects on tinnitus. In addition, we want to evaluate possible association between treatment response, and patient's and tinnitus specific characteristics including gender, THI basal score, laterality, tinnitus quality, duration, hearing loss class, etc.\n\n\nMethods/design\n\nThis is a part of a larger project which is designed to comprehensively investigate the efficacy of different tDCS protocols at different sites of brain for treatment of intractable chronic tinnitus. In this double blinded placebo controlled randomized trial will be conducted on intractable tinnitus patients (n=90) who will be randomly divided into three groups of anodal (anode/cathode on left/right AC), cathodal (cathode/anode on left/right AC, and sham (anode/cathode on left/right AC) tDCS. In the sham treatment, after 30 sec the device will be turned off without informing the patients. The experimental procedures of the study including tDCS interventions and the evaluations of the outcomes will be performed in the Bioelectromagnetic Clinic in Ahvaz Imam Hospital, Ahvaz, Iran. Patients with intractable chronic tinnitus (> 2 years) will be enrolled in this study. This is a single-center, non-stratified, with balanced randomization [1:1:1], double-blind, placebo-controlled, parallel-group study conducted in Bioelectromagnetic Clinic, Imam Khomeini Hospital, Ahvaz, Iran. Allocation concealment will be performed by random allocation cards using computer-generated random numbers. The patients will be randomly assigned into three groups of cathodal, anodal, or placebo tDCS.\n\nAll of the experimental procedures of this study were approved by the local ethics committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (registration code: IR.AJUMS.REC.1394.639) which were in complete accordance with the ethical standards and regulations of human studies of the Helsinki declaration (2014)25. After enrolment and before the start of the study, researcher will clearly explain the experimental procedures, the objectives, possible benefits, and side effects of the study to the patients and then all participants will fill and sign a written consent form for contribution in the study. The study was registered as a clinical trial in the Iranian registry of clinical trials (IRCT2016110124635N6).\n\nThe patients should meet specific criteria for inclusion in this project. The primary criteria will be evaluated during the neurological and otolaryngological examinations by experienced specialists. The patients will be selected among the tinnitus patients referred to the Tinnitus Clinic in the Khuzestan Cochlear Implant Center, Ahvaz, Iran. After the start of the study and anytime during the study, patients have the right to leave the study. In case of any significant adverse effects (AEs), the procedures for the respective patients will be terminated.\n\nInclusion criteria\n\n• Idiopathic chronic and medication-resistant tinnitus (more than 2 years)\n\n• Age range of 18 to 70 years old\n\n• No use of medications at the time of intervention\n\nExclusion criteria\n\n• History of epileptic seizures, brain trauma\n\n• Severe psychotic and psychiatric disorders\n\n• Concurrent severe vertigo\n\n• Meniere's disease\n\n• Severe organic comorbidity\n\n• Using pacemaker or defibrillator\n\n• Present pregnancy, neurologic disorders such as brain tumors,\n\n• Concurrent treatment for mental disorders.\n\nAll prospective subjects will undergo complete audiometric and neurologic examinations by experienced specialists. The experimental procedures of the study including tDCS interventions and the evaluations of the outcomes will be performed in the Bioelectromagnetic Clinic in Ahvaz Imam Hospital, Ahvaz, Iran.\n\nThis study is a double-blind randomized controlled clinical trial design and patients will be randomly assigned into three groups of anodal and cathodal tDCS and sham tDCS. The random allocation will be performed based on balanced randomization [1:1:1] where the allocation concealment will be applied by random allocation cards using computer-generated random numbers. The three groups will be matched for age, gender, ethnicity, and audiometric main characteristics. To reduce the procedure and subjective bias, the patients, the researchers who will evaluate the outcomes, and the researchers who perform data analyses will be blinded on the type of protocol. In addition, the blinding quality of the study will be assessed after completion of the intervention.\n\nThe direct current will be applied through a saline-soaked pair of surface electrodes (35 cm2) and delivered by a tDCS device which is a specially developed, battery-driven, constant current stimulator with a maximum output of 2 mA. The tDCS device used in this study will be an OASIS ProTM device by Mind Alive Inc (Edmonton, Alberta, Canada). Anodal and cathodal tDCS groups will consist of daily 20-min sessions of 2 mA current for five consecutive days per week and for 2 consecutive weeks (10 sessions in total). In anodal tDCS, the anode and cathode will be respectively centered over left AC (halfway T3 - F7) and right AC (halfway T4 - F8). In the cathodal tDCS the electrodes position will be reversed where cathode and anode will be respectively centered over left AC (halfway T3-F7) and right AC (halfway T4-F8). The site of electrodes will be selected according to the International 10-20 electroencephalography system. In the sham tDCS group, the electrode montage will be the same as the anodal tDCS, but the device will be turned off after 30 s without the knowledge of the participant. These parameters for sham stimulation were chosen based on previous reported findings where the perceived sensations of stimulation on the skin, such as tingling, usually disappear in the first 30 s of active tDCS23,26 (Table 1).\n\nNote: * 30 seconds after the start of the stimulation the device will be turned off without the knowledge of patient.\n\nThe previous studies on the safety and tolerability of tDCS have shown that the 2 mA current applied in 20 minutes are associated with no serious AEs24,26. The common reported AEs are itching or tingling sensation, mild headache or fatigue and local burns at the site of stimulation. However, most of the AEs and tolerability assessments have been performed on the tDCS were for single session ranging 20–30 minutes and current of 1 to 2 mA. In our study, we will use repeated sessions of tDCS (2 mA current for 20 minute, total 10 sessions, daily one session, five consecutive days per week and 2 consecutive weeks), thus the reported AEs in the previous studies may be intensified. Therefore, we will assess the AEs using a customized questionnaire (Supplementary File 1). The questionnaire will be performed during and after each tDCS session to record all the perceived AEs from the patients in all three tDCS groups.\n\n\nOutcome assessments\n\nThere will be different evaluations before the intervention. After enrollment, the patients will undergo complete audiometric and neurological assessments by expert specialties. The tinnitus severity for each patient will be determined through asking the patient and confirming the class by an expert otolaryngologist as buzzing, cicadas, high pitch whistling, hissing, humming, ringing, pulsating, thumping, and ticking. The tinnitus laterality and duration will be also determined.\n\nHearing assessments will be performed in an acoustically isolated chamber (ISO 8253–1:2010). Pure-tone audiometry will be performed using AC 40 dual channel Audiometer (Intracoustics Co., Denmark). The hearing thresholds will be recorded over the frequency ranges of 250 to 8000 Hz for air conduction and 500 to 4000 Hz for bone conduction pathways, using the modified Hughson–Westlake Method as per recommended by ANSI 199727. Pure-tone audiometry is considered normal when the hearing thresholds at all frequencies are below 20 decibels hearing level (dBHL). Hearing loss will be classified according to the type and degree28. The class of hearing loss in both ears will be assessed as normal hearing threshold (<20 dB), mild hearing loss (20 – 40 dB), moderate hearing loss (41 – 70 dB), severe hearing loss (70 – 90 db), and profound hearing loss (> 90 db). The hearing class is determined as the average of threshold in 250 Hz, 1000, 2000, and 4000 Hz. The possible interactions of the aforementioned variables and the amount of each primary or secondary outcome will be studied.\n\nThe primary and secondary outcomes will be measured in two main time points: pre- and post-intervention. The pre-intervention assessments of the outcomes will be performed at the first day of the intervention before tDCS stimulation (T1). There will be four time points of post-intervention assessments as T2, T3, T4, and T5 corresponding to the immediately after last session, one hour, one week, and one month after last tDCS session. Table 2 represents the SPIRIT schematic protocol of the study and timing of the assessments.\n\nNote: THI: tinnitus handicap inventory; AEs: adverse effects, -T1: one month before intervention, T0: one day before intervention, T1: 5 min before intervention, T2: immediately after last session, T3: at hour after last session, T4: one week, and T5: at month after last tDCS session. Real tDCS refers to anodal and cathodal tDCS groups.\n\nPrimary outcome. Tinnitus handicap inventory (THI) score will be the primary outcome. The THI is a 25-item questionnaire and commonly used as self-reporting inventory for disabling level of tinnitus. The THI items are grouped into three subscales: Functional (11 items), emotional (9 items), and catastrophic (5 items) subscales. Each item is three point Likert type scale with three alternative assessments: yes (4 points), sometimes (2 points), and no (0 point). The questionnaire yields scores for each subscale and a total score that ranges 0 to 100, where higher score means more severe tinnitus or a greater handicap29. The THI is widely used as the primary outcome measure in double-blind trials with a control group to assess the effect of pharmacological or non-pharmacological agents in tinnitus patients. The functional subscale items reflect the effect of tinnitus on mental, social, occupational, and physical functioning. The emotional subscale items probe the individual’s emotional reactions to the tinnitus and the catastrophic response items address whether tinnitus makes the respondent feel desperate, trapped, hopeless or out of control. In this study, the THI will be assessed prior to intervention, and immediately, and at one month after the last tDCS session. Response to treatment is defined as a reduction of equal or more than 20 points in THI score (Pre-treatment THI – Post-treatment THI ≥ 20). This cut-off value for treatment response to tDCS is generally considered as significant clinical improvement in the previous similar studies15,30.\n\nSecondary outcomes\n\nTinnitus loudness and distress. The tinnitus loudness and distress will be assessed using a numeral 0–10 visual analogue scale rating scale before intervention, and immediately, one hour, one week, and one month after last tDCS session. For tinnitus loudness and distress a 10 percent or 2 point reduction of 0–10 scale will be considered as treatment response31,32.\n\n\nBlinding assessment\n\nThe previous studies have shown that the experience of the sensations induced by tDCS are usually fade out after 30 seconds; thus, we will use this period in the sham tDCS group, to evaluate the blinding quality of the study design, after completion of the intervention we will ask the patients whether they know which type of stimulation, real or sham tDCS, they received. This assessment will be performed after each tDCS session in all groups.\n\nAdverse effects and tolerability\n\nPrevious studies have reported no severe AEs of tDCS for different protocols. However, most of the previous studies used single session tDCS. For this study, considering the multisession protocol of tDCS, the AEs of and tolerability to the repeated sessions of tDCS will be assessed using a customized questionnaire (Supplementary File 1) which will be applied after the last session.\n\n\nStatistical assessments\n\nAll statistical tests will be performed using SPSS (V20.0, IBM Corporation, New York, USA). To determine whether the anodal, cathodal, and sham tDCS groups are well-matched in terms of age, sex, quality of tinnitus, tinnitus laterality, THI score, and duration and class of hearing loss, these dependent variables will be compared between the three groups using independent samples t-tests.\n\nFor nominal data like gender and class of hearing loss, Chi-square tests will be conducted across groups. Independent sample t-tests were also used to determine if individual baseline measures differed between groups (THI, tinnitus loudness, and distress). Prior to conducting t-tests, Levene’s test for equality of variances was used. In no instance was Levene’s test violated; thus, equality of variances was assumed. The number of female and male participants and the quality of tinnitus symptoms, across each group will be compared using Pearson Chi-square tests. Mixed repeated-measure ANOVAs with the within‐subject factor time and the between‐subject factor stimulation were performed for evaluating changes in THI, tinnitus loudness, tinnitus distress, BDI and BAI scores. For the ANOVAs, sphericity was tested with the Mauchly’s test, and in the event of a violation of Mauchly’s test, the Greenhouse-Geisser correction was applied. In case of significant effects, follow-up post-hoc t-tests were carried out using LSD adjustments for multiple comparisons to examine if tDCS caused a significant difference relative to sham or baseline. An analysis of covariance will be performed with Time as within-subject factor, Stimulation as between-subject factor, and gender, laterality of tinnitus, duration of tinnitus as covariates. In addition, we will calculate Pearson’s correlations between change of THI and tinnitus loudness and distress values from pre- to post-stimulation.\n\nFinally, we will examine the number of responders versus non-responders under each treatment condition (anodal, cathodal, and sham). This will be accomplished by calculating change scores between the pre-treatment and post-treatment THI values (Pre-treatment – Post-treatment). Responders will be considered individuals who showed reductions in THI score of equal or more than 20 points in the THI score (Pre-treatment THI – Post-treatment THI ≥ 20). For tinnitus loudness and distress a 10 percent or 2-point reduction of 0–10 scale is considered as treatment response. Pearson’s chi-square tests will be used to evaluate whether there are differences in the proportion of responders versus non-responders under each condition. For all statistical tests the P-value < 0.05 is defined as statistically significant level.\n\n\nSample size calculation\n\nThe sample size of this study was calculated using the standard sample size formula (represented in Equation 1). The study of Shakhawat et al. was considered as the basic study to determine the sample size of our study with the predetermined power and level of significance. Considering the power of 85 percent and for confidence interval of 95 percent, the sample size of this study is 22 patients. We will use 25 patients in each group so that future attrition of the patients due to long term period of the tDCS sessions will be compromised.\n\n\n\n\nDiscussion\n\nRecent studies have shown beneficial effects of tDCS in improving cognitive functions in healthy individuals7 and also symptoms of different neuropsychiatric disorders such as depression8, obsessive compulsive disorder9, auditory-verbal hallucinations10, and chronic pain11. The therapeutic effects of tDCS in tinnitus have been reported by several studies; however, the findings are heterogeneous and controversial. In addition, most of the conducted studies have focused on single session tDCS, with short follow-up periods ranging some hours, to a few days13,16,33. The present study aims to investigate the therapeutic efficacies of repeated sessions of bilateral tDCS (anodal/cathodal over the left/right AC) on tinnitus symptoms, its loudness, and distress in patients with intractable chronic tinnitus. The main advantage of this study is that it is a double blinded which will remove the placebo effects of the tDCS. The other main advantage of this study is the one month follow-up which is relatively long period, compared to the previous studies. Moreover, we aim to evaluate the possible interactions between the disease specific features and treatment response to develop a more specific and disease oriented tDCS protocol. To our knowledge this will be the first study of repeated sessions of tDCS with one month follow-up. Considering the repeated sessions of tDCS in this study, we will investigate the AEs of and tolerability of the patients in this study.\n\n\nTrial status\n\nTo date, the enrollment of the patients has been performed and the allocation will be performed in the near future. The study is supposed to be started in end of January and finished in August 2018.\n\n\nDissemination of findings\n\nThe data collection of this study is supposed to be completed August 2018 and the results of this trial will be communicated to the external funding body through a formal report. In addition, the trial results will be communicated to the public through publishing as dataset and original research in the relevant scientific journals. There is no limit in the publication of the trial results.\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\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis study is financially supported by Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran (Grant No: HRC-9409). The AJUMS has no role in the design of the study, collection of data, analyses, and interpretation of data as well as in composing the manuscript. It, as the external funding body has reviewed the study in a double blinding peer review policy and it was approved by the editorial board member of the 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 would like to thank the staff of the Khuzestan cochlear center, Ahvaz, Iran for their assistance in performing the audiometric assessments.\n\n\nSupplementary materials\n\nSupplementary File 1: The customized questionnaire of adverse effects of and tolerability to Transcranial Direct Current Stimulation (tDCS). The assessments of AEs and tolerability will be performed during and after each session.\n\nClick here to access the data.\n\n\nReferences\n\nSanchez L: The epidemiology of tinnitus. Audiol Med. 2004; 2(1): 8–17. Publisher Full Text\n\nEggermont JJ: Pathophysiology of tinnitus. Prog Brain Res. 2007; 166: 19–35. 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}
|
[
{
"id": "31942",
"date": "23 Mar 2018",
"name": "Leonid S. Godlevsky",
"expertise": [
"Reviewer Expertise Neurophysiology"
],
"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\nProposed protocol of investigation aims for the clinical effectiveness of tDCS in patients suffered from resistant to current therapy tinnitus assessment. Both cathode and anode role is in interest to verify based on the double blind method exploration.\nHence, one month follow up along with filtering informative data from placebo effects and identification of adverse effects of tDCS are advantages of this protocol. Besides, two daily trials of tDCS during 5 days also points on the novelty of approach on the way of therapeutic potential of tDCS estimation in patients suffered from tinnitus.\nSuch advantages of the protocol correspond to perfectly described methods, design, criteria on inclusion and exclusion, study protocol and outcomes assessments. Technology of tDCS is clear and reliable; all steps are controlled by researches. Statistical assessments are explicit for data gained in observations. Authors carefully consider all adverse effects which might encounter in tDCS delivering and must identify in their investigation.\nTHI along with distress level will be used for the evaluation of the clinical effectiveness of tDCS. Meanwhile, background of the project contains data on quality of life (QL) worsening of patients with tinnitus. Despite QL is in good correlation with distress [1], it could be interesting to hear why just only distress was in scope and QL was not. QL was investigated by others cited authors just for estimation effectiveness of tinnitus treatment (N4 from list of literature- Khoramzadeh S.et al., 2016). The same question is for other disturbances mentioned in background chapter – such as sleep deteriorations, anxiety and depression development. It would be of worth to delineate criteria on resistance of patients suffered from tinnitus. Thus, it is interesting to hear details on their negative experience during previous drug treatment.\nSome notions on electrodes planned to use rose. In accordance to protocol the location of electrodes corresponds to 10-20 system of EEG electrode location. It is not enough for better imagine of the brain structures topography affected with your stimulations. Shape of electrodes is also important with such respect. Besides due to a flexibility of large electrodes the problem on non uniformity of currents under 3D electrodes leads to the increase of risks of burning of underlying tissues [2]. Hence, explanations on avoiding this problem are quite desirable. Also manufacturer of electrodes should be mentioned.\nIt should to note that authors [3] did not use intensity of current higher than 1.5 mA when delivered stimulations through 35 cm2 electrodes and emphasized on adverse effects of tDCS. What is the reason for treating patients with higher intensity of electric current in your protocol? Parameters and regimes of planned interventions should have better ground. Namely, why you have justified 10 sessions? Is it settled for other brain pathology treatment?\n30 s period, which is planned to use for sham-stimulated patients might be distinguishable by them in the course of delivering 10 trials. Hence, proposed control via asking patients if they know what type of influence is going on looks rather reliable procedure for verifying blind type of investigation. But it should be mentioned if the moment of turning of stimulator was soundless or not, just for prevention patients training for the distinguishing between types of stimulation.\nGeneral conclusion is that description of protocol on the tDCS treatment of patients suffered from tinnitus is rather convincing and deserves publication in highly ranked journal. Above mentioned minor remarks do not make serious impact upon very positive general estimation of proposed protocol of investigations. New and promising alternative for patients with tinnitus treatment is expected as result of its realization.\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": []
},
{
"id": "32356",
"date": "04 Apr 2018",
"name": "Daniel Robles-Camarillo",
"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\n1.\n\nSummary This protocol aims to research the therapeutic effect of tDCS, adverse effects and tolerability of bilateral tDCS over auditory cortex on tinnitus symptoms, applied on a very specific group of patients with intractable chronic tinnitus. It is proposed an interesting a double blinded experiment with three patient groups that consider the electrodes allocation: cathodal, anodal, or placebo. The three groups will be matched for age, gender, ethnicity, and audiometric main characteristics.\nIt is a novel treatment of daily 20-min sessions for five consecutive days per week and for 2 consecutive weeks. It is very well described the inclusion-exclusion criteria, the selection of patients’ groups and the analysis of electrophysiological and subjective findings, which are done by statistical methods. Authors consider baseline evaluations, hearing assessment, primary and secondary outcomes. In difference to other studies, authors gave one-month follow-up period, in order to study the AEs of and tolerability of the patients\n\n2.\n\nGeneral comments Protocol design: the inclusion and exclusion criteria are well structured. Pre-treatment complete audiometric and neurologic examinations by experienced specialists was a good establishment. The balanced random allocation performed by cards using computer-generated random numbers, is a good idea, because it considers the age, gender, ethnicity, and audiometric main characteristics. As a part of a larger experiment, final results will be deterministic in intractable tinnitus treatment. The sample size may be greater than n=90, to improve the statistical significance\n\n3.\n\nConstructive criticism It should be very interesting if authors show their data in corresponding plots to clearly visualize the patients’ distribution, electrophysiological and subjective readings and findings, pre- and post-treatment This study purposes the application of tDCS electric parameters. Authors use a specific device (by Mind Alive Inc.), it will be desirable if they show the precise electrical plot parameters (voltage, current, power and frequency) against time, performed with this device used.\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": []
}
] | 1
|
https://f1000research.com/articles/7-317
|
https://f1000research.com/articles/6-1728/v1
|
22 Sep 17
|
{
"type": "Research Note",
"title": "Rapid coral mortality following doldrums-like conditions on Iriomote, Japan",
"authors": [
"Andrew H Baird",
"Sally A. Keith",
"Erika Woolsey",
"Ryuta Yoshida",
"Tohru Naruse",
"Sally A. Keith",
"Erika Woolsey",
"Ryuta Yoshida",
"Tohru Naruse"
],
"abstract": "Coral bleaching can be induced by many different stressors, however, the most common cause of mass bleaching in the field is high sea temperatures (SST). Here, we describe an unusual bleaching event that followed very calm sea conditions combined with higher than average sea surface temperatures (SST). Patterns of mortality differed from typical thermal bleaching in four ways: 1) mortality was very rapid; 2) the suite of species most affected was different; 3) tissue mortality in Acropora spp. was often restricted to the center of the colony; 4) the event occurred early in the summer. The two weeks prior to the event included 8 days where the average wind speed was less than 3 ms-1. In addition, SSTs in the weeks preceding this event were 1.0-1.5°C higher than the mean for the last 30 years. We hypothesize that the lack of water movement induced by low wind speeds combined with high SST to cause colonies anoxic stress resulting in this unusual bleaching event.",
"keywords": [
"climate change",
"coral bleaching",
"coral reefs",
"disturbance"
],
"content": "Introduction\n\nCoral bleaching is a generalized response that can be induced by many different stressors1–3. Whilst the most common cause of large scale bleaching on coral reefs is unusually high sea surface temperatures (SSTs)4,5, prolonged periods of calm weather, known as the doldrums, have also been associated with mass bleaching events in the Caribbean6,7 and the Indo-Pacific8–10. Experimental work has also confirmed that low water flow can exacerbate thermal bleaching11,12.\n\nThe ecology of thermal coral bleaching is reasonably well documented. For example, colonies affected by high temperatures typically take between two to six weeks to bleach and bleached tissue can take another two to twenty weeks to die13. In addition, species vary in their susceptibility to thermal bleaching14,15, resulting in a predicable hierarchy of response16,17. Temporal patterns are also apparent with most high temperature induced mass bleaching events generally occurring towards the end of the summer months18,19. Any change in this predictable bleaching ecology suggests an alternative cause (i.e., not thermal stress) for a given bleaching event.\n\nHere, we describe an atypical bleaching event that we hypothesize was caused by an interaction of temperature with very calm sea conditions caused by an extended period of low winds. We identify a number of characteristic features of this doldrums bleaching that allow it to be distinguished from thermal bleaching in the field. Establishing the cause of specific bleaching events is vital in order to correctly attribute damage caused by climate change and other potential stressors.\n\n\nMethods\n\nThe study site was on the reef crest (1 m depth) at Nata Reef, Iriomote, Japan (24.4282°N, 123.7955°E). Initial observations at the site were made between 26 and 29 May, 2016 at which point in time no bleached corals were noted Surveys to quantify bleaching and mortality were conducted on 12 June, 2016. Twenty replicate 1m2 quadrats were placed haphazardly on the reef crest, and the condition and species identity of all hard coral colonies with a maximum diameter greater than 5cm were recorded. Species were identified in the field following20 and the names updated to the currently accepted names following21 Colonies were placed in one of six bleaching categories following21: (1) unbleached, (2) the entire colony pale, (3) 1–50% of the colony white, (4) 51–99% of the colony white, (5) 100% of colony white or fluorescent, or (6) recently dead. The data from the quadrats was pooled as the data was collected. The bleaching mortality index was calculated following16. Data on environmental conditions leading up to the bleaching episode were obtained from the Japan Meteorological Agency, which allows for these data to be used as long as due credit is given.\n\n\nResults\n\nBleaching and mortality was rapid. No colonies were bleached at the time of the first surveys (26 May, 2016) yet two weeks later (12 June, 2016), 5% of colonies were dead and a further 31% were bleached (Table 1).\n\nBMI = Bleaching Mortality Index.\n\nMortality was highest in Montipora aequituberculata and Montipora efflorescens (Figure 1A), and in an additional three species of the family Merulinidae, who were also badly affected (Table 1). Bleaching and tissue mortality were generally restricted to the center of colonies in the locally abundant species Acropora digitifera and Acropora hyacinthus (Figure 1B, C, D).\n\n(a) Dead and dying Montipora aequituberculata colonies (b) Acropora hyacinthus colony with bleached and dying tissue in the middle of the colony (c) a second A. hyacinthus colony (d) close up of the colony in (c).\n\nThe bleaching event occurred early in June, the first month of the northern summer, following a period of low wind and higher than average sea surface temperature (SST). Eight days in the previous two weeks had average wind speeds of under 3 ms-1 (Table 2). Winds were also mostly from the south, which is offshore at the study site and therefore likely to further reduce wave size and water motion (Table 2). Mean daily SSTs in the month preceding the second survey were 1.0–1.5°C higher than the mean for the previous 30 years (Table 3).\n\nData from Japan Meteorological Agency.\n\nValues are the degrees in centigrade above the 30 year average for this site in each time interval. Data from the Japan Meteorological Agency.\n\n\nDiscussion\n\nThis bleaching event was different to typical thermal bleaching in a number of important ways. In particular, rapid bleaching and tissue mortality restricted to the center of Acropora colonies, an atypical hierarchy of susceptibility, and the occurrence of the event in early summer, all distinguish this event from typical thermal bleaching. We hypothesize that unusually high SST combined with a lack of water flow due to low winds speeds resulted in anoxic stress to these colonies. This hypothesis is supported by doldrums like conditions (Table 2) combined with higher than average mean daily surface ocean temperatures (Table 3) in the weeks prior to the event.\n\nIn contrast to the typical thermal response, bleaching and mortality were very rapid, with a high proportion of colonies bleached and some dying within the two week period between the surveys (Table 1). Bleaching and, in particular, mortality typically take between 4–6 weeks to present in corals following thermal stress13. In addition, the hierarchy of susceptibility was very different to that following thermal bleaching. Here, the worst affected species included two Montipora spp. and a number of merulinids (Table 1), when typically Acropora spp. and Pocillopora spp. are the most severely affected following thermal bleaching5,15,22.\n\nThe pattern of tissue bleaching and mortality was also unusual. In Acropora colonies the typical pattern following thermal stress is for the whole colony to bleach13. In contrast, mortality was restricted to the center of most Acropora colonies in this event (Figure 1a, b, c). Tissue mortality beginning in the center of the colony typically indicates anoxia, which often occurs in aquaria with inadequate flow or oxygenation (pers obs). This pattern of mortality is also superficially similar to feeding scars caused by Acanthaster planci or Drupella spp.23 and a naïve observer might well have attributed this mortality to either of these corallivores24. A thorough search of the site, including underneath these and adjacent colonies, indicated that neither of these corallivores were present.\n\nThe timing of the bleaching event in early summer is also unusual. Thermal bleaching typically occurs much later in the summer. For example, recurrent seasonal bleaching on Magnetic Island, Australia, occurs in the last month of the austral summer i.e., February18. Similarly, the 1998 mass bleaching event in Japan was first noticed in the latter part of the summer i.e., late July25. In contrast, this doldrums event occurred early in June, the first month of the northern summer.\n\nDoldrums-like conditions (defined by NOAA as days with average wind speeds of less than 3 ms-1) have previously been linked to mass bleaching events6–9. However, the capacity of the doldrums to cause more localized damage outside of the typical thermal bleaching window in late summer has not previously been recognized. In addition, the potential link to anoxia, while tested in the laboratory26, has not been made in the field. This observation is especially important in the context of the continuing increase in the scale and frequency of mass bleaching events27 because it would generally be assumed that this small-scale phenomenon might presage a larger mass bleaching event. Determining the cause of specific bleaching events is vital in order to accurately distinguish the effects of climate change versus other causes of degradation on coral reefs.\n\n\nData availability\n\nThe pooled raw bleaching data is provided in Table 1.\n\nSource data for Table 2 are available from the Japan Meteorological Agency, at: http://bit.ly/2hck2G6, http://bit.ly/2wAVhcg.\n\nSource data to generate the values in Table 3 are available from the Japan Meteorological Agency, at: http://bit.ly/2y8qlBw.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the Australian Research Council Centre of Excellence for Coral Reef Studies (CE140100020) and VILLUM FONDEN (10114).\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 thank the staff at the Iriomote Tropical Biosphere Research Station, University of the Ryukyus, for their assistance.\n\n\nReferences\n\nGlynn PW: Coral reef bleaching in the 1980s and possible connections with global warming. Trends Ecol Evol. 1991; 6(6): 175–9. PubMed Abstract | Publisher Full Text\n\nBrown BE: Coral bleaching: causes and consequences. Coral Reefs. 1997; 16: S129–S38. Publisher Full Text\n\nBaird AH, Bhagooli R, Ralph PJ, et al.: Coral bleaching: the role of the host. Trends Ecol Evol. 2009; 24(1): 16–20. PubMed Abstract | Publisher Full Text\n\nGlynn PW: Coral reef bleaching: Ecological perspectives. Coral Reefs. 1993; 12(1): 1–17. Publisher Full Text\n\nHughes TP, Kerry JT, Álvarez-Noriega M, et al.: Global warming and recurrent mass bleaching of corals. Nature. 2017; 543(7645): 373–7. PubMed Abstract | Publisher Full Text\n\nJiménez C: Bleaching and mortality of reef organisms during a warming event in 1995 on the Caribbean coast of Costa Rica. Rev Biol Trop. 2001; 49(Suppl 2): 233–8. PubMed Abstract\n\nMiller J, Muller E, Rogers C, et al.: Coral disease following massive bleaching in 2005 causes 60% decline in coral cover on reefs in the US Virgin Islands. Coral Reefs. 2009; 28(4): 925–37. Publisher Full Text\n\nFisk DA, Done TJ: Taxonomic and bathymetric patterns of bleaching in corals, Myrimidon Reef (Queensland). Proc Fifth Inter Coral Reef Congress. 1985; 6: 149–54. Reference Source\n\nDeCarlo TM, Cohen AL, Wong GT, et al.: Mass coral mortality under local amplification of 2 °C ocean warming. Sci Rep. 2017; 7: 44586. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRaymundo LJ, Burdick D, Lapacek VA, et al.: Anomalous temperatures and extreme tides: Guam staghorn Acropora succumb to a double threat. Mar Ecol Prog Ser. 2017; 564: 47–55. Publisher Full Text\n\nNakamura T, van Woesik R: Water-flow rates and passive diffusion partially explain differential survival of corals during the 1998 bleaching event. Mar Ecol Prog Ser. 2001; 212: 301–4. Publisher Full Text\n\nNakamura T, Yamasaki H, van Woesik R: Water flow facilitates recovery from bleaching in the coral Stylophora pistillata. Mar Ecol Prog Ser. 2003; 256: 287–91. Publisher Full Text\n\nBaird AH, Marshall PA: Mortality, growth and reproduction in scleractinian corals following bleaching on the Great Barrier Reef. Mar Ecol Prog Ser. 2002; 237: 133–41. Publisher Full Text\n\nJokiel PL, Coles SL: Response of Hawaiian and other Indo-Pacific reef corals to elevated temperature. Coral Reefs. 1990; 8(4): 155–62. Publisher Full Text\n\nLoya Y, Sakai K, Yamazato K, et al.: Coral bleaching: the winners and the losers. Ecol Lett. 2001; 4(2): 122–31. Publisher Full Text\n\nMcClanahan TR, Baird AH, Marshall PA, et al.: Comparing bleaching and mortality responses of hard corals between southern Kenya and the Great Barrier Reef, Australia. Mar Pollut Bull. 2004; 48(3–4): 327–35. PubMed Abstract | Publisher Full Text\n\nSwain TD, Vega-Perkins JB, Oestreich WK, et al.: Coral bleaching response index: a new tool to standardize and compare susceptibility to thermal bleaching. Glob Chang Biol. 2016; 22(7): 2475–88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones RJ, Berkelmans R, Oliver JK: Recurrent bleaching of corals at Magnetic Island (Australia) relative to air and seawater temperature. Mar Ecol Prog Ser. 1997; 158: 289–92. Publisher Full Text\n\nFitt WK, McFarland FK, Warner ME, et al.: Seasonal patterns of tissue biomass and densities of symbiotic dinoflagellates in reef corals and relation to coral bleaching. Limnol Oceanogr. 2000; 45(3): 677–85. Publisher Full Text\n\nVeron JE: Corals of the World. The Australian Institute of Marine Science, Townsville, 2000. Reference Source\n\nHoeksema BW: Scleractinia. Bourne, 1900. Accessed through: World Register of Marine Species. Reference Source\n\nMarshall PA, Baird AH: Bleaching of corals on the Great Barrier Reef: differential susceptibilities among taxa. Coral Reefs. 2000; 19(2): 155–63. Publisher Full Text\n\nPratchett MS, Schenk TJ, Baine M, et al.: Selective coral mortality associated with outbreaks of Acanthaster planci L. in Bootless Bay, Papua New Guinea. Mar Environ Res. 2009; 67(4–5): 230–6. PubMed Abstract | Publisher Full Text\n\nBaird AH, Pratchett MS, Hoey AS, et al.: Acanthaster planci is a major cause of coral mortality in Indonesia. Coral Reefs. 2013; 32(3): 803–12. Publisher Full Text\n\nKayanne H, Harii S, Ide Y, et al.: Recovery of coral populations after the 1998 bleaching on Shiraho Reef, in the southern Ryukyus, NW Pacific. Mar Ecol Prog Ser. 2002; 239: 93–103. Publisher Full Text\n\nUlstrup KE, Hill R, Ralph PJ: Photosynthetic impact of hypoxia on in hospite zooxanthellae in the scleractinian coral Pocillopora damicornis. Mar Ecol Prog Ser. 2005; 286: 125–32. Publisher Full Text\n\nHughes TP, Barnes ML, Bellwood DR, et al.: Coral reefs in the Anthropocene. Nature. 2017; 546(7656): 82–90. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "26307",
"date": "05 Oct 2017",
"name": "Mikhail V. Matz",
"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 compact report of an unusual bleaching episode, presenting an interesting hypothesis that it could have been caused by anoxia during atypical doldrums conditions. Both are valuable for improving our understanding of factors affecting coral survival in times of changing climate. While there is no way to rigorously prove that the doldrums hypothesis is true, it certainly seems reasonable (and the wording throughout the paper is appropriate – there is never a claim that doldrums actually caused the observed bleaching).\nThat said, the paper would greatly benefit from expanded characterization of the doldrums episode, beyond tables 1 and 2. Is it possible to make wind speed and temperature graphs for broader range of dates (start in April) and compare that to conditions in the same time in previous years? This might show what was actually the most unusual – early temperature anomaly, low wind anomaly (by the way absolute values in Table 2 are not very informative without knowing how much below typical they are), or a combination of the two? Come to think of it, wave height could be a better proxy of water movement on the reef than wind speed, if it is possible to get that data (from satellite?).\nMinor things:\n“Tissue mortality beginning in the center of the colony typically indicates anoxia, which often occurs in aquaria with inade- quate ow or oxygenation (pers obs)” – I am not quite happy with the word “typically” used in a statement supported by nothing but personal observation… Can you please try once more to look up literature on this? (this is why my answer in \"partly\" to the literature citing question). This would also strengthen the doldrums case.\n“The bleaching mortality index was calculated following [16]” – as far as I can see, this index is not actually used for anything in the paper, so maybe it is not necessary? If keeping it, please expand a little: “The bleaching mortality index was calculated by [doing this and that], following [16].”\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": []
},
{
"id": "26305",
"date": "04 Dec 2017",
"name": "Robert van Woesik",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Baird and colleagues is a useful contribution to the literature. There are however a few minor edits that are necessary to make this short contribution publishable.\nFirstly, the title is inaccurate, or at least misleading. Doldrums is a maritime term that refers to the low-pressure area affected by the Intertropical Convergence Zone, where the prevailing winds are generally calm. The doldrums are considered to lie between 10oN and 10oS. Using the term doldrum-like conditions, instead of calm conditions, for a reef that is located at latitude 24 north confuses the terminology. I understand that NOAA, the US agency, has been using the term to refer to atypically calm periods irrespective of latitude, but again that doesn’t make it correct because they are also misusing the term. Change the title to reflect a short, atypically calm period.\nTable 1 should not simply have the corals in alphabetical order, but instead sort the table from most to least impacted.\nIntroduction Again, revise the use of the term doldrums.\n\nMethods Several periods are missing at the ends of sentences in the Methods section. Specify the aspect of the site. Which direction is the site facing? Data were pooled and were collected, not data was.\n\nResults Delete the word “who” in the sentence “who were also badly affected (Table 1)”\n\nDiscussion “was different from…”, not different to Revise doldrums-like conditions in several places. Again, different from, not different to. Periods are missing from the ends of sentences in several places of the Discussion. Split infinitive: change to : has not been previously…\n\nData availability Data are provided, not, data is provided\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1728
|
https://f1000research.com/articles/7-305/v1
|
12 Mar 18
|
{
"type": "Research Article",
"title": "Proprioception and vestibular alterations affect postural control in children with mild autism: A pilot study",
"authors": [
"Martin G. Rosario",
"Lizzette López",
"Michelle Méndez",
"Anas F Ababneh",
"Maryvi Gonzalez-Sola",
"Lizzette López",
"Michelle Méndez",
"Anas F Ababneh",
"Maryvi Gonzalez-Sola"
],
"abstract": "Background: Individuals diagnosed with Autism Spectrum Disorder (ASD) exhibit some type of motor control impairment, for instance, motor apraxia and history of gross motor delay that could lead to increased risk of fall. This pilot research was designed to assess and characterize static postural stability and create a starting point to better understand and describe postural control in children with mild autism. Method: We measured static postural control with center of pressure (COP) displacement in 10 children with mild autism during eight sensory conditions that challenge and cancel the visual, proprioceptive and vestibular systems. Results: Our results showed that children with autism demonstrated increased postural sway in response to challenges to the proprioceptive and vestibular systems. Conclusion: Therefore, under appropriate, challenging conditions, static postural control instability can be detected in children with mild autism.",
"keywords": [
"Autism Spectrum Disorder",
"Static Postural Control",
"Sway"
],
"content": "Introduction\n\nAutism is a developmental disorder characterized by deficits in language, social skills, environment interaction and motor function development; although the latter has not been thoroughly researched (Mari et al., 2003) (Fournier et al., 2010a). According to the \"Diagnostic and Statistical Manual of Mental Disorders\" (DSM-IV), classic autism, as well as Asperger's disorder, infantile disintegrative disorder and generalized not specified developmental disorder is now all classified under the Autism Spectrum Disorder (ASD) (American Psychiatric Association, 2013).\n\nIndividuals diagnosed with Autism Spectrum Disorder (ASD) have been shown to have deficiencies in motor control such as motor apraxia, clumsiness, reduced ankle movement, history of gross motor delay, and toe-walking (Ming et al., 2007; Teitelbaum et al., 1998). These deficiencies could lead to a risk of falls and lower quality of life in these individuals.\n\nKohen-Raz et al. (1992) found that children with autism have more difficulty maintaining postural control due to the gait pattern (walk on tip-toes) that they exhibit regularly. Also, children with ASD have difficulty in the integration of sensory information. Specifically, deficits may exist in the processing of vestibular and proprioceptive information when compared to children with normal development (Blanche et al., 2012; Fournier et al., 2010b; Shumway-Cook & Woollacott, 2001a).\n\nThe literature shows concurrent limitations and consensus in which sensory system specifically alter postural control in children with autism. Nevertheless, problems in motor control may be caused by some deficit in postural control mechanisms (visual, proprioceptive and vestibular systems) or problems in the integration/adaptation of these systems (Blanche et al., 2012; Fournier et al., 2010a; Fournier et al., 2010b; Kern et al., 2007; Kohen-Raz et al., 1992; Molloy et al., 2003; Shumway-Cook & Woollacott, 2001b).\n\nConsequently, it is necessary to study and describe how the sensory systems relate to postural control in children with ASD. The primary purpose of our research project is to identify postural deficiencies that may exist and to describe postural control in children who have autism with the objective to ascertain how these systems react when challenged or altered. Therefore, we hypothesized that when standing still (double legged), children with ASD will exhibit the following: 1) increased sway in one or more of the balance conditions, 2) challenges of the vestibular and proprioceptive systems will increase center of pressure (COP), and sway in a mediolateral (M-L) direction.\n\nThe long term goals of this pilot study are to provide the foundation to enhance the understanding of postural control, create new inquiry projects to help comprehend postural control in the different categories of autism and aid in developing targeted interventions for fall prevention in children with autism.\n\n\nMaterials and methods\n\nParents of children diagnosed with mild ASD who were interested in the study called the number on the recruitment flyer. The PI spoke with the parent to assess whether their child qualified for the study based on the following inclusion and exclusion criteria. The inclusion criteria were: (1) children from 7–12 years of age, male or female, (2) mild ASD diagnosis (as determined by a medical doctor (3) (5) capable of ambulating independently. Exclusion criteria were: (1) children with additional neurological problems, (2) children with visual problems, (3) children unable to tolerate walking or standing barefoot, (4) children who have fallen three or more times in the last three months, and (5) children with vestibular problems. We assessed the vestibular system utilizing the Fukuda Stepping Test (Fukuda, 1959). After hearing the qualifications, if the parent wished to volunteer their child for the study, an appointment was made for them to come to the Biomechanical Laboratory.\n\nTen children, diagnosed with a mild ASD that fulfilled the inclusion criteria participated in this study (Table 1). This project was conducted in the Biomechanical Laboratory of the Medical Science Campus of the University of Puerto Rico. The recruitment of participants was performed by posting flyers at the University of Puerto Rico, Medical Science Campus and other Centers in the Metropolitan Area.\n\nkg: kilogram; m: meter; SD: standard deviation\n\nAfter parents and children had given written consent, the following steps were followed: (1) A preparatory protocol was performed to familiarize the children with the study protocol, the staff and the children spent 5 minutes watching photos related to the materials, equipment, workplace, and team members. (2) Anthropometric measurements were taken (weight and height) as descriptive measures and to calibrate the pressure mat, MatScan. (3) The Fukuda test was performed to rule out vestibular system impairment (Fukuda, 1959).\n\nTo assess balance, we used a MatScan TM pressure mat (TekScan, Boston, MA). This mat contains sensors that measure body sway in centimeters (cm), anteriorly (forward), posteriorly (backward) (A–P), or laterally (sideways) (L–R). The mat provides information about the direction and amount of sway as well as area or center of pressure (COP). The data collected with the pressure mat, center of pressure (cm2) and sway (cm), was analyzed with Tekscan Sway Analysis Module (SAM) software designed for this purpose. This test was used to determine the effectiveness of an individual’s ability to use different sensory stimuli to examine the balancing of the body while standing under different conditions. The MatScan has an intra-rater reliability of .96-1.0 (Zammit et al., 2010).\n\nThe balance assessment procedure included the following; each subject was instructed to stand on both feet for 15 seconds on the pressure MatScan under eight different conditions. The first four conditions were executed over a stable surface and consisted of the following: (1) eyes open (EO) -evaluates visual, vestibular and proprioceptive systems, (2) eyes closed (EC) -eliminates visual input, evaluates vestibular and proprioceptive system, (3) eyes open while moving head up and down (at 60 beats per minute) (EO HUD) -evaluates visual and proprioceptive system and alters vestibular input, (4) eyes closed while moving the head up and down (EC HUD) -evaluates the effect of removing visual information, and the vestibular input being altered. The subjects then performed the same four tasks, this time standing on an unstable platform (high quality closed cell foam 19 inches long x 15 inches width x 2.25 inches) with the purpose of altering the proprioceptive input and increasing dependence on the visual and vestibular systems. (1) eyes open (EO MAT) -evaluates visual and vestibular system while the proprioceptive system is challenged and, (2) eyes closed (EC MAT) -removes visual input, evaluates the vestibular system and alters the proprioceptive system. (3) eyes open while moving the head up and down (EO MAT HUD) -evaluates visual system modifying the input of proprioceptive and vestibular systems, (4) eyes closed while moving the head up and down (EC MAT HUD) -evaluates altered proprioceptive and, vestibular systems in the absence of visual system.\n\n\nData analysis\n\nThe software used to analyze all the information was the statistical package for the social science version 19 (SPSS). P values <.05 were accepted as statistically significant. This study’s focus was to identify postural control deficiency in children diagnosed with mild ASD. We assessed how participants reacted when exposed to a stable and unstable surface and how altered proprioceptive information impacts postural control. We used a ANOVA analyses to compare differences in COP and sways (AP and ML) within the eight balance sensory conditions. A Bonferroni post hoc analysis was used to identify the specific variables or parameters to which significant differences could be attributed.\n\n\nResults\n\nWe recruited ten children diagnosed with mild ASD, eight male and two female with an age average of 8.58.5± 1.433 (anthropometric information are in Table 1).\n\nWe compared postural control between firm (stable) and foam (unstable) surface to identify postural instability. In detail, we used eye open on a firm surface test as a baseline and compared to the other three (eyes closed, eyes open head up and down, and eyes closed head up and down) conditions. Likewise, we examined eyes open on foam (unstable) to the other three conditions on the foam.\n\nResults of the comparison of average COP and sway movement under four different conditions (EO, EC, EO HUD, and HUD EC) on a stable surface and the mean excursion of the COP and sways under the same four conditions on an unstable surface are shown in Table 2. In our study, the most significant results were that children showed significantly more oscillations (instability) on the comparison of unstable surface versus the stable condition.\n\nCOP: Center of Pressure; EO: Eyes Open; EC: Eyes Close; HUD: Head Up and Down; MAT: Pressure Mat; AP: antero-posterior; ML: medio-lateral; SD: standard deviation; cm: centimeters\n\n\nDiscussion\n\nThe purpose of our study was to identify postural control deficiency, compare how participants react when exposed to a stable and unstable surface, and to describe how each system responds when altered. We identified postural instability and the systems that might be responsible for these alterations in these children with mild autism. Therefore, we discussed our findings in the following working hypothesis.\n\nHypothesis: children with ASD will exhibit altered postural instability in one or more of the balance conditions. Under stable and unstable conditions, we identified postural instability on unstable condition. Similar to Molloy and colleagues (2003) we found that children with ASD exhibited increased oscillations when standing on the foam, or unstable surface, compared to a stable platform, thus showing postural alterations (Molloy et al., 2003). Therefore, our hypothesis is accepted.\n\nParticipants showed increased COP excursion (postural instability) on the stable surface under EC HUD condition (when the proprioceptive is the only unaltered system, however not significant at this point. It is possible that the proprioceptive system could not compensate correctly when the input of the visual was canceled, and when altering the vestibular input, to maintain postural control.\n\nDuring stable surface under all conditions, AP oscillations predominated. Usually, this is expected because human oscillations occur in AP axis due to knee and ankle joints, similar to an inverted pendulum, thus allowing slight medio-lateral movements (Shumway-Cook & Woollacott, 2001b). However, when these oscillations exceed the stability limits, it might be due to weakness in the muscles of the lower extremities or delay of the integration or adaptation of the proprioceptive system (Shumway-Cook & Woollacott, 2001b).\n\nPostural control on an unstable surface shows that our participants exhibit greater COP excursion during the EO MAT HUD and EC MAT HUD condition. This condition was designed to evaluate the role of the visual system input. Therefore, we believe that the visual system was not capable helping to maintain proper balance when the vestibular and proprioceptive were stimulated. However, we believe that the visual system was not affected; it is just not enough to compensate for the other two tested systems. While the participants moved their heads up and down (in a coordinated fashion), they were requested to gaze at a fixed point during the entire task. Because of their low capability of concentration, it was not possible for these children to stare at a fixed point during the whole task. Our participants (when challenging the proprioceptive system) exhibited a trend of movements to the right and anterior directions showing further instability in each one of the tests. Similarly, when altering the visual and vestibular inputs, it was observed a tendency to move anterior and to the right. These results lead us to believe that proprioceptive input in children with autism could interfere with the ability of this system to send the correct information to maintain proper postural stability. (Blanche et al., 2012; Fournier et al., 2010b; Hansson et al., 2010; Shumway-Cook & Woollacott, 2001b). Contrary to a stable surface, ML oscillations predominated under all conditions on the unstable surface (Table 2). One possible explanation for this event may be that the alteration of the proprioceptive system is responsible for the adjustment of the activation of the musculature of the trunk, thus causing instability in the ML direction (Blanche et al., 2012; Fournier et al., 2010b; Hansson et al., 2010). Another possible justification might be related to balance control in the ML direction, mainly occurring due to weakness at the hip and trunk areas. Therefore, we believe that weakness in the trunk and hip muscles and deficits in weight bearing distribution might cause an increase in ML oscillations (O’sullivan, 2007; Shumway-Cook & Woollacott, 2001b).\n\nIn conclusion, children with mild autism spectrum disorder showed postural instability on the stable and unstable surfaces. Our study established that these children exhibited an increase in oscillations both for the AP and ML directions; however, the direction varied depending on what systems were altered. Thus, under controlled test conditions, children with autism exhibit diminished postural control or postural instability due to vestibular and proprioceptive alteration and postural instability in an ML direction.\n\n\nData availability\n\nPressure data collected was CoP, center of Pressure displacement in centimeters squared and sways. Antero-posterior (AP) and laterally sideways data was measured in centimeters. All measurements were collected during the 8 sensory balance conditions. Eyes open, eyes closed, eyes open head movements and eyes closed head movements. Same as the previous test were analyzed, however on standing on a foam/unstable surface. 10.5256/f1000research.14179.d197064 (Rosario et al., 2018)\n\n\nEthics and consent\n\nInstitutional Review Board (IRB) approval at the University of Puerto Rico, Medical Science Campus was obtained prior the recruitment of the participants. We obtained informed consent from all individuals (children and parents) included in the investigation.",
"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\nThanks to the University of Puerto Rico Medical Science Campus for providing the Biomechanical Laboratory and the MatScan equipment necessary for the development and completion of this study. Thanks to SER de Puerto Rico in San Juan, Puerto Rico for allowing us to recruit subjects in their facilities. This article was published with support from Texas Woman’s University Libraries’ Open Access Fund.\n\n\nReferences\n\nAmerican Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders: DSM-V. Washington, DC: American Psychiatric Association; 2013. Ayres, J. Sensory Integration and Praxis Test (SIPT).1996;6(112). Reference Source\n\nBlanche EI, Reinoso G, Chang MC, et al.: Proprioceptive processing difficulties among children with autism spectrum disorders and developmental disabilities. Am J Occup Ther. 2012; 66(5): 621–624. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFournier KA, Hass CJ, Naik SK, et al.: Motor coordination in autism spectrum disorders: A synthesis and meta-analysis. J Autism Dev Disord. 2010b; 40(10): 1227–1240. PubMed Abstract | Publisher Full Text\n\nFournier KA, Kimberg CI, Radonovich KJ, et al.: Decreased static and dynamic postural control in children with autism spectrum disorders. Gait Posture. 2010a; 32(1): 6–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFukuda T: The stepping test: two phases of the labyrinthine reflex. Acta Otolaryngol. 1959; 50(2): 95–108. PubMed Abstract | Publisher Full Text\n\nHansson EE, Beckman A, Håkansson A: Effect of vision, proprioception, and the position of the vestibular organ on postural sway. Acta Otolaryngol. 2010; 130(12): 1358–1363. PubMed Abstract | Publisher Full Text\n\nKern JK, Garver CR, Grannemann BD, et al.: Response to vestibular sensory events in autism. Res Autism Spectr Disord. 2007; 1(1): 67–74. Publisher Full Text\n\nKohen-Raz R, Volkmar FR, Cohen DJ: Postural control in children with autism. J Autism Dev Disord. 1992; 22(3): 419–432. PubMed Abstract | Publisher Full Text\n\nMari M, Castiello U, Marks D, et al.: The reach-to-grasp movement in children with autism spectrum disorder. Philos Trans R Soc Lond B Biol Sci. 2003; 358(1430): 393–403. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMing X, Brimacombe M, Wagner GC: Prevalence of motor impairment in autism spectrum disorders. Brain Dev. 2007; 29(9): 565–570. PubMed Abstract | Publisher Full Text\n\nMolloy CA, Dietrich KN, Bhattacharya A: Postural stability in children with autism spectrum disorder. J Autism Dev Disord. 2003; 33(6): 643–652. PubMed Abstract | Publisher Full Text\n\nO’sullivan SB: Examination of motor function: Motor control and motor learning. Physical rehabilitation. FA Davis Company Philadelphia. 2007; 233–234.\n\nRosario M, López L, Méndez M, et al.: Dataset 1 in: Proprioception and vestibular alterations affect postural control in children with mild autism: A pilot study. F1000Research. 2018. Data Source\n\nShumway-Cook A, Woollacott MH: Constraints of motor control. Motor control: Theory and practical applications. 2nd ed. Philadelphia, Pa.; London: Lippincott Williams & Wilkins. 2001a; 127–161.\n\nShumway-Cook A, Woollacott MH: Development of postural control. Motor control: Theory and practical applications. 2nd ed. Philadelphia, Pa.; London: Lippincott Williams & Wilkins. 2001b; 192–221.\n\nTeitelbaum P, Teitelbaum O, Nye J, et al.: Movement analysis in infancy may be useful for early diagnosis of autism. Proc Natl Acad Sci U S A. 1998; 95(23): 13982–13987. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZammit GV, Menz HB, Munteanu SE: Reliability of the TekScan MatScan(R) system for the measurement of plantar forces and pressures during barefoot level walking in healthy adults. J Foot Ankle Res. 2010; 3(11): 11. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "32378",
"date": "09 Apr 2018",
"name": "Amit Bhattacharya",
"expertise": [
"Reviewer Expertise Biomechanics",
"Quantitative posturagraphy Gait assessment",
"bone fragility quantification",
"effect of low level toxic chemicals on motor functions"
],
"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 pilot study with 10 subjects (8 males and 2 females) provided assessment of postural balance in children with mild autism using a Matscan pressure mat. Since the purpose of pilot study is to keep minimal variability among subjects’ demographics, what was the rationale of having only two female subjects? This small sample size of female subjects needs to be justified.\nSince COP measured with MatScan pressure mat sensors provides area (cm*cm) of pressure distribution under the sole, the results cannot be comparable to sway area measured by the “gold standard” force plate which is a measure of area contained within x-y distribution of center of pressure sway positions of whole body during the balance test. The discussion section needs to address the issues of concerns about the differences in measures of COP with a MatScan pressure mat vs with a Force Plate. Recent 2018 article by Goetschius et. al1 reports that the pressure-mat COP measurements are smaller than those of the force-plate, and the differences between devices appeared to increase as the measurement magnitude increased. Goetschius et. al (2018)\n\nalso reported that data collection period longer than 10 sec (15 sec test was used in the current manuscript) may demonstrate an even greater discrepancy in time dependent metrics between a MatScan pressure mat and Force plate devices. In other words, while a MatScan pressure mat data may be correlatable to those reported with a Force platform, the absolute values of postural balance outputs cannot be compared between two measurement systems. Therefore, measurements by a MatScan pressure mat and Force Plate should not be used interchangeably. Is it possible that under the feet pressure distribution pattern (instead or in addition to COP) captured by a MatScan pressure mat provides a unique signature of discrimination between mild autism and advanced autism? These issues need to be added as a limitations, as well as possible future utility of MatScan pressure mat metrics for quantifying proprioception feedback by the autism associated changes in pressure-receptors distribution patterns under the sole of feet.\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": "31806",
"date": "15 May 2018",
"name": "Juan Carlos Galloza-Otero",
"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 understand that this is a pilot study and that the task of recruiting patients with the appropriate inclusion criteria may be very difficult. Regarding the study design, it would have been valuable to have an age-matched control group similar as it was done on the referenced study by Fournier et. al.1 They studied the postural control of 13 children with Autism Spectrum Disorders and had 12 age-matched controls. Even though the sample was also small, having a control group in this study added more value to the findings and final conclusions. In the reviewed study by Rosario et. al. the absence of a control group in the study design should be mentioned as a limitation that may hinder the conclusions drawn from the results. This being said, there is significant scientific value to this study and more data is needed to evaluate potential postural control issues in patients with autism spectrum disorders.\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?\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/7-305
|
https://f1000research.com/articles/6-1969/v1
|
07 Nov 17
|
{
"type": "Software Tool Article",
"title": "PPInfer: a Bioconductor package for inferring functionally related proteins using protein interaction networks",
"authors": [
"Dongmin Jung",
"Xijin Ge",
"Dongmin Jung"
],
"abstract": "Interactions between proteins occur in many, if not most, biological processes. This fact has motivated the development of a variety of experimental methods for the identification of protein-protein interaction (PPI) networks. Leveraging PPI data available STRING database, we use network-based statistical learning methods to infer the putative functions of proteins from the known functions of neighboring proteins on a PPI network. This package identifies such proteins often involved in the same or similar biological functions. The package is freely available at the Bioconductor web site (http://bioconductor.org/packages/PPInfer/).",
"keywords": [
"graph mining",
"protein-protein interaction",
"pseudo-absence",
"semi-supervised learning",
"support vector machine"
],
"content": "Introduction\n\nThe functions of many proteins remain unknown. This is a big challenge in functional genomics. As proteins that consist of the same protein complex are often involved in the same cellular process, the pattern of protein-protein interactions (PPIs) can give information regarding protein function. Thus PPI databases can be useful to predict protein function, complementing conventional approaches based on protein sequence analyses.\n\nMany databases store information on protein-protein interactions as well as protein complexes. For example, the Biological General Repository for Interaction Datasets (BioGRID) includes over 500,000 manually annotated interactions1. The STRING database aims to provide a critical assessment and integration of protein-protein interactions, including direct (physical) as well as indirect (functional) associations. The basic interaction unit in STRING is the functional association, i.e. a specific and productive functional relationship between two proteins2.\n\nOnce the PPI has been obtained experimentally, there are numerous methods to analyze the network. A neighbor counting method for protein function prediction was developed3,4. The theory of Markov random fields was used to infer functions using protein-protein interaction data and the functional annotations of its interaction protein partners5. There are many algorithms that try to integrate multiple sources of data to infer functions6–8. We propose a method that can infer the putative functions of proteins by solving classification problems and thus identifying closely connected proteins known to be involved in a certain process.\n\nWe use the kernel method for the graph as a similarity measure between proteins, instead of using the first or second level neighbors, and thus our proposed method provides scores or distances derived from a graph kernel. The main advantage of the proposed method is that the neighbors of a set of proteins can be ranked in terms of scores. Generally, we need two or more classes for classification problem. Although we only know the target, we want to apply semi-supervised learning techniques to the PPI through this package. Thus, the main idea of this method is how to find another class so that two classes can be used for classification problem. Eventually, we can classify proteins to identify such closely related proteins by using this package. Finally, functional enrichment analyses such as ORA and GSEA are incorporated to predict the protein function from the closely related proteins.\n\n\nMethods\n\nThe support vector machine (SVM) is one of most widely used methods for classification9. Suppose we have a dataset in the real space and that each point in our dataset has a corresponding class label. A SVM is involved in a convex optimization problem to separate data points in the dataset according to their class, by maximizing distance between class and minimizing a penalty for misclassification for each class, at the same time. Unfortunately, the graph data is not in the real space. Cover’s theorem10 provides the useful idea behind a nonlinear SVM, which is to find an optimal separating hyperplane in high-dimensional feature space mapped by using a suitable kernel function, just as we did for the linear SVM in original space.\n\nGraph (network) data is ubiquitous and graph mining tries to extract novel and insightful information from data. Graph kernels are defined in the form of kernel matrices, based on the normalized Laplacian matrix for a graph. The best-known kernel in a graph is the diffusion kernel11. The motivation is that it is often easier to describe the local neighborhood than to describe the structure of the whole space12. Another method is called a regularized Laplacian matrix13 and is widely used in areas such as spectral graph theory, where properties of graphs are studied in terms of their eigenvalues and vectors of adjacency matrices14. Broadly speaking, kernels can be thought of as functions that produce similarity matrices15. In the package, we choose only the regularized Laplacian matrix as a graph kernel for the PPI.\n\nIn many biological problems, datasets are often compounded by imbalanced class distribution, known as the imbalanced data problem, in which the size of one class is significantly larger than that of the other class. Many classification algorithms such as a SVM are sensitive to data with an imbalanced class problem, leading to a suboptimal classification. It is desirable to compensate the imbalance effect in model training for more accurate classification. A possible solution to this problem is to use the one-class SVM (OCSVM) by learning from the target class only16. In one-class classification, it is assumed that only information of one of the classes, the target class, is available, and no information is available from the other class, known as the background. The OCSVM can be solely applied because we have only one class, the target. However, it is known that one-class classifiers seldom outperform two-class classifiers when the data from two class are available17.\n\nThe strategy of this package is to make use of the OCSVM and classical SVM, sequentially. First, we apply the OCSVM by training a one-class classifier using the data from the known class only. Let n be the number of proteins in the target class. This model is used to identify distantly related proteins among remaining N – n proteins in the background. Indeed, we do not know whether or not each of proteins in the background interacts with the proteins of the target. Thus, it does not always imply that proteins in the background do not interact with the target. In fact, their associations with the target are not observed to date yet. Proteins with zero similarity with the target class are extracted. Then they are potentially defined as the other class by pseudo-absence selection methods18 from spatial statistics. The target class can be seen as real presence data. The main idea of the proposed method is to adopt the pseudo-absence class. For the data to be balanced, assume that two classes contain the same number of proteins. Next, by the classical SVM, these two classes are used to identify closely related proteins whose scores ranging from -1 to 1 are close to 1 among remaining N – 2n proteins. Semi-supervised learning can be applied to make use of large unlabeled data and small labeled data. Some of these methods directly try to label the unlabeled data. Eventually, those found by this procedure can be functionally linked to the target proteins. This is usually based on the assumption that unannotated proteins have similar functions as their interacting proteins.\n\n\nUse cases\n\nWe need a list of proteins and the kernel matrix to infer functionally related proteins. With the STRING database for mouse, it is supposed that the target is the set of proteins in the Ras signaling pathway from the KEGG pathway (http://www.genome.jp/kegg-bin/show_pathway?mmu04014).\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nThe tyrosine kinase receptor binds to a ligand in the extracellular space. Then its cytoplasmic domain undergoes conformational change that forms dimerization, resulting in transphophotyrosine. The SH2 domain recognizes this phosphotyrosine. The Grb2 adaptor protein containing SH2 binds these receptors and recruits Sos, which is the Ras-GEF inducing Ras, to release its GDP and bind a GTP instead. The Ras protein is activated by this guanine nucleotide exchange factor. GTP-activated Ras can activate PI3K. Once PIP3 is formed by PI3K, an Akt/PKB kinase can become tethered via its PH domain. Once activated, Akt/PKB proceeds to phosphorylate a series of protein substrates, leading to aiding survival by reducing the possibility of apoptotic suicide program. For this reason, several GO terms about the RTK and PI3K pathways are shown in Figure 1. We can find cell migration due to the integrin and FAK. MAP kinase activity, which is a downstream of Ras signaling and causes cell proliferation, is statistically significant.\n\nThis figure was generated with Plotly (https://plot.ly/).\n\nIn the GSEA, the gene list is sorted by the standardized score showing how much they are functionally close to the target. Genes with higher scores are functionally more related to the target. The positive values of the enrichment score indicate enrichment at the top of the ranked gene list. Proteins that are closely related to the target are enriched in categories with high enrichment scores. In other words, these proteins are depleted in categories with low enrichment scores. We can see the RTK, MAPK and Wnt signaling pathways in Figure 2. One possible reason is that Akt can activate β-catenin both indirectly and indirectly.\n\nThis figure was generated with Plotly (https://plot.ly/).\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nIn Figure 3, the network visualization of the functional enrichment analysis is displayed. Nodes indicate GO terms and KEGG pathways. The connection between two nodes depends on the proportion of overlapping genes of corresponding two categories19. The size of nodes is proportional to the number of genes in their categories. The more significant categories are, the less transparent their nodes are. This network may be useful to see the overview of the functional enrichment.\n\nThis figure was generated with Plotly (https://plot.ly/).\n\n\nDiscussion\n\nThe proposed method is highly dependent on protein interaction networks. There are many prominent databases that include extensive information on protein interactions, and interactions are also reported in thousands of literature references. Therefore, the choice of data can be critical. Another issue is a technical problem. The one-class support vector machine can be sensitive to the choice of kernels and parameters. However, there is potential in inferring putative functions of proteins from PPIs, complementing conventional methods based on protein sequence analyses.\n\n\nSoftware availability\n\nThe PPInfer package is available at: http://bioconductor.org/packages/PPInfer/\n\nSource code is available at: https://github.com/Bioconductor-mirror/PPInfer\n\nArchived source code as at the time of publication: https://doi.org/doi:10.5281/zenodo.103512820\n\nLicense: Artistic-2.0",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis material is based upon work supported by the National Science Foundation/EPSCoR Grant Number IIA-1355423 and by the State of South Dakota.\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\nChatr-Aryamontri A, Breitkreutz BJ, Heinicke S, et al.: The BioGRID interaction database: 2013 update. Nucleic Acids Res. 2013; 41(Database issue): D816–D823. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzklarczyk D, Franceschini A, Wyder S, et al.: STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015; 41(Database issue): D447–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFellenberg M, Albermann K, Zollner A, et al.: Integrative analysis of protein interaction data. Proc Int Conf Intell Syst Mol Biol. 2000; 8: 152–161. PubMed Abstract\n\nSchwikowski B, Uetz P, Fields S: A network of protein-protein interactions in yeast. Nat Biotechnol. 2000; 18(12): 1257–1261. PubMed Abstract | Publisher Full Text\n\nDeng M, Zhang K, Mehta S, et al.: Prediction of protein function using protein-protein interaction data. J Comput Biol. 2003; 10(6): 947–960. PubMed Abstract | Publisher Full Text\n\nDeng M, Chen T, Sun F: An integrated probabilistic model for functional prediction of proteins. J Comput Biol. 2004; 11(2–3): 463–475. PubMed Abstract | Publisher Full Text\n\nJoshi T, Chen Y, Becker JM, et al.: Genome-scale gene function prediction using multiple sources of high-throughput data in yeast Saccharomyces cerevisiae. OMICS. 2004; 8(4): 322–333. PubMed Abstract | Publisher Full Text\n\nPeng W, Wang J, Cai J, et al.: Improving protein function prediction using domain and protein complexes in PPI networks. BMC Syst Biol. 2014; 8(1): 35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVapnik V: The nature of statistical learning theory. Springer-Verlag. 1995. Reference Source\n\nCover TM: Geometrical and statistical properties of systems of linear inequalities with applications in pattern recognition. IEEE Transactions on Electronic Computers. 1965; EC-14(3): 326–334. Publisher Full Text\n\nKondor R, Lafferty J: Diffusion kernels on graphs and other discrete structures. In Proceedings of the 19th International Conference on Machine Learning.2002; 315–322. Reference Source\n\nCook DJ, Holder LB (Eds.): Mining graph data. John Wiley & Sons. 2006. Publisher Full Text\n\nSmola AJ, Kondor R: Kernels and regularization on graphs. In Learning theory and kernel machines. Springer. 2003; 144–158. Publisher Full Text\n\nSamatova NF, Hendrix W, Jenkins J, et al.(Eds.): Practical graph mining with R. CRC Press. 2013. Reference Source\n\nKolaczyk ED, Csardi G: Statistical analysis of network data with R. Springer. 2014. Publisher Full Text\n\nScholkopf B, Burges CJ, Smola AJ: Advances in kernel methods: support vector learning. 1999. MIT press. Reference Source\n\nMa Y, Guo G: Support vector machines applications. Springer. 2014. Publisher Full Text\n\nSenay SD, Worner SP, Ikeda T: Novel three-step pseudo-absence selection technique for improved species distribution modelling. PLoS One. 2013; 8(8): e71218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYu G, Wang LG, Yan GR, et al.: DOSE: an R/Bioconductor package for disease ontology semantic and enrichment analysis. Bioinformatics. 2014; 31(4): 608–609. PubMed Abstract | Publisher Full Text\n\nJung D, Ge X: PPInfer: a Bioconductor package for inferring functionally related proteins using protein interaction networks. Zenodo. 2017. Data Source"
}
|
[
{
"id": "27695",
"date": "22 Nov 2017",
"name": "Cathy H. Wu",
"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 describes a new R package, PPInfer, for inferring functions of proteins based on their connectivity in a PPI network. Discovery of new functions for proteins is an important problem and the method as described is an interesting and theoretically sound approach.\n\nThe authors provide detailed example code for using PPInfer. Unfortunately, when trying to reproduce the use case, there was an error in the first step:\n> K.10090 <- readRDS(\"K10090.rds\") Error in gzfile(file, \"rb\") : cannot open the connection In addition: Warning message: In gzfile(file, \"rb\") :\n\ncannot open compressed file 'K10090.rds', probable reason 'No such file or directory'\n\nTherefore, we couldn't test the rest of the use case.\n\nAlso, in general, the use case would benefit from more text explanation so that readers understand the purpose of the tool and how to interpret the results. Specifically:\nWhat is the meaning of the gene ratio on the x-axis in Figure 1?\n\nIn Figure 3, are the nodes enriched KEGG pathways and GO terms as determined by GSEA? What was the threshold for deciding which nodes should appear in the graph? What is the biological message of this graph?\n\nA discussion of the functionally related proteins themselves (not just the enriched terms/pathways) would be helpful. How do these proteins functionally interact with the ras pathway proteins in the STRING network? Perhaps you could discuss the STRING confidence scores or types of evidence for these interactions.\n\nIt would be helpful to have more discussion of exactly what we learn from this approach. Can we infer that the proteins identified in the use case might be involved in the Ras pathway? It seems like based on the GO/KEGG annotation for the proteins, we could already have assumed they had something to do with signal transduction even without knowing that they are closely associated with Ras pathway proteins. Are there any examples of proteins whose association with the Ras pathway was surprising? Are there any proteins about which little is known or whose GO/KEGG annotation does not indiciate that they would be associated with the Ras pathway?\n\nPlease note that for questions 3, 4, and 5 on the referee report form: we were unable to fully evaluate this as we were unable to run the software. We have therefore assigned the answer \"no\" to reflect this.\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? No\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? No",
"responses": [
{
"c_id": "3216",
"date": "08 Dec 2017",
"name": "Dongmin Jung",
"role": "Author Response",
"response": "Thank you for taking the time to read and review our paper. In the revised version, we have improved the clarity of the figures and the text in several places, according to your suggestions. Necessary files are available at https://zenodo.org/record/1066236. Please, download K10090.rds and install the current version of this package. In Figure 1, The horizontal axis represents the proportion of our interesting genes in a certain functional category. In Figure 3, the 200 most significant categories from the GSEA are used, with the cutoff of 0.05 and 0.2 for p-values and edges, respectively. For GO terms, the two largest subnetworks are involved in transcription and signal transduction. We can see that Ras is related to tumorigenesis via the PI3K-Akt pathway from KEGG. For the discussion of the functionally related proteins themselves, two figures are added. One is the heatmap for the relationship between top proteins and pathways in Figure 4. The other is visualization of PPI among proteins in Figure 5. Please, see the text for further discussion."
}
]
}
] | 1
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https://f1000research.com/articles/6-1969
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https://f1000research.com/articles/7-45/v1
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11 Jan 18
|
{
"type": "Research Article",
"title": "Incorporating health workers’ perspectives into a WHO guideline on personal protective equipment developed during an Ebola virus disease outbreak",
"authors": [
"Saskia Den Boon",
"Constanza Vallenas",
"Mauricio Ferri",
"Susan L. Norris",
"Constanza Vallenas",
"Mauricio Ferri",
"Susan L. Norris"
],
"abstract": "Background: Ebola virus disease (EVD) health facility transmission can result in infection and death of health workers. The World Health Organization (WHO) supports countries in preparing for and responding to public health emergencies, which often require developing new guidance in short timelines with scarce evidence. The objective of this study was to understand frontline physicians’ and nurses’ perspectives about personal protective equipment (PPE) use during the 2014-2016 EVD outbreak in West Africa and to incorporate these findings into the development process of a WHO rapid advice guideline. Methods: We surveyed frontline physicians and nurses deployed to West Africa between March and September of 2014. Results: We developed the protocol, obtained ethics approval, delivered the survey, analysed the data and presented the findings as part of the evidence-to-decision tables at the expert panel meeting where the recommendations were formulated within eight weeks. Forty-four physicians and nurses responded to the survey. They generally felt at low or extremely low risk of virus transmission with all types of PPE used. Eye protection reduced the ability to provide care, mainly due to impaired visibility because of fogging. Heat and dehydration were a major issue for 76% of the participants using goggles and for 64% using a hood. Both gowns and coveralls were associated with significant heat stress and dehydration. Most participants (59%) were very confident that they were using PPE correctly. Conclusion: Our study demonstrated that it was possible to incorporate primary data on end-users’ preferences into a rapid advice guideline for a public health emergency in difficult field conditions. Health workers perceived a balance between transmission protection and ability to care for patients effectively while wearing PPE. These findings were used by the guideline development expert panel to formulate WHO recommendations on PPE for frontline providers caring for EVD patients in outbreak conditions.",
"keywords": [
"Guidelines",
"World Health Organization",
"Values",
"Preferences",
"Hemorrhagic Fever",
"Ebolavirus",
"Personal Protective Equipment"
],
"content": "Introduction\n\nHealth facility transmission is a hallmark of early Ebola virus disease (EVD) outbreaks and usually results in infection and death of health workers particularly before the identification of Ebola virus as responsible for the clinical presentation of one or a cluster of patients1–3. Contributing factors include non-specific clinical presentation, lack of local advanced diagnostic capabilities and suboptimal infection prevention and control (IPC) practices, amplified by poor surveillance in struggling health systems. The epidemiological pattern of the 2014–2016 EVD outbreak in West Africa revealed a similar story, but this time with an unprecedented scale and geographic spread, resulting in a record number of affected health workers, with 881 cases and 513 deaths by late 20154. Health workers are more likely than non-health workers to be infected: depending on the profession, the risk can be 21 to 32 times higher5.\n\nThe correct use of personal protective equipment (PPE) as part of comprehensive IPC measures contributes to the prevention of EVD transmission in healthcare settings by providing a protective barrier from contaminated fluids. However, the characteristics of the material and the configuration of the equipment may lead to health worker discomfort, overheating, and concerns about dexterity and safety to perform clinical tasks when PPE is used in the typical conditions of high heat and humidity present in West African EVD Treatment Centers6,7. As the United Nations’ international health agency, the World Health Organization (WHO) has the mandate to support Member States in preparing for and responding to a wide range of public health emergencies that often require that new technical guidance is developed in short timelines with scarce evidence base. Following an urgent request from affected Member States, WHO started the production of a PPE guideline for EVD outbreaks.\n\nA rapid review of the efficacy and comparative effectiveness of various components of PPE was commissioned in preparation for an expert panel meeting to develop recommendations on optimal PPE for health workers in Ebola treatment units (ETUs) in outbreak settings. It became clear very early in the process that high quality efficacy and comparative effectiveness studies addressing the use of specific PPE items for EVD in outbreak settings were lacking8. In addition to the paucity of data, it was critically important to gather and include the perspectives of health workers who had “real-life” experience in ETUs in West Africa. Early reports of the local conditions indicated that broader clinical questions than PPE performance as a transmission barrier were as important: usability, comfort, dexterity and impact on communication with patients, for example. The underlying principle was that evidence from efficacy and comparative effectiveness studies was necessary but insufficient for contextualization and adequate decision-making. This approach highlights the importance of understanding the way individuals exercise judgement (values and preferences) when selecting options with potential benefits, harms, and inconveniences in real life and is current best-practice in WHO standard guidelines9. Values and preferences are often informed mainly by the opinion of guideline expert panel members, however such proxies for persons affected by the recommendations in a guideline are often inadequate or even inaccurate. Thus, in the early stages of the 2014–2016 EVD outbreak in West Africa, in the context of time constraints and the absence of published data, it was crucial to incorporate the values and preferences of health workers into the guideline development process.\n\nThe purpose of this study was to support the development process of a WHO rapid advice guideline on PPE for EVD care in outbreaks. The specific objectives were to understand and describe frontline physician and nurses’ perspectives about PPE use, while providing direct care for EVD patients in the unprecedented conditions of the 2014–2016 EVD outbreak in West Africa and to incorporate these findings into the rapid advice guideline development process.\n\n\nMethods\n\nThe 2014–2016 EVD outbreak in West Africa was initially declared a Public Health Emergency of International Concern in early August 2014, coinciding with the decision to develop a WHO rapid advice guideline on the selection and use of PPE for EVD care in outbreaks. We electronically surveyed international frontline physicians and nurses who participated in foreign medical teams deployed to the affected countries in early stages of the EVD outbreak. The pragmatic approach was necessary given that this survey was developed and delivered at the height of outbreak and that WHO had very limited time available in which to produce guidance.\n\nThe online, 23-item survey was developed specifically for this study (Supplementary File 1). The first section consisted of multiple-choice questions examining participant demographic characteristics, role, and experience with PPE in West Africa. The next section addressed health worker exposure to the following specific components of PPE: eye protection (goggles/face shields), nose and mouth protection (medical mask/particulate respirator), gloves (single/double gloves), body covering (gowns/coveralls), foot wear (boots/closed shoes), and head covering (hair cover/hoods). In subsequent sections, we used a four or five-point Likert-scale to examine participants’ perceptions about the impact of each PPE item on the following domains: safety, communication, ability to provide patient care, personal wellbeing (heat and dehydration), and comfort. In addition, for each of the items, participants could provide free-text comments on open-ended questions to describe any difficulties or to provide suggestions on how PPE could be improved. The final section explored specific training needs and confidence in PPE. The last question asked participants to compare two sets of PPE available in West Africa shown side-by-side in a picture: one was composed of lighter items and the other had more robust components.\n\nFive experts reviewed the study protocol and questionnaire during the development phase. Subsequently, three clinicians with experience in the EVD outbreak in West Africa similar to that of the sampling frame field-tested the survey for consistency, readability, completeness, and question sequencing. The final version of the online survey incorporated all relevant feedback and comments. We obtained expedited approval of the study protocol and survey from the WHO Ethics Review Committee (RPC690).\n\nWe contacted potential participants via email. The first email explained the objectives, expected time commitment, and provided a link to the informed consent form and online survey on Survey Monkey®. Participation was voluntary and implied informed consent. A follow-up email in 5 days reminded potential participants of the deadline (10 days after launching). Participants could withdraw from the study at any time without providing any justification.\n\nThe study population consisted of international frontline physicians and nurses with direct field experience caring for EVD patients in West Africa. Our sampling frame targeted international physicians and nurses deployed by WHO and Médecins Sans Frontières (MSF) to West Africa between March and September 2014. We used maximum variation purposeful sampling, a non-probability sampling strategy, to capture a wide range of health worker perspectives and experiences in two organizations and four different countries affected by the EVD outbreak. Health workers were reached through a contact individual in each organization (MSF and WHO) who directly emailed potential participants. Physicians and nurses from the affected countries and from other international organizations were not included for pragmatic reasons given the extreme time constraints and infeasibility of obtaining additional organizational approvals in the available timeline. An initial communication error lead to the contact of other groups of health workers that did not have frontline clinical experience. The perspectives of these workers were considered for WHO quality improvement efforts, but were excluded from this analysis as these groups were not part of the approved sampling frame for this study.\n\nParticipants could indicate their experience with more than one item for each PPE component (e.g., both goggles and face shields for eye protection). For the purpose of statistical analysis, we considered each participant’s experience with a PPE item unique and independent. We analysed closed-ended questions with STATA 10 (StataCorp. 2007. College Station, TX) using counts, proportions, and the Chi-square test when comparisons were appropriate.\n\nTwo independent researchers analysed the answers to the open-ended questions using an iterative and reflexive process. This encompassed close reading and re-reading of the answers using constant comparison within and across different participants to identify key topics. The researchers then grouped the interpretations and understanding of the participant’s ideas and selected quotes to represent these findings, discussing discrepancies to achieve agreement.\n\nImmediately after data collection with the Survey Monkey® instrument, all information was downloaded to an anonymized spreadsheet, removed from the online database. All analyses were performed on de-identified data.\n\nThe rapid advice guideline was developed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach9,10. With this approach, clinical and public health recommendations are based on a systematic review and critical appraisal of the evidence on benefits and harms of an intervention, and an assessment of the balance between the two. Other considerations are also taken into account when an expert panel formulates recommendations, including feasibility, acceptability and resource implications of the intervention options, and the effects on equity across subpopulations. The relative value of the potential outcomes of the intervention options and the values and preferences of persons affected by the intervention are also important considerations. The findings of the survey were presented at the guideline development meeting and incorporated into evidence-to-decision tables (Supplementary File 2) to inform the formulation of recommendations for PPE components in the context of an EVD outbreak. Evidence-to-decision tables followed the GRADE-DECIDE11 approach and were populated by the WHO guideline development team in preparation for the expert panel meeting. These tables were key instruments used to present multiple sources of information to the guideline expert panel, helping to structure the discussion and to document the final judgements and decisions that underpin each recommendation.\n\n\nResults\n\nWe developed the study protocol, obtained WHO ethics approval, contacted the participants, delivered the survey, analysed the data, and presented the findings as part of the evidence-to-decision tables at the expert panel meeting where the recommendations were formulated in a period of 8 weeks.\n\nWe invited 192 health workers (166 from MSF and 26 from WHO) to participate in the survey and 74 (39%) responded. Respondents from MSF included 30 logisticians and water, sanitation and hygiene experts who were excluded because they were not part of the sampling frame. Thus 44 participants (33 physicians and 11 nurses) were included in the final analysis and their characteristics are described in Table 1.\n\n* n=42 because of missing data for 2 survey participants.\n\n* *Other tasks included: triage (n=3), medical rounds (2), nursing or direct patient care (3), outreach activities (7), checking or decontaminating colleagues (2), low risk activities such as teaching, training, administrative, pharmacy, informing family members (3), high risk activities such as carrying or lifting patients or corpses, disinfecting or spraying corpses, birth assistance, intra-osseous line insertion (7).\n\nFor each of the different components of PPE, one item was used by the majority of survey participants (Table 2). For example, 42 (95%) of participants had experience using goggles, while only seven (16%) had used a face shield (some participants had experience with both types of eye protection). Generally, health workers felt at low or extremely low risk regardless of the type of PPE used. PPE, particularly goggles, particulate respirators, and medical masks or hoods, impaired communication (Table 2). A reduction in the ability to provide care was predominantly related to eye protection equipment - both face shields and goggles. Heat and dehydration were a significant or major issue for 31 participants using goggles (76%) compared to two (29%) using a face shield (p=0.02), and for 27 (64%) using a hood compared to none using a hair cover (p=0.02). Heat and dehydration also were a significant or major issue for the majority of individuals using a gown (n=11, 73%) or coverall (n=26, 87%); however, there was no significant difference between the two groups (p=0.41). Goggles were considered more uncomfortable (n=29, 71%) than face shields (n=2, 29%, p=0.08) (Table 2).\n\nPercentages reflect total minus missing values for that specific question. n/a, not applicable. * Indicates p-value < 0.05.\n\nParticipants indicated that fogging of goggles or face shields was a major issue, affecting visibility and potentially creating a hazard for health workers as well as patients. There was some indication that fogging was a bigger issue with goggles and a few participants indicated that they would have preferred a face shield. Two participants indicated that the goggles caused pain after using them for extended periods. A number of participants noted that goggles did not cover sufficient skin of the face and there were requests for larger goggles, which would have the added advantage of greater visibility. Other issues were the poor quality of face shield and goggles, poor fit of goggles, and the logistical challenges of waiting to clean and dry re-usable goggles. One respondent summarized it as follows: “The goggles (are) not so comfortable and (they) felt like the \"unsafe\" part of the PPE. They move easily, hurt on the head, and affect vision in a negative way due to sweat, etc.”.\n\nMedical mask and the particulate respirator were reported to cause difficulty breathing when wet (due to sweat or condensation). One participant doubted the mask’s effectiveness when wet. Two participants were of the opinion that respirators were excessive since EVD is not airborne.\n\nThe main problem regarding gloves was the risk of having them slip down, allowing fluids to contact the skin as illustrated by the following respondent: “Some people found using tape over gloves (the second pair) useful as sometimes they did roll down during arduous patient care activity and in the end I also did this”. Other participants also attempted to solve this problem by taping gloves to the coverall, however this occasionally resulted in the tearing of gloves or the coverall. It was also mentioned that gloves were not long enough and that they tore easily.\n\nMany participants indicated the need for lighter suits with better ventilation. As one respondent commented: “During the dry season and if it was a sunny day it became quickly unbearable to stay too long (in the ETU). Ebola patients need lots of care and support, full PPE hinders this process. We need lighter and cooler PPE to be able to provide better care and stay longer inside (the ETU). Full PPE causes heat exhaustion and dehydration”. Difficulties included finding the right size coverall – in several instances the available coveralls were too small, leaving the health worker to opt for a coverall of lesser quality or have difficulties removing the coverall. A number of health workers indicated that they had difficulty taking off the coverall. Specific issues included having to remove the face shield first, leaving the eyes and face unprotected while undressing from the coverall, and problems taking off the coverall over large rubber boots. One respondent mentioned that coveralls with attached shoe covers could increase the risk of tripping. One respondent commented that boots were too big causing difficulty walking on irregular ground. As for reusable items (goggles and boots), it was mentioned that the time required to fully decontaminate and dry them sometimes brought challenges and put pressure on the team.\n\nA third of survey participants had received formal training over 2 to 3 days (n=15, 34%) and four (9%) reported training duration of more than 3 days. On the other hand, 20% (n=9) had received no formal or on-the-job training and another 20% (n=9) reported training for 2 hours or less. The remaining 15% of study participants (n=7) had training of one day or less. A number of participants commented that they would have liked to have had training, more formal training, or longer training. Others indicated that they would have liked to receive training before their departure, or before arriving at the treatment centre. The training topics that the survey participants would have liked included: removal of PPE and how to manage eye glasses. One health worker recommended weekly refresher training, especially in the light of frequent equipment changes, which may impact the order items are put on and taken off. Another health worker commented: “I believe that only experienced people can teach about Ebola. Teaching on the use of PPE is not about dressing and undressing. It is about using a set of behaviours with it and the understanding of all the underlying water and sanitation principles and applying them”. Regarding hand hygiene, alcohol-based hand-rub was not always available and there was conflicting information in different settings about which product to use.\n\nThe majority of participants (n=26, 59%) were very confident that they were using PPE correctly, 17 (39%) were reasonably confident and 1 (2%) was not very confident. Generally, participants were least confident about goggles (fogging, moving/displacing), medical masks and particulate respirators (difficulty breathing, becoming uncomfortable), and gloves (rolling down, tearing). Removing PPE was also an area that people felt less confident about (e.g., taking arms and feet out of a coverall, lack of face protection during undressing if the face shield was worn outside the hood). As one health worker illustrated: “Taking off the (Tyvek suit) coverall was difficult due to my height; it required me to wiggle out of it more than the average person”. A respondent also mentioned feeling less confident working in the screening area where much lighter PPE was worn, while possibly also being exposed to infectious patients.\n\nWhen asked to indicate their preference regarding two sets of PPE depicted in a picture, 8 (18%) participants preferred the PPE that was composed of lighter items, 33 (77%) participants preferred the more robust components, 2 (5%) did not have a preference and one participant did not respond to the question.\n\n\nDiscussion\n\nThe 2014–2016 EVD outbreak in West Africa required extensive local and international response and for the first time since EVD was described in 1976, a large number of organizations were directly involved in clinical and laboratory activities in the field. These interactions highlighted differences in the selection and use of PPE across the organizations. Early on in the outbreak, when the cases of health worker transmission were numerous and confusion about the best available equipment was wide-spread, WHO was asked to provide technical guidance in a short period of time. When a public health emergency involves a new disease, or a known disease with a different presentation, there may be scarce or no evidence on the benefits and harms of potential interventions. Indirect evidence (e.g., from related diseases such as other blood-borne pathogens and simulation), expert opinion, and data acquired and analysed in real-time may become the best available evidence for the guideline panel. In addition, factors other than the effectiveness of interventions may have a significant influence on the direction and strength of the recommendations. Such was the situation in 2014 during the height of the EVD outbreak in West Africa; a rapid review of the effectiveness of different types of PPE for protecting health workers revealed insufficient evidence upon which to draw conclusions about optimal PPE8.\n\nIn this context and within a period of 8 weeks, we developed and executed a survey, the results of which formed a critical part of the evidence upon which the recommendations developed by the expert panel were based12. To the best of our knowledge, this approach of collecting primary data regarding the values and preferences of persons affected by clinical or public health recommendations in a guideline is novel in the extremely challenging setting of a public health emergency.\n\nOverall, our findings showed that health workers perceive a balance between transmission protection and the ability to effectively care for complex patients while using PPE. The survey highlighted a slight preference of health workers for face shields compared to goggles because of less fogging, easier communication and better fit. There was no strong preference for one item of PPE over the other for all other PPE components. Given the variation in preferences for different components of PPE and the absence of data on comparative effectiveness, it may be important to provide a choice for health workers. This was, in fact, a guiding principle during the development of the PPE guidelines. Several issues raised by survey participants should be relatively straightforward to address, making a major contribution to health worker safety and comfort, such as providing a sufficient range of sizes, choice of equipment, and adequate training on how to put on and take off PPE in the conditions that will be faced in the field.\n\nWe experienced a number of challenges planning and executing this study. We had to develop a survey questionnaire de novo with limited time for field testing. Although this likely had a minimal impact on the results, we noted two questions that participants appeared to have difficulty comprehending (questions 11 and 23; see Supplementary File 1); if we had had more time for field testing we could have revised the questionnaire before formal data collection began. While our aim was to include only health workers who had provided direct patient care, such as nurses and physicians, given a communication error early in the study, we invited to participate and consequently received responses from workers without direct clinical experience who had been deployed to the EVD outbreak. Because these workers were not part of our pre-defined sampling frame, we excluded their responses from the analysis. Similarly, our survey failed to take into account the fact that PPE consists of different components such as eye protection, nose and mouth protection, gloves and body coverings that work together to protect the health worker from the risk of infection. In the first part of our questionnaire we asked how the survey participant experienced individual components of PPE (e.g., goggles or face mask). However, it is difficult to review these components as isolated items, separate from the rest of the PPE. As one survey participant noted: “It is the combination of the respirator and the face shield which is difficult. One or the other would be manageable but, both together meant major impairment”. Another survey participant commented: “The coverall would probably be better tolerated if we could breathe easier and see without problems”. In addition, although we compared gowns and coveralls, we did not specify or ask about the materials the body coverings were made of, its level of fluid resistance, or whether the head cover was attached or not. Such issues can have a significant impact on health workers’ experiences. It also became clear that solutions to an issue with one component of PPE could compromise the safety of another element of PPE. For example, participants mentioned that they would improvise and tape gloves to the coverall in order to prevent them from slipping down, but then the coverall would tear when removing the tape. Finally, the combination of different components of PPE may change the order in which PPE items are put on and taken off, thus end-users may perform donning and doffing procedures that are different than the training they received. This is particularly relevant if there are frequent changes in the availability of specific types of PPE, as was the case early in the outbreak response.\n\nMost of the limitations of this study were caused by pragmatic decisions the research team had to make in order to complete the study in the available time. This was in and of itself an invaluable learning experience for undertaking similar projects in the future. Specifically, we had to include only international health workers deployed by WHO and MSF in our study; therefore, we did not collect information on the values and preferences of local health workers and health workers deployed by other organizations. There were two important reasons as to why we selected our sampling frame. First, we carried out the survey at the height of the EVD epidemic when local doctors and nurses were fully engaged in the response efforts and we refrained from removing them from their primary work. Internationally recruited health workers on the other hand, were usually deployed for shorter periods and could thus participate when they returned home. Second, we had little time in which to execute the survey before the guideline meeting and we anticipated that it would be a lengthier and more complex process to identify and recruit local health workers. Thus, the findings of this survey may not be applicable to local health workers. In addition, generalizability of our findings to other international health workers involved in the Ebola response may be limited due to the small size of our purposive sample.\n\nIn the context of the most challenging of research settings, our study proceeded very efficiently and effectively in several regards. Peer reviewers for both the study protocol and draft survey made very helpful comments within 1 to 2 days. The WHO Ethics Review Committee approved the survey in less than two weeks. By reaching out to several key managers and opinion leaders from the two organizations, we were quickly able to identify frontline clinicians that were part of the sampling frame. The online format of the survey allowed us to quickly reach a larger number of health workers in different countries who had recent personal experience with different types of PPE in the EVD outbreak. The combination of different types of questions in our survey also worked well. Closed and Likert-scale questions made analysis of trade-offs and comparisons of health workers’ preferences possible while open-ended questions allowed the survey participants to share additional thoughts and perspective in more depth.\n\n\nConclusion\n\nOur study highlights some of the challenges and potential limitations and demonstrates the feasibility of generating and incorporating primary data on end-users’ values and preferences into a rapid advice guideline developed during the height of a public health emergency with extreme field conditions. Our survey showed that health workers perceive a balance between transmission protection and their ability to effectively care for patients while wearing PPE. These findings were a critical part of the information used by the guideline development expert panel when formulating recommendations on PPE for frontline health workers caring for EVD patients in outbreak conditions.\n\n\nEthical statement\n\nWe obtained expedited approval of the study protocol and survey from the World Health Organization Ethics Review Committee (RPC690). As approved by the ethics committee, we provided a link to the informed consent form with the survey. Participation was voluntary and implied informed consent. Participants could withdraw from the study at any time without providing any justification.\n\n\nData availability\n\nDue to the small number of survey participants, the detailed information collected, and the terms in the consent form approved by the WHO Ethics Review Committee, which guaranteed participant anonymity, the individual-level data cannot be made available. Requests for raw data can be dealt with on a case-by-case basis by contacting the corresponding author Dr den Boon, who will facilitate enquiries to the WHO Ethics Review Committee.",
"appendix": "Competing interests\n\n\n\nSDB and CV declare no competing interests. MF declares that his spouse is an employee at Bristol Myers Squibb and owns company stock as part of her remuneration plan. SLN declares that she is a member of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) Working Group, has published numerous papers related to GRADE, and that her career has benefited from this relationship. GRADE is the guideline process used by her employer, the World Health Organization, to develop guidelines.\n\n\nGrant information\n\nThis study was funded by WHO core funds. No external funding was obtained.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments.\n\nWe thank the following people for providing invaluable comments on the project proposal and questionnaire: Patricia Hudelson, Gordon Guyatt, Martine Verwey, Doris Bacalzo and Elie Akl. We are grateful to Armand Sprecher from Médecins Sans Frontières for sending the questionnaire to his staff during the peak of the outbreak response. We are also grateful to the survey participants for making this study possible.\n\n\nSupplementary material\n\nSupplementary File 1: Study questionnaire.\n\nClick here to access the data.\n\nSupplementary File 2: Evidence-to-decision tables used in the formulation of recommendations for the WHO Rapid Advice Guideline: Personal Protective Equipment in the context of filovirus disease outbreak response.\n\nClick here to access the data.\n\n\nReferences\n\nEbola haemorrhagic fever in Zaire, 1976. Report of a WHO/International Study Team. Bull World Health Organ. 1978; 56(2): 271–293. PubMed Abstract | Free Full Text\n\nEbola haemorrhagic fever in Sudan, 1976. Report of a WHO/International Study Team. Bull World Health Organ. 1978; 56(2): 247–270. PubMed Abstract | Free Full Text\n\nWHO: Outbreak of Ebola haemorrhagic fever, Uganda, August 2000–January 2001. Wkly Epidemiol Rec. 2001; 76(6): 41–46. PubMed Abstract\n\nWHO:Ebola Situation Report - 18 November 2015. Accessed 7 Nov 2016. Reference Source\n\nWHO: Health worker Ebola infections in Guinea, Liberia and Sierra Leone: Preliminary report. Geneva: World Health Organization; 2015. Accessed 7 Nov 2016. Reference Source\n\nBrearley MB, Heaney MF, Norton IN: Physiological responses of medical team members to a simulated emergency in tropical field conditions. Prehosp Disaster Med. 2013; 28(2): 139–144. PubMed Abstract | Publisher Full Text\n\nBah EI, Lamah MC, Fletcher T, et al.: Clinical presentation of patients with Ebola virus disease in Conakry, Guinea. N Engl J Med. 2015; 372(1): 40–47. PubMed Abstract | Publisher Full Text\n\nHersi M, Stevens A, Quach P, et al.: Effectiveness of Personal Protective Equipment for Healthcare Workers Caring for Patients with Filovirus Disease: A Rapid Review. PLoS One. 2015; 10(10): e0140290. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO handbook for guideline development-2nd edition. Geneva: World Health Organization; 2014. accessed 5 May 2017. Reference Source\n\nGuyatt GH, Oxman AD, Kunz R, et al.: Going from evidence to recommendations. BMJ. 2008; 336(7652): 1049–1051. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlonso-Coello P, Schünemann HJ, Moberg J, et al.: GRADE Evidence to Decision (EtD) frameworks: a systematic and transparent approach to making well informed healthcare choices. 1: Introduction. BMJ. 2016; 353: i2016. PubMed Abstract | Publisher Full Text\n\nWHO: Personal Protective Equipment in the Context of Filovirus Disease Outbreak Response: Rapid Advice Guideline. Geneva: World Health Organization; 2016. Accessed 7 Nov 2016. PubMed Abstract"
}
|
[
{
"id": "30348",
"date": "29 Jan 2018",
"name": "Patrick Mbah Okwen",
"expertise": [
"Reviewer Expertise Clinical care",
"Public health and health economics in LMICs"
],
"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\nWell written paper on an important and largely ignored subject: ‘health workers perspectives for guidelines’; Also on top global health issue ‘Ebola virus disease’. Study process was speedy and appropriate for the urgency needed for guidelines to be developed making this a good learning experience. However, there are a few points of attention listed below. I have also highlighted the sections relevant to my comments here.\nMethods\nAQ1: ‘The 2014–2016 EVD outbreak in West Africa was initially declared a Public Health Emergency of International Concern in early August 2014, coinciding with the decision to develop a WHO rapid advice guideline on the selection and use of PPE for EVD care in outbreaks.’ This statement will fit more within the background section, consider moving into background.\nAQ2: ‘We electronically surveyed international frontline physicians and nurses who participated in foreign medical teams deployed to the affected countries in early stages of the EVD outbreak.’ Clearly stating time frame in the methods section within which survey was done will also be helpful for readers, although a time frame is given later under participants, it is not clear if this was for survey or the sampling. this time frame is also very early in the outbreak\nAQ3: Settings is not well described, consider discussing setting in more detail under a separate title.\nResults AQ4: Clinicians express discomfort and safety, it may be interesting to know if at some point in the interviews they weighed in on safety versus comfort e.g. will the feeling of safety make them cope with discomfort? Or does discomfort make safety inconsequential?\nI have answered ‘Partly’ to the question “Is the work clearly and accurately presented and does it cite the current literature?” as a small part of the methods may benefit clarity if texts are moved around.\nI have answered ‘Partly’ to the question “Are the conclusions drawn adequately supported by the results?” as it will be important to discuss discomfort versus safety of risk or clearly state if this was not evaluated by the study.\n\nMiriam N. Nkangu comments This is an interesting piece and important in the context of infectious diseases. I will like to appreciate the authors for taking the initiative during such an emergency to collect such data. I will recommend the paper to be considered for indexing especially as it contributes towards developing guidelines for PPE which was more of a challenge to health workers during the outbreak. Understanding their challenges and experiences especially in very humid temperatures is important. Most importantly, the 2014 outbreak was a remarkable and most catastrophic outbreak. Thus, using the outbreak as a point of focus adds value to the work considering that it pulled health workers from various countries.\nMethods, Background/limitation\nWhy only physicians and nurses perspectives regarding PPE? I understand the relative risk for physicians and nurses as frontline workers is high, but other health workers are involved, and have recorded fatality rates, their experiences with PPE may also add value especially in the context of developing guidelines. Maybe the authors should consider adding this to limitations.\nMethods\nFour or five Likert is not explicit; it does not tell which questions were measured using scales of four and which used five and how they way categorize for-example., 1 indicating low or high? Agree or somewhat agree? Understand the sample size was small and is actually mentioned as a limitation, however, any data on number of nurses and physicians that were deployed by WHO and MSF during the period of data collection for background purposes and to justify the limitation? The sentence under data analysis is not clear to me, maybe rephrasing to better explain to the audience “For the purpose of statistical analysis, we considered each participant’s experience with a PPE item unique and independent.” The survey assumes that all the participants speak and write English? Language characteristic not mentioned considering that these affected countries some are French countries. If all participants were not English speaking how was it translated? Especially as the authors mentioned that respondents could not comprehend some questions due to time constraint.\n\nThe literature highlights some gender differences for PPE amongst physicians and nurses especially in African context-assuming nurses are mostly women and physicians men- -it would have been good to explore differences between nurses and physicians with regards to the specific PPE used. Were physicians exposed to more sophisticated PPE than nurses?\nOther comments that may be of interest to the authors: I understand the limitation of the paper is focused on participants in Ebola treatment centers and only foreign deployed. However, guidelines should take into consideration local reality in terms of culture? Based on previous outbreaks, most families prefer to care for patient at home and given the limited resources in this context; Local materials were used at home in 2014 as PPE http://www.cnn.com/2014/09/25/health/ebola-Fatu-family/index.html.\nGiven the reality of limited resources, and the fact that most families prefer to care for patient at home it would add more value also to consider experiences of those who cared for patient at home, the type of PPE used and opportunities in incorporating local reality into evidence-based guidelines for PPE.\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": [
{
"c_id": "3482",
"date": "09 Mar 2018",
"name": "Saskia Den Boon",
"role": "Author Response",
"response": "Well written paper on an important and largely ignored subject: ‘health workers perspectives for guidelines’; Also on top global health issue ‘Ebola virus disease’. Study process was speedy and appropriate for the urgency needed for guidelines to be developed making this a good learning experience. However, there are a few points of attention listed below. I have also highlighted the sections relevant to my comments here. Author’s response: Thank you for reviewing and approving our paper. See below our responses to your comments. We have made a number of changes in the text in response to your comments. Methods AQ1: ‘The 2014–2016 EVD outbreak in West Africa was initially declared a Public Health Emergency of International Concern in early August 2014, coinciding with the decision to develop a WHO rapid advice guideline on the selection and use of PPE for EVD care in outbreaks.’ This statement will fit more within the background section, consider moving into background. Author’s response: We removed this sentence from the methods section and have added it, slightly modified, to the background section. AQ2: ‘We electronically surveyed international frontline physicians and nurses who participated in foreign medical teams deployed to the affected countries in early stages of the EVD outbreak.’ Clearly stating time frame in the methods section within which survey was done will also be helpful for readers, although a time frame is given later under participants, it is not clear if this was for survey or the sampling. this time frame is also very early in the outbreak Author’s response: We send out the request for participation in September 2014 and have added this to the methods section. Later we indicate that the survey was open for a 10-day period. The health workers eligible for participation were those who were deployed to West Africa between March and September 2014 which was already stated in the methods section under participants. AQ3: Settings is not well described, consider discussing setting in more detail under a separate title. Author’s response: We are not sure how to respond to this question of the reviewer. We did an online survey among health workers who were deployed by MSF or WHO to respond to the Ebola outbreak in West Africa early on in the epidemic. Health workers worked in local hospitals, clinics or Ebola Treatment Centers, but because we did not ask further information about these settings we cannot provide a more detailed description. Results AQ4: Clinicians express discomfort and safety, it may be interesting to know if at some point in the interviews they weighed in on safety versus comfort e.g. will the feeling of safety make them cope with discomfort? Or does discomfort make safety inconsequential? Author’s response: We assume that the reviewer is referring to question 11 which asked, “please indicate how safe you felt by ticking a box for each aspect of Personal Protective Equipment”. As we have stated in the discussion, survey participants had difficulty answering this question because of the way the answer categories were phrased, e.g. “extremely low risk, I felt comfortable”. In this answer category we wanted comfortable to mean “I am not worried about safety”, but this was sometimes interpreted as “I am physically comfortable (e.g. not overheated, etc.)”. If we had had more time for piloting, we would have been able to pick this up before sending out the survey. However, through comments from health workers it became clear that they indeed cope with discomfort because the PPE makes them feel safe and we have added the following sentence to the discussion: “Health workers accept a certain degree of discomfort in return for the protection provided by PPE”. I have answered ‘Partly’ to the question “Is the work clearly and accurately presented and does it cite the current literature?” as a small part of the methods may benefit clarity if texts are moved around. Author’s response: we hope that our amendments have improved the methods section. I have answered ‘Partly’ to the question “Are the conclusions drawn adequately supported by the results?” as it will be important to discuss discomfort versus safety of risk or clearly state if this was not evaluated by the study. Author’s response: we hope that our amendment has taken away the concern of the reviewer. Miriam N. Nkangu comments This is an interesting piece and important in the context of infectious diseases. I will like to appreciate the authors for taking the initiative during such an emergency to collect such data. I will recommend the paper to be considered for indexing especially as it contributes towards developing guidelines for PPE which was more of a challenge to health workers during the outbreak. Understanding their challenges and experiences especially in very humid temperatures is important. Most importantly, the 2014 outbreak was a remarkable and most catastrophic outbreak. Thus, using the outbreak as a point of focus adds value to the work considering that it pulled health workers from various countries. Author’s response: Thank you for reviewing our paper and for making helpful comments and suggestions. See below our responses. Methods, Background/limitation Why only physicians and nurses perspectives regarding PPE? I understand the relative risk for physicians and nurses as frontline workers is high, but other health workers are involved, and have recorded fatality rates, their experiences with PPE may also add value especially in the context of developing guidelines. Maybe the authors should consider adding this to limitations. Author’s response: We agree with the reviewer about the importance of PPE for other health workers, for example cleaners, laboratory workers, burial teams and other workers. However, the focus of the WHO guideline which our study aimed to inform, was on healthcare workers and therefore we also focused our survey on this group. Methods Four or five Likert is not explicit; it does not tell which questions were measured using scales of four and which used five and how they way categorize for-example., 1 indicating low or high? Agree or somewhat agree? Author’s response: We agree with the reviewer that it would have been better to have used a comparable (e.g. 5-point scale) for all the questions. If we had more time for piloting, we may have picked this up before sending out the survey. Now, the questions on safety and comfort had a 4-point scale and questions on communication, ability to provide care, and heat and dehydration had a 5-point scale. As can be seen in the questionnaire which is included in the supplementary material, we did not use coding in the answer categories. Understand the sample size was small and is actually mentioned as a limitation, however, any data on number of nurses and physicians that were deployed by WHO and MSF during the period of data collection for background purposes and to justify the limitation? Author’s response: As stated in the results section, we invited 192 health workers (166 from MSF and 26 from WHO) to participate in the survey, but this included health workers outside the sampling frame (e.g. logisticians and water, sanitation and hygiene experts). Unfortunately we do not have more detailed information on numbers deployed. The sentence under data analysis is not clear to me, maybe rephrasing to better explain to the audience “For the purpose of statistical analysis, we considered each participant’s experience with a PPE item unique and independent.” Author’s response: We have now added the following clarification to the methods section: “i.e. we did not account for the fact that the experience came from one and the same health worker”. The survey assumes that all the participants speak and write English? Language characteristic not mentioned considering that these affected countries some are French countries. If all participants were not English speaking how was it translated? Especially as the authors mentioned that respondents could not comprehend some questions due to time constraint. Author’s response: Yes, this is correct. We assumed that all participants could speak and write English and we did not translate the questionnaire. The miscomprehension was due to the fact that two questions were not phrased clearly, rather than the language skills of the survey participants. The literature highlights some gender differences for PPE amongst physicians and nurses especially in African context-assuming nurses are mostly women and physicians men- -it would have been good to explore differences between nurses and physicians with regards to the specific PPE used. Were physicians exposed to more sophisticated PPE than nurses? Author’s response: This is a very interesting question. Although our study was not designed to answer this question and the number of participants was too small to do any stratified analysis, I had a brief look at the data. We indeed found a higher proportion of physicians among males (91%) than among females (57%), but there were no obvious differences in robustness of PPE, when I compared gown or coverall use between males and females, or between physicians and nurses (varying between 32-36% using a gown). Other comments that may be of interest to the authors: I understand the limitation of the paper is focused on participants in Ebola treatment centers and only foreign deployed. However, guidelines should take into consideration local reality in terms of culture? Based on previous outbreaks, most families prefer to care for patient at home and given the limited resources in this context; Local materials were used at home in 2014 as PPE http://www.cnn.com/2014/09/25/health/ebola-Fatu-family/index.html. Given the reality of limited resources, and the fact that most families prefer to care for patient at home it would add more value also to consider experiences of those who cared for patient at home, the type of PPE used and opportunities in incorporating local reality into evidence-based guidelines for PPE. Author’s response: We acknowledge the importance of this issue brought up by the reviewer but it fell outside the scope of the study and the WHO guideline that we were aiming to inform."
}
]
},
{
"id": "30694",
"date": "20 Feb 2018",
"name": "Elizabeth L. Beam",
"expertise": [
"Reviewer Expertise Infection control",
"personal protective equipment",
"education 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\nI had difficulty reading the tables in the article. I thought maybe it was the way they were displaying on my computer, but nothing seemed to change when I clicked on them. Please make these charts simple to read and clear. I need to see the tables to make sure your findings are adequately described.\nThe article is really well written. I was very pleased with the quality of the writing and the honesty of the authors about their challenges. This is important work in the area of PPE use.\nWhile I know that this was quick work in a difficulty setting, I still feel like the article needs to do justice to personal protective equipment research of the past 20 years (at least since SARS). The major section that needs more referencing is the discussion section. How do your findings compare to what we have found in epidemiological studies, simulation studies, and others on PPE. Even if these studies were not done in the context of an outbreak of EVD in Africa, they should still be discussed. There is literature on some of these areas that would bring worthwhile context to your findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3481",
"date": "09 Mar 2018",
"name": "Saskia Den Boon",
"role": "Author Response",
"response": "I had difficulty reading the tables in the article. I thought maybe it was the way they were displaying on my computer, but nothing seemed to change when I clicked on them. Please make these charts simple to read and clear. I need to see the tables to make sure your findings are adequately described. Author’s response: Thank you for reviewing and approving our paper. We assume that you are referring to table 2. We have revised the table by adding the categories that were previously omitted from the table. For example, in version 1 we only presented in the table the number health workers that indicated they felt at extremely low or low risk, but in version 2 we have added a column indicating the number of health workers feeling at high or extremely high risk. We have also added a foot note explaining how the denominator in each cell reflects missing values for that particular question. We hope that this has improved the readability of the tables. The article is really well written. I was very pleased with the quality of the writing and the honesty of the authors about their challenges. This is important work in the area of PPE use. Author’s response: Thanks for these kind words about our study. While I know that this was quick work in a difficulty setting, I still feel like the article needs to do justice to personal protective equipment research of the past 20 years (at least since SARS). The major section that needs more referencing is the discussion section. How do your findings compare to what we have found in epidemiological studies, simulation studies, and others on PPE. Even if these studies were not done in the context of an outbreak of EVD in Africa, they should still be discussed. There is literature on some of these areas that would bring worthwhile context to your findings. Author’s response: Thanks for this suggestion. We have added a number of references to the literature to our discussion section."
}
]
}
] | 1
|
https://f1000research.com/articles/7-45
|
https://f1000research.com/articles/6-506/v1
|
18 Apr 17
|
{
"type": "Research Note",
"title": "Mitochondrial dysfunction in diabetic neuropathy may be involved in the development of neuropathic pain via a reduction in neurosteroid synthesis",
"authors": [
"Stephen R. Humble"
],
"abstract": "Background: Recent work in a model of diabetic neuropathy revealed that layer 2/3 cortical pyramidal neurones of the pain pathway exhibited reduced endogenous neurosteroid modulation of the GABAAR and exogenously applied neurosteroids had an exaggerated impact. It is postulated that this is related to reduced precursor synthesis, due to mitochondrial dysfunction in diabetic neuropathy. Benzodiazepines are also known to activate neurosteroidogenesis by binding to mitochondrial translocator protein (TSPO). This study explored the differential effect of diazepam on GABAAR modulation via neurosteroidogenesis in diabetic and wild type (WT) mice. Methods: Whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from layer 2/3 cortical pyramidal neurons of the pain pathway from mice with type-II diabetic neuropathy (ob/ob) and WT controls aged 60-80 days. Results: There was a key difference in the response of the WT and ob/ob cortical neurons to simultaneous incubation with diazepam and flumazenil. In contrast, diazepam and the 5a-reductase inhibitor finasteride, individually or in combination, produced the same response in both strains. Conclusions: The exaggerated effect of diazepam on GABAergic inhibitory tone in the ob/ob, despite the presence of the GABAAR benzodiazepine antagonist flumazenil is likely observed due to physiological upregulation of key neurosteroidogenic enzymes in response to the reduced pregnenolone synthesis by the mitochondria. By increasing pregnenolone via TSPO activation, it is possible to promote enhanced neurosteroidogenesis and increase GABAergic inhibitory tone via an alternate route. In diabetic neuropathic pain, mitochondrial dysfunction may play an important role. Enhancing the GABAergic neurosteroid tone could be of potential therapeutic benefit.",
"keywords": [
"Diabetes",
"neuropathic",
"pain",
"neurosteroid",
"benzodiazepine",
"ob/ob",
"mitochondria",
"TSPO",
"GABA"
],
"content": "Introduction\n\nDiabetic neuropathy is a common cause of painful neuropathy, and treatment is often suboptimal because the underlying aetiology is poorly understood. Peripheral and central sensitisation are implicated in the development of neuropathic pain with neuroplasticity occurring at multiple levels of the pain pathway (Harvey & Dickenson, 2008). GABAergic neurones at all levels of the pain pathway have a vital role in the transmission of painful stimuli in the perception of pain itself (D’Mello & Dickenson, 2008). Endogenous and exogenous neurosteroids may act as potent positive allosteric modulators of GABAA receptors (GABAARs) and consequently exhibit analgesic, anxiolytic, anticonvulsant, and sedative properties (D'Hulst et al., 2009).\n\nWithin inhibitory synapses, the presynaptic fusion of a single vesicle releases the inhibitory neurotransmitter GABA to activate synaptic GABAARs. Under voltage-clamp conditions this causes a miniature inhibitory postsynaptic current (mIPSC). Drugs that enhance GABAAR function cause a prolongation of the mIPSC decay phase. Recent work in a model of type-II diabetic neuropathy (ob/ob) revealed that layer 2/3 cortical pyramidal neurones of the pain pathway exhibited reduced endogenous neurosteroid modulation of the GABAAR, and exogenously applied neurosteroids had an exaggerated impact (Humble, 2016a). The mechanism responsible appeared unrelated to GABAAR sensitivity, but instead was associated with a reduction of neurosteroid precursors, such as pregnenolone, which is metabolised sequentially to the active compound allopregnanolone (Figure 1) (Humble, 2013; Humble, 2016a). Pregnenolone is synthesised in the mitochondrion from its precursor cholesterol by the side chain cleavage enzyme P450 located in the inner mitochondrial membrane (Do Rego et al., 2009; Schumacher et al., 2012), and it is postulated that diabetic neuropathy may be associated with a reduction in mitochondrial activity. Cholesterol is translocated across the mitochondrial membrane by the 18 kDa translocator protein (TSPO) in a coordinated fashion with the steroidogenic acute regulatory (StAR) protein (Do Rego et al., 2009; Gatliff & Campanella, 2016; Rupprecht et al., 2010; Stocco et al., 2017; Figure 1).\n\nCholesterol is taken through the mitochondrial membrane by the translocator protein (TSPO) where it is converted to pregnenolone by the cytochrome P450 side chain cleavage enzyme. Pregnenolone is converted to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD), which is in turn reduced to dihydroxyprogesterone by 5α-reductase (5α-R). Dihydroxyprogesterone is converted to allopregnanolone by 3α-hydroxysteroid dehydrogenase (3α-HSD). Postsynaptic GABAARs are activated by GABA that has been released from vesicles in the presynaptic nerve terminal. GABA induces a conformational change of the GABAAR, opening its central channel and thereby allowing the passage of chloride ions and the subsequent generation of miniature inhibitory postsynaptic currents (mIPSCs). The negative chloride ions induce hyperpolarisation of the neuronal membrane, which mediates neuronal inhibition. Neurosteroids, such as the active compound allopregnanolone, modulate GABAAR function and facilitate inhibition of the neuronal membrane. (Humble, 2013)\n\nThe present study explored the impact of the benzodiazepine diazepam, a positive allosteric modulator of the GABAAR (D’Hulst et al., 2009), on GABAAR modulation via neurosteroidogenesis in diabetic and wild type (WT) mice. Benzodiazepines are also known to activate neurosteroidogenesis by binding to TSPO (Rupprecht et al., 2010; Tokuda et al., 2010).\n\n\nMethods\n\nThe methods are identical to those published by the same author previously (Humble, 2016a), with the exception of the drugs diazepam and flumazenil, which were not used in the previous study. Diazepam and flumazenil were purchased (Tocris, Bristol UK) and prepared as concentrated stock solutions in dimethyl sulfoxide before being added to the artificial extracellular solution as per the previous study (Humble, 2016a).\n\n\nResults\n\nWhole-cell voltage-clamp recordings were made in L2/3 cortical neurones of WT and ob/ob mice after at least two hours of incubation with diazepam, flumazenil and finasteride. Diazepam alone had the same effect on both strains of mice. In the WT mice, flumazenil inhibited the effect of diazepam (τW: control = 4.0 ± 0.1 ms, n = 35; finasteride 50 μM = 4.2 ± 0.1 ms, n = 7; diazepam 1 μM = 5.9 ± 0.2 ms, n = 6; flumazenil 10 μM & diazepam 1 μM = 4.0 ± 0.2 ms, n = 7; flumazenil 10 μM, finasteride 50 μM & diazepam 1 μM = 4.0 ± 0.3 ms, n = 6; One-way ANOVA, P <0.05; post hoc Newman Keul’s test revealed a difference only for diazepam 1 μM, P <0.05; Figure 2). By contrast, in the ob/ob mice, flumazenil only partially inhibited the effect of diazepam, and the persisting effect of diazepam in the presence of flumazenil in the ob/ob mice could be prevented by the presence of the 5α-reductase enzyme inhibitor finasteride (τW: ob/ob control = 3.5 ± 0.1 ms, n = 25; finasteride 50 μM = 3.7 ± 0.2 ms, n = 6; diazepam 1 μM = 5.7 ± 0.3 ms, n = 6; flumazenil 10 μM & diazepam 1 μM = 4.9 ± 0.3 ms, n = 6; flumazenil 10 μM, finasteride 50 μM & diazepam 1 μM = 3.7 ± 0.1 ms, n = 5; One-way ANOVA, P <0.05; post hoc Newman Keul’s test revealed significant intergroup differences for the flumazenil groups, P <0.05; Figure 2).\n\n(A) Superimposed exemplar averaged GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs) acquired from a representative WT cortical neurone and from equivalent neurones after ~2 hours pre-incubation of the brain slice with diazepam (1μM), flumazenil (10 μM) and finasteride (50 μM). (B) Superimposed exemplar averaged GABAAR-mediated mIPSCs acquired from a representative ob/ob cortical neurone and from equivalent neurones after ~2 hours pre-incubation of the brain slice with diazepam (1μM), flumazenil (10 μM) and finasteride (50 μM). (C) Histogram illustrating that flumazenil is able to prevent the effect of diazepam to prolong the duration of the GABAAR-mediated mIPSC in WT cortical neurones, but only has a partial efficacy in ob/ob cortical neurones (τw in ms; one-way ANOVA, P >0.05; Post hoc Newman Keul’s test). The persisting effect of diazepam in the presence of flumazenil in the ob/ob mice could be prevented by the presence of the 5α-reductase enzyme inhibitor finasteride (τw in ms; one-way ANOVA, P >0.05; Post hoc Newman Keul’s test). Ctrl = control; Finast = finasteride; Diaz = diazepam; Flumaz = flumazenil.\n\n\nDiscussion\n\nLayer 2/3 cortical neurones from mature type-II diabetic ob/ob are known to have a reduced endogenous pregnane-derived neurosteroid tone in comparison to strain matched WT controls (Humble, 2016a). The present data indicate that by promoting the uptake of pregnenolone’s precursor cholesterol by the mitochondria, via TSPO, diazepam may rescue the reduced neurosteroid tone. The restored neurosteroid tone could re-establish GABAAR-mediated neuro-inhibitory tone in cases of neuropathic hypersensitivity. With specific reference to these data, the key result is the difference in response of the WT and ob/ob to simultaneous incubation with diazepam and flumazenil. In contrast, diazepam and the 5α-reductase inhibitor finasteride individually or in combination produced the same response in both WT and ob/ob. This may be interpreted as follows: in the WT, the primary effect of diazepam incubation is direct allosteric modulation of the GABAAR, with negligible contribution from neurosteroidogenesis via mitochondrial TSPO activation. In comparison, diazepam has an exaggerated effect on GABAergic inhibitory tone in the ob/ob, despite the presence of the GABAAR benzodiazepine antagonist flumazenil. This effect is likely observed due to physiological upregulation of the key rate-limiting enzymes involved in neurosteroidogenesis in response to the reduced pregnenolone synthesis by the mitochondria (Figure 1; Humble, 2016a). Thus by increasing the availability of the neurosteroid precursor pregnenolone via TSPO activation, it is possible to promote enhanced neurosteroidogenesis and thereby increase GABAergic inhibitory tone via an alternate route. Benzodiazepines modulate the GABAAR by binding to the α-γ subunit interface (D'Hulst et al., 2009), while neurosteroids bind the GABAAR from a cavity within the α-subunit domain and modulate it directly via the α-β subunit interface (Hosie et al., 2006). There have already been a number of other studies of ligands for the mitochondrial TSPO, as this is a promising target (Gatliff & Campanella, 2016; Giatti et al., 2009; Papadopoulos & Lecanu, 2009; Rupprecht et al., 2010; Zhang et al., 2016). With reference to diabetic neuropathic pain and hypersensitivity, mitochondrial dysfunction may play an important role, and enhancing the GABAergic neurosteroid tone directly or indirectly could be of potential therapeutic benefit.\n\nOpen Science Framework: Dataset of ‘Neurosteroids are reduced in diabetic neuropathy and may be associated with the development of neuropathic pain’, doi: 10.17605/osf.io/bk3tw (Humble, 2016b). Raw data for the present study can be found in Diazepam.zip.\n\nPlease refer to (Humble, 2016a) for details of standard software used for data analysis.",
"appendix": "Author contributions\n\n\n\nDr Stephen Humble is responsible for this all work, including planning the experiments, performing the experiments and writing the paper. Prof Hales and Lambert, and Dr Belelli assisted Dr Humble with regards to the planning of some experiments. However, after discussion it was decided that their contributions merited being listed in the Acknowledgements section rather than as co-authors.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by the Wellcome Trust (090667).\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 am indebted to the generous support of the Wellcome Trust and would also like to thank: Prof Hales, Dr Belelli, Dr McCrimmon, Prof Peters, Prof Sillar, Dr Connolly, Dr Miles, Prof Poisbeau, Prof Lambert for their scientific advice, Miss Gallacher, Miss Wright, Dr McLeod, Mr McLeod, Dr Newman, Dr Cooper, Dr Brown, Dr Panetta, Dr Livesey for their technical assistance, Prof Matthews, Dr Moffat, Mr F Kafka, Prof P Anand, Dr P Donatien, Dr R Privitera, Dr Y Yangou, Prof A Dickenson, Dr Platt, Dr Ladas, Dr Jenner, Dr Bhaskar, Dr Kontouli, Dr Feynman, Mrs E Humble for their support.\n\n\nSupplementary material\n\nSupplementary Tables 1 and 2: Analysed raw data. Click here to access the data.\n\n\nReferences\n\nD'Hulst C, Atack JR, Kooy RF: The complexity of the GABAA receptor shapes unique pharmacological profiles. Drug Discov Today. 2009; 14(17–18): 866–875. PubMed Abstract | Publisher Full Text\n\nD'Mello R, Dickenson AH: Spinal cord mechanisms of pain. Br J Anaesth. 2008; 101(1): 8–16. PubMed Abstract | Publisher Full Text\n\nDo Rego JL, Seong JY, Burel D, et al.: Neurosteroid biosynthesis: enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides. Front Neuroendocrinol. 2009; 30(3): 259–301. PubMed Abstract | Publisher Full Text\n\nGatliff J, Campanella M: TSPO: kaleidoscopic 18-kDa amid biochemical pharmacology, control and targeting of mitochondria. Biochem J. 2016; 473(2): 107–21. PubMed Abstract | Publisher Full Text\n\nGiatti S, Pesaresi M, Cavaletti G, et al.: Neuroprotective effects of a ligand of translocator protein-18 kDa (Ro5-4864) in experimental diabetic neuropathy. Neuroscience. 2009; 164(2): 520–529. PubMed Abstract | Publisher Full Text\n\nHarvey VL, Dickenson AH: Mechanisms of pain in nonmalignant disease. Curr Opin Support Palliat Care. 2008; 2(2): 133–139. PubMed Abstract | Publisher Full Text\n\nHosie AM, Wilkins ME, da Silva HM, et al.: Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature. 2006; 444(7118): 486–489. PubMed Abstract | Publisher Full Text\n\nHumble S: Dataset of ‘Neurosteroids Are Reduced in Diabetic Neuropathy and May Be Associated with the Development of Neuropathic Pain’. Open Science Framework. 2016b. Publisher Full Text\n\nHumble SR: Neurosteroids; endogenous analgesics? PhD Thesis, University of Dundee. 2013. Reference Source\n\nHumble SR: Neurosteroids are reduced in diabetic neuropathy and may be associated with the development of neuropathic pain [version 1; referees: 1 approved, 2 approved with reservations]. F1000Res. 2016a; 5: 1923. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPapadopoulos V, Lecanu L: Translocator protein (18 kDa) TSPO: an emerging therapeutic target in neurotrauma. Exp Neurol. 2009; 219(1): 53–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRupprecht R, Papadopoulos V, Rammes G, et al.: Translocator protein (18 kDa) (TSPO) as a therapeutic target for neurological and psychiatric disorders. Nat Rev Drug Discov. 2010; 9(12): 971–988. PubMed Abstract | Publisher Full Text\n\nSchumacher M, Hussain R, Gago N, et al.: Progesterone synthesis in the nervous system: implications for myelination and myelin repair. Front Neurosci. 2012; 6: 10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStocco DM, Zhao AH, Tu LN, et al.: A brief history of the search for the Protein(s) involved in the acute regulation of steroidogenesis. Mol Cell Endocrinol. 2017; 441: 7–16. PubMed Abstract | Publisher Full Text\n\nTokuda K, O'Dell KA, Izumi Y, et al.: Midazolam inhibits hippocampal long-term potentiation and learning through dual central and peripheral benzodiazepine receptor activation and neurosteroidogenesis. J Neurosci. 2010; 30(50): 16788–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang LM, Qiu ZK, Chen XF, et al.: Involvement of allopregnanolone in the anti-PTSD-like effects of AC-5216. J Psychopharmacol. 2016; 30(5): 474–81. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "22166",
"date": "30 May 2017",
"name": "Slobodan M. Todorovic",
"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\nMy major concern with this study is that title and discussion are not relevant to the data presented. There is no justification to link this study to neuropathic pain or painful diabetic neuropathy. Perhaps more appropriate title would be:\n“Mitochondrial dysfunction in an animal model of Type 2 diabetes may be involved reduction in neurosteroid synthesis “\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? Partly",
"responses": [
{
"c_id": "2745",
"date": "09 Mar 2018",
"name": "Stephen Humble",
"role": "Author Response",
"response": "Dear Prof Todorovic, Thank you for taking the time to review the paper. I agree that the title should be changed. I have reworded it as follows in relation to your comments: “Mitochondrial dysfunction in an animal model of diabetic neuropathy is associated with a reduction of neurosteroid synthesis” With the benefit of hindsight I realize that linking these data in isolation directly to neuropathic pain and painful diabetic neuropathy is not justified. For the purposes of brevity full rationale was not explained in as much depth as was required to explain the link fully. If this paper is viewed alongside this other recent paper then the scientific rationale and justification may be understood: Humble SR: Neurosteroids are reduced in diabetic neuropathy and may be associated with the development of neuropathic pain [version 1; referees: 1 approved, 2 approved with reservations]. F1000Res. 2016a; 5: 1923. I will make some modifications to the abstract and discussion in relation to your comments as follows: Abstract section Old (final sentence):In diabetic neuropathic pain, mitochondrial dysfunction may play an important role. Enhancing the GABAergic neurosteroid tone could be of potential therapeutic benefit. New:In diabetic neuropathy, mitochondrial dysfunction may play an important role. Enhancing the GABAergic neurosteroid tone could be of potential therapeutic benefit. Discussion sectionOld (final sentence):With reference to diabetic neuropathy, mitochondrial dysfunction may play an important role, and enhancing the GABAergic neurosteroid tone directly or indirectly could be of potential therapeutic benefit. New:Considering the findings in this paper alongside previous work (Humble, 2016a) it appears that mitochondrial dysfunction may play an important role in the development of type 2 diabetic neuropathy. In this context, it follows that enhancing the GABAergic neurosteroid tone directly or indirectly could be of potential therapeutic benefit for diabetic neuropathic pain and hypersensitivity. Yours sincerely Stephen Humble"
}
]
},
{
"id": "23003",
"date": "06 Jun 2017",
"name": "Pascal Darbon",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present article is a follow-up of Humble’s previous paper in F1000Research (Humble SR. Neurosteroids are reduced in diabetic neuropathy and may be associated with the development of neuropathic pain [version 1; referees: 1 approved, 2 approved with reservations]. F1000Research 2016, 5:1923 doi: 10.12688/f1000research.9034.1). The current article could be considered as an extended version providing further clarification on involved mechanisms. Therefore to fully understand the presented results, I recommend reading first the previous article.\nHowever, in order to reach a broader leadership illustration and description could be improved. Figure 1 could be improved by highlighting the difference with Humble’s 2016 paper; action sites of Diazepam, Flumazenil and Finasterid could be indicated. Likewise, results description focuses on difference between both mice strains without fully presenting individual results (Finast, Diaz, Flumaz…). This required a strong background in the field.\nMy last comment concerns the primary effect of diazepam on GABAAR. The experiment consists in a 2 h incubation and it has been shown (in vivo in other brain structure, Zeitler et al. Eur J Neurosci, 2016) that Diazepam acts within 15-30 min then is relayed by neurosteroidogenesis. Therefore, it is difficult to conclude on the primary effect alone.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "2791",
"date": "15 Jun 2017",
"name": "Stephen Humble",
"role": "Author Response",
"response": "Dear Prof Darbon,Thank you for your comments and for taking the time to review the paper. I agree that the illustration and description could be improved and I will do so as per your suggestions. I agree that the brief background description makes the research harder for a broad readership- this is as a result of the article’s word limits.Regarding the 2 hour incubation period for diazepam. This issue was considered at length during the experimental design stage and this is a relevant concern. It is of course true that in vivo diazepam acts within 15-30 minutes and that in vivo propofol and etomidate may act within seconds when given intravenously. However, previous work (Benkwitz et al., 2007; Gredell et al., 2004) has shown that in brain slice preparations the duration of onset may be much longer in lipophilic compounds such as these because the drug has to diffuse through the slice to reach the GABAA receptor on the neurone located within the slice. This contrasts to rapid drug delivery via an intact in vivo circulation, which may deliver the drug directly to the relevant neurone within seconds to minutes. Taking all this into account it was decided to use a 2 hour incubation period in order to maximise the impact of diazepam and highlight the difference between the diabetic neurones and the wild type. i.e. that diazepam in the presence of flumazenil (benzodiazepine receptor antagonist) had a greater response in the diabetic neurones, which appears to be mediated via upregulated neurosteroidogenesis and could be blocked by finasteride. Finally, I think that these results are not inconsistent with the Zeitler et al., 2016 paper. Yours sincerelyStephen Humble"
}
]
}
] | 1
|
https://f1000research.com/articles/6-506
|
https://f1000research.com/articles/7-301/v1
|
09 Mar 18
|
{
"type": "Research Article",
"title": "The performance of select universities of medical sciences based on the components affecting medical education",
"authors": [
"Mehdi Tayebi Arasteh",
"Behrooz Pouragha",
"Masume Bagheri Kahkesh",
"Mehdi Tayebi Arasteh",
"Masume Bagheri Kahkesh"
],
"abstract": "Background: Every educational institution requires an evaluation system in order to find out about the quality and desirability of its activities, especially if it is a complex and dynamic environment. The present study was conducted to evaluate the educational performance of schools affiliated to Alborz University of Medical Sciences to help improve their performance. Methods: This descriptive analytical study was conducted in six schools affiliated to Alborz University of Medical Sciences in April 2016-October 2016 and October 2016-April 2017. The evaluation was carried out in two stages: self-assessment by service executives across schools, and external assessment in person by the university’s expert staff. The study tools included the components, criteria and desirable standards of educational performance in ten categories. Data were analyzed in SPSS. Results: The results obtained showed that, in April-October 2016, the highest performance evaluation scores pertained to the \"secure testing\" and \"rules and regulations\" components and the lowest to the \"packages for reform and innovation in education\" and \"the school action plan\" components. In October 2016-April 2017, the highest scores pertained to \"workforce empowerment\" and \"secure testing\" and the lowest to \"faculty affairs\" and \"electronic education management system\". Conclusions: Offering a balanced portrayal of the actual performance of schools using the right performance indicators in two consecutive periods can help further motivate the superior schools and encourage the weaker schools to strive harder. Competition among schools to get a higher score in the components affecting medical education helps mobilize them to move toward reform and improvement.",
"keywords": [
"Performance Evaluation",
"School",
"Medical Sciences Education",
"Alborz University of Medical Sciences"
],
"content": "Introduction\n\nHigher education institutions have a vital role in educating human resources and thereby the economic development and growth of developing countries1. Given the role of education in the future of countries, the coherent and ongoing monitoring and evaluation of the performance of higher education institutions has a great effect in their efficacy2. The use of performance evaluation indicators including input, process and output has become common in higher education institutions since 19803–6.\n\nLike any organization, higher education institutions require a careful ongoing evaluation system to learn of the quality and desirability of their activities in today’s complex and dynamic environment. Performance evaluation can therefore be a motivational (like some higher education institutions in the UK) or compulsory (like some higher education institutions in the US) tool7 for identifying opportunities, threats, strengths and improvement areas8. Using the right indicators is crucial to the successful performance evaluation of educational institutions. These indicators have to be compatible and standard (i.e. strong, valid and comparable among institutions and over time), purposeful (i.e. present evidence on their alignment with the institution's mission and accountability), simple and clear (i.e. have a clear methodology and specific non-heavy output that can also be used for wider evaluations). In fact, the main purpose of these indicators is to provide criteria that enable various institutions to have a meaningful comparison of their performance against that of similar institutions7,9.\n\nSince different institutions can have different goals depending on whether they are public or non-public, they can have different performance indicators and activity evaluations. As one of Iran's public higher education institutions, Alborz University of Medical Sciences is not so newly-established that only its input data can be used as the evaluation criteria but is not so old that only its output data are used either. Although only a few years have elapsed since the university’s establishment, its affiliated schools do not have adequate knowledge about the processes needed for the realization of educational outputs despite having many of the necessary inputs. This study seeks to guide the schools affiliated to this university toward specific outputs in accordance with the university’s educational objectives and the existing expectations and while defining process indicators6,10 along with output and input indicators in different areas of education and their evaluation at specific intervals7,11.\n\nThe present study was conducted to reflect the strengths and weaknesses of the schools affiliated to Alborz University of Medical Sciences and create a competitive atmosphere with the help of ten components of educational performance evaluation among the schools over short consecutive intervals through self-assessment and external assessment at the end of two six-month periods in the attempt to improve the performance of the new schools of Alborz University of Medical Sciences.\n\n\nMethods\n\nThe present descriptive analytical study was conducted in six schools affiliated to Alborz University of Medical Sciences over one year in April 2016–October 2016 (First six months) and October 2016 to April 2017 (Second six months) in two stages, including \"self-assessment\" by the schools' service executives and \"external assessment\" by the university’s expert staff. In the self-assessment stage, the activity executives in each school assessed their performance based on ten defined components. In the external assessment stage, the university’s education office experts visited the six schools and assessed their external performance based on the same components. Feedback was given on the results of the first assessment period to the school deputies in the university’s educational council and to the deans of the schools at the dean’s council, and recommendations for reaching more favorable results in future performance evaluations were presented.\n\nCensus sampling was carried out in all the six schools of Alborz University of Medical Sciences. Some schools, such as the schools of dentistry and pharmacy, were newly established (two and one years respectively since establishment) and therefore did not obtain good scores in some of the components, but had entered the evaluations voluntarily so as to be part of this ongoing performance evaluation from the start. For the performance evaluation of the schools, ten key components in the field of medical education were extracted (Table 1) according to a specific model12 and assessed by evaluators according to the conditions prevailing in the schools and the defined standards for scores of 0 to 100. Each component includes one \"criterion\" that expresses the best possible form of executing the component effectively. Each criterion includes one or several \"markers\" that illustrate the most important aspects of each criterion in the best possible quality. Each marker is defined by a \"scale\" that measures it, and each scale by a \"standard\" that determines the favorability of the indicator (Box 1 presents a sample of the scoring).\n\nFor simplicity and less administrative bureaucracy, data were collected electronically by a system installed for this very purpose. In the self-assessment stage, the data and documentation related to the ten components of performance evaluation were uploaded in the university’s performance evaluation system by the activity executives, and after the schools' internal evaluation period, the university’s expert staff from five different departments, including \"university education office\", \" Education Development Center (EDC)\", \"university education director\", \"faculty affairs office\" and \"electronic education management system (SAMA)\", first reviewed the uploaded data in the performance evaluation system, then registered the external performance evaluation scores in the system along with the needed explanations after visiting the schools and observing the real performance of the units and comparing them to the uploaded documentation.\n\nThe validity of the study tool was assessed and confirmed through interviews and consultation with the education staff directors, school deans, faculty members, education experts and students of different disciplines in the form of joint committees, and its reliability was also confirmed with a test-retest coefficient of 0.89 in all six schools. Data were analyzed for each assessment period and compared between the different assessment periods in SPSS.\n\n\nResults\n\nAlborz University of Medical Sciences is one of Iran's medical sciences universities with six schools (Nursing and Midwifery, Health, Paramedical Sciences, Medicine, Pharmacy and Dentistry), 22 fields of study at the bachelor, master and general and assistant practitioner levels, 157 faculty members, 190 visiting professors and 180 education, administrative and financial personnel. The results obtained in each evaluating department follows (also see Dataset 1).\n\nAccording to the results from April–October 2016, the highest external performance evaluation scores in education pertained to the nursing and midwifery (900 points) and health (800 points) schools (out of 1000) and the lowest to the pharmacy (320 points) and dentistry (360 points) schools. In October 2016 to April 2017, the highest external performance evaluation scores in education again pertained to the nursing and midwifery (940 points) and health (880 points) schools and the lowest to the dentistry (480 points) and pharmacy (740 points) schools.\n\nThe results showed little change between the schools' ranking from the first to the second period of evaluation, although the pharmacy and dentistry schools switched their ranks. In general, the external performance evaluation score increased in the second period compared to the first in all the schools. Of the maximum attainable score of 6000 points for the entire university, 3700 points (62%) were obtained in the first period and 4660 points (78%) in the second period (Figure 1).\n\nAccording to the results of the study, the difference between the schools' internal and external performance evaluation scores in April–October 2016 was 540 in the dentistry school, 480 in the pharmacy school, 320 in the school of medicine, 280 in the paramedical sciences school, 200 in the health school and 100 in the nursing and midwifery school, making for a total of 1920 points; in October 2016–April 2017, the difference was 520 in the dentistry school, 200 in the paramedical sciences school, 180 in the school of medicine, 160 in the pharmacy school, 120 in the health school and 60 in the nursing and midwifery school, making for a total of 1240 points (Figure 2).\n\nThe results from April–October 2016 revealed the highest performance evaluation scores (as strengths) to pertain to the \"secure testing\" (480 out of 600 points) and \"rules and regulations\" (440 out of 600 points) components, while \"packages for reform and innovation in education\" (260 out of 600 points) and \"the school action plan\" (300 out of 600 points) obtained the lowest scores (as improvable components). In October 2016–April 2017, \"workforce empowerment\" (600 out of 600 points) and \"secure testing\" (560 out of 600 points) obtained the highest scores (as strengths), and \"faculty affairs\" (380 out of 600 points) and \"electronic education management system\" (360 out of 600 points) obtained the lowest scores (as improvable components); (Figure 3).\n\nAccording to the results from October 2016–April 2017 compared to April–October 2016, the highest improved-upon score belonged to \"packages for reform and innovation in education\" (220 points) followed by \"workforce empowerment\" (180 points), such that all six schools obtained full scores in the \"workforce empowerment\" component. Clearly, the components that received high scores in the first period could not improve significantly in the second period (Figure 3).\n\n\nDiscussion\n\nThe infancy and inadequate experience and knowledge of Alborz University of Medical Sciences in some key performance indicators of medical education had led to routineness and poor performance in its affiliated schools. To give a detailed account of their expectations and create a dynamic environment in the schools, the education directors and experts of the university extracted the components affecting the main educational activities of the university. Next, the schools were internally and externally assessed with regard to these components and were ranked based on their scores in all the components. The present article examines the improvement of the scores of these various components and the outcome of creating competition between the schools to obtain higher scores in April–October 2016 and October 2016–April 2017.\n\nAccording to the results, the school of nursing and midwifery obtained the highest external evaluation scores in both periods, which could have been due to the proper software infrastructures in this school compared to others, which made the components flourish. It is therefore possible for this school to still be the standard-bearer of all schools in future performance evaluations. Other schools will need more time to catch up with this school and improve their components, because scoring higher in some of the components requires greater effort over time. Some studies have criticized that the measures and indicators used for performance evaluation should be generalizable to all institutions8. In the present study, attempts were made to choose the most basic indicators, but the new schools, especially the dentistry and pharmacy schools, were unable to obtain a good score in some of the components.\n\nIn the present study, performance components were not defined merely on the basis of the available data, and the components were chosen such that their improvement resulted in meeting higher education goals13, were not correlated with one another7 and could not be deliberately and easily manipulated by the schools14.\n\nSome studies have pointed out that the results of the performance evaluation of educational institutions can be presented in general institution rankings and also as indicator rankings across different institutions15. In the present study, the results are presented as institution ranking, although it was also possible to present them as indicator ranking for each school. In Canada, the directors of higher education institutions generally oppose this kind of ranking and claim that such rankings remove educational institutions from their main goal7.\n\nAlthough Alborz University of Medical Sciences aimed to obtain higher scores in every component and improve the educational performance of the university, the field study showed that each school sought to score higher than the others rather than score higher in each of the components16. The competition among the schools to rank higher than their first evaluation increased their total performance evaluation score in Oct. 2016–April 2017 by 26%. Since all the schools tried to obtain higher scores, no particular changes were observed in the schools' ranking in the second evaluation period, except in the school of pharmacy, which moved from the 6th rank to the 5th. According to the law of diminishing returns, scoring will be harder and less ascending on later occasions, especially for schools that have scored higher on the previous occasion17.\n\nThe purpose of the self-assessment (internal evaluation) of the components affecting medical education by the schools was to identify their strengths and weaknesses by self-checking against the standards and expectations. The present study showed a big gap between the internal and external evaluation scores in the first assessment occasion, which reduced by 35% in the second assessment occasion; in other words, the gap between \"what they really are\" and \"what they think they are\" decreased6. Perhaps the higher scores obtained for the schools in the external evaluation in the second assessment period has contributed to the smaller gap between these two evaluations. Nevertheless, it is possible for the schools to have sought to encourage the external evaluators to give them higher scores by giving themselves higher scores in the self-assessments18,19.\n\nIn general, of the ten components affecting medical education in the first assessment period, the schools were more successful and efficient and scored higher in the \"secure testing\" and \"rules and regulations\" components, as they had been routine duties of the schools for years. The lowest scores in the first assessment period pertained to the \"packages for reform and innovation in education\" component20 due to its infancy and \"the school action plan\" component due to the schools not being plan-oriented. With an improved awareness of their weaknesses in the first assessment period, the schools targeted these components in the second period and obtained higher scores in them so as to remove them from the list of low-scoring components, and they were thus replaced by the next low-scoring components. This trend is expected to continue in future assessments, as greater attention will be paid by the schools to the lowest-scoring components.\n\nWhat prevents universities and schools to monitor, evaluate and get accredited is their belonging to the public sector21. Most medical science universities in Iran are public and receive state budget and therefore lack the motivation to compete in the marketplace for obtaining non-governmental resources22. In general, universities that are non-governmentally funded are more strict about attracting funds and new students by entering national and international accreditation programs and demonstrating their features and capacity in key accreditation indicators10. It seems that, in public universities, improving performance indicators is meant to display the management capability rather than absorbing funds from the market.\n\nThe limitations of this study include the poor generalizability of the results to other educational institutions. It is therefore recommended to extract and assess components that are more specific to each institution7 and generalize the components to other educational institutions with more caution. Also, attending to these ten components in performance evaluation led to a neglect of the tasks unforeseen in these components. In fact, institutions “carry out that which is evaluated\". It is therefore important to update and reform the components in consecutive evaluations7 and emphasize the indicators23,24 and. Also, evaluation errors are likely in the measurement of data. Training evaluators and increasing their skills for the precise evaluation of performances together with the use of valid, reliable, structured, simple, clear and justifiable measures that can be extracted from reliable sources and used in different institutions25 can have a significant effect on the validity of the results.\n\n\nConclusion\n\nThe performance evaluation of schools by studying the components affecting the medical education they provide and specific standards can provide a good alternative to general and intuitive judgments. Although the results of these performance evaluations can benefit different groups differently, a technical and balanced performance evaluation is used in this study to extract the schools' strengths and weaknesses in two consecutive periods and motivate the superior schools for further improvement and encourage the weaker ones to strive harder26. The competition among schools mobilizes them to improve these components and their software infrastructures7,11; however, these competitions might be more beneficial if they are held at national and international levels27.\n\n\nData availability\n\nDataset 1: The key components, criteria, standards and evaluating departments of performance evaluation in the schools, in the first and second six months 10.5256/f1000research.13938.d19562228\n\nSeries*: the first column shows the number of evaluated components.\n\nComponent **: The second column shows the title of the component that is evaluated.\n\nCriterion ***: The third column shows the expected performance criterion for each component or index for evaluation.\n\nDesirable Standard ****: The fourth column shows the expected functional standard of each component or Standard setting criteria.\n\nEvaluating Department*****: The fifth column shows the Department that performs the external evaluation or Project location.\n\nInternal evaluation and External evaluation: The sixth columns show the scores for internal and external evaluation of schools.\n\nDifference: the seventh columns show the average of internal and external evaluation and its standard deviation for all schools.",
"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 present study was supported by Alborz University of Medical Sciences and the researchers wish to express their gratitude to the university dean, the school directors and experts and the evaluators.\n\n\nReferences\n\nMundial B: Higher education in developing countries. Peril and promise. Washington, D.C. 20433, U.S.A.: The International Bank for Reconstruction and Development/THE WORLD BANK; 2000. Reference Source\n\nParmenter D: Key performance indicators: developing, implementing, and using winning KPIs. John Wiley & Sons. 2015. Publisher Full Text\n\nGreen G, Keim R: After implementation whats next-evaluation. Journal of Systems Management. 1983; 34(9): 10–15.\n\nMorteza M, Davoud H, Roghayeh H: A conceptual framework for assessing the university performance. J Syst Manage. 2009; 3(3): 145–161.\n\nLancaster University: Key performance indicators. 2017; [cited 2017 21 June]. Reference Source\n\nSencan H, Karabulut AT: Monitoring of Educational Performance Indicators in Higher Education: A Comparison of Perceptions. Educ Sci Theory Pract. 2015; 15(2): 359–376. Publisher Full Text\n\nPollard E, Williams M, Williams J, et al.: How should we measure higher education? A fundamental review of the Performance Indicators. 2013. Reference Source\n\nSursock A, Smidt H, Davies H: Trends 2010: A decade of change in European Higher Education. European University Association Brussels. 2010; 1. . Reference Source\n\nEducation NCoIiH: Higher education in the learning society: report of the National Committee. 1997: The Committee. Reference Source\n\nGibbs G: Implications of ‘Dimensions of quality’in a market environment. York: Higher Education Academy. 2012. Reference Source\n\nJalaliyoon N, Taherdoost H: Performance evaluation of higher education; a necessity. Procedia Soc Behav Sci. 2012; 46: 5682–5686. Publisher Full Text\n\nDochy F, Segers M: Selecting indicators on the basis of essential criteria and appropriate assessment methods for a quality assurance system. In CHEPS Conference,\"Quality Assessment in Higher Education\" at Utrecht. 1990.\n\nOswald A: Public Money Manage. 2001; 21(3): 5–6. Publisher Full Text\n\nBaty P: Measured, and found wanting more. Times Higher Education. 2010; 35–8. Reference Source\n\nHEFCE: Counting what is measured or measuring what counts? League tables and their impact on higher education institutions in England. Higher Education Funding Council for England. 2008. Reference Source\n\nKarsten S, Visscher AJ, Dijkstra AB, et al.: Towards standards for the publication of performance indicators in the public sector: The case of schools. Public Adm. 2010; 88(1): 90–112. Publisher Full Text\n\nBoyes W, Melvin M: Fundamentals of economics. Cengage Learning. 2013. Reference Source\n\nRoss JA: The reliability, validity, and utility of self-assessment. Practical Assessment, Research & Evaluation. 2006; 11(10): 1–13. Reference Source\n\nBoud D, Falchikov N: Quantitative studies of student self-assessment in higher education: A critical analysis of findings. High Educ. 1989; 18(5): 529–549. Publisher Full Text\n\nThe Educational Deputy of Ministry of Health and Medical Education: Packs of evolution and innovation in education. T.E.D.E.o.M.o.H.a.M. Education, Editor, Ministry of Health and Medical Education: Tehran. 2015; 1–110.\n\nAbramo G, Cicero T, D'Angelo CA: The dangers of performance-based research funding in non-competitive higher education systems. Scientometrics. 2011; 87(3): 641–654. Publisher Full Text\n\nEsmaieli Kia Q, Molla Nazari M: A Framework for Evolution in the Financial and Operational Accountability Systems of Iranian Public Universities from the Experts' Perspective. Health Accounting. 2016; 4(4): 1–25. Reference Source\n\nCoates H: Excellent measures precede measures of excellence. J High Educ Pol Manage. 2007; 29(1): 87–94. Publisher Full Text\n\nMoosakhani M, Haghkhah D, Hassanzadeh R: Provide a conceptual framework for assessing university performance. Quarterly Journal Of Educational Leadership & Administration. 2009; 3(3): 145–161.\n\nHicks D: Performance-based university research funding systems. Res Policy. 2012; 41(2): 251–261. Publisher Full Text\n\nHeinrich CJ, Marschke G: Incentives and their dynamics in public sector performance management systems. J Policy Anal Manage. 2010; 29(1): 183–208. Publisher Full Text\n\nSantiago P, Donaldson G, Herman J, et al.: OECD Reviews of Evaluation and Assessment in Education: Australia. OECD Publishing (NJ1), 2011. Reference Source\n\nPouragha B, Tayebi Arasteh M, Bagheri Kahkesh M: Dataset 1 in: The performance of select universities of medical sciences based on the components affecting medical education. F1000Research. 2018. Data Source"
}
|
[
{
"id": "31757",
"date": "19 Mar 2018",
"name": "Ahmad Rahbar",
"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\nTayebi Arasteh et al performed an interesting subject about the performance of select universities of medical sciences based on the components affecting medical education. This is one of the most important factors in education.\nI think the paper is suitable for publication. I have some comments that if it was necessary please do it:\n\nAbstract:\nPlease correct the aim as a title 'subjects'.\nIntroduction:\nPlease write the established year of Alborz University. Please replace the end of two six months with the end of graduation term.\nResults:\n\nPlease write the acceptable P-value.\nDiscussion:\nDiscussion was written very well but if it is possible compare it more. Please write the main result in the first paragraph of discussion\nStudy limitation:\nPlease write the limitation of tools and manpower separately Please omit the reference of this part. In limitation doesn’t need the reference.\nConclusion:\nPlease omit the reference of this part. In conclusion doesn’t need the reference\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": "32793",
"date": "30 Apr 2018",
"name": "Roghayeh Khabiri",
"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 evaluated faculties of Alborz University of Medical Sciences for two sets of six months. They evaluated schools according to two stages, including \"self-assessment\" by the schools' service executives and \"external assessment\". It is very common in Iranian Universities and useful in helping improve their performance.\n\nThey found that the highest scores pertained to \"workforce empowerment\" and \"secure testing\" and the lowest to \"faculty affairs\" and \"electronic education management system.\n\nThis subject is important because they found the power point and weakness of their university and they informed that how they work. The strengths of this research are the tools that the researcher used according components, criteria and desirable standards of educational performance in ten categories. It can be used for another universities too.\n\nSome additional comments:\n\nI think introduction is long and they can shorten it\n\nThe Ethical code can be written in a separate part of method\n\nIn the abstract, if it is possible, write the p-value\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
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https://f1000research.com/articles/7-301
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https://f1000research.com/articles/7-44/v1
|
11 Jan 18
|
{
"type": "Research Article",
"title": "Insulin resistance indices and coronary risk in adults from Maracaibo city, Venezuela: A cross sectional study",
"authors": [
"Juan Salazar",
"Valmore Bermúdez",
"Luis Carlos Olivar",
"Wheeler Torres",
"Jim Palmar",
"Roberto Añez",
"Maria Gratzia Ordoñez",
"José Ramón Rivas",
"María Sofía Martínez",
"Juan Diego Hernández",
"Modesto Graterol",
"Joselyn Rojas",
"Luis Carlos Olivar",
"Wheeler Torres",
"Jim Palmar",
"Roberto Añez",
"Maria Gratzia Ordoñez",
"José Ramón Rivas",
"María Sofía Martínez",
"Juan Diego Hernández",
"Modesto Graterol",
"Joselyn Rojas"
],
"abstract": "Background: Insulin resistance (IR) is a metabolic disorder related to atherosclerosis. Its measurement is of great importance not only as a marker of diabetes but also for cardiovascular disease. The aim of this research study was to evaluate the relationship between various IR indices and coronary risk in an adult population from Maracaibo city, Venezuela. Methods: The Maracaibo City Metabolic Syndrome Prevalence Study is a descriptive, cross-sectional study with random and multi-stage sampling. In this sub study, 1272 individuals of both genders were selected with the measurement of basal insulin and coronary risk according to the Framingham-Wilson formula calibrated for our population. The insulin resistance indices evaluated were HOMA2-IR, triglycerides and glucose index (TyG) and triglycerides/HDL ratio (TG/HDL). The predictive capacity and association between each index and the coronary risk event in 10 years were determined. Results: Of the evaluated population, 55.2% were female, 34.8% had a coronary risk ≥5% in 10 years, with the TG/HDL and TyG indices showing the highest AUC 0.712 (0.681-0.743) and 0.707 (0.675-0.739), respectively; compared to HOMA2-IR. Both were also the indices most associated with increased coronary risk, especially TG/HDL ≥3 with a higher association [OR = 2.83 (1.74-4.61); p<0.01] after multivariable adjustment. Conclusions: TyG (≥4.5) and TG/HDL (≥3) indices showed a great predictive capacity of higher coronary risk, with being TG/HDL more associated even after adjusting for abdominal obesity and hs-CRP. Therefore, these represent useful tools for determining IR.",
"keywords": [
"insulin resistance",
"coronary risk",
"triglycerides",
"HOMA2-IR",
"HDL-C"
],
"content": "Introduction\n\nInsulin resistance (IR) is defined as a metabolic disorder in which the insulin effects in the target tissue are diminished, leading to derangements in carbohydrate, lipid and protein metabolism1. IR has been related to the development of several pathologies such as type 2 Diabetes Mellitus (DM2)2, Metabolic Syndrome (MS)3 and neurodegenerative diseases4. Therefore, the diagnosis and treatment of IR before the expression of the clinical disease are highly important for clinicians5. However, most methods to evaluate IR are costly and difficult to operate, such as the euglycemic-hyperinsulinemic clamp, which is considered the gold standard method for its detection6.\n\nThere is a need to implement new markers that are easier to interpret and more affordable, one being the Homeostasis Model Assessment (HOMA-IR), one of the most widely used, which is calculated from the measurement of fasting glucose and insulin concentration in the plasma7. However, the need to determine plasma insulin is a characteristic that limits its use in low-income populations. This limitation has been curbed by the use of the Triglycerides-Glucose (TyG) index, which is obtained from the fasting product of plasma glucose and triglycerides (TG) levels, and has shown an excellent predictive capacity to determine IR even in 2004 subjects of both genders ≥18 years from Maracaibo city in a previous report of our research team8.\n\nThe potential role of IR in the development of cardiovascular diseases (CVD) as a consequence of metabolic derangements, such as hyperglycemia, dyslipidemia, and inflammation together with hypertension, are involved in the progression of atherosclerosis9. The aim of this study was to evaluate the relationship between IR indices and coronary risk in an adult population from Maracaibo city, Venezuela.\n\n\nMethods\n\nThe study was approved by the Bioethics Committee of the Endocrine and Metabolic Research Center – University of Zulia (approval number: BEC-006-0305). This ethical approval included all future studies that used the data from the Maracaibo City Metabolic Syndrome Prevalence Study (MMSPS). All participants signed written consent for participation in the study before being questioned and physically examined by a trained team.\n\nThe MMSPS is a descriptive, cross-sectional study performed in Maracaibo city, Venezuela, during the period May 2007 - December 2009, with the aim to determine the prevalence of MS and cardiovascular risk factors in the adult population of this county. The study include a total of 2230 individuals of both gender, older than 18 years old, and the study protocol was previously reported10. The most important aspects of the protocol are presented here. Maracaibo city was divided into parishes, which were sampled proportionally through a multistage random sampling, defining conglomerates in two phases: In the first phase, the conglomerates represented the sectors of the 18 parishes, selecting 4 areas per parish by means of simple random sampling; in the second phase, the conglomerates were represented by the neighborhood of each chosen area, to which a random number was assigned.\n\nFor this present sub-analysis, individuals between 30–74 years of age were included, excluding those with a past medical history of ischemic heart disease (requirements necessary for the calculation and calibration of the Framingham-Wilson equation), as well as those individuals without available basal insulin determination necessary for HOMA2-IR calculation, thus the final total sample was 1272 individuals.\n\nAll individuals were evaluated, with a complete clinical history performed by trained assistants. Personal past medical and family history were gathered, with an emphasis on metabolic, endocrine and cardiovascular diseases history, as well as sociodemographic characteristics, such as age, gender and race.\n\nAfter 8 hours of fasting, determination of glucose, total cholesterol, triglycerides, and HDL-C was done with an automated analyzer (Human Gesellschaft fur Biochemica und Diagnostica mbH, Germany). The intra-assay coefficient of variation for total cholesterol, TAG, glucose and insulin was 3%, 5%, 3%, and <10%, respectively. Insulin was determined using an ultrasensitive ELISA double-sandwich method (DRG Instruments GmbH, Germany), with a detection limit <1 mU/L. Likewise, serum hs-CRP levels were quantified employing immunoturbidimetric assays (Human Gesellschaft für Biochemica and Diagnostica, mH).\n\nThe calculation of the Triglycerides and Glucose index (TyG) was performed according to Simental-Mendía et al.11, using the equation = ln [fasting triglycerides (mg / dl) x basal glucose (mg / dl)] / 2; thus expressing itself on a logarithmic scale. Previously, we proposed for a 4.5 cut-off point for this index to define IR for this population8. The Triglycerides/HDL ratio (TG/HDL) was calculated from the division of TG between HDL-C; 3 was used as a as cut-off point12. The HOMA2-IR was calculated using the HOMA-Calculator v2.2.2 software provided by the Oxford Center for Diabetes Endocrinology and Metabolism. The proposed cut-off point for our population was 2.00 to define IR13.\n\nFor the evaluation of coronary risk, the Framingham-Wilson formula calibrated for the population of Maracaibo was used14. The coronary risk was classified into two categories: 1) <5% in 10 years; 2) ≥5% in 10 years. Type 2 diabetic patients were defined with one of the following criteria: a) previous diagnosis of DM2; b) no previous personal history of DM2, but with fasting glucose greater than or equal to 126 mg/dl in two different measurements15. Abdominal obesity was defined according to the cut-off points for abdominal circumference previously obtained in our population (Women: 91cm; Men: 98cm)16, as well as the levels of high Sensitivity C-Reactive Protein (hs-CRP), which were measured randomly in 842 subjects, elevated hs-CRP defined as ≥0.765 mg/L17.\n\nQualitative variables were expressed in absolute and relative frequencies, evaluating the association between them using the χ2 test (Chi square). Quantitative variables were expressed as arithmetic means±SD, after analysis of normality by Geary test. The variables with non-normal distribution were subjected to logarithmic transformation.\n\nROC curves were constructed to analyze the predictive capacity of coronary risk of IR index. ROC curves were constructed for the total and specific sample by gender using R software version 3.4.1. The Area Under the Curve (AUC) was used to establish the predictive ability of the IR indices, where an AUC of 1.00 is considered a perfect diagnostic test18. The comparisons between AUC were made through the Delong's test19. Likewise, a logistic regression model was performed for which index was associated with a coronary risk ≥5% (dependent variable), adjusted by age group, sex and IR indices. In successive models, these were adjusted according to abdominal obesity and hs-CRP. All data were analyzed with SPSS v.21 for Windows (IBM Chicago, IL). The results were considered statistically significant when p<0.05.\n\n\nResults\n\nA total of 1272 individuals were evaluated, 55.2% (n=702) were female; 65.2% (n=829) had coronary risk <5% in 10 years, while 34.8% (n=448) had coronary risk ≥5% in 10 years. The frequency of DM2 for the two coronary risk groups was 29.6% (n=45) and 70.4% (n=107), respectively. The mean age of the sample population was 47.13±10.92 years. Other general characteristics of the sample population are presented in Table 1.\n\n*High sensititve C-reactive protein values were measured in n=842. TyG: Tryglycerides-glucose index; TG/HDL: Tryglycerides/HDL ratio; SD: Standard deviation.\n\nWhen evaluating the predictive capacity of the IR indices for coronary risk (Figure 1), it was evidenced that the TyG index had an AUC=0.735 (CI95%: 0.707-0.763), higher than that observed for the HOMA2-IR [(AUC=0.589; CI95%: 0.556-0.622; p=2.2x10-26)]. Likewise, the TG/HDL index showed a greater predictive capacity compared to HOMA2IR (AUC=0.772 vs. AUC=0.589; p=1.20x10-12). There were no significant differences in the predictive capacity between the TyG index and the TG/HDL (p=0.079), (Table 2).\n\nAUC, area under the curve; TyG: Tryglycerides-glucose index; TG/HDL: Tryglycerides/HDL ratio.\n\nAUC, area under the curve; TyG: Tryglycerides-glucose index; TG/HDL: Tryglycerides/HDL ratio; CI: Confidence Interval\n\naDelong’s test between female and male gender TyG = 0.002; HOMA2-IR = 0.729; TAG/HDL = 7.86x10-5\n\nIn the evaluation by gender, both women and men had a TyG index with significantly higher AUC than HOMA2-IR (Women: AUC=0.770 vs AUC=0.590; p=1.68x10-12; Men: AUC=0.682 vs AUC=0.578; p=8.03x10-5), whereas only in men the TyG index had a statistically greater predictive capacity than the TG/HDL index (AUC=0.682 vs. AUC=0.649; p=0.005). Likewise, in women a greater predictive capacity compared to men was observed for both the TyG index (AUC=0.770 vs AUC=0.682; p=0.002), and TG/HDL (AUC=0.767 vs. 0.649; p=7.86x10-5) (Table 2).\n\nSimilarly, when evaluating the indices in individuals without diagnosis of DM2 (Figure 2), it was demonstrated that TG/HDL and TyG indices were the greatest predictors of individuals without DM2, with AUC values of 0.712 (CI95%: 0.681-0.743) and 0.707 (CI95%: 0.675-0.739), respectively. These were significantly superior to those exhibited by HOMA2-IR, even in the analysis by gender (Table 2).\n\nAUC, area under the curve; TyG: Tryglycerides-glucose index; TG/HDL: Tryglycerides/HDL ratio.\n\nDuring the evaluation of subjects’ distribution according to IR indices and coronary risk in 10 years (Table 3), a statistically significant association was found. However, it was observed in the multivariate model that only individuals with a TG/HDL ≥3 were more likely to present ≥5% coronary risk in 10 years (OR: 3.17 CI95%: 2.15-4.68; p<0.01), even after model adjusting for abdominal obesity and hs-CRP (OR: 2.83 CI95%: 1.74-4.61; p<0.01).\n\nTG: triglycerides; TyG: triglycerides-glucose index.\n\naPearson's chi-square test\n\nModel 1: adjusted by indices of insulin resistance, age groups and sex. Excluding diabetic subjects\n\nModel 2: Model 1 + abdominal obesity\n\nModel 3: Model 2 + Elevated hs-CRP\n\n\nDiscussion\n\nIR is currently well-known for its physiopathological role in conditions such as obesity, arterial hypertension, dyslipidemia, DM2 and CVD2,3,9. The detection of IR in primary care is a potential prevention strategy. For this reason, the use of low cost and easy use indices, such as TyG and TG/HDL, could be valuable tools in daily clinical practice, even over HOMA2-IR.\n\nDifferent physiopathological mechanisms has been linked to IR, including an increase in cardiovascular risk by promoting endothelial dysfunction, inhibiting the synthesis of nitric oxide, and stimulating the secretion of endothelin-1 and the expression of cell adhesion molecules20. It also affects the smooth muscle cells by promoting proliferation, migration and apoptosis, making the atherosclerotic plaque more unstable, which is associated with secretion of pro-inflammatory mediators by macrophages21. These morphological and functional changes in the vascular wall and the heart could explain the increased cardiovascular risk in IR states9.\n\nSeveral studies have evaluated the predictive capacity of TyG index for CVD, but there are few reports for Latin American populations. In this analysis, it was found that TyG is a better predictor of coronary risk compared with HOMA2-IR. Similarly, Martínez-Larrad et al.22, in a combined analysis conducted in non-diabetic individuals from the Mexico City Diabetes Study, the San Antonio Heart Study and Spanish Insulin Resistance Study, observed that the TyG index had higher AUC (between 0.673-0.875) to discriminate ≥10% risk coronary in 10 years than the HOMA-IR (between 0.579-0,746), as well as other less used IR indices, such as the McAuleys index and the insulin sensitivity index of Avignon.\n\nThis behavior has also been determined in two Asian populations, where the TyG index was a better predictor of coronary arterial calcification than the HOMA-IR23,24, although in one of these populations, it was observed that by including another predictive variable, such as the abdominal circumference, the predictive capacity of this marker increased24. On the other hand, Sánchez-Íñigo et al.25 showed that in 5014 patients belonging to The Vascular Metabolic CUN cohort, the association of the TyG index categorized in quintiles and the risk of developing a coronary event was twice the risk in the upper quintile. In addition, Irace et al.26 analyzed the relationship between the TyG and HOMA indices with the risk of carotid atherosclerosis, and demonstrated that only the former was significantly associated, even after adjustment with the presence of metabolic syndrome.\n\nOur results coincide with these previously reported findings that reveal a significant superiority of TyG over HOMA2-IR in the prediction of coronary risk, which was also more marked in the female population, probably associated with a greater distribution of intra-abdominal fat and proatherogenic factors. This interaction between the proinflammatory molecule and the TyG was evident in multivariable analysis after the adjustment by hs-CRP.\n\nIn regards to the TAG/HDL ratio, most studies in Latin America have compared the predictive capacity for cardiovascular risk or association with cardiovascular risk factors in children27. Only those reports of Salazar et al.28,29 in the Argentinean population have linked the TAG/HDL ratio with cardiovascular events in Latin American adults. In this sense, our findings coincide with those shown in the Argentinean population, evidencing not only the high predictive capacity of individuals with coronary risk ≥5% in 10 years by the TAG/HDL ratio, but the close relationship that exists between the variables even after adjustment for covariates such as abdominal obesity and hs-CRP, higher than the TyG index. These results differ from those exhibited by Du et al.30 in more than 7000 Chinese individuals, who showed that TyG index is the best predictor of IR, whereas TAG/HDL ratio is body fat distribution dependent.\n\nThe TyG and TG/HDL indices superiority over HOMA2-IR in the prediction of coronary risk demonstrates the importance of serum TAG in the pathophysiology of IR. Although one of the disadvantages posed for HOMA-IR is that it only reflects IR in the liver, given the ability of basal insulin to suppress gluconeogenesis in this organ during the fasting period26. In this study, the mathematical model HOMA2-IR was used, whose advantage with its predecessor is the estimation of peripheral resistance to insulin31. Therefore, the greater prediction of the indices that have serum TG in their calculation does not depend specifically on the ability to measure peripheral IR in skeletal muscle or adipose tissue, but possibly, the greater contribution of TG in the development of IR32.\n\nAdditionally, the differences in regards of IR according to gender and its potential influence on cardiovascular risk is a controversial topic in the endocrinology field33. Our findings reflect that this difference is shown even in the measurement methods, with a greater predictive capacity of coronary risk in women, possibly associated with differences in serum levels of TG and HDL-C according to gender, an aspect that must be taken into consideration when making determinations.\n\nIt should be noted that because of the cross-sectional design of the study, causality cannot be defined, so future prospective design studies should evaluate these IR indices for the prediction of cardiovascular mortality. However, our findings demonstrate the importance of the TyG and TG/HDL indices, especially the latter in the prediction of coronary risk, and these are tools to determine IR of easier application in regions where the economic aspect is a limitation of measurement for serum insulin.\n\n\nData availability\n\nDataset 1: MMSPS Insulin resistance indices and coronary risk dataset. BMI: Body Mass Index, WaistC: Waist Circumference, BP: Blood Pressure; hs-CRP: high Sensitivity C Reactive Protein. DOI, 10.5256/f1000research.13610.d18958734",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by research grant Nº CC-0437-10-21-09-10 from the Technological, Humanistic, and Scientific Development Council (Consejo de Desarrollo Científico, Humanístico y Tecnológico; CONDES), University of Zulia.\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\nRojas J, Bermúdez V, Leal E, et al.: Insulinorresistencia e hiperinsulinemia como factores de riesgo para enfermedad cardiovascular. Archivos Venezolanos de Farmacología y Terapéutica. 2008; 27(1Suppl): 29–39. Reference Source\n\nGastaldelli A: Role of beta-cell dysfunction, ectopic fat accumulation and insulin resistance in the pathogenesis of type 2 diabetes mellitus. Diabetes Res Clin Pract. 2011; 93(Suppl 1): S60–S65. PubMed Abstract | Publisher Full Text\n\nAminot-Gilchrist DV, Anderson HD: Insulin resistance-associated cardiovascular disease: potential benefits of conjugated linoleic acid. Am J Clin Nutr. 2004; 79(6 Suppl): 1159S–1163S. PubMed Abstract | Publisher Full Text\n\nDe Felice FG, Lourenco MV, Ferreira ST: How does brain insulin resistance develop in Alzheimer's disease? Alzheimers Dement. 2014; 10(1 Suppl): S26–32. PubMed Abstract | Publisher Full Text\n\nFonseca VA: Early identification and treatment of insulin resistance: impact on subsequent prediabetes and type 2 diabetes. Clin Cornerstone. 2007; 8 Suppl 7: S7–18. PubMed Abstract | Publisher Full Text\n\nDeFronzo RA, Tobin JD, Andres R: Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol. 1979; 237(3): E214–223. PubMed Abstract | Publisher Full Text\n\nMatthews DR, Hosker JP, Rudenski AS, et al.: Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia. 1985; 28(7): 412–419. PubMed Abstract | Publisher Full Text\n\nSalazar J, Bermúdez V, Calvo M, et al.: Optimal cutoff for the evaluation of insulin resistance through triglyceride-glucose index: A cross-sectional study in a Venezuelan population [version 2; referees: 2 approved]. F1000Research. 2017; 6: 1337. Publisher Full Text\n\nAbel ED, O'Shea KM, Ramasamy R: Insulin resistance: metabolic mechanisms and consequences in the heart. Arterioscler Thromb Vasc Biol. 2012; 32(9): 2068–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBermúdez V, Marcano RP, Cano C, et al.: The Maracaibo city metabolic syndrome prevalence study: design and scope. Am J Ther. 2010; 17(3): 288–294. PubMed Abstract | Publisher Full Text\n\nSimental-Mendía LE, Rodríguez-Morán M, Guerrero-Romero F: The product of fasting glucose and triglycerides as surrogate for identifying insulin resistance in apparently healthy subjects. Metab Syndr Relat Disord. 2008; 6(4): 299–304. PubMed Abstract | Publisher Full Text\n\nMcLaughlin T, Abbasi F, Cheal K, et al.: Use of metabolic markers to identify overweight individuals who are insulin resistant. Ann Intern Med. 2003; 139(10): 802–9. PubMed Abstract | Publisher Full Text\n\nBermúdez V, Rojas J, Martínez MS, et al.: Epidemiologic Behavior and Estimation of an Optimal Cut-Off Point for Homeostasis Model Assessment-2 Insulin Resistance: A Report from a Venezuelan Population. Int Sch Res Notices. 2014; 2014: 616271. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBermúdez V, Salazar J, Bello L, et al.: Coronary Risk Estimation According to a Recalibrated Framingham-Wilson Score in the Maracaibo City Metabolic Syndrome Prevalence Study. The Journal for Cardiology Photon. 2014; 107: 160–170. Reference Source\n\nAmerican Diabetes Association: Standards of Medical Care in Diabetes-2017: Summary of Revisions. Diabetes Care. 2017; 40(Suppl 1): S4–S5. PubMed Abstract | Publisher Full Text\n\nBermúdez V, Rojas J, Salazar J, et al.: Sensitivity and Specificity Improvement in Abdominal Obesity Diagnosis Using Cluster Analysis during Waist Circumference Cut-Off Point Selection. J Diabetes Res. 2015; 2015: 750265. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBermudez V, Cabrera M, Mendoza L, et al.: Epidemiological behavior of high–sensitivity C-Reactive Protein (hs-CRP) in adult individuals in the Maracaibo city, Venezuela. Revista Latinoamericana de Hipertensión. 2013; 8: 16–24. Reference Source\n\nAkobeng AK: Understanding diagnostic tests 3: Receiver operating characteristic curves. Acta Pediatre. 2007; 96(5): 644–7. PubMed Abstract | Publisher Full Text\n\nDemler OV, Pencina MJ, D'Agostino RB Sr: Misuse of DeLong test to compare AUCs for nested models. Stat Med. 2012; 31(23): 2577–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuniyappa R, Iantorno M, Quon MJ: An integrated view of insulin resistance and endothelial dysfunction. Endocrinol Metab Clin North Am. 2008; 37(3): 685–711. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBornfeldt KE, Tabas I: Insulin resistance, hyperglycemia, and atherosclerosis. Cell Metab. 2011; 14(5): 575–585. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartínez-Larrad MT, Lorenzo C, González-Villalpando C, et al.: Associations between surrogate measures of insulin resistance and waist circumference, cardiovascular risk and the metabolic syndrome across Hispanic and non-Hispanic white populations. Diabet Med. 2012; 29(11): 1390–4. PubMed Abstract | Publisher Full Text\n\nKim MK, Ahn CW, Kang S, et al.: Relationship between the triglyceride glucose index and coronary artery calcification in Korean adults. Cardiovasc Diabetol. 2017; 16(1): 108. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim JH, Lee DY, Park SE, et al.: Triglyceride glucose index predicts coronary artery calcification better than other indices of insulin resistance in Korean adults: the Kangbuk Samsung Health Study. Precision and Future Medicine. 2017; 1(1): 43–51. Publisher Full Text\n\nSánchez-Íñigo L, Navarro-González D, Fernández-Montero A, et al.: The TyG index may predict the development of cardiovascular events. Eur J Clin Invest. 2016; 46(2): 189–97. PubMed Abstract | Publisher Full Text\n\nIrace C, Carallo C, Scavelli FB, et al.: Markers of insulin resistance and carotid atherosclerosis. A comparison of the homeostasis model assessment and triglyceride glucose index. Int J Clin Pract. 2013; 67(7): 665–672. PubMed Abstract | Publisher Full Text\n\nQuijada Z, Paoli M, Zerpa Y, et al.: The triglyceride/HDL-cholesterol ratio as a marker of cardiovascular risk in obese children; association with traditional and emergent risk factors. Pediatr Diabetes. 2008; 9(5): 464–71. PubMed Abstract | Publisher Full Text\n\nSalazar MR, Carbajal HA, Espeche WG, et al.: Identifying cardiovascular disease risk and outcome: use of the plasma triglyceride/high-density lipoprotein cholesterol concentration ratio versus metabolic syndrome criteria. J Intern Med. 2013; 273(6): 595–601. PubMed Abstract | Publisher Full Text\n\nSalazar MR, Carbajal HA, Espeche WG, et al.: Comparison of two surrogate estimates of insulin resistance to predict cardiovascular disease in apparently healthy individuals. Nutr Metab Cardiovasc Dis. 2017; 27(4): 366–373. PubMed Abstract | Publisher Full Text\n\nDu T, Yuan G, Zhang M, et al.: Clinical usefulness of lipid ratios, visceral adiposity indicators, and the triglycerides and glucose index as risk markers of insulin resistance. Cardiovasc Diabetol. 2014; 13: 146. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWallace TM, Levy JC, Matthews DR: Use and abuse of HOMA modeling. Diabetes Care. 2004; 27(6): 1487–95. PubMed Abstract | Publisher Full Text\n\nWatt MJ: Storing up trouble: does accumulation of intramyocellular triglyceride protect skeletal muscle from insulin resistance? Clin Exp Pharmacol Physiol. 2009; 36(1): 5–11. PubMed Abstract | Publisher Full Text\n\nKim SH, Reaven G: Sex differences in insulin resistance and cardiovascular disease risk. J Clin Endocrinol Metab. 2013; 98(11): E1716–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalazar J, Bermúdez V, Olivar LC, et al.: Dataset 1 in: Insulin resistance indices and coronary risk in adults from Maracaibo city, Venezuela: A cross sectional study. F1000Research. 2018. Data Source"
}
|
[
{
"id": "30640",
"date": "22 Feb 2018",
"name": "Miguel Murguía-Romero",
"expertise": [
"Reviewer Expertise Biomedical 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 “Insulin resistance indices and coronary risk in adults from Maracaibo city, Venezuela: A cross sectional study”, supports the use of TyG and TG/HDL formulas as a reliable way to estimate the coronary risk in a Venezuelan population, contributing with the use of these indices in the study of Latin American populations.\nThis article is valuable specifically because:\n\nAddresses the research problem in an appropriate way using the Framingham-Wilson formula to evaluate coronary risk, and including relevant IR indices in the analysis.\n\nThe results are valuable because the analysis by gender is included, as well as ROC curves are used to estimate the predictive capacity of the indices.\n\nThe statistical analysis is well applied: the descriptive statistical is informative and contribute to adequately contextualize the sample characteristics; also the chosen tests, and the statistical parameter reported are adequate.\n\nThe supplementary material is well structured and the data are congruent with the text (“General characteristics of the sample”) and with Table 1 (according to a rapid analysis I made importing the file into a database).\n\nThere are, however, several inconsistencies that may be corrected to improve it:\nAn inconsistency is evident in the formula of TyG; Simental et al. (2008) cite the TyG formula as:\nln[fasting triglycerides (mg/dL) x fasting glucose (mg/dL)/2] While this article cite TyG as: ln[fasting triglycerides (mg/dL) x fasting glucose (mg/dL) ]/2\nAre the differences between formulas only a typing error? I think authors have an opportunity to clarify which one of this formulas makes better sense for biomedical research along with the relationships between the physiological parameters included in this index.\n\nAt the header of Table 1, an “a” is missing in “Maracaibo”.\n\nThere is no correspondence between the declared quantity of participants with coronary risk bigger than 5% in the text (n=448; Page 4, 2nd column, 1st line) vs. the one reported in Table 1 (n=443)\n\nThe range of cell 1 in Table 1, written as “0.735 (0.707-0.63)”, is incongruent with the one reported in the text. Authors must clarified which one is the correct one.\n\nTable 3, first column: “TG/HDL” instead “TAG/HDL”. Please, review it for homogeneity also in “Discussion”.\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
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https://f1000research.com/articles/7-44
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https://f1000research.com/articles/7-297/v1
|
08 Mar 18
|
{
"type": "Research Article",
"title": "A draft genome sequence for the Ixodes scapularis cell line, ISE6",
"authors": [
"Jason R. Miller",
"Sergey Koren",
"Kari A. Dilley",
"Derek M. Harkins",
"Timothy B. Stockwell",
"Reed S. Shabman",
"Granger G. Sutton",
"Sergey Koren",
"Kari A. Dilley",
"Derek M. Harkins",
"Timothy B. Stockwell",
"Reed S. Shabman",
"Granger G. Sutton"
],
"abstract": "Background: The tick cell line ISE6, derived from Ixodes scapularis, is commonly used for amplification and detection of arboviruses in environmental or clinical samples. Methods: To assist with sequence-based assays, we sequenced the ISE6 genome with single-molecule, long-read technology. Results: The draft assembly appears near complete based on gene content analysis, though it appears to lack some instances of repeats in this highly repetitive genome. The assembly appears to have separated the haplotypes at many loci. DNA short read pairs, used for validation only, mapped to the cell line assembly at a higher rate than they mapped to the Ixodes scapularis reference genome sequence. Conclusions: The assembly could be useful for filtering host genome sequence from sequence data obtained from cells infected with pathogens.",
"keywords": [
"tick",
"genome",
"cell line",
"ISE6",
"Ixodes scapularis"
],
"content": "Introduction\n\nThe Ixodes scapularis embryonic 6 (ISE6) cell line is a widely used resource that is permissive to pathogens including human pathogens transmitted by ticks. Two decades ago, a collection of I. scapularis cell lines were derived from embryonated eggs including IDE lines derived from northern ticks and ISE lines derived from southern ticks (Munderloh et al., 1994). Recent proteomics analysis suggested that the ISE6 line is derived from neuronal cells (Oliver et al., 2015). ISE6 cells have been used to isolate and analyze bacterial pathogens including: the causative agent of human granulocytic ehrlichiosis (HGE) (Munderloh et al., 1999); Borrelia burgdorferi, the causative agent of Lyme disease (Obonyo et al., 1999); the causative agent of southern tick-associated rash illness (STARI) (Varela et al., 2004); and Rickettsia felis, the causative agent of spotted fever (Pornwiroon et al., 2006). ISE6 cells have been used to study viral pathogens including: Semliki Forest virus (SFV) and Hazara virus (arbovirus, family Bunyaviridae, genus Nairovirus) (Garcia et al., 2005); and Langat virus (LGTV), a Flavivirus (Grabowski et al., 2016). The cells have also been used to study RNAi and genome engineering in ticks; reviewed in (Oliver et al., 2015).\n\nThe Ixodes scapularis (black-legged tick) genome had been estimated to harbor 70% repeat content (Ullmann et al., 2005) when our lab participated in a community effort to sequence and assemble a tick genome. A reference sequence built from 3.8X Sanger sequencing and the Celera Assembler (Istrail et al., 2004; Myers et al., 2000) software was fractured into 570,640 contigs (369,495 scaffolds) with a contig N50 of only 2,942 bp. The total contig span was 1.388 Gbp though the genome size was estimated at 2.1 Gbp. The assembly supported the annotation of 20,486 protein-coding genes and an extensive analysis of tick biology (Gulia-Nuss et al., 2016). The assembly is maintained at VectorBase (Giraldo-Calderón et al., 2015) under the name IscaW1.\n\nA genome assembly for the ISE6 genome would assist investigations of ISE6 as a biological system. It would also provide a host subtraction tool for ISE6-based sequencing assays. Host subtraction is the bioinformatics process of filtering reads whose origin is host DNA and RNA (Daly et al., 2015). Host subtraction enriches the non-host component of sequence datasets and is especially attractive for assays involving high-throughput sequencing technologies that generate short reads in high volume where data reduction can realize cost savings. Following host subtraction, remaining reads can be mapped to references and counted, or used as queries to sequence databases, or assembled to reconstruct novel transcript or genome sequences. With an expectation that the IES6 genome would be as challenging as the tick genome, we sequenced IES6 with high coverage and long reads that, taken together, might generate a high quality reference genome assembly.\n\n\nMethods\n\nISE6 cells were obtained from the American Type Culture Collection (ATCC), cell line CRL-11974, lot number 100005, patent 5,869,335. This cell line had been isolated from ticks collected in Georgia, USA. As generally described by ATCC technical bulletins and personal communications, cells were grown in Leibovitz L-15B media pH 7.0 (Leibovitz, 1963) (ThermoFisher Scientific) supplemented with 80 mM glucose, 10% tryptose phosphate broth, 0.1% bovine lipoprotein cholesterol concentrate and 2% heat inactivated fetal bovine serum (Munderloh & Kurtti, 1989). An addition of 0.7% non-essential amino acids (NEAA) concentrate (ThermoFisher Scientific) was also added prior to incubation. The cells were incubated in a gently shaking flask at 31°C with no C02.\n\nFor short-read sequencing, genomic DNA was isolated from the cell line using a Qiagen genomic DNA isolation kit. Bioanalyzer analysis confirmed high molecular weight DNA was recovered. The library was size selected using Pippin Prep and prepared using the NextGen paired end barcoded genomic library construction protocol. Library quantification and normalization was performed by qPCR. The library was sequenced on the Illumina NextSeq 500 platform to generate 2x150 paired reads. Reads were demultiplexed which removed barcodes and sequencing adapters, and further treated with CutAdapt 1.8.1 to remove any remaining adapter.\n\nFor long-read sequencing, cells were grown until they attached to the flask. Genomic DNA was extracted from ISE6 cells using a Qiagen Genomic DNA isolation kit, stopping prior to the G2 isolation step. Frozen pellets and frozen cells were shipped to the Icahn School of Medicine at Mount Sinai for library construction and sequencing using standard SMRTbell template preparation kits (Pacific Biosciences). A total of 52 SMRTcells were run on the PacBio Sequel platform using standard PacBio protocols.\n\nThe long reads were corrected and assembled with the Canu assembler (Koren et al., 2017) version 1.6. Canu was run with the SGE grid engine and Java 1.8 using default parameters except: minOverlapLength = 1000 bp, corMhapSensitivity = \"low\", and genomeSize = 1 Gbp. The contig consensus sequences were polished using SMRT Link version 5.0.1.9585 which includes Arrow version 2.2.1, blasr 5.3, and pbalign 0.3.1 (Pacific Biosciences).\n\nShort reads were mapped with bowtie2 (Langmead & Salzberg, 2012) version 2.2.5 using either end-to-end or local-alignment mode as indicated in the text. Using default settings, the mapper reported at most one mapping per read and reported read maps individually though it used reads as pairs to select alignments. Mappings were analyzed with samtools (Li et al., 2009) version 1.2.1 and bedtools (Quinlan, 2014; Quinlan & Hall, 2010) version 2.26. K-mers were counted using Jellyfish (Marçais & Kingsford, 2011) version 2.2.6 for several values of K: 11, 15, 21, 41, 51, and 61. Each computed K-mer histogram was analyzed with GenomeScope (Vurture et al., 2017). Single-copy gene analysis used BUSCO (Simão et al., 2015) version 3.0.2 with Arthropoda OrthoDB version 9. Alignments between the tick and cell line assemblies were computed on contigs from each assembly using nucmer, the local aligner in the MUMmer package (Kurtz et al., 2004) version 3.1. The BLAST analysis used TBLASTN in NCBI BLAST+ (Camacho et al., 2009) version 2.2.31. The Ixodes protein predictions were downloaded from UniProt in Nov 2017.\n\n\nResults\n\nPacBio sequencing yielded 192.5 Gbp in 27.3 million unpaired reads, providing approximately 92X coverage of the estimated 2.1 Gbp tick genome. This dataset included 190.7 Gbp in reads >= 1 Kbp and 115.9 Gbp in reads >= 10 Kbp. To overcome high base call error observed in single-molecule long-read data, the long reads were subjected to the Canu correction process which filtered, trimmed, and polished reads based on alignment with other long reads (Koren et al., 2017). Correction yielded 36.7 Gbp in 2.1 million reads with a 17,680 bp N50. Read counts are shown in Table S1. Read length distributions, before and after correction, are shown in Figure S1.\n\nThe corrected long reads were assembled in isolation with the Canu long-read assembler (Koren et al., 2017). The initial assembly, named Ise6_asm0, contained 18,717 contigs. The uncorrected long reads were used to polish the contig consensus sequences using the Arrow process. This was run in two iterations to produce assemblies Ise6_asm1 and Ise6_asm2 respectively. The released assembly, named Ise6_asm2, contained 2,691,078,110 bases in 18,717 contigs with a 269,660 bp contig N50. Statistics for each assembly are shown in Table S2.\n\nSince the tick contigs are generally smaller than the cell line contigs, it seemed likely that some tick contigs would be wholly contained by cell line contigs. The IscaW1 and Ise6_asm0 assemblies were compared with the MUMmer local alignment software; see Table S3. A high-confidence alignment subset (filtered with delta-filter -1) was used. The average identity of aligned bases was 96.58%. Both assemblies were almost fully covered by alignments. Using alignments of 1 Kbp or more, IscaW1 contigs with at least one alignment made up 78.6% of IscaW1 bases. The set of IscaW1 contigs with at least 90% coverage in any one alignment made up 35.5% of IscaW1 bases.\n\nTo provide an orthogonal dataset for assembly assessment, the genome was also sequenced on a short-read platform. Illumina sequencing yielded 25 Gbp in 171 million 2x150 bp paired reads, providing approximately 24X short-read coverage of the 2.1 Gbp estimated genome size of the tick. Comparative map statistics are shown in Table S4. When paired reads were mapped to Ise6_asm0 using a local alignment algorithm, 97.83% of reads mapped as a concordant pair. For comparison, when the reads were mapped to the IscaW1 tick reference assembly, 91.68% of reads mapped as a concordant pair. This result indicates that the ISE6 assembly is more representative of ISE6 genome structure than the Ixodes reference.\n\nFor consensus quality assessment, the paired reads were mapped to Ise6_asm2 and IscaW1 contigs using a more stringent, global (end-to-end) alignment algorithm; see Table S4. Among these alignments, the read sequence disagreement with the contig consensus was 1.79% for Ise6_asm2 and 5.03% for IscaW1. This demonstrates that the ISE6 consensus is more representative of ISE6 genome sequence than the Ixodes reference.\n\nThe rates of concordant pair mapping to zero, one, or multiple sites were 23%, 29%, and 48% respectively for Ise6_asm2 and 44%, 30%, and 25% for IscaW1; see Table S4. Thus, by paired-read mappability, both assemblies contain 29%–30% unique sequence while the ISE6 assembly captures an additional 23% of reads and these align to repeat sequences in the assembly.\n\nThe global alignment 23% unmapped rate in Ise6_asm2 is an order of magnitude larger than the unmapped rate among the local alignments. It is possible that the long and short read sequencing captured genuine differences at unstable regions of the cell line genome. It seems more likely that the genome harbors repeat instances that are similar-but-not-identical to those in the assembly.\n\nUsing the global alignments and accepting all mapped reads (whether mapped as a pair or not), the Ise6_asm2 assembly mapped 81% of reads while IscaW1 mapped 65%. Thus, the Ise6_asm2 assembly outperformed the IscaW1 assembly as a host subtraction tool using pairwise local, pairwise global, and read-wise global alignments.\n\nThe assembly was assessed for completeness using gene content analysis. The latest UniProt protein predictions on the IscaW1 tick genome assembly were used as TBLASTN query sequences against the cell line assembly. Out of 20,473 predicted proteins: 20,290 (99.1%) had at least one hit in Ise6_asm0 while 183 predictions had no hit. The Ise6_asm2 assembly was analyzed for gene content using the BUSCO collection of genes thought to be single-copy in arthropod genomes; Table S5. Of 1066 genes searched, 1.4% were fragmented, 3.6% were missing, and 95% were complete. These results indicate that the assembly is fairly complete for single-copy genes.\n\nThe Ise6_asm2 contig span is 2.8 Gbp which exceeds the 1.4 Gbp contig span of the IscaW1 tick reference assembly as well as the 2.1 Gbp estimated genome size for tick. The discrepancy could be due to several factors. It is possible that the cell line genome is larger than the tick genome, or that the assembly contains dual representations of heterozygous loci that assembled separately, or that the IscaW1 reference assembly underrepresents repeats present in the tick and ISE6 genomes. These possibilities were explored with several analyses.\n\nK-mer analysis (Vurture et al., 2017) provides an assembly-free genome size estimate extrapolated from the frequency distributions of short, contiguous sequences extracted from the sequencing reads and counted using an exact-match algorithm (Marçais & Kingsford, 2011). In K-mer analysis of our short-read data, the observed distribution could not be fit to a model. Analysis of the corrected long read K-mers was similarly inconclusive. As illustrated by the representative plot in Figure S2, the distribution does not include a strong peak other than the mode at 1X. These results are possibly due to the low coverage in both datasets, which would be especially low if heterozygous haplotypes were represented separately in contigs.\n\nNext, genome size estimation was attempted using coverage analysis of paired reads mapped to contigs. Pairs mapped end-to-end provided 39.9 Gbp of mapped bases. In the distribution of read coverage per contig base, there is a smooth peak with a 9X mode and a tail at higher coverage; see Figure S3. Using the mapped base count divided by 9X to represent the average coverage of unique sequence in the genome, the extrapolated genome size is 4.43 Gbp, which is about twice as large as the tick estimated genome size. This suggests that the assembly process separated the haplotypes of a 2.22 Gbp diploid genome.\n\nLong-read coverage was analyzed next. The uncorrected long reads had been mapped to Ise6_asm1 contigs for the final iteration of Arrow consensus polish. Based on those results, the per-base coverage peaks at 34X with a shoulder at almost twice that level; see Figure 1. This suggests that 34X represents the coverage mode for haplotype-separated sequence. With 175.48 Gbp in mapped reads, 158.47 Gbp of read sequence aligned to 151.93 Gbp of contig sequence. These mapping and coverage results combine to indicate 151.93 / 34 = 4.47 Gbp size for the combined haplotypes (diploid) and 2.24 Gbp for the haploid genome. Thus, the completeness of the 2.8 Gbp assembly is uncertain and the assembly appears to harbor double-representation of loci that are haplotype-separated and under-representation of genomic repeats, as indicated in Figure 1.\n\nRead coverage per base was computed by mapping uncorrected long reads to assembled contigs. Colors were added to highlight the following interpretation. Green: the minor peak of low-coverage bases suggests 87 Mbp of contig bases lies near breakpoints such as contig ends and false joins. Blue: the dominant peak, with mode at 34X, suggests that 1769 Mbp of the assembly is haplotype-separated sequence possibly representing 885 Mbp of the diploid genome. Yellow: the shoulder near 64X suggests that about 654 Mbp of the diploid genome is captured as diploid-consensus sequence. Red: high coverage (including bases not shown with coverage over 100X) suggests that under-represented genomic repeats occupy 54 Mbp of the assembly but more of the genome.\n\nLocal alignments between assemblies were examined for support of the haplotype separation hypothesis. The cell line Ise6_asm0 contigs were aligned to the tick reference IscaW1 contigs using the nucmer local alignment software. As shown in Figure S4, the largest IscaW1 contig aligns full-length to two contigs of Ise6_asm0. The full set of alignments was filtered to retain one best alignment per Ise6_asm0 position (with delta-filter -q) and to retain only IscaW1 contigs with at least 50% coverage in one such alignment. Using these alignments to distinguish IscaW1:ISE6 multiplicities, 43% of IscaW1 bases are in contigs with 1:1 relation to ISE6, while 17% of bases are in contigs with 1:2 relations, and 3% of bases are in contigs with relations of 1:3 or higher; see Table S3. Not all contigs could be categorized. Further work would be required to fully partition and phase all duplicated contigs with high confidence.\n\nOf 1066 BUSCO genes searched against Ise6_asm2, 67% were complete and single-copy, and 28% were complete and duplicated. This indicates that almost a third of single-copy genes are duplicated in the assembly, presumably due to haplotype separation. If this rate of sequence duplication applies to the assembly overall, the 2.67 Gbp assembly indicates a 2.29 Gbp genome size.\n\nDuring the assembly submission to GenBank, NCBI flagged one contig for sequence similarity to Rickettsia, a genus of endosymbionts common to Ixodes scapularis (Gillespie et al., 2012; Labruna et al., 2007; Steiner et al., 2008; Zeringóta et al., 2017). The long read coverage in the flagged region differs from the flanking regions; see Figure S5. The contig was split and re-submitted without the flagged region.\n\n\nDiscussion\n\nThe ISE6 cell line, derived from the Lyme disease tick Ixodes scapularis, is widely used but has so far lacked a genome reference sequence. With this report, the genome has been sequenced, assembled, and released. The genome sequence was generated from high coverage in PacBio Sequel long reads, assembled with the Canu assembler, and polished with Arrow. Gene content analysis indicated that the assembly is largely complete though read mapping analysis indicated that some genomic repeats are under-represented in the assembly. Read mapping and single-gene analysis indicated that portions of the genome are represented as a diploid consensus while other portions are represented in haplotype-separated copies. The new assembly provides a more accurate representation of the cell line genome compared to the previous closest reference, which was an assembly of the I. scapularis tick genome. In our mapping of cell line gDNA short read pairs that were not used for the assembly, the cell line assembly was more effective for identifying host reads compared to the tick reference. Thus, the new assembly provides a resource for analysis of the cell line and for host subtraction to assist the detection of pathogens present in the cells.\n\nComparable genome size estimates were obtained by three methods. Short-read coverage analysis indicated 2.22 Gbp. Long-read coverage indicated 2.24 Gbp. Single-copy gene analysis indicated 2.29 Gbp. The tick genome was previously estimated to be 2.1 Gbp so the cell line may harbor some ISE6-specific sequence. Identification of such sequences is left for future work. Our local alignments of the tick and ISE6 assemblies covered nearly all of both assemblies, so any cell-line-specific sequence is likely to involve genomic repeats.\n\nWe hope to enhance the assembly resource in several ways. We hope to generate a second version of the sequence using different assembly parameters. A larger genome size estimate, in particular, should lead to higher generated coverage in corrected reads. Additional corrected reads would necessarily be shorter than the ones used here, but they could provide additional depth for repeat detection and repeat resolution. We hope to provide gene annotation, repeat content analysis, and more specific haplotype separation analysis of the second assembly.\n\n\nData availability\n\nThe gDNA Illumina and PacBio reads are available at NCBI SRA under BioSample SAMN06329993. This entire assembly has been deposited at GenBank under the accession GCA_002892825.1. Whole Genome Shotgun sequencing project is available under accession PKSA00000000.",
"appendix": "Competing interests\n\n\n\nSK has received travel support to speak at Oxford Nanopore conferences.\n\n\nGrant information\n\nJCVI staff was supported by DHS contract HSHQDC-15-C-B0059. SK was supported by the Intramural Research Program of the National Human Genome Research Institute, 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\nAcknowledgements\n\nThis study utilized the computational resources of the Biowulf system at the National Institutes of Health, Bethesda, MD (https://biowulf.nih.gov). Vinita Puri assisted the sample preparation at JCVI. PacBio sequencing was performed at the Icahn School of Medicine at Mount Sinai by Robert P. Sebra, Melissa L. Smith, and Ying-Chih Wang.\n\n\nSupplementary material\n\nFigure S1. Length distributions for PacBio long reads before (blue) and after (green) correction by the Canu assembler. The comparison indicates that the correction step removed most short reads while reducing the number and lengths of long reads. Each blue or green vertical bar represents the number of reads in a 10 bp window. Reads longer than 30 Kbp (not shown) provided 1,623,767,067 bp in 47,967 reads before correction and 333,923,002 bp in 10,211 reads after correction.\n\nClick here to access the data.\n\nFigure S2. K-mer frequency distribution plotted by GenomeScope from K-mers found in PacBio long reads by Jellyfish with K=41. Similar plots were generated by GenomeScope for selected values of K ranging from 11 to 61 from the corrected long reads and also the (uncorrected) short reads. The distribution is dominated by K-mers that occur once (coverage = 1X), which can be attributed to random base call error.\n\nClick here to access the data.\n\nFigure S3. Histogram of short-read coverage of large contigs. The mode of the distribution is at 9X. The minor peak at 0X indicates uncovered contig bases, which are frequent at contig ends. The pseudo-peak at 51X represents all bases covered at 51X or greater. Reads were mapped to all contigs but only large contigs (length >= 10Kbp) were plotted here. The mapping used end-to-end alignments and reported at most one map per read. The mapping used reads as pairs but reported all mapped reads. A map report produced with ‘samtools stats’ reported 277,543,724 reads mapped including 261,690,992 properly paired in the mapping, and 39,913,302,735 based mapped and no clipping. Assuming 9X is the average read coverage of unique sequence in the genome, the genome size is estimated as 278 Gbp / 9X = 4.4 Gbp, or roughly twice the tick estimated genome size. This suggests that the assembly captures many diploid loci twice and that 9X is the average coverage of a haplotype-separated sequence in the assembly.\n\nClick here to access the data.\n\nFigure S4. Alignment of two ISE6 contigs to one IscaW1 contig. Contig ABJB010274751.1, the longest IscaW1 contig, has length 117,687 bp and is represented full-length on the X-axis. This contig has nearly-complete, full-length coverage in alignments to two ISE6_ASM1 contigs, each represented full-length on the Y-axis. Alignments were computed with nucmer and plotted with mummerplot using minimum length 1000 bp. ISE6_ASM1 contig tig00013410 (top) has length 309,049 bp and 19 local alignments including the 13 large alignments shown. ISE6_ASM1 contig contig tig00009157 has length 719,239 bp (bottom) and 30 local alignments including the 20 large alignments shown.\n\nClick here to access the data.\n\nFigure S5. Sequence identified as Rickettsia has unusual read coverage distribution. As part of the Ise6_asm2 assembly submission to GenBank, NCBI flagged one contig for having sequence similarity to Rickettsia, A search using NCBI megablast and the nt database confirmed that tig00009859, of length 618,170 bp, had a portion of sequence that was most similar to Rickettsia (various species). Uncorrected PacBio reads had been mapped to the Ise6_asm1 version of the assembly as part of the Arrow polish process that generated Ise6_asm2. Read coverage at every base was extracted from that mapping using samtools. Read coverage was plotted for the three coordinate ranges 1–250,000 (orange, upstream), 250,000–350,000 (blue, contig middle), and 350,000–600,000 (downstream, green). The flagged region falls within the contig middle coordinates. Because the contig middle appeared to have a different coverage profile from both flanking sequencings, the flagged region (268 Kbp to 330 Kbp) of the contig was removed and the upstream and downstream contigs were re-submitted to GenBank.\n\nClick here to access the data.\n\nTable S1. ISE6 sequencing results.\n\nClick here to access the data.\n\nTable S2. ISE6 contig size statistics for several assemblies.\n\nClick here to access the data.\n\nTable S3. Nucleotide local alignments between the contigs of IscaW1 and Ise6_asm0. Alignments were generated and analyzed with the MUMmer package including the delta-filter alignment filter.\n\nClick here to access the data.\n\nTable S4. Alignment rates for paired reads mapped to contigs. Short reads were mapped with bowtie2 in local mode or global (end-to-end) mode.\n\nClick here to access the data.\n\nTable S5. Single-gene content analysis with BUSCO. Analysis of Ise6_asm2 using the Arthropoda OrthoDB version 9 single-copy genes.\n\nClick here to access the data.\n\n\nReferences\n\nCamacho C, Coulouris G, Avagyan V, et al.: BLAST+: architecture and applications. BMC Bioinformatics. 2009; 10: 421. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDaly GM, Leggett RM, Rowe W, et al.: Host Subtraction, Filtering and Assembly Validations for Novel Viral Discovery Using Next Generation Sequencing Data. PLoS One. 2015; 10(6): e0129059. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarcia S, Billecocq A, Crance JM, et al.: Nairovirus RNA sequences expressed by a Semliki Forest virus replicon induce RNA interference in tick cells. J Virol. 2005; 79(14): 8942–8947. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGillespie JJ, Joardar V, Williams KP, et al.: A Rickettsia genome overrun by mobile genetic elements provides insight into the acquisition of genes characteristic of an obligate intracellular lifestyle. J Bacteriol. 2012; 194(2): 376–394. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGiraldo-Calderón GI, Emrich SJ, MacCallum RM, et al.: VectorBase: an updated bioinformatics resource for invertebrate vectors and other organisms related with human diseases. Nucleic Acids Res. 2015; 43(Database issue): D707–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrabowski JM, Perera R, Roumani AM, et al.: Changes in the Proteome of Langat-Infected Ixodes scapularis ISE6 Cells: Metabolic Pathways Associated with Flavivirus Infection. PLoS Negl Trop Dis. 2016; 10(2): e0004180. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGulia-Nuss M, Nuss AB, Meyer JM, et al.: Genomic insights into the Ixodes scapularis tick vector of Lyme disease. Nat Commun. 2016; 7: 10507. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIstrail S, Sutton GG, Florea L, et al.: Whole-genome shotgun assembly and comparison of human genome assemblies. Proc Natl Acad Sci U S A. 2004; 101(7): 1916–1921. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoren S, Walenz BP, Berlin K, et al.: Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017; 27(5): 722–736. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKurtz S, Phillippy A, Delcher AL, et al.: Versatile and open software for comparing large genomes. Genome Biol. 2004; 5(2): R12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLabruna MB, Pacheco RC, Richtzenhain LJ, et al.: Isolation of Rickettsia rhipicephali and Rickettsia bellii from Haemaphysalis juxtakochi ticks in the state of São Paulo, Brazil. Appl Environ Microbiol. 2007; 73(3): 869–873. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLangmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4): 357–359. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeibovitz A: The growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere. Am J Hyg. 1963; 78(2): 173–180. PubMed Abstract | Publisher Full Text\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–2079. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarçais G, Kingsford C: A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics. 2011; 27(6): 764–770. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMunderloh UG, Jauron SD, Fingerle V, et al.: Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture. J Clin Microbiol. 1999; 37(8): 2518–2524. PubMed Abstract | Free Full Text\n\nMunderloh UG, Kurtti TJ: Formulation of medium for tick cell culture. Exp Appl Acarol. 1989; 7(3): 219–229. PubMed Abstract | Publisher Full Text\n\nMunderloh UG, Liu Y, Wang M, et al.: Establishment, maintenance and description of cell lines from the tick Ixodes scapularis. J Parasitol. 1994; 80(4): 533–543. PubMed Abstract | Publisher Full Text\n\nMyers EW, Sutton GG, Delcher AL, et al.: A whole-genome assembly of Drosophila. Science. 2000; 287(5461): 2196–2204. PubMed Abstract | Publisher Full Text\n\nObonyo M, Munderloh UG, Fingerle V, et al.: Borrelia burgdorferi in tick cell culture modulates expression of outer surface proteins A and C in response to temperature. J Clin Microbiol. 1999; 37(7): 2137–2141. PubMed Abstract | Free Full Text\n\nOliver JD, Chávez AS, Felsheim RF, et al.: An Ixodes scapularis cell line with a predominantly neuron-like phenotype. Exp Appl Acarol. 2015; 66(3): 427–442. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPornwiroon W, Pourciau SS, Foil LD, et al.: Rickettsia felis from cat fleas: isolation and culture in a tick-derived cell line. Appl Environ Microbiol. 2006; 72(8): 5589–5595. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuinlan AR: BEDTools: The Swiss-Army Tool for Genome Feature Analysis. Curr Protoc Bioinformatics. 2014; 47: 11.12.1–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuinlan AR, Hall IM: BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–842. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimão FA, Waterhouse RM, Ioannidis P, et al.: BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31(19): 3210–3212. PubMed Abstract | Publisher Full Text\n\nSteiner FE, Pinger RR, Vann CN, et al.: Infection and co-infection rates of Anaplasma phagocytophilum variants, Babesia spp., Borrelia burgdorferi, and the rickettsial endosymbiont in Ixodes scapularis (Acari: Ixodidae) from sites in Indiana, Maine, Pennsylvania, and Wisconsin. J Med Entomol. 2008; 45(2): 289–297. PubMed Abstract | Publisher Full Text\n\nUllmann AJ, Lima CM, Guerrero FD, et al.: Genome size and organization in the blacklegged tick, Ixodes scapularis and the Southern cattle tick, Boophilus microplus. Insect Mol Biol. 2005; 14(2): 217–222. PubMed Abstract | Publisher Full Text\n\nVarela AS, Luttrell MP, Howerth EW, et al.: First culture isolation of Borrelia lonestari, putative agent of southern tick-associated rash illness. J Clin Microbiol. 2004; 42(3): 1163–1169. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVurture GW, Sedlazeck FJ, Nattestad M, et al.: GenomeScope: fast reference-free genome profiling from short reads. Bioinformatics. 2017; 33(14): 2202–2204. PubMed Abstract | Publisher Full Text\n\nZeringóta V, Maturano R, Luz HR, et al.: Molecular detection of Rickettsia rhipicephali and other spotted fever group Rickettsia species in Amblyomma ticks infesting wild birds in the state of Minas Gerais, Brazil. Ticks Tick Borne Dis. 2017; 8(1): 81–89. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "31696",
"date": "22 Mar 2018",
"name": "Scott Emrich",
"expertise": [
"Reviewer Expertise Genome informatics",
"vector genomics",
"sequence-based bioinformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMiller et al. present a long-read based assembly of the ISE6 tick cell line. Although mostly in the background/discussion sections of this paper, the Ixodes genome has been hard to assemble given a large number of repeats and heterogeneity between individuals. Similar to other recent long read efforts of mosquitoes, continuity / completeness has been improved by at the cost of residual haplotypes; only ~66% of the genome is single copy.\n\nOn the one hand this does not impact the main use case of this effort, namely an improved reference for host extraction. As a nice twist the authors included an Illumina experiment, for example, and showed more host (tick) DNA aligned to their reference. Given this cell line is most used for pathogen-related work this is a valuable contribution even though the assembly itself contains haplotypes.\nOn the other hand, this paper in of itself does not really provide any new solutions or even discussion on how to improve assembly or a reference tick genome, which makes it more of a data note (and fine for F1000Research). For example, getting DNA from a single individual is sometimes hard so other projects have resorted to using full sibs. Given that the cell line was derived from ticks (plural) is it even feasible to dilute the cell line to get what amounts to clonal flasks and try and sequence that? That would not help the repeat issue but may reduce the amount of heterogeneity.\nI have a few remaining specific questions/comments listed below:\n1. On page 3 you mention because 6.7% more pairs are concurrent you state \"This result indicates that the ISE6 assembly is more representative of ISE6 genome structure than the Ixodes reference.\" This is not wrong, but I have two followup questions. First, in the next section you observe that there is over 5% divergence between your line and the Ixodes reference (which makes sense). Could this affect your short-read alignment parameters and cause less pairs to map? Second, what if your contigs are 2X bigger that map these additional pairs but they are repeated in your assembly but not the genome (residual heterogyosity). I guess my issue is using \"structure\" vs. a more generic \"contents\" given the issues reported in the build.\n2. Similarly, you state on page 3 that the 25% of reads that map to multiple sites are repeats, but couldn't they map to un merged alleles given the later results in the manuscript? I realize repeats is ambiguous and the haplotype issue is discussed later but maybe generalize to \"repeated sequences present in the assembly\" (or similar) to allow for assembly artifacts and \"real\" repetitive sequences present in tick\n3. I was curious as to the BUSCO results of the old assembly ... the Ixodes reference was done long enough ago I am not sure they would have used it and a reader shouldn't have to look it up. In short, if we ignore the allele duplication issue are genes assembled better? The answer was yes for Aedes and I assume so here but it is unclear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "31976",
"date": "03 Apr 2018",
"name": "Chan Heu",
"expertise": [
"Reviewer Expertise Rickettsiology",
"tick-associated bacteria",
"molecular biology",
"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\nThe authors assembled a draft genome of ISE6 using long-read sequencing. In addition, they used short-read sequencing to validate and distinguish the ISE6 assembly through differences in genome size and mapping coverage from the already sequenced, albeit incomplete, Ixodes scapularis genome. The primary aim is to generate the genome of ISE6 for host genomic for potential application such as the subtraction from genomic analysis of pathogens and/or symbionts cultured in ISE6. The authors demonstrated very well that the draft genome of ISE6 showed more coverage than the tick genome. It was also a great approach to use three different methods to estimate the genome size.\nThere were a few areas that could have been improved upon. This article would have benefited from demonstrating if there is any difference in using the ISE6 genome assembly vs. the tick genome for subtracting host genomic background, or in some other application in which using the assembled ISE6 genome would be more beneficial than the tick genome. In addition, the authors could discuss further the origin of the contig that was similar to Rickettsia. For instance, the authors could hypothesize whether this contig came from a lateral gene transfer event, rickettsial contamination of the starting material, or technical error in the assembly process. Nevertheless, having a draft genome of ISE6 will be useful for in vitro research involving tick-associated bacteria, protozoans, and viruses.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-297
|
https://f1000research.com/articles/6-1599/v1
|
30 Aug 17
|
{
"type": "Study Protocol",
"title": "Continuous training and certification in neonatal resuscitation in remote areas using a multi-platform information and communication technology intervention, compared to standard training: A randomized cluster trial study protocol",
"authors": [
"Carlos Alberto Delgado",
"Enrique M. Gómez Pomar",
"Pablo Velásquez",
"Víctor Sánchez",
"Roberto Shimabuku",
"Luis Huicho",
"RCPNEOPERU Study Group",
"Enrique M. Gómez Pomar",
"Pablo Velásquez",
"Víctor Sánchez",
"Roberto Shimabuku",
"Luis Huicho"
],
"abstract": "Background: About 10% of all newborns may have difficulty breathing and require support by trained personnel. In Peru, 90% of deliveries occur in health facilities. However, there is not a national neonatal resuscitation and certification program for the public health sector. In addition, the Andes and the Amazon regions concentrate large rural remote areas, which further limit the implementation of training programs and the accomplishment of continuous certification. Neonatal resuscitation training through the use of Information, Communication and Technology (ICT) tools, running on computers, tablets or mobile phones, may overcome such limitations. This strategy allows online and offline access to educational resources, paving the way to more frequent and efficient training, and certification processes. Objective: To evaluate the effects of a neonatal resuscitation training and certification program that uses a Multi-Platform ICT (MP-ICT) strategy on neonatal health care in remote areas. Methods: We propose to conduct the study through a cluster-randomized trial, where the study and analysis unit is the health care facility. Eligible facilities will include primary and secondary health care level facilities that are located in provinces with neonatal mortality rates higher than 15 per 1,000 live births. We will compare the proportion of newborns with a heart rate ≥100 beats per minute at two minutes after birth in health care facilities that receive MP-ICT training and certification implementation, with those that receive standard training and certification. Discussion: We expect that the intervention will be shown as more effective than the current standard of care. We are prepared to include it within a national neonatal resuscitation training and certification program to be implemented at national scale together with policymakers and other key stakeholders. Trial registration: ClinicalTrials.gov Nº NCT03210194 Status of the study: This study is ongoing. Study protocol version 1.1 – March 31st, 2017",
"keywords": [
"In service training",
"Neonatal resuscitation",
"Cluster randomized trial"
],
"content": "Introduction\n\nApproximately 10% of newborns may have difficulty breathing at birth and require urgent support from trained personnel1. The first 60 seconds of life in those conditions require immediate and effective interventions to help infants survive2. Unfortunately, some newborns in low- and middle-income countries do not have access to appropriately trained personnel, making asphyxia one of the leading causes of neonatal death in those settings3.\n\nIn Peru, about 550,000 births occur per year (Peruvian Institute for Statistics and Informatics 2015; https://www.inei.gob.pe), and this means that approximately 6 newborns each hour may require respiratory support at birth. Problems in training health workers for adequate support of newborns is aggravated by a high turnover rate of personnel, particularly in small cities and rural areas, which hampers regular conduction of local training programs, even if they are perceived as a positive incentive4. Peru is a high-middle-income country that has made significant progress in reducing neonatal and infant mortality in the last decade, despite inequities in access to health services, especially in rural areas of the Amazon and the Andes5,6. In the years 2011–2012, the Peruvian neonatal mortality rate was estimated at 12.8 per thousand live births7, with an estimated 20 per thousand live births for Pasco and Cusco regions, 15 for Ayacucho and Amazonas, 12 for Huancavelica and 8 for Lima8. From 2000 to 2013, neonatal mortality in Peru fell by 51% from 16.2 deaths per 1000 livebirths to 8.09.\n\nThe Neonatal Resuscitation Program (NRP), from the American Academy of Pediatrics, includes evidence-based clinical guidelines and was designed to train health care providers and instructors in hospital settings1,10. A variant of the NRP program is the Helping Babies Breath (HBB) program, which was designed to provide neonatal resuscitation training in resource-poor settings. The HBB has shown to reduce neonatal mortality and it is proposed as a successful global intervention11. However, the greatest limitation of these programs is the need for recurrent training with a frequency of less than 12 months to maintain the acquired skills and knowledge12.\n\nOne of the few neonatal resuscitation programs that has been successfully implemented outside the United States is the Neonatal Resuscitation Program from the Brazilian Society of Pediatrics, which was implemented in 1991 (Brazilian Society of Pediatrics. Neonatal Resuscitation Program (Brazilian NRP); http://www.sbp.com.br/reanimacao). In Peru, despite several attempts made by the Social Security (Essalud) and the Ministry of Health13–16, there is still no national neonatal resuscitation program in place.\n\nA training system using Multi-Platform Information, Communication and Technology (MP-ICT) offers reliable and easier access to information and training packages from different locations, which can be implemented even in rural settings without access to the internet17. Platforms may be available online, and users can download the training content through a PC, tablet or mobile phone18. The training content may be repeatedly available after download, even in remote areas without access to internet.\n\nOur study aims to evaluate the performance of a continuous process of training and certification in neonatal resuscitation in primary and secondary health facilities in the departments of Ayacucho and Cusco, through the use of MP-ICT. It aims at providing proof of concept of the intervention, which then could be incorporated as a component of a national neonatal resuscitation program to be implemented nationwide.\n\nThe traditional way to train and certify health care professionals is through administration of a conventional theoretical course, reinforced subsequently by a practical component. This modality requires face-to-face contact between trainers and trainees. Health workers need to move from their job site to the instruction sites, usually located in the capital city of the department or in the capital city of the country. This poses economic and logistic challenges to both health authorities and health workers, and results in erratic frequency and low uptake of training courses.\n\nThis traditional training was considered as standard control, and it has been selected as the comparator for our MP-ICT intervention, because a comparison using placebo or no-training was considered to be unethical.\n\nThe use of MP-ICT for training and certification in neonatal resuscitation will increase the proportion of children with heart rate equal or greater than 100 beats per minute at two minutes of life, as compared to standard training.\n\nTo evaluate the effects of the use of a MP-ICT for training and certification on neonatal resuscitation in first and second level health facilities of Ayacucho and Cusco departments.\n\nTo assess intervention and control efficacy in selected health facilities, in order to compare: 1) Time to start positive pressure ventilation after birth; 2) Time to achieve heart rate greater or equal than 100 per minute; 3) Apgar at 1 minute and at 5 minutes; 4) Use of supplemental oxygen after 10 minutes of life; 5) Inspiratory oxygen fraction needed at 30 minutes after birth; 6) Mortality rate during the first 7 days of life; 7) Number of referrals to health facilities with greater resolution capacity during the first 7 days of life; 8) Number of certified health professionals as providers.; and 9) Number of certified health professionals as instructors.\n\nThe efficacy of the evaluation will be evaluated at 6 months after the intervention.\n\nStatus of the study: This study is ongoing.\n\nStudy protocol version 1.0 – March 31th, 2017.\n\nTrial registration data can be found in Supplementary File 1.\n\n\nResearch methodology and experimental plan\n\nAyacucho and Cusco are two Peruvian regions located to the south of the country. Both regions were selected for presenting a Neonatal Mortality Rate above 15 per 1,000 live births and for their accessibility for air travel. These regions cover areas of Andean highlands, but their territory is mainly on the Amazon rainforest (Peru Export and Tourism Promotion Board, 2017; http://www.peru.travel). Also, in those departments there is the \"Valley of the Apurímac, Ene and Mantaro Rivers\", also known as the VRAEM, which is a geopolitical extremely poor area and one of the major areas of coca growing in Peru19.\n\nAccording to Peruvian Registry of Institutions that Provide Health Services (RENIPRESS), Ayacucho has 418 and Cusco 848 registered health facilities (Registro Nacional de Instituciones Prestadoras de Servicios de Salud 2017 website; http://www.minsa.gob.pe/portalweb/02estadistica/estadistica_2.asp?sub5=2). However, last year (2016) almost 15,000 deliveries were attended at 80 public health facilities of primary and secondary level, which represents 50% of all deliveries in both regions (Sistema de Registro del Certificado de Nacido Vivo en Linea 2017 website; http://www.minsa.gob.pe/cnv/).\n\nBasic teams are composed by physicians (p), nurses (n) midwives (m) and technical nurses (t), who are registered in a database updated by the health personnel observatory reports (Observatorio de recursos humanos en salud 2017 website; http://observatorio.inforhus.gob.pe/). This observatory states that, up until June 11th, 2017, Ayacucho has 337p, 726n, 466m and 743t; and, Cusco has 661m, 942n, 486m and 806t.\n\nThe proposed study design is a randomized cluster trial. The units of study and analysis will be twelve health facilities from Ayacucho and Cusco departments. Each health facility will be a cluster. Clusters will be randomly assigned either to the MP-ICT course and certification package (intervention) or to the standard training and certification package (control). Additional details are shown in Table 1.\n\nLegend: Intervention ICT=Training and certification using Information and Communications Technology\n\nTemplate reference: Chan A-W, Tetzlaff JM, Gøtzsche PC, Altman DG, Mann H, Berlin JA, et al. SPIRIT 2013 explanation and elaboration: guidance for protocols of clinical trials. BMJ: British Medical Journal. 2013;346.\n\nThe objective of the intervention is not the patient, but the health provider. Training will be given to groups of health professionals who work in a health facility. Also, because of the geographical distribution of health facilities, their selection as units of study helps to minimize the possibility of contamination between interventions.\n\nPrimary and secondary level facilities located in Ayacucho and Cusco that have a neonatal mortality rate higher than 15 per 1,000 livebirths will be eligible. Health facilities whose authorities refuse participation of their health professionals, facilities with less than 290 births a year, facilities located at more than 210 kilometres from the department capital, and those located in high-risk areas due to social unrest will be excluded. Health professionals who attend deliveries and accept their voluntary participation will be included after signing informed consent forms.\n\nAssuming the occurrence of about 4,000 births during a period of 6 months of filed study observation, we aim at discriminating a 4% difference in the proportion of newborns with heart rate equal or greater 100 beats per minute at two minutes of life between the intervention and the control group18. We would require 12 clusters (health facilities) divided in two arms and assigning six for each arm, with an average of 334 deliveries per facility in the observation period. The proposed power of study is 80% and the acceptable alpha error is 5%. The percentage for heart rate >100 bpm at two minutes of life, during suitable resuscitation using self-inflated bag is 90%20, and it is the expected value for the intervention group. For the control group, it is expected at least 4 points below the value achieved in the intervention group (i.e. no more than 86%). There is no sample size for how many medical professionals should undertake the training. Just the professionals who attend deliveries will be invited to participate.\n\n\nDescription of interventions\n\nHealth facilities will not be blinded to the type of intervention they receive. The health professionals of each centre will be informed about the training they will receive after the allocation for Standard or MP-ICT training.\n\nStandard training will be conducted through a theoretical-practical course, which will be administered once during the study period, to all health professionals of the selected facilities, according to the randomization. It is an 8-hour course, with 3 hours of theory (based on suggested readings) and 5 hours of practice, which will take place during a single day, from 9:00 a.m. to 6:00 p.m. The course will be performed by staff of the Neonatal Unit of the National Institute of Child Health, and will be coordinated by an NRP instructor accredited by the American Academy of Pediatrics. Suggested readings will include the following sequential topics: 1) Initial Steps, 2) Positive Pressure Ventilation, 3) Cardiac Massage, 4) Intubation and Medications, and 5) General Review. Practical sessions will be carried out in groups, with 10 or more people per group, and will include all the health professionals of the facility. The practical content part will consist of simulations based on predetermined scenarios using neonatal mannequins and supplies needed for basic and advanced resuscitation, covering each component of the theoretical content. One instructor will be assigned for each group. A baseline and a follow-up theoretical test will be administered.\n\nParticipants who have completed their attendance to theoretical sessions, participated in the simulated practices and approved the printed exams taken the same day will we granted a Standard Certification. The practice will be evaluated qualitatively in terms of assistance, participation and performance of activities\n\nThe continuous process of training and certification in neonatal resuscitation will be developed as a multi-platform format, rechargeable online and accessible offline, and will be complemented with simulated practices. The preliminary format of this system is already in place, and is based on an interactive web platform (www.rcpneoperu.org), which was initially developed as part of the activities proposed in another project (Project first breath; http://www.grandchallenges.ca/grantee-stars/0690-01-10, adapted and used with permission). The platforms will contain training packages, and links to review and download neonatal resuscitation tools, technical documents, and regulatory information. During the first three months of the project, the platform will be improved and adjusted to the needs of the fieldwork in Ayacucho and Cusco. The adaptations include increasing hosting, ameliorating usability, uploading packages and videos, improvement of examination and certifications, and tests for readiness. The MP-ICT resource is user-friendly and can be accessed from remote locations through computers, personal portable devices and cell phones. It allows downloading of the different documents, which facilitates learning and theoretical evaluation in an interactive and virtual way, without the presence of instructors or teachers.\n\nAuthorized participants will be able to download the information for the theoretical activities in four packages: Package A, B, C and D: each one containing interactive tools as videos and action mazes (as Quandary), and other videos, documents or short text to read in Spanish. Through the website virtual meetings, as forums or chats, can be initiated. The interactivity is expressed in 360º videos, which allows the user to change the observation point in a recorded environment. The interactivity for action mazes, like Quandary (a software application suite for creating action mazes Version 2.3/Windows; http://www.halfbakedsoftware.com/quandary.php) has demonstrated that feedback-oriented interactive exercises increase higher-order cognitive skills21. The learning is completed according to the student's pace and needs, and the platforms offer didactic material. The learning process can be done in groups, but the exam is online and individual, and requires Internet connection. Staff can be trained to become a provider and then qualify to become an instructor. At least one instructor will be certified at each facility in order to train the trainers for continuing education in every facility. Instructors will be given access to the web platform to include news of their activities and to evaluate the practices of aspiring providers in their health facilities.\n\nThe theoretical online exam consists of 40 questions distributed in four modules encompassing basic and advanced neonatal resuscitation. Basic modules include a) Initial Steps of Resuscitation, and b) Positive Pressure Ventilation. Advanced modules include c) External cardiac massage, and d) Intubation and Medications.\n\nExamination for each module allows unlimited attempts, and it is necessary to achieve at least 60% success to be approved. According to approved modules and practices, health personnel can obtain a supplier or instructor certification in both basic neonatal resuscitation and advanced neonatal resuscitation. Two types of examination have been prepared, according to the objective of the applicant. One is an aimed at applicants to the qualification of Neonatal Resuscitation Provider. The other one, an advanced exam, is for applicants to the qualification of Neonatal Resuscitation Instructor.\n\nAll participants need to complete both the Theoretical and the Practical training components. The platform also will allow the use of an agenda-type tool for scheduling practices for aspiring providers or instructors who have passed their online exam. The Theoretical Component is available after the candidate has been registered in the multi-platform portal, to which they can access through a PC, tablet or cell phone with Internet connection. Access to the content is online the first time, but it may also be offline, after downloading the corresponding training modules.\n\nThe Practical Component can be taken only after the Theoretical Component is approved at the participants’ facilities. It consists of face-to-face simulations of predetermined clinical scenarios using mannequins and the needed equipment (bag and mask, suction bulbs, towels, etc). This component has 5 hours of practice, which will take place from 8:00 a.m. to 1:00 p.m. in the morning, or from 2:00 p.m. to 7:00 p.m. in the afternoon. Staff of the Neonatal Unit of the National Institute of Child Health and/or a local trained instructor will guide the practice. An NRP instructor accredited by the American Academy of Pediatrics will coordinate all practices.\n\nTrained instructors will rate the trainees’ performance on the Practical Component, according to assistance and participation in practical activities and qualitative qualification of approving performance. The Practical Component is intended only for health professionals who approved the theoretical exam online and who scheduled their participation with a selected instructor. Practical simulations will be carried out in groups of 3 to 4 people, and an instructor will be assigned for each group. They do not require theoretical presentations, pre-test or post-tests.\n\nTo be granted an MP-ICT Certification the trainee needs to have passed the online theoretical exam and the practical skills assessment.\n\nAn example of MP-ICT training can be found in Supplementary File 2.\n\nOur primary outcome is the percentage of infants with heart rate equal or greater than 100 per minute at two minutes of life. This outcome is an adequate proxy for effective response to neonatal resuscitation measures in the delivery room20,22,23.\n\nThe secondary outcomes are described in our secondary objectives.\n\nComputer-generated random numbers will be used to assign the selected health facilities to either MP-ICT training (Intervention Group) or to standard training (Control Group). There will be a matching process of health facilities by proportion of groups of non-medical professionals (nurses and obstetricians) and by availability of basic equipment and maternal and newborn care supplies (the information for which will be available to the health regional directorates), to ensure that health facilities are comparable. Once paired, they will be randomly allocated through a blocked randomization, to ensure a balanced distribution of facilities in each group.\n\nAfter randomization of each facility, standard training or MP-ICT training will be conducted. Evaluations will include a baseline assessment of available equipment and supplies for neonatal resuscitation, immediately after enrolment and before training. To determine the duration of the effects of the intervention, the proposed outcome indicators will be evaluated six months after the training. The primary and secondary outcomes will be assessed intrapartum (within 5 minutes after birth) and postpartum (at 24 hours and 7 days, to know the outcome of the infant). The assessments will be based on an observation sheet (see Supplementary File 3), in which trained research assistants (registered nurses or equivalent local personnel) will record the activities conducted during the delivery and the birth, and the results of the neonatal resuscitation. The observation of performance in neonatal resuscitation will also be recorded randomly through videos, to verify the validity of the data collected by the research assistant. The videos will be reviewed by the field monitor and by one of the investigators. In order to comply with confidentiality requirements, the videos will be deleted after verification of information.\n\nAn intention to treat analysis will be performed, including all clusters as they were initially assigned, including premature withdrawals. Additionally, a per-protocol analysis will be carried out, where only clusters that have completed the study protocol will be considered. The primary outcome will be compared in each arm using Chi-square test if the variable of distribution is normal, or alternatively through the Fisher's exact test. Secondary outcomes will be compared using the Student's t-test or the Kruskal-Wallis test. For supplementary oxygen use, the Chi-square test or Fisher's exact test will be used.\n\nWe will resort to local staff as research assistants, including the field supervisor. A small monetary incentive will be available for field workers and study monitors. They will conduct the preparatory work and the study procedures, including information presentations, informed consent administration, observation of case management, and completion of case report forms. Such personnel will be trained in the conduction of studies complying with the standards of good practice, before the study initiation.\n\n\nEthical aspects and confidentiality\n\nThe proposal, instruments and consent forms have been approved by the Institutional Ethics Committee of the Universidad Peruana Cayetano Heredia (record, 273-10-17). The Project Lead will communicate major protocol amendments to relevant parties. Written authorization will be obtained from the authorities of the regional health departments (DIRESAS) involved and from the authorities of the health facilities. Written informed consent will be obtained from the health professionals who will participate in the neonatal resuscitation training (Supplementary File 4). Written informed consent of the mothers will also be obtained for the collection of birth and newborn data (Supplementary File 5). On a random basis, the observation of performance in neonatal resuscitation will be recorded in video, which will be deleted after verification of the information. This information is included on the consent forms.\n\nThe information obtained will be coded and personal identification data, like names and dates of birth, will be deleted to protect confidentiality. Data access will be limited to the Principal Investigator and authorized Co-Investigators. The study documents will be stored in locked environments at the Centro de Investigación para el Desarrollo Integral y Sostenible (CIDIS) at Universidad Peruana Cayetano Heredia, to which access will be granted only to the Principal Investigator and the Co-Investigators authorized by the Principal Investigator.\n\n\nExpected results\n\nThe project proposes to streamline the process of training providers and trainers in neonatal resuscitation in Peru, a country with 31 million inhabitants and a current national neonatal mortality rate of 8 per 1,000 livebirths9. However, neonatal mortality amounts 20 per 1,0000 livebirths in rural areas6. This context poses a huge challenge needing innovative interventions that build on previous success factors that allowed Peru to remarkably reduce maternal and neonatal deaths5,6. We expect that our proposed intervention will show effectiveness amenable to scaling up at a national level, as part of a national neonatal resuscitation program, which should prioritize the most remote rural areas of the country, who are lagging behind and need urgent action.\n\nWe also anticipate that the continuous process of certification of health personnel in remote areas using multi-platform technologies will become a key component of the know-how to develop similar strategies in other low and middle-income countries, where national neonatal resuscitation programs are absent or are still incipient. The MP-ICT strategy is well suited to close the urban-rural gap in maternal and neonatal gap present in these countries, provided its design and implementation are developed, by taking into account an indispensable equity lens.\n\nIf the intervention proves to be effective, we will have made substantial progress in interaction with Peruvian policy-makers to ensure a smooth process of incorporation of the MP-ICT strategy into a National Neonatal Resuscitation Training and Certification Program that could be implemented for reaching health personnel at national level. This program would take advantage on the MP-ICT contributions, including the use of its interactive portal (www.rcpneoperu.org). In the meantime, we aim to reach health care personnel currently registered in Peru, which until June 2017 included 258 neonatologists and 3554 pediatricians, 23,101 general physicians, 28,363 nurses and 13,634 midwives, as well as students of different professional careers (updated Peruvian Physicians Registry http://cmp.org.pe/servicios/conoce-a-tu-medico; and Observatorio de recursos humanos en salud 2017 http://observatorio.inforhus.gob.pe/).\n\n\nEthical approval\n\nThe Institutional Ethics Committee, Universidad Peruana Cayetano Heredia (record 273-10-17; May 4th, 2017).",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nStars in Global Health, Round 8: Reproductive, Maternal, Newborn and Child Health. Grand Challenges Canada (grant number R-ST-POC-1707-06330).\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 RCPNEOPERU study group is composed of Peruvian paediatricians, neonatologists and nurses devoted to the care of newborns. Many of them work at the Instituto Nacional de Salud del Niño (INSN) in Lima, Peru, and perform clinical care activities, clinical teaching and scientific research. The authors are grateful for their participation and support: Jessica Niño De Guzmán, Erika Córdova, Lisset Aguilar, Karen Diaz, Frida Abad, Luz Salgado, Rosa de la Cruz, Nancy Arce, Juana La Rosa, Carlos Lomparte, Percy Martinez, Irene Valencia, Ana López, Karina Orbegoso, Jorge Aquije and all staff from Neonatal Unit from INSN. We also acknowledge Dr Luis Cifuentes, Chief of Experimental Surgical Unit at INSN, and all his staff, and Dr Alfonso Tapia (past - INSN Director) and Dr. Oswaldo Nuñez, current INSN Director, for his invaluable support.\n\n\nSupplementary material\n\nSupplementary File 1: Trial registration data.\n\nClick here to access the data.\n\nSupplementary File 2: An example of MP-ICT\n\nClick here to access the data.\n\nSupplementary File 3: Observation sheet.\n\nClick here to access the data.\n\nSupplementary File 4: Consent form for health professionals (version 2.0).\n\nClick here to access the data.\n\nSupplementary File 5: Consent form for mothers (version 2.0).\n\nClick here to access the data.\n\nSupplementary File 6: Completed SPIRIT checklist.\n\nClick here to access the data.\n\n\nReferences\n\nWyckoff MH, Aziz K, Escobedo MB, et al.: Part 13: Neonatal Resuscitation: 2015 American Heart Association Guidelines Update for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation. 2015; 132(18 Suppl 2): S543–S60. PubMed Abstract | Publisher Full Text\n\nVan Heerden C: An introduction to Helping Babies Breathe: the \"Golden Minute\" is here for South African newborn babies: review. Prof Nurs Today. 2012; 16(3): 6–7. Reference Source\n\nLawn JE, Kerber K, Enweronu-Laryea C, et al.: 3.6 Million Neonatal Deaths--What Is Progressing and What Is Not? Semin Perinatol. 2010; 34(6): 371–86. PubMed Abstract | Publisher Full Text\n\nHuicho L, Canseco FD, Lema C, et al.: [Incentives to attract and retain the health workforce in rural areas of Peru: a qualitative study]. Cad Saude Publica. 2012; 28(4): 729–39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuicho L, Segura ER, Huayanay-Espinoza CA, et al.: Child health and nutrition in Peru within an antipoverty political agenda: a Countdown to 2015 country case study. Lancet Glob Health. 2016; 4(6): e414–26. PubMed Abstract | Publisher Full Text\n\nHuicho L, Huayanay-Espinoza CA, Herrera-Perez E, et al.: Examining national and district-level trends in neonatal health in Peru through an equity lens: a success story driven by political will and societal advocacy. BMC Public Health. 2016; 16(Suppl 2): 796. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAvila J, Tavera M, Carrasco M: [Epidemiological characteristics of neonatal mortality in Peru, 2011–2012]. Rev Peru Med Exp Salud Publica. 2015; 32(3): 423–30. PubMed Abstract\n\nÁvila J, Tavera M, Carrasco M: Mortalidad Neonatal en el Perú y sus departamentos, 2011–2012. Lima: Ministerio de Salud, Dirección General de Epidemiologia. 2013; 1. Reference Source\n\nHuicho L, Segura ER, Huayanay-Espinoza CA, et al.: Child health and nutrition in Peru within an antipoverty political agenda: a Countdown to 2015 country case study. Lancet Glob Health. 2016; 4(6): e414–26. PubMed Abstract | Publisher Full Text\n\nPerlman J, Kattwinkel J, Wyllie J, et al.: Neonatal resuscitation: in pursuit of evidence gaps in knowledge. Resuscitation. 2012; 83(5): 545–50. PubMed Abstract | Publisher Full Text\n\nNiermeyer S: From the Neonatal Resuscitation Program to Helping Babies Breathe: Global impact of educational programs in neonatal resuscitation. Semin Fetal Neonatal Med. 2015; 20(5): 300–8. PubMed Abstract | Publisher Full Text\n\nWyllie J, Bruinenberg J, Roehr CC, et al.: European Resuscitation Council Guidelines for Resuscitation 2015: Section 7. Resuscitation and support of transition of babies at birth. Resuscitation. 2015; 95: 249–63. PubMed Abstract | Publisher Full Text\n\nElsensohn AN, Ricks DJ, Ota A, et al.: The success of Peru's Neonatal Resuscitation Initiative. Arch Dis Child Fetal Neonatal Ed. 2013; 98(4): F375–6. PubMed Abstract | Publisher Full Text\n\nHernández JA: Reflexiones sobre la organización de servicios de neonatología en el marco de la situación neonatal en el país. Perú. Ministerio de Salud. Proyecto 2000; 1998. Reference Source\n\nOliveros M, Chirinos J, Costa R, et al.: El recién nacido de muy bajo peso: proyecto multicéntrico. Diagnóstico (Perú). 2005; 44(2): 54–9. Reference Source\n\nHuicho L, Trelles M, Gonzales F, et al.: Mortality profiles in a country facing epidemiological transition: an analysis of registered data. BMC Public Health. 2009; 9: 47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBastawrous A, Armstrong MJ: Mobile health use in low- and high-income countries: an overview of the peer-reviewed literature. J R Soc Med. 2013; 106(4): 130–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHawkes CP, Walsh BH, Ryan CA, et al.: Smartphone technology enhances newborn intubation knowledge and performance amongst paediatric trainees. Resuscitation. 2013; 84(2): 223–6. PubMed Abstract | Publisher Full Text\n\nFerreira F: De-demonizing the VRAEM: A Peruvian-Cocalero Area. Subst Use Misuse. 2016; 51(1): 41–53. PubMed Abstract | Publisher Full Text\n\nSzyld E, Aguilar A, Musante GA, et al.: Comparison of devices for newborn ventilation in the delivery room. J Pediatr. 2014; 165(2): 234–9.e3. PubMed Abstract | Publisher Full Text\n\nCarnegie J: Use of Feedback-Oriented Online Exercises to Help Physiology Students Construct Well-Organized Answers to Short-Answer Questions. CBE Life Sci Educ. 2015; 14(3): pii: ar25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDawson JA, Kamlin CO, Wong C, et al.: Changes in heart rate in the first minutes after birth. Arch Dis Child Fetal Neonatal Ed. 2010; 95(3): F177–81. PubMed Abstract | Publisher Full Text\n\nYam CH, Dawson JA, Schmölzer GM, et al.: Heart rate changes during resuscitation of newly born infants <30 weeks gestation: an observational study. Arch Dis Child Fetal Neonatal Ed. 2011; 96(2): F102–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "25481",
"date": "28 Sep 2017",
"name": "Waldemar A. Carlo",
"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 This is a cluster randomized controlled trial to compare two educational techniques for NRP training. The sample size is rather small even though the outcome measures are surrogates rather than major outcome measures.\nBackground The NRP program is already web-based. The knowledge part is completed on line with subsequent local skills training. This is not clear from the facts given in the Background section.\nIt is stated that the comparator background section that training has to be done in the capital city but these training programs usually have local hospital based instructors. A local based instructor is used in all Helping Babies Survive programs and would be the best comparator.\nThe study design states that the objective of the intervention is the health care provider not the individual patient. However, the sample size is based on patient outcomes so this is unclear.\nTraining will be done in groups of 10 or more. The group is larger than the usual recommended by resuscitation programs.\nThe masking of the assessors of the practical component is not addressed.\nThe sample size does not take into consideration that only about 10% of the babies will need resuscitation. Comparable studies such as the study by Szyld quoted by the authors have had 10 times as many patients or even more.\nOther countries such as Chile introduce NRP as a nationwide program and reported decreases in neonatal mortality using standard methods for training.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "3147",
"date": "09 Nov 2017",
"name": "Carlos Delgado",
"role": "Author Response",
"response": "We appreciate the reviewer's comments. This evaluation is done by comparing two forms of training: The innovative way is to encourage self-learning participation through information and communication technologies, where participants can review the theory and then give an online exam. Self-learning includes strategies such as videos, action mazes and a virtual reality 360 video, only available for logged trainees <https://youtu.be/-rhMJWqijbU>. The traditional form has been called \"Standard\", but doing so does not take into account that there is no official program and there are no local instructors that have been implemented to train health personnel. Maybe it's better to call this training as “Traditional” instead of “Standard”. There is no Neonatal Resuscitation Training Program in the area, although from time to time some local efforts are made for training with various origins and formats (even some training are not updated). The control comparison could have been not to give training, but in order to offer a minimum training, a traditional, face-to-face and mainly expository form was proposed. In the following paragraphs we respond to specific observations that we highlight in quotes. \"The NRP program is already web-based. The knowledge part is completed on line with subsequent local skills training. This is not clear from the facts given in the Background section.\" Thanks you for your observation. We have edited the third paragraph of the Background section to clarify this. \"It is stated that the comparator background section that training has to be done in the capital city but these training programs usually have local hospital based instructors. A local based instructor is used in all Helping Babies Survive programs and would be the best comparator.\" We agree that these programs can be taught by a local based instructor, which is our goal. However, we are going to use hospital based instructors because most of the deliveries in our study region are being attended by nurses and midwifes and there is a scarcity of local doctors who can be used as local based instructors. We have clarified this in the second paragraph of the Choice of Comparator section. \"The study design states that the objective of the intervention is the health care provider not the individual patient. However, the sample size is based on patient outcomes so this is unclear.\" Thanks for the observation. While it is true that the primary target of the intervention is the health care provider, we used heart rate of babies as a quick reflection of the adequacy of the resuscitation provided by the health worker, in line with other interventions targeted primarily at providers to improve their resuscitation performance (please, see ref. 23). We have clarified this in the Justification for design section. \"Training will be done in groups of 10 or more. The group is larger than the usual recommended by resuscitation programs.\" We agree with your observation. It is our goal to have better trainer/trainee ratios. We were just trying to be realistic in terms of the availability of our resources. We are happy to inform that, once we have started the field work, we have been able to keep the trainer/trainee ratios lower than the expected originally, as we have had more trainers available to teach the program. \"The masking of the assessors of the practical component is not addressed.\" Since the whole health facility is considered for receiving th intervention or the standard training, it is unrealistic to expect that we could mask the assessors of the practical component. We have added a statement that clarifies this in the Description of the Interventions section. \"The sample size does not take into consideration that only about 10% of the babies will need resuscitation. Comparable studies such as the study by Szyld quoted by the authors have had 10 times as many patients or even more.\" Actually, our study is being conducted at a pilot scale. Thus we are looking for the minimum sample size for such study level. Accordingly, we have selected 12 paired health facilities, 6 on each arm and made our sample size calculation, as described in the paper. We clarified this paragraph in the revised paper. \"Other countries such as Chile introduce NRP as a nationwide program and reported decreases in neonatal mortality using standard methods for training.\" We agree with your comment. Our long-term goal is for the neonatal certification training and certification program using ICT to become a nationwide program. We have stated this in Expected Results section."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1599
|
https://f1000research.com/articles/7-289/v1
|
07 Mar 18
|
{
"type": "Opinion Article",
"title": "Defining “FGF21 Resistance” during obesity: Controversy, criteria and unresolved questions",
"authors": [
"Kathleen R. Markan"
],
"abstract": "The term “FGF21 resistance” was first used to describe increased circulating FGF21 levels concomitant to decreased FGF21 receptor complex expression in white adipose tissue of obese mice. Since this initial report, the term has been associated with a wide range of pathological states, including human obesity, in which circulating FGF21 levels are elevated. However, the notion of “FGF21 resistance” has been controversial partly due to difficulty in delineating the mechanisms underlying the physiological versus pharmacological effects of FGF21. Here, key aspects of the term “FGF21 resistance” are discussed including; the origin and experimental context surrounding the term “FGF21 resistance”, new criteria for evaluating FGF21 sensitivity in vivo and finally, crucial unresolved questions regarding the function of FGF21 during obesity.",
"keywords": [
"FGF21 resistance",
"fibroblast growth factor 21",
"obesity",
"insulin senstivity",
"FGF21",
"beta-klotho",
"insulin resistance"
],
"content": "\n\nFibroblast Growth Factor 21 (FGF21) has pleiotropic metabolic effects including increasing insulin sensitivity and energy expenditure, while decreasing body weight and sugar intake1,2. Paradoxical to these beneficial metabolic effects, circulating FGF21 is increased during obesity potentially suggesting a state of “FGF21 resistance”3. This hypothesis, however, has generated controversy within the field. In light of this and in response to a recent call for a unified definition of “FGF21 resistance”4, I would like to discuss the controversy surrounding “FGF21 resistance” during obesity, highlight unresolved questions and outline additional criteria for its definition.\n\n“FGF21 resistance” was first used to describe decreased expression of the FGF21 receptor complex in epididymal white adipose tissue, increased plasma FGF21, blunted ERK phosphorylation, and attenuated reduction in plasma glucose following low dose administration of FGF21 that occurred in obese mice3. Shortly thereafter, an independent group also reported decreased FGF21 co-receptor expression in white adipose tissue and increased plasma FGF21 levels in obese mice5. However, based on dose response studies, these investigators concluded that circulating FGF21 is increased during obesity to maintain insulin sensitivity, and not due to “FGF21 resistance”5. Hence, the existence of “FGF21 resistance” during obesity has remained controversial with the prevailing question: how can “FGF21 resistance” exist if pharmacological dosing is still efficacious1? Potentially, FGF21 sensitivity during obesity may be akin to insulin resistance whereby the biological effect of endogenous FGF21 is lacking yet pharmacological dosing elicits an effect. Therefore, although effects of exogenous FGF21 should be evaluated in testing FGF21 sensitivity4, consideration of the dose, functional readout, and time-course of FGF21 action should be taken.\n\nAlthough plasma FGF21 levels, FGF21 co-receptor expression and downstream signaling should be evaluated in defining “FGF21 resistance”4, it is difficult to interpret tissue-specific decreases in receptor and signaling activation without understanding how that specific tissue mediates FGF21’s effects. For example, what is the relevance of decreased co-receptor expression in white adipose tissue? Adipose tissue is necessary for FGF21’s acute insulin sensitizing effect6 yet, different results have been reported in vivo when either overexpressing or maintaining physiological levels of β-klotho during obesity7,8. Furthermore, brown adipocytes mediate the acute insulin sensitizing action of FGF216; meaning, that although decreased white adipose co-receptor expression has been used as a marker of “FGF21 resistance” we still do not understand the pathological relevance of this event.\n\nHow then, should “FGF21 resistance” during obesity be defined? Different experimental designs are required when evaluating the acute insulin sensitizing action of physiological levels of FGF21 versus the chronic effects on body composition of pharmacological doses of FGF21. Acute FGF21 sensitivity should be determined via insulin tolerance test following co-injection of insulin and FGF21 in addition to assessing tissue specific glucose uptake and activation of the FGF21 signaling cascade in brown adipose tissues6. To test chronic FGF21 sensitivity, weight loss and energy expenditure should be evaluated, although FGF21 signaling is difficult to assess since the specific tissue(s) mediating these effects of chronic FGF21 treatment remain undetermined.\n\nFinally, caution is warranted in translating rodent studies to man. Although plasma FGF21 is elevated in obese and diabetic humans9, human FGF21 is proteolytically cleaved in vivo10. Therefore, the bioactivity of increased circulating FGF21 in humans remains unknown. It is possible that increased circulating FGF21 during obesity could serve a yet uncharacterized role. FGF21 has been shown to have central effects11–13, however whether or not central FGF21 co-receptor expression and signaling are altered during obesity remains unreported.\n\nThere is still much to discover regarding FGF21 action but consideration of the points outlined here can help avoid ambiguity in defining “FGF21 resistance” during obesity. Undoubtedly, the definition of “FGF21 resistance” will continue to evolve as new physiological and pharmacological studies help unravel the mechanisms underlying FGF21’s metabolic actions.\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 is funded through a NIH K01 DK111758 award.\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\nKharitonenkov A, Shiyanova TL, Koester A, et al.: FGF-21 as a novel metabolic regulator. J Clin Invest. 2005; 115(6): 1627–35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPotthoff MJ: FGF21 and metabolic disease in 2016: A new frontier in FGF21 biology. Nat Rev Endocrinol. 2017; 13(2): 74–76. PubMed Abstract | Publisher Full Text\n\nFisher FM, Chui PC, Antonellis PJ, et al.: Obesity is a fibroblast growth factor 21 (FGF21)-resistant state. Diabetes. 2010; 59(11): 2781–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTanajak P: Letter to the Editor: Parameters, Characteristics, and Criteria for Defining the Term \"FGF21 Resistance\". Endocrinology. 2017; 158(5): 1523–1524. PubMed Abstract | Publisher Full Text\n\nHale C, Chen MM, Stanislaus S, et al.: Lack of overt FGF21 resistance in two mouse models of obesity and insulin resistance. Endocrinology. 2012; 153(1): 69–80. PubMed Abstract | Publisher Full Text\n\nBonDurant LD, Ameka M, Naber MC, et al.: FGF21 Regulates Metabolism Through Adipose-Dependent and -Independent Mechanisms. Cell Metab. 2017; 25(4): 935–944.e4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSamms RJ, Cheng CC, Kharitonenkov A, et al.: Overexpression of β-Klotho in Adipose Tissue Sensitizes Male Mice to Endogenous FGF21 and Provides Protection From Diet-Induced Obesity. Endocrinology. 2016; 157(4): 1467–80. PubMed Abstract | Publisher Full Text\n\nMarkan KR, Naber MC, Small SM, et al.: FGF21 resistance is not mediated by downregulation of beta-klotho expression in white adipose tissue. Mol Metab. 2017; 6(6): 602–610. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarkan KR, Potthoff MJ: Metabolic fibroblast growth factors (FGFs): Mediators of energy homeostasis. Semin Cell Dev Biol. 2016; 53: 85–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCoppage AL, Heard KR, DiMare MT, et al.: Human FGF-21 Is a Substrate of Fibroblast Activation Protein. PLoS One. 2016; 11(3): e0151269. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvon Holstein-Rathlou S, BonDurant LD, Peltekian L, et al.: FGF21 Mediates Endocrine Control of Simple Sugar Intake and Sweet Taste Preference by the Liver. Cell Metab. 2016; 23(2): 335–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTalukdar S, Owen BM, Song P, et al.: FGF21 Regulates Sweet and Alcohol Preference. Cell Metab. 2016; 23(2): 344–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSøberg S, Sandholt CH, Jespersen NZ, et al.: FGF21 Is a Sugar-Induced Hormone Associated with Sweet Intake and Preference in Humans. Cell Metab. 2017; 25(5): 1045–1053.e6. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "31641",
"date": "27 Mar 2018",
"name": "Moosa Mohammadi",
"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 article, Dr. Markan expresses her opinion on the ongoing controversy surrounding FGF21 resistance in obesity. This issue has not been explicitly addressed in the past publications so the commentary piece should be a refreshing read for the researchers in FGF signaling field in particular and obesity in general. Having said so, I recommend revising the text along the points below:\n\n1) In the Abstract, I suggest changing the verb “used” to “introduced”.\n\n2) First paragraph: I am wondering if the word “paradoxical” is the appropriate term to use here. Surely one could argue that the observed increases in FGF21 serum level in obese mice/human subjects represent a natural feedback mechanism in response to the progressive worsening of insulin resistance. Are there any published literature which correlate the severity of obesity with serum levels of FGF21? If there are, they should be discussed.\n\n3) Clearly articulate/elaborate the difference between the two studies under scrutiny by putting particular emphasis on the different interpretations of dose response curves of FGF21 in these two studies. Also, please discuss how differences in experimental approach might possibly have influenced the disparate conclusions reached by the authors of these two studies. Explain whether only b-klotho co-receptor or both b-klotho and FGFR1c show reduced expression in obesity. Clearly identify tissues that lose expression of b-Klotho and/or FGFR1c and provide information on the extent of these expression losses if known.\n\n4) For the sake of general readership, please introduce b-Klotho co-receptor and FGFR1c, the cognate receptor of FGF21, early on in the text perhaps even in the abstract. Please state that unlike classical paracrine FGFs, FGF21 operates through a dual receptor system.\n\n5) The point brought up on the inactivating proteolytic cleavage of FGF21 is interesting and worthy of further discussion. Please identify whether the reported increases in FGF21 levels measure the levels of full length bioactive form. I.e. are the assays used capable to differentiating between full length “active” and cleaved “inactive” forms.\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": []
},
{
"id": "34680",
"date": "08 Jun 2018",
"name": "Junichiro Sonoda",
"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\nMarkan KR discusses the controversial “FGF21 resistance” in obesity. It is an important topic for discussion given the crucial role of FGF21 signaling in health and disease. However, I feel that the discussion should also incorporate more recent findings, especially the results of tissue-specific CKO studies which have shed light on the sites of FGF21 action (instead of just stating “the specific tissue(s) mediating these effects of chronic FGF21 treatment remain undetermined”). Also, highlighting examples of local FGF21 resistance (e.g., pERK as a marker) vs. systemic FGF21 resistance (e.g., whole body metabolic effects) would be necessary since the receptors for FGF21 are located in various cell types.\n\nWhen the notion of “FGF21 resistance” was first introduced, it was an attractive idea to explain the “paradoxical” elevation in plasma FGF21 in obesity by analogy to other more established examples of hormone resistance where hormone insensitivity is associated with a feedback hormone production (e.g., insulin resistance, growth hormone resistance, leptin resistance, thyroid hormone resistance etc.). Around that time, the site of receptor expression important for the metabolic action of FGF21 was poorly understood, but the receptor complex expressed in adipocytes was thought to be responsible for many of the chronic metabolic effects. A decrease in Beta-Klotho protein expression in white adipose tissues in obese mice was thus considered to be a potential mechanism for “FGF21 resistance”. More recently, mouse genetic studies have uncovered that the induction of weight loss, glucose lowering, stimulation of energy expenditure, and increased water consumption, are mediated by the receptor complex expressed in the nervous system, rather than adipocytes (Owen et al. 2014 Cell Metab, etc.). This notion is completely overlooked by Markan KR, but in my view, is important when discussing “FGF21 resistance”.\n\nOther comments:\nBeta-Klotho was introduced in the 3rd paragraph without an explanation. For readers who are not familiar with FGF21 signaling, it should be explicitly noted that FGF21 acts by activating membrane-bound FGFR/Beta-Klotho receptor complex.\n\nIn the second paragraph, “blunted ERK phosphorylation” – which tissues were examined?\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": []
},
{
"id": "34679",
"date": "26 Jun 2018",
"name": "Andrew C. Adams",
"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 opinion presented by Dr. Markan discusses an important topic in the FGF21 field, which has persisted as an unresolved issue for quite some time. While the report presents a balanced view, I would suggest the following potential additions/revisions:\nIn the context of a discussion of FGF21 resistance and proteolytic cleavage, it is important to discuss recent developments as they pertain to the various circulating forms of FGF21. Specifically, it would be warranted to mention that it has been recently demonstrated that the ratio of ‘active’ FGF21 to total FGF21 can be modulated in humans (for example, in the GTT setting) and that in certain disease states, expression of the protease FAP are altered.\n\nIt would be helpful to mention the composition of the FGF21 receptor complex early in the manuscript, prior to discussion of tissue specific effects later in the article. Indeed, it is possible that there is local FGF21 resistance in specific tissues, as measured by pERK vs. traditional systemic hormonal resistance.\n\nWhen discussing translation of FGF21 results to man, it is important to consider that many of the proposed clinical candidates in this area have the site of FAP cleavage mutated, thus negating C terminal truncation (likely the most dramatic inactivation by endogenous proteases). While N terminal cleavage may still occur, it is likely that truncation would not impair action, unless the treatment was with wild type human FGF21.\n\nInclusion of discussion of more current findings from numerous groups on tissue specific ablation/overexpression of FGF21 receptor components would add significantly to the manuscript, specifically, detailed discussion of central vs. peripheral action might be relevant to the topic of ‘FGF21 Resistance’.\n\nAnother potential explanation for increased FGF21 in states such as obesity is that it may be a sustained homeostatic response to chronic insult. Indeed, FGF21 appears is elevated by a number of stressors (both acute and chronic), including oxidative stress, lipopolysaccharide (LPS), ethanol/alcohol and dietary stress (Fructose etc). Interestingly, when these stressors are removed, FGF21 levels typically normalize relatively rapidly.\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/7-289
|
https://f1000research.com/articles/7-90/v1
|
19 Jan 18
|
{
"type": "Research Article",
"title": "Badges for sharing data and code at Biostatistics: an observational study",
"authors": [
"Anisa Rowhani-Farid",
"Adrian G. Barnett",
"Adrian G. Barnett"
],
"abstract": "Background: Reproducible research includes sharing data and code. The reproducibility policy at the journal Biostatistics rewards articles with badges for data and code sharing. This study investigates the effect of badges at increasing reproducible research, specifically, data and code sharing, at Biostatistics. Methods: The setting of this observational study is the Biostatistics and Statistics in Medicine (control journal) online research archives. The data consisted of 240 randomly sampled articles from 2006 to 2013 (30 articles per year) per journal, a total sample of 480 articles. Data analyses included: plotting probability of data and code sharing by article submission date, and Bayesian logistic regression modelling to test for a difference in the probability of making data and code available after the introduction of badges at Biostatistics. Results: The probability of data sharing was higher at Biostatistics than the control journal but the probability of code sharing was comparable for both journals. The probability of data sharing increased by 3.5 times (95% credible interval: 1.4 to 7.4 times, p-value probability that sharing increased: 0.996) after badges were introduced at Biostatistics. On an absolute scale, however, this difference was only a 7.3% increase in data sharing (95% CI: 2 to 14%, p-value: 0.996). Badges did not have an impact on code sharing at the journal (mean increase: 1.1 times, 95% credible interval: 0.45 to 2.14 times, p-value probability that sharing increased: 0.549). Conclusions: The effect of badges at Biostatistics was a 7.3% increase in the data sharing rate, 5 times less than the effect of badges on data sharing at Psychological Science (37.9% badge effect). Though the effect of badges at Biostatistics did not impact code sharing, and was associated with only a moderate effect on data sharing, badges are an interesting step that journals are taking to incentivise and promote reproducible research.",
"keywords": [
"Reproducibility",
"incentives",
"rewards",
"data sharing",
"code sharing",
"meta-research"
],
"content": "Introduction\n\nHistorically, the replication of a scientific experiment has been the measure of its validity, however, not all experiments can be replicated in their totality1. ‘Replicability’ is the ability of a researcher to duplicate the results of a prior study if the same procedures are followed but new data are collected2. In 2009, Roger Peng mentioned in an editorial in Biostatistics that the minimum standard that could bridge the gap between replicability and nothing is “reproducible research”1. ‘Reproducibility’ is the ability of a researcher to duplicate the results of a prior study using the same materials as were used by the original investigator2. Reproducibility was defined by Peng in terms of sharing the data and computer code used to analyse the data and he described it as the “cornerstone of the scientific method”1. In a perspective piece in 2011, Peng likened reproducibility to a spectrum, at one end being the gold standard of full replication, and at the other, publication only3. Given the expectation that data will be accessible, researchers who refuse to share the evidentiary basis behind their conclusions, or the materials needed to reproduce published experiments, fail to maintain the standards of science4. Although in some instances highly-sensitive data cannot be shared for legal or privacy reasons.\n\nScientific journals are critical to changing the culture of research. Many journals are introducing data sharing policies, but studies have shown that policies alone are not effective in promoting a culture of sharing and that scientists potentially need to be rewarded for good behaviour5. Ioannidis et al. discuss changing the reward criteria to include ‘reproducible’ and ‘sharing’ using the PQRST criteria – productive, high-quality, reproducible, shareable, and translatable6. A systematic review of incentives that motivated researchers to share their data in the health and medical research community, uncovered only one evidence-based incentive that increased data sharing at the journal Psychological Science from 1.5% pre-incentive (2012) to 39.4% post-incentive (2015)7,8. This incentive was an open data badge developed by the Center of Open Science (COS) and introduced at the journal in January 20148.\n\nBadges for reproducible research were not an innovative creation of COS however. The journal Biostatistics introduced badges, or what they called kitemarks (named after the UK kitemark system of establishing product safety), on 1 July 2009 as part of their policy to reward reproducible research1. The policy was introduced by Roger Peng, the then Associate Editor for reproducibility (AER)1. Sharing was not enforced, rather authors were encouraged to consider the reproducibility of their research1. From here on, kitemarks will be referred to as badges, using common terminology.\n\nThe reproducibility policy at the journal instructed authors to indicate in their submission if they intend to submit supplementary materials that include data, code, or both1. The policy rewarded articles with data available with the letter D on the front page of the published article PDF, articles with code available with a C, and articles with data and code available and which were tested for reproducibility by the AER an R for reproducibility1. It is important to note that data refers to raw data and not simulated data, which are commonly used in statistics.\n\nThe policy change at Biostatistics provided an ideal opportunity to replicate the findings of the Kidwell et al. badge study by examining sharing rates at another journal that offered a reward or incentive for reproducible research8. We note that Kidwell et al. examined data and material sharing only, as badges were not offered for code.\n\nA survey conducted by Nature in 2016 indicates that the scientific community is in the midst of a reproducibility crisis9. The current culture in science provides strong incentives for innovation and relatively weak incentives for certainty and reproducibility10. Within the current ‘post-truth’ era there is much public scrutiny and suspicion around the validity of science. Such a debate, compounded by the reproducibility crisis, signals a time for a cultural shift in the scientific research process11. The sharing of data, as well as the computer code used to analyse the data, should, where possible, be integral components of the research process, however data sharing rates have been as low as 0%12. Of course, not all data can be shared due to legal and ethical constraints, but these are neither the only, nor main reasons behind low sharing rates13. Scientists are still exploring the barriers towards sharing and a key concern is that researchers are not incentivised to share3.\n\nOur aim is to investigate the effect of badges at increasing reproducible research, specifically, data and code sharing, at Biostatistics.\n\n\nMethods\n\nThis is an observational study with two journals, intervention and control, using a pre-post study design, with 30 randomly selected papers per year from 2006 to 2013 for each journal. We chose Statistics in Medicine as the control journal as it did not have a badges or any type of reproducible research reward scheme during those years, but is in the same field of research with similar goals of publishing papers on statistical methods development in health and medicine. The study setting is the Biostatistics and Statistics in Medicine research archive. All the information required was publicly available online, as such participant consent was not required and an ethics exemption (exemption number: 1700001051) was granted by the Office of Research Ethics and Integrity at the Queensland University of Technology.\n\nA sample of only 19 papers per journal would have given us a 90% power to detect a difference in data sharing of 37.9%, based on the effect of badges from the Kidwell et al. study8. This uses a two-sided 5% significance level. We felt this sample was unrealistically small, hence we instead based our sample size on the practical considerations of reading papers and examining their data and code sharing choices, given the time constraints of the first author’s (ARF) PhD. Thirty papers per year from 2006 to 2013 for two journals is a total sample of 480 papers, which is practically possible, and provides good coverage over the time of the policy change at Biostatistics.\n\nFor each year and journal a random number generator was used to select the research articles (in Microsoft Excel 2016l). Articles were included if they:\n\nGenerated and analysed original data (article had data and code to share), or\n\nConducted secondary analyses on a pre-existing dataset from another study (article had data and code to share), or\n\nGenerated simulated data (article did not have data to share but had code to share)\n\nArticles were excluded if:\n\nThey were meta-analyses, meta-regressions, or systematic reviews, as these papers usually contain the data within the paper\n\nThey were case series, opinion pieces or some other publication type without data or code\n\nIf an article was excluded then we sampled another article from the same year and journal to maintain the sample size. ARF read the research papers and extracted the details of the articles included in the study. Each article was screened using these search terms: “data”, “code”, “package”, “available”, “https”, “www”, “figshare”, and “github”. For the included articles, the following variables were documented: submission date, data sharing statement, data availability, hyperlink to dataset, code sharing statement, code availability, hyperlink to code, and badge allocation (for Biostatistics articles).\n\nThe second author (AGB) independently assessed data and code sharing for 20 randomly selected articles. There were minor discrepancies between the authors, which were resolved by discussion.\n\nUsing definitions from our previous work5, each research article was categorised for data and code sharing as:\n\nData sharing\n\navailable: articles that had a functioning link to a publicly available dataset deposited at a third-party site or attached as supplementary material to the electronic version of the article\n\npotentially available: articles that indicated that the dataset was potentially available upon request from the authors\n\nnot available: articles that did not indicate the availability of the dataset analysed in the article or where the link to the data was no longer working\n\nnone to share: articles that used simulated data and so did not have a raw dataset to share\n\nCode sharing\n\navailable: articles that had a functioning link to publicly available code deposited at a third-party site, or attached as supplementary material to the electronic version of the article or available within the article itself\n\npotentially available: articles that indicated that the code was potentially available upon request from the authors\n\nnot available: articles that did not indicate the availability of the code used to analyse the data (raw or simulated) or where the link to the code was no longer working\n\nWe defined the intervention period based on the policy change date at Biostatistics and using the article’s submission date as this is when authors are thinking about the journal requirements and perhaps becoming aware of the badge. Since the policy change was on 1 July 2009, papers submitted to Biostatistics after that date were in the intervention period. We included a six month gap before the policy change as an interim phase because papers submitted during this time (1 January 2009 to 1 July 2009) could experience the badge policy upon re-submission, so papers submitted in this period were categorized into the interim period. Any papers submitted to Biostatistics before 1 January 2009 were in the control period and all papers submitted to Statistics in Medicine were controls.\n\nThe first analysis examined data and code availability and probability of sharing over time using submission date. As a sensitivity analysis, we used the articles’ publication dates extracted from PubMed in place of submission date. We conducted this sensitivity analysis to examine whether the policy was associated with a change based on the very latest date that authors could make changes to their papers.\n\nWe plotted the binary data and code sharing over time and included a smooth curve to estimate the mean sharing rate over time in each journal. The smooth curves were made using a LOESS smooth with a span of 0.9, and we also plotted the 95% confidence intervals. Papers where there was no data to share (i.e., using simulated data) were excluded from these plots.\n\nTo test for a difference in the probability of making data and code available after the introduction of badges, we used logistic regression and presented the results as prevalence ratios rather than odds ratios, as prevalence ratios are generally easier to understand14. Due to possible convergence issues with a standard logistic regression model using a log-link to estimate prevalence ratios, we ran a Bayesian logistic regression model using WinBUGS (version 1.3.4). Using a Bayesian model has the added advantage of giving 95% credible intervals and Bayesian p-values that are far easier to interpret than frequentist confidence intervals and p-values. The Bayesian p-values used here estimate the probability that sharing increased after the policy change at Biostatistics. As well as showing the change in data and code sharing probability, on the relative scale, of the prevalence ratio, we also show the absolute increase in sharing probability after the policy change together with 95% credible intervals.\n\nIn a sensitivity analysis we used a strong control for time by including year as a random effect, assuming that each year has its own data sharing rate. This essentially matches papers from Biostatistics and Statistics in Medicine from the same year. We did this to adjust for other changes over time, for example a potential increase over time in data and code depositories such as GitHub, Figshare, and Dryad, and a potential decrease in data and code availability for papers published many years ago because of broken links15.\n\nThe data analysis was made using the statistical software R (version 3.2.3).\n\n\nResults\n\nFlow charts show the frequency of data and code availability for each journal (Figures 1a and 1b). Biostatistics had 8 articles with no data to share, bringing the sample with possible data available to 232; 20 of which had data available, 3 had data potentially available and 209 had no data available. Statistics in Medicine had 31 articles with no data to share, bringing the sample with possible data available to 209; 3 of which had data available, 4 had data potentially available and 202 had no data available.\n\na: Flow chart of data availability. Randomly selected Biostatistics articles from 2006 to 2013, b: Flow charts of data availability. Randomly selected Statistics in Medicine articles from 2006 to 2013.\n\nThe frequency of code availability for each journal is in Figures 2a and 2b, which were comparable for the two journals. Statistics in Medicine had 24 articles with code available, 27 potentially available, and 189 with no code available, while Biostatistics had 14 articles with code available, 22 potentially available, and 204 with no code available.\n\na: Flow charts of code availability. Randomly selected Biostatistics articles from 2006 to 2013, b: Flow charts of code availability. Randomly selected Statistics in Medicine articles from 2006 to 2013.\n\nThe data available and probability of sharing by submission date together with a smooth curve and 95% confidence intervals are in Figure 3a. The vertical red lines are at 1 July 2009, the date badges were introduced at Biostatistics, and 1 January 2009, six months prior to the policy change (interim period). It is clear that data availability and probability of sharing were greater over time in Biostatistics than in the control journal, Statistics in Medicine, but the probability of sharing data at Biostatistics was still low, at well below 0.25. Interestingly an increase in data sharing at Biostatistics took place before badges were introduced at the journal. The results of the sensitivity analysis using publication date are shown in Figure 3b. The smooth curves in Figure 3b are similar to those in Figure 3a and show that data availability and probability of sharing were increasing at Biostatistics before badges were introduced.\n\na: Plot of data availability over time by submission date. The dots at ‘No’ or ‘Yes’ are individual articles and the lines are a smoothed mean using a LOESS together with 95% confidence intervals (grey areas). The red lines indicate the interim period: 1 January 2009 to 1 July 2009. b: Plot of data availability over time by publication date. The dots at ‘No’ or ‘Yes’ are individual articles and the lines are a smoothed mean using a LOESS together with 95% confidence intervals (grey areas). The red lines indicate the interim period: 1 January 2009 to 1 July 2009.\n\nThe code availability and probability of sharing by submission date together with a smooth curve and 95% confidence intervals are in Figures 4a. The smooth curves for Biostatistics and Statistics in Medicine are mostly on top of each other in this graph, except for a drop-off in sharing at Biostatistics in later years. This indicates no great difference in code sharing at these journals. Figure 4b shows the results of the sensitivity analysis, where publication date was used instead of submission date. In this graph (Figure 4b), the smooth curve for Biostatistics and Statistics in Medicine are again mostly on top of each other, showing an increase in code sharing over time at both journals, but around mid-2011 the two curves diverged, with Statistics in Medicine showing an increase in code sharing and Biostatistics a drop.\n\na: Plot of code sharing over time. The dots at ‘No’ or ‘Yes’ are individual articles and the lines are a smoothed mean using a LOESS together with 95% confidence intervals (grey areas). The red lines indicate the interim period: 1 January 2009 to 1 July 2009. b: Plot of code sharing over time. The dots at ‘No’ or ‘Yes’ are individual articles and the lines are a smoothed mean using a LOESS together with 95% confidence intervals (grey areas). The red lines indicate the interim period: 1 January 2009 to 1 July 2009.\n\nThe logistic regression model estimated that the probability of data sharing increased by 5.1 (95% CI for prevalence ratio: 0.6 to 14.8, p-value: 0.942) times that of the control period in the interim period of 1 January 2009 to 1 July 2009. This Bayesian p-value gives an estimated 94.2% probability that the mean rate of sharing increased. After the interim period, the probability of data sharing increased by an estimated 3.5 (95% CI: 1.4 to 7.4, p-value: 0.996) times after badges were introduced. On an absolute scale, this difference was only a 7.3% increase in data sharing (95% CI: 2 to 14%). After controlling for time, badges increased the probability of data sharing at the journal by an estimated 3.9 times (95% CI: 1.2 to 9.5, p-value: 0.991). This is comparable to the prevalence ratio of 3.5 when time was not added as a random effect, which shows that controlling for time only slightly increased the effect badges had on the probability of data sharing.\n\nDuring the interim period, badges did not have an effect on code sharing (prevalence ratio of approximately 1). After the interim period there was an estimated 0.6% increase (95% CI: –5 to 8%, p-value: 0.549) in sharing. After adjusting for time, this absolute difference reduced to –1.4% (95% CI: –7 to 5%, p-value: 0.286). This suggests that badges did not have an impact on the probability of sharing code.\n\nWe often encountered issues with broken hyperlinks at both journals. Forty-nine out of 76 (64%) articles that provided links to data and code at Biostatistics had broken links and at Statistics in Medicine, 21 out of 50 (42%) articles that provided links to data and code had broken links.\n\n\nDiscussion\n\nThe results of this observational study and those of the related Kidwell et al. badge study8 cannot accurately deduce the effectiveness of badges because of the biases of the non-randomised study design. The Kidwell et al. 2016 badge study received criticism from Hilda Bastian on its study design, analyses, and claims16. One of the criticisms was that the badges scheme was not the only intervention offered at the journal, there were four other co-interventions offered in 2014, and so any effect could not be attributed to badges alone16. Bastian reasonably argued that to isolate the impact of badges, groups that had the same conditions except badges were needed16. Our study is also exposed to similar limitations with regard to confounding as other changes may have occurred that we were not aware of. However, we can derive some insight into the effect badges had on data and code sharing from the results of both observational studies.\n\nAfter the introduction of badges at Biostatistics, the probability of data sharing increased 3.5 times. This prevalence ratio might seem like a large increase but on an absolute scale it is only a 7.3% increase in the rate of data sharing, which is much lower than the 37.9% effect of badges at Psychological Science8. The large difference between the effect of badges at Biostatistics and Psychological Science could be related to differences in the culture of sharing between the two fields, and the timeframes of the studies: 2006 to 2013 for our study, versus 2012 to 2015 for Kidwell et al. Our study analysed incentives for data and code sharing at an earlier time when the reproducibility crisis was not yet a testified reality, hence researchers may have been more primed to change behaviour in the Kidwell et al. study. Also, since statisticians typically re-analyse existing datasets, it might be harder for them to share the data as they might not have the rights. This is contrary to research in psychological science where original data is normally collected and analysed, making sharing a potentially simpler task.\n\nThere was an apparent increase in data sharing before badges were introduced at Biostatistics (Figure 3a). One possibility is that articles that were submitted before the policy change could still have experienced the policy because of the time needed for peer review and resubmission. We used submission date to determine if articles were prepared before or after the policy change because we know that sharing data often takes preparation time and we believed that authors were therefore more likely to react to the policy when they were writing their first draft. However, data sharing seemed to be increasing before badges were introduced even when we used publication date in a sensitivity analysis. The reproducibility policy at Biostatistics was built on the existing framework that “allowed and encouraged authors to place supplementary materials online”1. Such an option of depositing supplementary material could have contributed to the rise in data sharing before badges. Also, Roger Peng assumed the role as the Associate Editor for reproducibility at Biostatistics in 2006, which might have catalysed a change in the culture of reproducibility at the journal.\n\nBadges did not appear to have an effect on code sharing as the prevalence ratio was close to 1 with a 95% credible interval that included 1. This is an unexpected outcome as code is of great importance in the field of biostatistics. A possible explanation behind the lack of badge effect on code sharing could be our definition of code sharing, which might seem traditional compared to the reproducibility policy at Biostatistics. We defined code sharing as the availability of the code used to analyse the data (original or simulated) in the article. The policy at Biostatistics included referencing “…software that is widely available from central repositories (e.g. CRAN, Statlib)”. It is true that providing a link to a third-party repository where software packages are deposited, such as vignettes, typically contain some general code, but it often takes specialized skills to work out the code at these repositories, and they might not always explain the analyses covered in the actual published article. This is in line with what Stodden et al. recommended in their piece on reproducibility in Science, “Data and code underlying discoveries must be discoverable from the related publication, accessible, and reuseable”17.\n\nThe effect of badges on data and code sharing could have been higher if we did not encounter issues with broken links. For Biostatistics the “Supplementary Material” link did not work for the majority of articles. The current editors of Biostatistics indicated that when the publisher (Oxford) switched to a new publishing platform in January 2017, some of the supplemental material was lost in the transfer (personal communication, J Leek, 8 November 2017). Our results could have looked different if we had started collecting data in 2006, as the links could have been working back then.\n\nBadges have been promoted as a simple solution because they are low cost. However, while collecting data for our study, we noticed that articles did not always appear to be allocated with badges correctly, implying that assigning badges is not always clear cut and journal staff may need to spend more time on verification. An alternative approach is that peer-reviewers check for data and code availability and assign badges as part of the standard peer review process. It could be that peer-reviewers prefer to have access to the data and code in order to review the article anyway, so this model might work, but it still requires additional time and effort on their part and as they receive little recognition for their work, plus it might be unfair to expect all peer-reviewers to check for data and code sharing.\n\n\nConclusion\n\nEfforts are underway by the global meta-research community to strengthen the reliability of the scientific method18. Data and code sharing is an indispensable part of the movement towards science that is open; where scientific truth is not a questionable commodity, but is easily accessible, replicable, and verifiable19. The cultural shift towards reproducible science is complex and it calls for a twofold change in the attitudes of individual researchers toward reproducibility, and the leadership provided by the systems and services that support scientific research. As such, journals, universities, government bodies, and funders are key players in promoting this culture. Transparency and reproducibility are elements central to strengthening the scientific method, and data and code provide the key to scientific truth12. As Peng argued in Science, the culture of reproducibility will not drastically change overnight, but simply bringing the notion of reproducibility to the fore and making it routine will make a difference3. Badges are already being used by journals including Biostatistics, Psychological Science, British Medical Journal Open Science, and Association for Computing Machinery to encourage researchers to share the evidence behind their work1,20. Based on this observational study and a previous study, it appears that badges do help to increase data sharing, but a randomised trial is needed to better estimate their true effect, as well as studies of the additional time needed to implement and maintain them.\n\n\nData availability\n\nAnonymised data and the code used in the analyses are publicly available at: https://doi.org/10.6084/m9.figshare.5687548\n\n\nConsent\n\nAn ethics exemption was granted by the Office of Research Ethics and Integrity at the Queensland University of Technology for this study (exemption number: 1700001051). No consent was needed as all data collected and analysed in this study were publicly available.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed\n\n\nGrant information\n\nThis study was supported in kind by the Institute of Health and Biomedical Innovation at the Queensland University of Technology.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThanks to one of the current Editors of Biostatistics, Professor Jeffrey Leek, for his prompt response to our enquiry. Sincere thanks goes to my PhD colleague, Victoria McCreanor, for reading through the draft manuscript and providing feedback.\n\n\nReferences\n\nPeng RD: Reproducible research and Biostatistics. Biostatistics. 2009; 10(3): 405–408. PubMed Abstract | Publisher Full Text\n\nGoodman SN, Fanelli D, Ioannidis JP: What does research reproducibility mean? Sci Transl Med. 2016; 8(341): 341ps12. PubMed Abstract | Publisher Full Text\n\nPeng RD: Reproducible research in computational science. Science. 2011; 334(6060): 1226–1227. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNational Academy of Sciences, National Academy of Engineering (US) and Institute of Medicine (US) Committee on Science, Engineering, and Public Policy: On Being a Scientist: A Guide to Responsible Conduct in Research: Third Edition. Washington, DC: The National Academies Press (US); 2009. PubMed Abstract | Publisher Full Text\n\nRowhani-Farid A, Barnett AG: Has open data arrived at the British Medical Journal (BMJ)? An observational study. BMJ Open. 2016; 6(10). Publisher Full Text\n\nIoannidis JP, Khoury MJ: Assessing value in biomedical research: The PQRST of appraisal and reward. JAMA. 2014; 312(5): 483–484. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRowhani-Farid A, Allen M, Barnett AG: What incentives increase data sharing in health and medical research? A systematic review. Research Integrity and Peer Review. 2017; 2(1): 4. Publisher Full Text\n\nKidwell MC, Lazarević LB, Baranski E, et al.: Badges to Acknowledge Open Practices: A Simple, Low-Cost, Effective Method for Increasing Transparency. PLoS Biol. 2016; 14(5): e1002456. PubMed Abstract | Publisher Full Text | Free Full Text\n\n1,500 scientists lift the lid on reproducibility: Nature News & Comment.\n\nEbersole CR, Axt JR, Nosek BA: Scientists' Reputations Are Based on Getting It Right, Not Being Right. PLoS Biol. 2016; 14(5): e1002460. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarbui C: Sharing all types of clinical data and harmonizing journal standards. BMC Med. 2016; 14(1): 63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIqbal SA, Wallach JD, Khoury MJ, et al.: Reproducible Research Practices and Transparency across the Biomedical Literature. PLoS Biol. 2016; 14(1): e1002333. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Panhuis WG, Paul P, Emerson C, et al.: A systematic review of barriers to data sharing in public health. BMC public health. 2014; 14: 1144. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeddens JA, Petersen MR: Approaches for estimating prevalence ratios. Occup Environ Med. 2008; 65(7): 501–506. PubMed Abstract | Publisher Full Text\n\nVines TH, Albert AY, Andrew RL, et al.: The Availability of Research Data Declines Rapidly with Article Age. Curr Biol. 2014; 24(1): 94–97. PubMed Abstract | Publisher Full Text\n\nBastian H: Bias in Open Science Advocacy: The Case of Article Badges for Data Sharing. In., PLOS Blogs; 2017; 2017. Reference Source\n\nStodden V, McNutt M, Bailey D, et al.: Enhancing reproducibility for computational methods. Science. 2016; 354(6317): 1240–1241. PubMed Abstract | Publisher Full Text\n\nIoannidis JP, Fanelli D, Dunne DD, et al.: Meta-research: Evaluation and Improvement of Research Methods and Practices. PLoS Biol. 2015; 13(10): e1002264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReproducibility and reliability of biomedical research: improving research practice. In.: The Academy of Medical Sciences. 2015. Reference Source\n\nSena ES: Inaugural editorial: advancing preclinical and translational research of relevance to medicine. BMJ Open Science. 2017; 1(1). Publisher Full Text"
}
|
[
{
"id": "30171",
"date": "24 Jan 2018",
"name": "Gustav Nilsonne",
"expertise": [
"Reviewer Expertise Neuroscience",
"Meta-science"
],
"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 an observational investigation of the effect of badges/kitemarks on sharing of data and code. The authors compared one journal that introduced badges in 2009 (Biostatistics) to one that did not (Statistics in Medicine). The main finding was that badges were associated with an increase in open data publication, although with a considerably smaller effect than that observed in the one previous investigation of this question. The finding is important and may influence publishing policies.\nThe main limitation of this study is that only two journals were included. The authors provide a strong reason for this, namely that Biostatistics is unique in having introduced badges sufficiently long ago to have a period of follow-up of several years.\nStatistical methods are sound; descriptive statistics are very clear and inferential statistics are appropriate. The choice to use the six months before badges came into effect (the \"interim period\") as a reference period is arbitrary, and it is not possible for a reader to assess whether six months would capture most papers undergoing review and revision at the time the policy was introduced. If papers took longer, that could contribute to the increasing rates of data sharing observed in the run-up to the introduction of badges, seen in figure 3. Thus, the choice of reference period is likely to yield a conservative estimate of the effect of the policy.\nPapers were coded by one person only, and that is a minor weakness. My experience with coding articles for open data and similar outcomes leads me to think that the main reason to have two coders in this context is to reduce the risk of error, rather than to handle interrater differences in assessments. It is not likely, in my opinion, that errors in the coding have effects on a magnitude approaching that seen for the intervention.\nI have cursorily reviewed the data and analysis code also. The data variable names are self-explanatory. The code, written in Markdown, is sufficiently documented with inline headings. However, the code requires some modification before it can be run independently, and this is not documented. For example, on line 18, the code attempts to load the data, by calling a file that is not provided. The data must instead be loaded by opening the provided R workspace file. I suggest that the data be made available in a csv or txt file, which is safer for accessibility in the long term and across softwares. The code could then call this file.\nThe ancillary finding of high rates of broken hyperlinks to data at both journals is interesting, as is the explanation that supplementary data were lost by one of the journals during a platform migration. I have several times advanced the argument that this risk is one motivation for publishing data and code using dedicated repositories, but I have not previously seen an empirical observation such as this one. I suggest that this finding should be mentioned in the abstract.\nOne minor detail: In figure 4, the legend does not explain the difference between panels a and b, as in figure 3.\nIn summary, this article makes an important contribution towards our understanding of effects of badges on publication of open data and open code. I am happy to approve this article.\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": "3473",
"date": "07 Mar 2018",
"name": "Anisa Rowhani-Farid",
"role": "Author Response",
"response": "We have updated the Rmarkdown code and added an R data set so anyone can independently run the analysis. Although ARF coded all the data, AGB verified 20 observations and the two authors debated a handful of cases where it was not clear. In general the sharing of data or not was relatively clear-cut and hence we believe that one coder should be sufficient. We agree that the six month date window prior to the introduction of badges is arbitrary, and this is why we also plotted the sharing rates over time so that readers could see when any changes in sharing took place, and why we tried the alternative time scale of submission date. We have now mentioned broken links in in the abstract. We have corrected this: \"One minor detail: In figure 4, the legend does not explain the difference between panels a and b, as in figure 3\"."
}
]
},
{
"id": "30239",
"date": "12 Feb 2018",
"name": "Bastian Greshake",
"expertise": [
"Reviewer Expertise Bioinformatics",
"meta-science"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study evaluates the effects that badging has on availability of both data and source code. Badges that highlight and reward articles that share data/code have been described as a simple way to increase sharing behavior and thus reproducibility. This makes this study highly relevant and interesting to the field of meta-research. To measure whether badging has this desired effect the authors compare the availability of data/code in the journal Biostatistics - which introduced badges in 2009 - with a control journal, Statistics in Medicine. The main finding is that there seems to be a small effect on data sharing associated with the introduction, while badges appear to have no effect on the sharing of code.\n\nWhile the statistical methods are appropriate and sound, the study has some limitations given by the data set on how the authors coped with missing data. First of all, and this is probably the main limitation, there is only a small set of publications in the authors' set of 480 that have data/code associated with them. Only around 8% of all publications have code attached, a number that drops to less than 5% for data. Given this overall low rate of sharing - and the large time span covered - there is a lot of fluctuation in the observed sharing behavior for both code and data (c.f. Figure 3 & 4) between the two journals. Given that only Statistics in Medicine is used as a control journal it is unclear how much of these differences are cultural between the audiences of the two journals (also c.f. that code sharing is more prevalent in SIM than Biostatistics and increases more despite the lack of badges). A larger set of control-journals would potentially minimize this 'culture'-effect (though requiring a potentially unreasonably large time investment).\nIn addition to this there are further effects that complicate a clear inference of the effect of badges:\nThere is a general trend that \"Open Science\" and data sharing are becoming more popular (c.f. Tenopir et al. (2015)1). In line with this the authors find that the sharing of data in Biostatistics already increased prior to the introduction of badges, even when going for conservative measures. Additionally there is an observed increase in data sharing in Statistics in Medicine in later years, albeit from a lower baseline. I think this general trend should be included in the discussion.\n\nThe authors additionally find that the links for 64% of articles that provide data/code are broken in Biostatistics and highlight the issue that OUP had with losing (not only) supplementary data. The authors treat these articles as having data/code not publicly available. This not only leads to a marked decrease overall decrease of articles with data/code available but can furthermore be a potential source of bias. For OUPs Molecular Biology and Evolution it was seen that these losses on behalf of OUP not only affected supplementary materials but even individual years worth of full text articles (c.f. http://rossmounce.co.uk/2017/04/02/open-letter-to-oxford-university-press/). If OUP also lost the supplementary materials of certain publication date ranges for Biostatistics this will heavily bias the analysis presented here.\n\nGiven these limitations I would be cautious to assume whether badges have a positive effect or not on data availability at this point.\nTo improve the resolution by adding more data and to decrease the biases mentioned in 2) I recommend taking the original data/code availability statements at face value and not treat broken links as publications without data/code. I think it is defendable to assume that the data/code was available at the time of publication when the main text suggests so. Doing this the potential effect of badges should become more pronounced as it is not hidden by the 'link-rot' that OUP is responsible for.\n\nOverall, I think this is a valuable contribution to the field of meta-research and our understanding of incentives around sharing and reproducibility. I hope that the minor improvements suggested above can be useful to make this data set even more useful.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "3472",
"date": "07 Mar 2018",
"name": "Anisa Rowhani-Farid",
"role": "Author Response",
"response": "We looked for additional control journals to minimise the ‘culture’-effect described above. The journals that met the eligibility criteria of being in the field of applied statistics and/or biostatistics and which had a similar relative high prestige in the field as per Biostatistics were the following: Statistical Methods in Medical Research Biometric Journal Biometrics Royal Statistical Society – Series C Applied Statistics However, none of these journals were suitable as controls as they all had reproducibility policy changes during the time frame of our study (2006 to 2013) or had a reproducibility policy in place before that timeframe. We have added an explanation of this in our methods. The general trend towards more open science is now discussed in the Discussion. We used sensitivity analyses that adjusted for the effects of time (including trends), and the effects of badges were relatively similar to the unadjusted results. We have conducted sensitivity analyses for data and code sharing to account for the broken links at the journal. These results have been added and discussed in the article."
}
]
}
] | 1
|
https://f1000research.com/articles/7-90
|
https://f1000research.com/articles/6-877/v1
|
13 Jun 17
|
{
"type": "Data Note",
"title": "Data of physiological measurements and quality of life in subjects with mild to moderate COPD compared to asymptomatic current smokers, former smokers and never-smokers",
"authors": [
"Nveed Chaudhary",
"Karsta Luettich",
"Michael J. Peck",
"Elena Pierri",
"Loyse Felber-Medlin",
"Gregory Vuillaume",
"Patrice Leroy",
"Julia Hoeng",
"Manuel C. Peitsch",
"Nveed Chaudhary",
"Karsta Luettich",
"Michael J. Peck",
"Elena Pierri",
"Loyse Felber-Medlin",
"Patrice Leroy",
"Julia Hoeng",
"Manuel C. Peitsch"
],
"abstract": "Chronic obstructive pulmonary disease (COPD) is a common inflammatory airway disease predominantly associated with cigarette smoking, and its incidence is increasing worldwide. According to the Global Initiative for Obstructive Lung Disease (GOLD) guidelines, spirometry is used to diagnose the disease. However, owing to its complexity, spirometry alone may not account for the multitude of COPD phenotypes or the early, asymptomatic lung damage seen in younger smokers. In addition, suitable biomarkers enabling early diagnosis, guiding treatment and estimating prognosis are still scarce, although large scale ‘omics analyses have added to the spectrum of potential biomarkers that could be used for these purposes. The aim of the current study was to comprehensively profile patients with mild-to-moderate COPD and compare the profiles to i) a group of currently smoking asymptomatic subjects, ii) a group of healthy former smokers, and iii) a group of healthy subjects that had never smoked. The assessment was conducted at the molecular level using proteomics, transcriptomics, and lipidomics and complemented by a series of measurements of traditional and emerging indicators of lung health (ClinicalTrials.gov identifier: NCT01780298). In this data note, we provide a comprehensive description of the study population’s physiological characteristics including full lung function, lung appearance on chest computed tomography, impulse oscillometry, and exercise tolerance and quality of life (QoL) measures.",
"keywords": [
"COPD",
"lung function",
"gas transfer",
"impulse oscillometry",
"lung sound analysis",
"biomarker"
],
"content": "Introduction\n\nChronic obstructive pulmonary disease (COPD) is a respiratory disease characterized by progressive airflow limitation and is associated with an abnormal inflammatory response of the lung to noxious particles and gases. Globally, airflow obstruction can be seen in approximately 25% of adults aged 40 and over1, and the prevalence of COPD is on the rise worldwide, leading to predictions of COPD becoming the third leading cause of death by 20302.\n\nThe clinical assessment of a patient with suspected obstructive lung disease relies on symptoms such as shortness of breath and persistent cough, medical history, history of risk factors (e.g. cigarette smoking) and spirometry. The latter, based on the latest recommendations of the Global Initiative for Obstructive Lung Disease (GOLD) guidelines (www.goldcopd.org), is required for confirming a COPD diagnosis in case of a post-bronchodilator ratio of forced expiratory volume in 1 second over forced vital capacity (FEV1/FVC) of less than 0.7 or 70%. The efforts of GOLD to simplify the diagnosis of COPD to a single repeatable test that uses inexpensive equipment in the physician’s office have proved critical and invaluable in the day-to-day diagnosis and management of the disease. However, it has become clear that COPD is a very complex, heterogeneous disease consisting of a multitude of different phenotypes and syndromes, even among subjects with a similar degree of airflow limitation, and with highly variable rates of progression3. Spirometry alone may also not be sufficiently sensitive to account for early lung damage that remains asymptomatic, particularly in the younger smoker4. Moreover, airflow obstruction does not correlate well with clinical outcomes such as frequency of exacerbations and mortality5. It is not surprising then that pharmacological interventions are rather modestly successful and long-term, positive patient-centric outcomes are infrequently achieved6. Similarly, and although our mechanistic understanding of COPD pathophysiology is ever-increasing, the identification of suitable biomarkers for the diagnosis, treatment and prognosis of the disease is still lagging behind compared to other areas of clinical research7. However, these gaps in our knowledge have long been recognized and were recently highlighted as key areas for further research, together with recommendations for how to address them6. In addition, there is a clear call for the application of novel, sophisticated approaches to precision medicine to aid in answering some of these questions7.\n\nThe study that we conducted was designed with at least some of these aspects in mind, aimed at the identification of a biomarker or a panel of biomarkers for the differentiation of subjects with mild to moderate COPD, current smokers, former smokers and never-smokers, using gene and protein expression analyses in various biological samples together with the assessment of traditional and emerging indicators of lung health. In this data note, we provide a comprehensive description of the study population and their physiological characteristics, including full lung function, lung appearance on chest computed tomography, impulse oscillometry, exercise tolerance and different quality of life measures. We also introduce lung sound analysis (stethographics) as a potential approach to identify subjects with subclinical disease that would be missed by considering spirometry outcomes on their own.\n\nThe results of proteomics and lipidomics analyses were published here8 and here9, and transcriptomics results are currently in press10.\n\n\nMaterials and methods\n\nThis study used a parallel-group, case-controlled study design to assess a number of established and potentially novel biomarkers in smokers with COPD and in various control populations (never-smokers, former smokers, and asymptomatic current smokers). In this data note, we provide data of physiological measurements and quality of life (QoL) for the 240 subjects who completed the study. Following approval from a UK National Health Service (NHS) Ethics Committee (The Black Country Ethics Committee), the study was conducted as a single center study in strict compliance with Good Clinical Practice (GCP) guidelines.\n\nThe study has been registered on ClinicalTrials.gov with identifier NCT01780298 (trial registration date: 21 January 2013).\n\nPotentially suitable subjects were identified for inclusion into the study via the study center’s database and by media advertising. Subjects of both genders (41–70 years old) were enrolled in this study, starting with the COPD study group. Never-smokers, former and current smokers were then enrolled aiming to match the subjects with COPD by age (±5 years), ethnicity, and gender. The smoking history of all smoking subjects was at least 10 pack-years. Former smokers had to have quit smoking at least one year prior to the study. Subjects that discontinued participation (for medical or personal reasons) were replaced.\n\nAdditional information regarding the recruitment process, including inclusion and exclusion criteria for this study and information regarding withdrawal or removal of subjects are provided in the Supplementary Material (sections S1 to S3).\n\nThe study comprised a maximum of 5 out-patient visits to the study center. The initial visit served to inform the potential participant about the study and the potential risks as well as to obtain informed consent. Once informed consent was obtained, subject screening including recording of demographic data, vital signs, weight and height, determination of medical and surgical history, physical examination, and measurement of forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) took place. Further, a 12-lead electrocardiogram (ECG), laboratory assessments and urinalysis were performed. Females of childbearing potential underwent a serum pregnancy test, and all subjects underwent alcohol breath test, plasma cotinine test and a drugs of abuse test. Smokers were provided with information about smoking cessation. Finally, subjects were asked to provide ≥0.1 g of sputum sample. For subjects identified as having COPD based on screening assessments and who had their diagnosis documented by the investigator, the subject’s general practitioner was notified in writing of the assessment results and clinical conclusions. This first visit was followed by visits 1a or 1b to give subjects another opportunity to produce an adequate sputum sample and to reassess non-smokers previously deemed ineligible based on sputum neutrophil counts, respectively.\n\nAt visit 2 and prior to all other procedures, eligibility was reassessed and the questionnaires were completed. Vital signs and differential fractional exhaled nitric oxide (FENO) were recorded; full lung function including transfer factor (carbon monoxide diffusing capacity [TLCO]) and exhaled breath temperature (EBT) were measured, and subjects were asked to undergo impulse oscillometry (IOS) and computerized multichannel lung sounds analysis (stethographics). In addition, during this visit, induced sputum, blood, and nasal samples were collected for subsequent analysis of inflammatory markers and ‘omics including transcriptomics, proteomics and lipidomics.\n\nDuring visit 3, subjects underwent a high-resolution computerized tomography (HRCT) lung scan and cardio-pulmonary exercise testing with an electronically braked cycle ergometer. Twelve-lead ECG, vital signs and physical examination were performed before and after the exercise test.\n\nAssessments at visit 4 included measurement of vital signs, differential FENO, full lung function, IOS, stethographics, EBT, nasal sampling, and sputum induction.\n\nA follow-up telephone call approximately 3 to 10 days after visit 4 concluded the study, and a summary letter was sent to their general practitioner by the investigator. Throughout the study, smokers received smoking cessation advice, and any adverse events (AEs) were recorded.\n\nThe full schedule of events is provided in the Supplementary Material (section S4).\n\nThe summary of the study enrolment and recruitment success, as well as a summary of the samples collected from each of the 240 subjects and the physiological and clinical measurements that were taken are depicted in Figure 1, following the 2010 Consolidated Standards of Reporting Trials (CONSORT) guidelines11.\n\nSpirometry was performed pre- and post-bronchodilator administration in all subjects at visit 1 to meet ATS/ERS criteria12. Predicted values for FEV1 and FVC were calculated according to the formula of the European Coal and Steel Community (ECSC)13.\n\nThe multiple-breath inert gas washout (helium dilution) technique measuring the functional residual capacity (FRC) of the lungs was employed to determine lung volume. The subjects were asked to breathe normally into a closed circuit spirometer connected to a re-breathing bag filled with 9% helium and 21% oxygen. After tidal breathing, subjects performed an expiratory reserve volume (ERV) maneuver over the next 30 seconds, and a trend of the helium wash-in curve and the helium value was obtained.\n\nFor gas transfer measurements, after a few tidal breaths, each subject was asked to take a breath in and then exhale as far as possible, continuing until they felt that the lungs were completely empty. This maximal exhalation was considered the residual volume (RV). The subject was then asked to inhale as far as possible which estimated at least 90% of the subject’s vital capacity (VC) followed by holding their breath for 10 seconds without straining. Finally, the subjects were asked to exhale as far as possible. On completion of the maneuver, the subject was allowed to rest for at least 4 minutes and remain seated before repeating the test at least twice.\n\nIOS was performed twice during the study, at visits 2 and 4. The subject was asked to put the mouthpiece between his/her teeth and to keep their lips firmly sealed around the mouthpiece and breathe normally while wearing a nose-clip and sitting upright with his/her head straight or slightly extended. Once the subject’s breathing baseline was established, measurements were recorded up to 90 seconds.\n\nStethographics analysis was performed at visits 2 and 4, using the 16-channel lung sound analyzer system STG1602 (Stethographics, Boston, MA, USA) following the supplier’s recommendations14. Lung sound data were obtained from a normal deep-breathing protocol (pattern 1; P1), cough recording at FVC, and an intermediate deeper-than-normal breath pattern (pattern 2; P2). Each breath pattern was recorded for a minimum of 30 seconds allowing 3 to 6 breaths to be taken, with measurements completed over 3 to 4 minutes. Measurements were taken in a quiet room to minimize the likelihood of movement artefacts and ambient noise and analyzed using the proprietary stethographics software. Each of the 16 parameters derived from one measurement were evaluated and a score from 0 to 10 was assigned based on the value of the individual parameter. The total standard Acoustic COPD Score (ACOPDS) was calculated as the sum of the individual score for each parameter. The maximum score for each parameter was 10; therefore, the maximum possible ACOPDS for each subject was 160.\n\nChest scans were obtained using a 64-slice Discovery VCT scanner (GE Healthcare, Little Chalfont, Buckinghamshire, UK) without contrast medium. Scan duration was less than 5 minutes, including positioning the subject. When the subject was acclimatized in a supine position on the CT scanner table, they were instructed on use of the breath-holding procedure required for the scan. A standard HRCT scan of the chest was acquired at 1.25 mm slices every 10 mm on inspiration. Any deviation from standard CT scan parameters was recorded. CT scans were assessed separately by 2 radiologists who were blinded to the subjects’ details and study group assignments. Each HRCT scan was analyzed within 24 hours, and any abnormalities including lung nodules and extrathoracic abnormalities, were reported. A scoring system was applied to grade the various features visible on the scans15–18:\n\n1. Extent of disease: Emphysema was defined as areas of decreased attenuation, usually without discrete walls, and of non-uniform distribution causing permeative destruction of lung parenchyma. The extent of emphysema was estimated visually to the nearest 5%. The mean figure was taken as the extent of emphysema. The following score was used: 0 = absent, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe.\n\n2. Severity of bronchial dilatation was measured in morphologically normal lung and graded semi-quantitatively by comparison with the homologous pulmonary artery. The scores assigned were: 0 = none; 1 = mild (1.5x to 2.5x diameter of pulmonary artery); 2 = severe (>2.5x diameter of pulmonary artery).\n\n3. Traction bronchiectasis was defined as bronchial dilatation within areas of reticular pattern and was graded by comparison with the homologous pulmonary artery using the same scoring system as for severity of bronchial dilatation.\n\n4. A total bronchiectasis score was derived by adding up the severity of bronchial dilatation and traction bronchiectasis scores.\n\n5. Bronchial wall thickening was graded as follows: 0 = none; 1 = 0.5x; 2 = 0.5-1x; or 3 = >1x the diameter of the adjacent pulmonary artery.\n\n6. Small airways disease was defined as areas of decreased attenuation associated with a reduction in the number and caliber of pulmonary vessels, but without vascular distortion that is seen in centrilobular emphysema17. The extent of decreased attenuation ascribable to small airways disease was estimated visually to the nearest 5% and scored as follows: 0 = absent, 1 = mild, 2 = moderate, 3 = severe and 4 = very severe.\n\n7. The total COPD score was derived from the means of the component scores.\n\nThe cardiopulmonary exercise tolerance test following the modified Bruce protocol19 was conducted at visit 3 by a clinical physiologist. All phases of the exercise test were completed if possible and if the subject did not experience clinically significant findings or significant fatigue. Maximal oxygen uptake or VO2max was recorded.\n\nAll subjects completed three questionnaires during the study: 1) A general questionnaire considering education, lifestyle, smoking history and family history of disease; 2) the SF-36 questionnaire - a multi-purpose, short-form QoL health survey20; and 3) the modified Medical Research Council (MMRC) Dyspnoea Scale, a self-grading scale of the degree of breathlessness during activities. The MMRC Dyspnoea Scale uses a simple grading system to assess a subject's level of dyspnoea (shortness of breath)21. Subjects were asked to grade the degree of breathlessness during activities by choosing one of the following:\n\n1. Not troubled by breathlessness except on strenuous exercise.\n\n2. Short of breath when hurrying or walking up a slight hill.\n\n3. Walks slower than contemporaries on the level because of breathlessness, or has to stop for breath when walking at own pace.\n\n4. Stops for breath after about 100 meters or after a few minutes on the level.\n\n5. Too breathless to leave the house, or breathless when dressing or undressing.\n\nThe modified BODE (mBODE) index was derived from the BMI (B), the degree of airflow obstruction indicated by FEV1 % predicted (O), functional dyspnea as measured by the MMRC Dyspnoea Scale (D), and exercise capacity reflected by VO2max (E)22,23, and scored based on the following matrix (Table 124):\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nData availability\n\nThe main data are provided for the 240 subjects who completed the study (60 subjects per group). For some assessments, parameters were measured at 2 visits and the measurement for each visit is provided. Missing data were not replaced.\n\nClinical study data were transferred from the clinical site as locked SAS datasets, formatted according to the Study Data Tabulation Model or SDTM (https://www.cdisc.org/standards/foundational/sdtm, accessed 16 May 2017). The data presented here was extracted from these standardized study datasets.\n\nDataset 1. Demographics. List of all unique study subject IDs (USUBJID) and demographic information including sex, age, COPD GOLD stage (if any), cigarette consumption (expressed as pack-years and cigarettes per day), study group (ARMCD), and the matching ID (MATCHID) allowing the pairing of control subjects to their ‘matched’ COPD subject.\n\nDOI, 10.5256/f1000research.11698.d16378125\n\nDataset 2. Vital Signs. Table of body mass index, diastolic and systolic blood pressure, heart and respiration rate measures. The data for each measurement (TEST) are presented as value (standard result, STRESN) and unit (standard unit, STRESU) for each subject, and visit number (VISITNUM) at which measurements were obtained.\n\nDOI, 10.5256/f1000research.11698.d16378226\n\nDataset 3. Lung Function. Table of lung function data for each subject, including percent predicted values for:\n\n- Forced expiratory volume in one second (FEV1 %Pred)\n\n- Forced vital capacity (FVC %Pred)\n\n- Ratio of FEV1 to maximum vital capacity (FEV 1 % VC MAX)\n\n- Functional residual volume by helium dilution technique (FRC-He %Pred)\n\n- Maximum expiratory flow at 25% of expired volume (MEF 25 %Pred)\n\n- Maximum expiratory flow at 75% of expired volume (MEF 75 %Pred)\n\n- Diffusion capacity (transfer factor) for carbon monoxide (TLCO %Pred)\n\nThe data for each measurement (TEST) are presented as value (standard result, STRESN), together with the visit number (VISITNUM) at which measurements were obtained.\n\nDOI, 10.5256/f1000research.11698.d16378327\n\nDataset 4. Impulse Oscillometry. Table of impulse oscillometry data for each subject, including resistance at 5 (R at 5Hz) and 20 Hertz (R at 20Hz), reactance at 5 (X at 5Hz), and Resonant Frequency. The data for each measurement (TEST) are presented as value (standard result, STRESN), together with the visit number (VISITNUM) at which measurements were obtained.\n\nDOI, 10.5256/f1000research.11698.d16378428\n\nDataset 5. Stethographics. Stethographics measures for each subject were obtained for 2 breathing patterns, also referred to as normal breathing pattern or P1(P1 : normal) and deeper breathing or P2 (P2 : deeper). Data presented here include scores for:\n\n- Expiratory crackle rate for P2 breathing pattern [Expir Crackle Rate(P2 : deeper)]\n\n- Inspiratory crackle rate for P2 breathing pattern [Insp Crackle Rate(P2 : deeper)]\n\n- Expiratory wheeze rate for P2 breathing pattern [Expir Wheeze Rate(P2 : deeper)]\n\n- Inspiratory wheeze rate for P2 breathing pattern [Insp Wheeze Rate(P2 : deeper)]\n\n- Inspiratory chest amplitude for P2 breathing pattern [Insp chest RMS score(P2 : deeper)]\n\n- Average lead and lag of chest channels compared to the tracheal channel for P2 breathing pattern [Lead score(P2 : deeper); Lag score(P2 : deeper)]\n\n- Inter-channel asynchrony at the beginning and end of inspiration for P2 breathing pattern [Lead STDev score (channel asynchrony independent of trachea sound) (P2 : deeper); Lag STDev score (channel asynchrony independent of trachea sound)(P2 : deeper)]\n\n- Lead time-integrated amplitude for P2 breathing pattern [Lead time-integrated amplitude(P2 : deeper)]\n\n- Lag time-integrated amplitude for P2 breathing pattern [Lag time-integrated amplitude(P2 : deeper)]\n\n- Ratio of low frequency energy to high frequency energy for P2 breathing pattern [Max R4 (low freq/high freq)(P2 : deeper)]\n\n- Ratio of duration of inspiration to the duration of expiration for P2 breathing pattern [R1 (Insp.Dur/Expir.Dur)(P2 : deeper)]\n\n- Ratios of peak inspiratory amplitude to peak expiratory amplitude for P2 breathing pattern [Ratio(peak insp amplitude/peak expir amplitude)(P2 : deeper)]\n\n- Dynamic range score(P2 : deeper)\n\n- Slope of the chest versus tracheal sound function during inspiration for P2 breathing pattern [Slope of chest vs trachea during Insp(P2 : deeper)]\n\n- Non-weighted total acoustic COPD scores for both breathing patterns [COPD total score NOTweighted(P1 : normal)], [COPD total score NOTweighted(P2 : deeper)]\n\n- Weighted total acoustic COPD scores for both breathing patterns [COPD total score weighted(P1 : normal)], [COPD total score weighted(P2 : deeper)]\n\n- The individual scores for each stethographics parameter (TEST) are presented as value (standard result, STRESN) for each subject, together with the visit number (VISITNUM) at which stethographics was performed.\n\nDOI, 10.5256/f1000research.11698.d16378629\n\nDataset 6. Lung HRCT. HRCT data for each subject are presented as scores for:\n\n- Bronchial wall thickening\n\n- Emphysema and Emphysema Type\n\n- Extent of Disease\n\n- Interstitial disease and Interstitial disease distribution\n\n- Interstitial Score\n\n- Nodules + other abnormality\n\n- Severity of bronchial dilatation\n\n- Small airways disease\n\n- Traction bronchiectasis\n\n- The sum of the severity of bronchial dilatation and traction bronchiectasis scores (Total Bronchiectasis Score)\n\n- The representative mean of the 5 component scores, i.e. Extent of Disease score, Severity of bronchial dilatation score, Traction bronchiectasis, Bronchial wall thickening score, and Small airways disease score (Total COPD CT Score)\n\nThe individual scores for each CT parameter (TEST) are presented as value (standard result, STRESN) together with the visit number (VISITNUM) at which HRCT scans were obtained.\n\nDOI, 10.5256/f1000research.11698.d16378830\n\nDataset 7. Cardiopulmonary Exercise Tolerance Test. Cardiopulmonary exercise tolerance test data, i.e. heart rate, gas exchange ratio or respiratory exchange ratio (RER), absolute and relative rates of maximum oxygen consumption (VO2; VO2/kg), absolute and relative percent predicted oxygen consumption (VO2 %Pred; VO2/kg %Pred), and carbon monoxide output (VCO2) for each subject.\n\nThe data for each measurement (TEST) are presented as value (standard result, STRESN) and unit (standard unit, STRESU) for each subject, together with the visit number (VISITNUM) at which measurements were obtained.\n\nDOI, 10.5256/f1000research.11698.d16378931\n\nDataset 8. Questionnaires. Table listing individual scores for:\n\n- Modified Medical Research Council (MMRC) Dyspnoea Scale (MMRCDS for BODE)\n\n- Bodily Pain component of SF-36 questionnaire (SF-36_Bodily Pain)\n\n- General Health component of SF-36 questionnaire (SF-36_General Health)\n\n- Mental Health component of SF-36 questionnaire (SF-36_Mental Health)\n\n- Physical Functioning component of SF-36 questionnaire (SF-36_Physical Functioning)\n\n- Social Functioning component of SF-36 questionnaire (SF-36_Social Functioning)\n\n- Emotional Role Functioning component of SF-36 questionnaire (SF-36_Role Emotional)\n\n- Physical Role Functioning component of SF-36 questionnaire (SF-36_Role Physical)\n\n- Vitality component of SF-36 questionnaire (SF-36_Vitality)\n\n- Modified BODE index (mBODE Index)\n\nThe score for each assessment (TEST) is presented as value (standard result, STRESN) for each subject.\n\nDOI, 10.5256/f1000research.11698.d16379032\n\n\nConsent\n\nThe study protocol number QASMC202 was reviewed and approved by The Black Country Ethics Committee, a UK National Health Service (NHS) Ethics Committee (reference number 11/WM/0114).\n\nWritten informed consent to collect and use personal data such as age, gender, ethnicity and medical/surgical history, and to publish these results was obtained from all study participants. None of the study participants can be identified by the sponsor by using any of the data provided in this data note.",
"appendix": "Author contributions\n\n\n\nNIC, MJP, MCP conceived and designed the study. EP and LFM managed and coordinated the study. GV and PL prepared and analysed the data. KL wrote the first draft of the manuscript, and MJP and GV contributed to the writing of the manuscript. MJP, GV, and JH made critical revisions of the manuscript. All authors reviewed and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNIC, KL, MJP, EP, LFM, GV, PL, MCP and JH are full-time employees of Philip Morris Products SA, Philip Morris International. KL, MJP, EP, LFM, GV, PL, MCP and JH declared no other potential conflicts of interest with respect to the research, authorship, and/or publication of this article. NIC discloses royalties related to the sales of OFEV (nintedanib; Boehringer Ingelheim), which is marketed for the treatment of pulmonary fibrosis.\n\n\nGrant information\n\nPhilip Morris International was the sole source of funding for this study.\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 are indebted to the clinical staff for their extraordinary clinical experience and diligence in conducting this study. The authors would further like to acknowledge Filippo Toni and Jeremie de Torrente for their project management support throughout the course of the study. Special thanks also goes to Kishor Lad for assistance with clinical data management activities.\n\n\nSupplementary material\n\nSupplementary Material S1: Inclusion and exclusion criteria.\n\nClick here to access the data.\n\nSupplementary Material S2. Removal/withdrawal of subjects from the study.\n\nClick here to access the data.\n\nSupplementary Material S3. Study restrictions.\n\nClick here to access the data.\n\nSupplementary Material S4. Schedule of events.\n\nClick here to access the data.\n\n\nReferences\n\nDiaz-Guzman E, Mannino DM: Epidemiology and Prevalence of Chronic Obstructive Pulmonary Disease. Clin Chest Med. 2014; 35(1): 7–16. PubMed Abstract | Publisher Full Text\n\nWHO: World health statistics 2008. World Health Organization; 2008. Reference Source\n\nAgusti A, Calverley PM, Celli B, et al.: Characterisation of COPD heterogeneity in the ECLIPSE cohort. Respir Res. 2010; 11(1): 122. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCelli BR, Halbert RJ, Isonaka S, et al.: Population impact of different definitions of airway obstruction. Eur Respir J. 2003; 22(2): 268–273. PubMed Abstract | Publisher Full Text\n\nCoxson HO, Leipsic J, Parraga G, et al.: Using Pulmonary Imaging to Move Chronic Obstructive Pulmonary Disease beyond FEV1. Am J Respir Crit Care Med. 2014; 190(2): 135–144. PubMed Abstract | Publisher Full Text\n\nCelli BR, Decramer M, Wedzicha JA, et al.: An official American Thoracic Society/European Respiratory Society statement: research questions in COPD. Eur Respir Rev. 2015; 24(136): 159–172. PubMed Abstract | Publisher Full Text\n\nRooney C, Sethi T: Biomarkers for precision medicine in airways disease. Ann N Y Acad Sci. 2015; 1346(1): 18–32. PubMed Abstract | Publisher Full Text\n\nTitz B, Sewer A, Schneider T, et al.: Alterations in the sputum proteome and transcriptome in smokers and early-stage COPD subjects. J Proteomics. 2015; 128: 306–320. PubMed Abstract | Publisher Full Text\n\nTitz B, Luettich K, Leroy P, et al.: Alterations in Serum Polyunsaturated Fatty Acids and Eicosanoids in Patients with Mild to Moderate Chronic Obstructive Pulmonary Disease (COPD). Int J Mol Sci. 2016; 17(9): pii: E1583. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTalikka M, Martin F, Sewer A, et al.: Mechanistic evaluation of the impact of smoking and chronic obstructive pulmonary disease on the nasal epithelium. Clin Med Insights Circ Respir Pulm Med. 2017; In press. Publisher Full Text\n\nSchulz KF, Altman DG, Moher D, et al.: CONSORT 2010 Statement: Updated guidelines for reporting parallel group randomised trials. J Clin Epidemiol. 2010; 63(8): 834–840. PubMed Abstract | Publisher Full Text\n\nMiller MR, Hankinson J, Brusasco V, et al.: Standardisation of spirometry. Eur Respir J. 2005; 26(2): 319–338. PubMed Abstract | Publisher Full Text\n\nQuanjer PH, Tammeling GJ, Cotes JE, et al.: Lung volumes and forced ventilatory flows. Eur Respir J. 1993; 6(Suppl 16): 5–40. PubMed Abstract | Publisher Full Text\n\nBergstresser T, Ofengeim D, Vyshedskiy A, et al.: Sound transmission in the lung as a function of lung volume. J Appl Physiol (1985). 2002; 93(2): 667–674. PubMed Abstract | Publisher Full Text\n\nWells AU, King AD, Rubens MB, et al.: Lone cryptogenic fibrosing alveolitis: a functional-morphologic correlation based on extent of disease on thin-section computed tomography. Am J Respir Crit Care Med. 1997; 155(4): 1367–1375. PubMed Abstract | Publisher Full Text\n\nWells AU, Desai SR, Rubens MB, et al.: Idiopathic Pulmonary Fibrosis: a composite physiologic index derived from disease extent observed by computed tomography. Am J Respir Crit Care Med. 2003; 167(7): 962–969. PubMed Abstract | Publisher Full Text\n\nHansell DM, Rubens MB, Padley SP, et al.: Obliterative bronchiolitis: individual CT signs of small airways disease and functional correlation. Radiology. 1997; 203(3): 721–726. PubMed Abstract | Publisher Full Text\n\nRoberts HR, Wells AU, Milne DG, et al.: Airflow obstruction in bronchiectasis: correlation between computed tomography features and pulmonary function tests. Thorax. 2000; 55(3): 198–204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcInnis KJ, Balady GJ: Comparison of submaximal exercise responses using the Bruce vs modified Bruce protocols. Med Sci Sports Exerc. 1994; 26(1): 103–107. PubMed Abstract | Publisher Full Text\n\nWare JE Jr, Sherbourne CD: The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care. 1992; 30(6): 473–483. PubMed Abstract\n\nMahler DA, Wells CK: Evaluation of clinical methods for rating dyspnea. Chest. 1988; 93(3): 580–586. PubMed Abstract | Publisher Full Text\n\nCelli BR, Cote CG, Marin JM, et al.: The body-mass index, airflow obstruction, dyspnea, and exercise capacity index in chronic obstructive pulmonary disease. N Engl J Med. 2004; 350(10): 1005–1012. PubMed Abstract | Publisher Full Text\n\nLopez-Campos JL, Cejudo P, Marquez E, et al.: Modified BODE indexes: Agreement between multidimensional prognostic systems based on oxygen uptake. Int J Chron Obstruct Pulmon Dis. 2010; 5: 133–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCardoso F, Tufanin AT, Colucci M, et al.: Replacement of the 6-min walk test with maximal oxygen consumption in the BODE Index applied to patients with COPD: an equivalency study. Chest. 2007; 132(2): 477–482. PubMed Abstract | Publisher Full Text\n\nVuillaume G, Chaudhary N, Luettich K, et al.: Dataset 1 in: Data of physiological measurements and quality of life in subjects with mild to moderate COPD compared to asymptomatic current smokers, former smokers and never-smokers. F1000Research. 2017. Data Source\n\nVuillaume G, Chaudhary N, Luettich K, et al.: Dataset 2 in: Data of physiological measurements and quality of life in subjects with mild to moderate COPD compared to asymptomatic current smokers, former smokers and never-smokers. F1000Research. 2017. Data Source\n\nVuillaume G, Chaudhary N, Luettich K, et al.: Dataset 3 in: Data of physiological measurements and quality of life in subjects with mild to moderate COPD compared to asymptomatic current smokers, former smokers and never-smokers. F1000Research. 2017. Data Source\n\nVuillaume G, Chaudhary N, Luettich K, et al.: Dataset 4 in: Data of physiological measurements and quality of life in subjects with mild to moderate COPD compared to asymptomatic current smokers, former smokers and never-smokers. F1000Research. 2017. Data Source\n\nVuillaume G, Chaudhary N, Luettich K, et al.: Dataset 5 in: Data of physiological measurements and quality of life in subjects with mild to moderate COPD compared to asymptomatic current smokers, former smokers and never-smokers. F1000Research. 2017. Data Source\n\nVuillaume G, Chaudhary N, Luettich K, et al.: Dataset 6 in: Data of physiological measurements and quality of life in subjects with mild to moderate COPD compared to asymptomatic current smokers, former smokers and never-smokers. F1000Research. 2017. Data Source\n\nVuillaume G, Chaudhary N, Luettich K, et al.: Dataset 7 in: Data of physiological measurements and quality of life in subjects with mild to moderate COPD compared to asymptomatic current smokers, former smokers and never-smokers. F1000Research. 2017. Data Source\n\nVuillaume G, Chaudhary N, Luettich K, et al.: Dataset 8 in: Data of physiological measurements and quality of life in subjects with mild to moderate COPD compared to asymptomatic current smokers, former smokers and never-smokers. F1000Research. 2017. Data Source"
}
|
[
{
"id": "23964",
"date": "20 Jul 2017",
"name": "Amany F. Elbehairy",
"expertise": [
"Reviewer Expertise Respiratory Physiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented detailed data across groups of smokers with and without COPD and healthy non-smokers. Data are well presented and methodology is detailed. I have 3 comments/suggestions.\nThe title is very long and need to be more concise. Suggestions: \"Physiological and biological characterization of smokers with and without COPD\" .\n\nThe authors used FEV1/FVC fixed ratio of 0.7 to diagnose COPD among their smokers. Would the data be different if they used both criteria of having FEV1/FVC less than 0.7 and less than the lower limit of normal to diagnose COPD?\n\nData presented for CPET are the peak values. Data at standardized work rate or ventilation can give better characterization of in-between groups differences.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "3466",
"date": "07 Mar 2018",
"name": "Gregory Vuillaume",
"role": "Author Response",
"response": "We are grateful for the reviewer’s feedback and followed the advice to shorten the title accordingly. We are uncertain whether we understand this comment correctly. The reference population of never-smokers in this study was rather small (N=60), and there was considerable variation in the lung function data. Considering the lower limit of normal, i.e. the 5th percentile of the FEV1/FVC observed in never-smokers, in addition to the 2009 GOLD criterion of FEV1/FVC <0.7, we believe we would not have misclassified the current smokers as being “healthy”. In brief, therefore, we do not think that the data would have been different. This is valuable feedback, which we appreciate. Unfortunately, we only have the peak values for the CPET and cannot study potential between-group differences further."
}
]
},
{
"id": "25445",
"date": "14 Sep 2017",
"name": "David Mannino",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide data from a Phillip Morris sponsored study that looks at biomarkers of lung health in a groups of people with COPD and \"healthy\" current, former and never smokers. The data is provided- but no analysis is done of the raw data.\nI believe summary tables of the key features of the database would be a useful addition.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "3465",
"date": "07 Mar 2018",
"name": "Gregory Vuillaume",
"role": "Author Response",
"response": "We appreciate the positive feedback. While our intention was to provide the data describing the physiological characteristics of the study population, we understand the reviewer’s point of view and prepared a table of summary statistics for all of the reported endpoints."
}
]
}
] | 1
|
https://f1000research.com/articles/6-877
|
https://f1000research.com/articles/7-282/v1
|
06 Mar 18
|
{
"type": "Research Article",
"title": "The effect of a health literacy approach to counselling on the lifestyle of women with gestational diabetes: A clinical trial",
"authors": [
"Mehrafza Gharachourlo",
"Zohreh Mahmoodi",
"Mahnaz Akbari Kamrani",
"Maryam Tehranizadeh",
"Kourosh Kabir",
"Mehrafza Gharachourlo",
"Mahnaz Akbari Kamrani",
"Maryam Tehranizadeh",
"Kourosh Kabir"
],
"abstract": "Background: Gestational diabetes is a common pregnancy disorder that affects the mother’s and neonate’s health. The present study was conducted to investigate the effect of a health literacy approach to counselling on the lifestyle of women with gestational diabetes. The present randomized controlled clinical trial was conducted in 2017 using a parallel design. The subjects included 84 eligible women presenting to Alborz and Kamali Hospitals, Karaj, Iran. Methods: Convenience sampling was first used to select the subjects. They were then assigned to an intervention or control group based on randomized blocks of four. Both groups attended counselling sessions. The mothers in the intervention group attended six sessions of counselling with a health literacy approach in addition to counselling on routine pregnancy care. The control group attended counselling sessions on safe pregnancy care and received a training package containing all the subjects discussed in the intervention group. The Lifestyle Questionnaire and the Iranian Health Literacy Questionnaire were completed by the mothers at the beginning and at the end of the sessions as well as three weeks after the sessions. The data obtained were analyzed in SPSS-19. Results: According to the study findings, the scores of lifestyle (P=0.8) and health literacy (P=0.423) showed no significant differences between the intervention and control groups before the intervention. Significant differences were, however, observed in the mean scores of lifestyle and health literacy between the two groups immediately and three weeks after the intervention. Comparing the means showed a higher increase in the mean scores in the intervention group (P<0.001). Conclusions: Providing counselling services by midwives can significantly help modify mothers’ unhealthy lifestyle choices and increase their health literacy; therefore, reducing maternal and neonatal consequences, especially in high-risk pregnancies. Trial registration number: IRCT2017021427728N3 Trial registry: Iranian Registry of Clinical Trials Trial registration date: 5th April 2017",
"keywords": [
"Gestational diabetes",
"health literacy",
"lifestyle",
"counselling"
],
"content": "Introduction\n\nAlthough pregnancy is a normal physiologic phenomenon, it can deviate from its normal path and cause disability, or even maternal and fetal mortality1. Gestational diabetes is a major disorder that causes numerous complications in the mother and her baby. Gestational diabetes is a metabolic disorder that emerges or is diagnosed for the first time with carbohydrate intolerance during pregnancy and is considered the most common gestational complication2. The prevalence of gestational diabetes differs in different regions, which can be explained by factors, such as ethnic diversity, differences in lifestyle and nutrition and different screening protocols3. This disease influences maternal and fetal health and causes the emergence of numerous undesirable outcomes4. These complications include hypertension, preeclampsia, urinary tract infections, hydramnios, surgical interventions, the risk of developing type 2 diabetes in mothers in the future, and macrosomia followed by an increased risk of birth trauma, congenital anomalies, childhood obesity and growth disorders in neonates5.\n\nMany factors are associated with gestational diabetes, including social determinants of health6. Lifestyle is one of the main factors that can play a key role in preventing or treating gestational diabetes. Research suggests serious relationships between developing diabetes and lifestyle risk factors, such as smoking, inappropriate nutrition, drinking, obesity and limited physical activity7,8. Nowadays, more than 70% of diseases are believed to be somewhat related to people’s lifestyle and many illnesses appear to be directly or indirectly caused by lifestyle or at least be exacerbated or sustained by lifestyle9,10. Gestational diabetes prevention strategies have recently focused on promoting healthy lifestyles in patients by encouraging physical activity and promoting healthy nutrition11. Nevertheless, modifying lifestyle requires adequate information in this regard12. In fact, the decisions made by people and their performance regarding lifestyle behaviors depends on their level of literacy, which exerts a key effect on preventing and controlling chronic diseases such as diabetes13. Today, health literacy improves health behaviors, creates a healthy lifestyle and promotes the quality of life14–16. Health literacy refers to one’s capacity for acquiring, interpreting and understanding primary health information and services required for proper health decision making17. Enhancing the awareness and preparedness during pregnancy helps the mother pass this stage of life with fewer complications. As a result, pregnancy provides a good opportunity for teaching and counseling pregnant women and making them aware of the advantages of having a healthy lifestyle18. Given the importance of pregnant women as a vulnerable social class, the fifth goal of United Nations Millennium Development Goals, i.e. a 75% reduction in maternal mortality through improving mothers’ health19, and the significance of gestational diabetes as the most common pregnancy complication and a threat to the mother’s and baby’s health, the present study was performed to examine the effect of a health literacy approach to counselling on the lifestyle of women with high-risk pregnancy and gestational diabetes.\n\n\nMethods\n\nThe present randomized controlled clinical trial was conducted using a parallel design. The study population comprised pregnant women with gestational diabetes presenting to Alborz and Kamali hospitals in Karaj, Iran in April 2017.\n\nThe present research was approved by the Ethics Committee of Alborz University of Medical Sciences and Health Services on 4 March 2017 (Abzums.Rec.1395.146), and registered in the Iranian Registry of Clinical Trials (IRCT2017021427728N3) on 5th April 2017. A completed CONSORT checklist can be found in Supplementary File 1.\n\n\nSubjects\n\nInclusion criteria: The eligible candidates comprised 18–35-year-old Iranian women with gestational diabetes, whose disease was first diagnosed using the FBS, OGTT or GCT tests, as per the criteria of the World Health Organization20. The exclusion criteria consisted of being absent from, at most, two counseling sessions, having other physical or mental co-morbidities, such as cardiac, renal and thyroid disorders, and unwillingness to participate in the study.\n\nAfter going to prenatal clinics of Alborz and Kamali Hospitals for routine check-ups, the researcher (MG) identified eligible mothers and explained the study objectives to them and asked them to sign an informed consent form if they were willing to participate. Convenience sampling was first used to select the subjects. They were then assigned to the intervention and control groups based on randomized blocks of four. Each “block” had a specified number of randomly ordered treatment assignments.\n\nBased on the formula for calculating the difference between two ratios and given a probability of proper lifestyle in 10% of the subjects in the intervention group (with gestational diabetes) and its improvement to 30%, the sample size was calculated as 84, i.e. n=42 in each group.\n\n\n\nα=5 % β=20 % P1=10% P 2=3% n1/n2=1 n=42\n\nThe intervention group received counselling on routine pregnancy care and a health literacy approach to counselling for modifying lifestyle. The control group received counselling on routine pregnancy care as per the safe national maternal protocol of Ministry of Health and Medical Education of Iran as well as a training package containing all the subjects discussed in the intervention group. The sessions were taken by MG, MT. More information about the training sessions are included in Supplementary File 2.\n\nBoth groups attended six 1.5-hour sessions, once a week. To prevent cross talk by participants, the counselling sessions for the control and intervention groups were held on different days. The researcher followed up the mothers by phone.\n\nThe content of sessions in the intervention group was presented using a health literacy approach according to the protocol for the care of mothers with gestational diabetes approved by Ministry of Health and Medical Education of Iran as follows:\n\nSession one: Introducing the members of the group to one another and the group leader, establishing a positive relationship, stating the group rules and creating motivations for the active participation and timely attendance in the sessions, explaining gestational diabetes and attitudes towards it and the role of a proper lifestyle in coping with gestational diabetes.\n\nSession two: Counselling on self-awareness skills, understanding the status quo, explaining the suffering and difficulties they experienced, introducing and learning how to use insulin, injection-associated problems, attitudes towards insulin treatment and the ways of coping with these attitudes and illustrating a favorable status.\n\nSession three: Counselling on health-related skills, including understanding basic information about nutrition, essential diets, understanding the effects of nutrition on improving the status, understanding basic information about physical exercise and its effects on improving maternal and fetal status, and explaining the capacity of performing exercises and the types of exercise allowed during pregnancy.\n\nSession four: Counselling on the concepts associated with emotional skills, including understanding the fear and stress caused by the effect of the disease on the fetus and the body, the subject’s appearance and the disease survival after pregnancy and the ways of coping with it.\n\nSession five: Understanding and counselling on the concepts associated with communication skills, understanding the effect of family and social supports, comprehending basic information about incorrect health behaviors and their effects on the mother and the fetus, understanding the effect of the support provided by the spouse, family and society on these behaviors and introducing services associated with pregnancy and screening.\n\nSession six: Summarizing the presented subjects, practicing the counseled skills, responding to questions and receiving feedback on the classes held.\n\nAt the beginning, the end and three weeks after the sessions, Iranian Health Literacy Questionnaire (IHLQ) and Lifestyle Questionnaire (LSQ) were distributed among both groups and they were asked to complete them. If the patient was illiterate, the researcher completed the questionnaire by an interview.\n\nThe data collection tools comprised the IHLQ, the LSQ and a sociodemographic checklist (Supplementary File 3).\n\nThe 53-item IHLQ contains 9 subscales, including access to health information access, health information use, reading skills, comprehension skills, assessment and judgment skills, decision-making and communication skills, health knowledge, individual empowerment and social empowerment. The validity and reliability of IHLQ were confirmed by Haghdoost et al. in 201421.\n\nThe 70-item LSQ contains 10 dimensions of health, physical health, sports and fitness, weight management and nutrition, disease prevention, mental health, spiritual health, social health, avoidance of drugs, alcohol and opiates, accident prevention and environmental health. Lali et al. confirmed the validity and reliability of this questionnaire in Isfahan, Iran, in 2008–2009 academic year10.\n\nThe data collected were analyzed in SPSS-19 using independent t-test, Mann-Whitney test, Fisher’s exact test and Chi-square test.\n\n\nResults\n\nA total of 100 subjects were initially recruited, and 16 were excluded during the study: 8 subjects were excluded in the intervention group, including 2 for failing to complete the questionnaires, 4 for being absent for more than two sessions, and 2 for preterm delivery at a 25-week and 29-week gestational age; 8 subjects were also excluded from the control group, including 4 for failing to complete the questionnaires, 3 for failing to attend the sessions and 1 for abortion at 16 weeks of pregnancy. Therefore, the study ended with 84 subjects (Figure 1).\n\nThe present study investigated 84 mothers with gestational diabetes in two groups, who were examined until the end of the study. After examining the normality of the study variables, no significant differences were observed between the two groups in terms of mean age, level of education, body mass index (BMI), occupational status and gestational age, and the two groups were therefore matched in terms of the cited variables (Table 1).\n\nAccording to the study findings, no significant differences were observed between the two groups in terms of the mean score of lifestyle before the intervention. Therefore, the two groups were matched before the intervention in terms of lifestyle. The two groups exhibited significant differences in the mean score of lifestyle after the intervention. This mean score showed a significant increase immediately and three weeks after the intervention, and this increase was higher in the intervention group (Table 2).\n\n*P-values are comparing the difference in scores of lifestyle in three phases, before, immediately after and 3 weeks after intervention, between the intervention and the control groups.\n\nNo significant differences were observed between the two groups in terms of the mean score of health literacy before the intervention, and the two groups were therefore matched in this regard. The two groups exhibited significant differences in the mean score of health literacy immediately and three weeks after the intervention, and this increase was significant in the intervention group (Table 3).\n\n*P-value is comparing the difference in scores of health literacy in three phases, before, immediately after and 3 weeks after intervention, between the intervention and the control groups.\n\n\nDiscussion\n\nGestational diabetes is a common disorder during pregnancy, which can negatively affect prenatal outcomes and is associated with lifestyle risk factors. Modern prevention methods of gestational diabetes emphasize modifying lifestyle risk factors18,22.\n\nThe present study findings suggest no significant differences between the two groups before counselling in terms of the score of lifestyle. In other words, the two groups presented the same level of lifestyle upon entering the study; however, immediately after and three weeks after the intervention, a higher increase was observed in the mean scores of variables, such as physical health, sports and fitness, weight management and nutrition, disease prevention, mental health, avoidance of drugs, alcohol and opiates, accident prevention and the overall lifestyle of the mothers, in the intervention group compared to those in the control group, although no significant differences were observed between the two groups in spiritual health and environmental health.\n\nIn 2011, Luoto et al. found that interventions such as counselling can improve lifestyle quality in pregnant patients at risk of developing gestational diabetes23. Numerous studies have explained the effect of interventions on modifying lifestyle, and their results are consistent with those obtained in the present study. In 2015, Khadivzadeh et al. found that educational lifestyle interventions can help promote self-care in patients with gestational diabetes24. Babaei et al. (2016)25 and Korpi-Hyövälti et al. (2012) reported that individual counseling conducted by experts as part of lifestyle interventions modifies nutrition and sports dimensions of lifestyle26,27. A study conducted by Babei et al. titled “the effect of educational intervention of lifestyle modification on blood pressure control in patients with hypertension” showed that educational programs can modify and increase the score of lifestyle dimensions, including sports and fitness, weight management and nutrition and mental health, although these interventions were found not to affect spiritual health25.\n\nBefore the intervention, no significant differences were observed between the two groups in the score of health literacy, and the two groups were matched in terms of the level of health literacy. The mean score of health literacy, however, showed a higher increase immediately and three weeks after the intervention in the intervention group compared to that in the controls. Given that the two groups were matched in terms of demographic characteristics and level of health literacy, the findings suggest the effect and role of counseling and the counselor midwife in improving the level of health literacy in mothers with gestational diabetes. This finding is consistent with the results of the studies conducted by Tol et al.28 and Kandula et al.29. These researchers found that education and counselling increase the score of health literacy in diabetics with any levels of health literacy. Luoto et al. reported that interventions at the level of patient communication skills can improve the awareness, literacy and clinical indicators of diabetics23. Today, many health problems and human social needs can be solved using advisors as facilitators for knowledge based on useful relationships.30.\n\nThe strength of the present study was the fact that the intervention and control groups matched in terms of some confounding demographic variables affecting the results, including age, level of education, BMI and gestational age, and the results obtained were not therefore affected by these variables. The researchers made efforts to use random allocation and match the subjects in both groups; however, the subjects’ lifestyle was impossible to be completely controlled and this was a limitation of the present research.\n\n\nConclusion\n\nThe findings obtained from the present research showed that both routine pregnancy counselling and a health literacy approach to counselling causes an increase in the overall score of lifestyle and health literacy. As members of the health system and the main supervisors of pregnant women, midwives have the closest relationship with these women and are in charge of providing them with care services and necessary recommendations during pregnancy. They can therefore take more effective steps towards improving mothers’ health and pregnancy outcomes by applying correct counselling principles for resolving mothers’ problems.\n\n\nEthical statement\n\nThe present research was approved by the Ethics Committee of Alborz University of Medical Sciences and Health Services on 4 March 2017 (Abzums.Rec.1395.146). Written informed consent was obtained from all subjects for participation in the study.\n\n\nData availability\n\nDataset 1: Sociodemographic characteristics, and results from the IHLQ and LSQ questionnaires, before, immediately after and 3 weeks after intervention for control and intervention groups. DOI, 10.5256/f1000research.13838.d19505431",
"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 funding this work.\n\n\nAcknowledgements\n\nThis study was extracted from a Master’s thesis (MG) on midwifery counselling. The authors would like to express their gratitude to the esteemed authorities of the Deputy of Research of Alborz University of Medical Sciences for funding this study, as well as all midwives working in prenatal clinics of Alborz and Kamali hospitals and all pregnant women who participated in this study.\n\n\nSupplementary material\n\nSupplementary File 1: CONSORT checklist.\n\nClick here to access the data.\n\nSupplementary File 2: Information about the control and intervention group sessions.\n\nClick here to access the data.\n\nSupplementary File 3: Sociodemographic checklist.\n\nClick here to access the data.\n\n\nReferences\n\nJames DK, Steer PJ, Weiner CP, et al.: High Risk Pregnancy E-Book: Management Options-Expert Consult. Elsevier Health Sciences; 2010. Reference Source\n\nCunningham F, Leveno K, Bloom S, et al.: Williams Obstetrics, 24e. Mcgraw-hill; 2014. Reference Source\n\nO'Sullivan EP, Avalos G, O'Reilly M, et al.: Atlantic DIP: the prevalence and consequences of gestational diabetes in Ireland. Ir Med J. 2012; 105(5 Suppl): 13–5. PubMed Abstract\n\nJain R, Davey S, Davey A, et al.: Can the management of blood sugar levels in gestational diabetes mellitus cases be an indicator of maternal and fetal outcomes? The results of a prospective cohort study from India. J Family Community Med. 2016; 23(2): 94–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmerican Diabetes Association: Standards of medical care in diabetes--2014. Diabetes care. 2014; 37(Suppl 1): S14–S80. PubMed Abstract | Publisher Full Text\n\nSharifi N, Dolatian M, Mahmoodi Z, et al.: Gestational diabetes and its relationship with social determinants of health according to world health organization model: Systematic review. 2017. Reference Source\n\nMahmoodi Z, Karimlou M, Sajjadi H, et al.: A Communicative Model of Mothers’ Lifestyles During Pregnancy with Low Birth Weight Based on Social Determinants of Health: A Path Analysis. Oman Med J. 2017; 32(4): 306–314. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang C: Risk factors for gestational diabetes: from an epidemiological standpoint. Gestational diabetes during and after pregnancy. Springer; 2010. 71–81. Publisher Full Text\n\nSedighi S, Amir Ali Akbari S, Afrakhteh M, et al.: Comparison of lifestyle in women with polycystic ovary syndrome and healthy women. Glob J Health Sci. 2015; 7(1): 228–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLali M, Abedi A, Kajbaf MB: Construction and validation of the lifestyle questionnaire (LSQ). 2012. Reference Source\n\nGerstein HC: Do lifestyle changes reduce serious outcomes in diabetes? N Engl J Med. 2013; 369(2): 189–90. PubMed Abstract | Publisher Full Text\n\nJavadzade SH, Sharifirad G, Radjati F, et al.: Relationship between health literacy, health status, and healthy behaviors among older adults in Isfahan, Iran. J Educ Health Promot. 2012; 1(1): 31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvon Wagner C, Knight K, Steptoe A, et al.: Functional health literacy and health-promoting behaviour in a national sample of British adults. J Epidemiol Community Health. 2007; 61(12): 1086–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdams RJ, Piantadosi C, Ettridge K, et al.: Functional health literacy mediates the relationship between socio-economic status, perceptions and lifestyle behaviors related to cancer risk in an Australian population. Patient Educ Couns. 2013; 91(2): 206–12. PubMed Abstract | Publisher Full Text\n\nEyüboğlu E, Schulz PJ: Do health literacy and patient empowerment affect self-care behaviour? A survey study among Turkish patients with diabetes. BMJ Open. 2016; 6(3): e010186. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSouza JG, Apolinario D, Magaldi RM, et al.: Functional health literacy and glycaemic control in older adults with type 2 diabetes: a cross-sectional study. BMJ Open. 2014; 4(2): e004180. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBerkman ND, Davis TC, McCormack L: Health literacy: what is it? J Health Commun. 2010; 15(Suppl 2): 9–19. PubMed Abstract | Publisher Full Text\n\nSharma R, Biedenharn KR, Fedor JM, et al.: Lifestyle factors and reproductive health: taking control of your fertility. Reprod Biol Endocrinol. 2013; 11(1): 66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTirkesh F, Bahrami N, Bahrami S: Assessment of Achievement to Improving Maternal Health from Third Millennium Development Goal in Dezful University of Medical Sciences. Community Health. 2016; 2(2): 98–105. Reference Source\n\nAgarwal MM: Gestational diabetes mellitus: An update on the current international diagnostic criteria. World J Diabetes. 2015; 6(6): 782–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaghdoost AA, Rakhshani F, Aarabi M, et al.: Iranian Health Literacy Questionnaire (IHLQ): An Instrument for Measuring Health Literacy in Iran. Iran Red Crescent Med J. 2015; 17(6): e25831. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang C, Tobias DK, Chavarro JE, et al.: Adherence to healthy lifestyle and risk of gestational diabetes mellitus: prospective cohort study. BMJ. 2014; 349: g5450. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuoto R, Kinnunen TI, Aittasalo M, et al.: Primary prevention of gestational diabetes mellitus and large-for-gestational-age newborns by lifestyle counseling: a cluster-randomized controlled trial. PLoS Med. 2011; 8(5): e1001036. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhadivzadeh T, Azhari S, Esmaily H, et al.: Effects of self-care education on perceived stress in women with gestational diabetes under insulin treatment. Evidence Based Care. 2015; 5(3): 7–18. Publisher Full Text\n\nBabaei-Sis M, Ranjbaran S, Mahmoodi H, et al.: The Effect of Educational Intervention of Life Style Modification on Blood Pressure Control in Patients with Hypertension. J Educ Community Health. 2016; 3(1): 12–9. Publisher Full Text\n\nKorpi-Hyövälti E, Heinonen S, Schwab U, et al.: Effect of intensive counselling on physical activity in pregnant women at high risk for gestational diabetes mellitus. A clinical study in primary care. Prim Care Diabetes. 2012; 6(4): 261–8. Publisher Full Text\n\nKorpi-Hyövälti E, Schwab U, Laaksonen DE, et al.: Effect of intensive counselling on the quality of dietary fats in pregnant women at high risk of gestational diabetes mellitus. Br J Nutr. 2012; 108(5): 910–7. PubMed Abstract | Publisher Full Text\n\nTol A, Pourreza A, Rahimi Foroshani A, et al.: Assessing the effect of educational program based on small group on promoting knowledge and health literacy among women with type2 diabetes referring to selected hospitals affiliated to Tehran University of Medical Sciences. Razi Journal of Medical Sciences. 2013; 19(104): 10–9. Reference Source\n\nKandula NR, Nsiah-Kumi PA, Makoul G, et al.: The relationship between health literacy and knowledge improvement after a multimedia type 2 diabetes education program. Patient Educ Couns. 2009; 75(3): 321–7. PubMed Abstract | Publisher Full Text\n\nEtherington K: Rehabilitation counselling in physical and mental health. Jessica Kingsley Publishers; 2002. Reference Source\n\nGharachourlo M, Mahmoodi Z, Akbari Kamrani M, et al.: Dataset 1 in: The effect of a health literacy approach to counselling on the lifestyle of women with gestational diabetes: A clinical trial. F1000Research. 2018. Data Source"
}
|
[
{
"id": "31588",
"date": "14 Mar 2018",
"name": "Shirin Djalalinia",
"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\nGharachourlo et al performed an interesting clinical trial about the effect of a health literacy approach to counselling on the lifestyle of women with gestational diabetes, enrolled 42 eligible 18–35-yearold Iranian women with gestational diabetes in each groups. They concluded that providing counselling services by midwives can significantly promote the lifestyle patterns and increase their health literacy, especially in high-risk pregnancies.\nThe field of gestational diabetes consider as one of the most important priorities of maternal health and designing and development of the manuscript are interesting.\nI think the paper may therefore be suitable for publication after some minor changes that I suggested below:\nPlease rename 'subjects' to 'participant'. In the Introduction section; please provide the gap of related evidence and briefly point to justification of conduction of present study in these regards. In the Methods section; refer to your approaches in data analyzing even used soft wares. The strengths aspects of investigation could be discussed in more details. Further subjects could be providing for more complementary researches in related domains.\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": []
},
{
"id": "31590",
"date": "15 Mar 2018",
"name": "Mojgan Javadnoori",
"expertise": [
"Reviewer Expertise Sexual and reproductive health",
"midwifery",
"adolescent health",
"maternal and child health",
"women health"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear authors, Your manuscript addresses an important aspect of caring for high risk pregnancies, however some suggestions may be considered:\n\nIntroduction should clarify the gap in literature about interventions on lifestyle and their effects on pregnancy outcome. In the other words, what is gap in knowledge about this topic that your study aimed to answer it?\n\nPlease give explanations about this counselling approach (health literacy approach). Is your intervention a counselling or an education?\n\nWhat does the “parallel design” mean? Please explain it.\n\nThe value of P for comparing before and after of each group is not mentioned in the tables.\n\nIt is important that, \"no significant differences were observed between the two groups in terms of demographic variables\", or life style, or health literacy, “Therefore, the two groups were matched before the intervention in terms of lifestyle” …… IS NOT “matching process”. Matching process is used in case-control design. “Random allocation” is utilized to control such confounding variables in randomized control trials. So it should be correct throughout the results and discussion; sentences such as: “The strength of the present study was the fact that the intervention and control groups matched in terms of …”\n\nThe first sentence in conclusion part is not highlighted in results because the p value In tables, is for comparing two groups no before and after in one group.\n\nThe first paragraph of discussion can be deleted.\n\nThe title of tables is very long and contains unnecessary explanations.\n\nIt is suggested to change the word “subjects” to “participants”.\n\nThe last sentence of the background in abstract should be remove to methods.\n\nThe content of counselling approach for intervention group, vs routine care and package for control group is not clear. “The control group received counselling on routine pregnancy care as per the safe national maternal protocol of Ministry of Health and Medical Education of Iran”. Is this protocol the same that was used as counselling for routine pregnancy care? Please describe it more clearly.\n\nWhat is the novelty of your study findings?\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? Partly",
"responses": []
},
{
"id": "31591",
"date": "03 Apr 2018",
"name": "Fariba Ebtekar",
"expertise": [
"Reviewer Expertise Midwifery",
"community health"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe problem statements agreed with the title and seemed to be significance. Several appropriate references were used in the introduction section, but the literature was not clearly visible to the reader, and it required several conflicting findings about the effect of Health literacy on life style.\n\nThe aim of this study is clearly stated, however, it is better to remove \"high risk pregnancy \"in the last sentences of the introduction section.\n\nIn methodology section, the instruments were explained, and their reliability and validity were stated. The population used was adequate. The counselling sessions were explained clearly. It is better to clarify the number of participants in intervention and control groups from each hospital. The statistical techniques were stated but no discussion was given in this particular section.\n\nIn the results section, the findings were presented by appropriate figure and tables according to aim of the study.\n\nIn the discussion section, the findings of other studies supported the results of the present study but the conflicting findings were not stated.\n\nThe conclusion section was stated clearly that included summary of the main findings and recommendation but the recommendation for further researches was not given.\n\nIn the Socio Demographic check list it was better “weight gain” written instead of increase weight.\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? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-282
|
https://f1000research.com/articles/7-281/v1
|
06 Mar 18
|
{
"type": "Software Tool Article",
"title": "Seqfam: A python package for analysis of Next Generation Sequencing DNA data in families",
"authors": [
"Matthew Frampton",
"Elena R. Schiff",
"Nikolas Pontikos",
"Anthony W. Segal",
"Adam P. Levine",
"Elena R. Schiff",
"Nikolas Pontikos",
"Anthony W. Segal",
"Adam P. Levine"
],
"abstract": "This article introduces seqfam, a python package which is primarily designed for analysing next generation sequencing (NGS) DNA data from families with known pedigree information in order to identify rare variants that are potentially causal of a disease/trait of interest. It uses the popular and versatile Pandas library, and can be straightforwardly integrated into existing analysis code/pipelines. Seqfam can be used to verify pedigree information, to perform Monte Carlo gene dropping, to undertake regression-based gene burden testing, and to identify variants which segregate by affection status in families via user-defined pattern of occurrence rules. Additionally, it can generate scripts for running analyses in a “MapReduce pattern” on a computer cluster, something which is usually desirable in NGS data analysis and indeed “big data” analysis in general. This article summarises how seqfam’s main user functions work and motivates their use. It also provides explanatory context for example scripts and data included in the package which demonstrate use cases. With respect to verifying pedigree information, software exists for efficiently calculating kinship coefficients, so seqfam performs the necessary extra steps of mapping pedigrees and kinship coefficients to expected and observed degrees of relationship respectively. Gene dropping and the application of variant pattern of occurrence rules in families can provide evidence for a variant being causal. The authors are unaware of other software which performs these tasks in familial cohorts, so seqfam fulfils this need. Gene burden rather than single marker tests are often used to detect rare causal variants due to greater power. Seqfam may be an attractive alternative to existing gene burden testing software due to its flexibility, particularly in grouping and aggregating variants.",
"keywords": [
"python",
"bioinformatics",
"NGS",
"DNA",
"pedigree-information",
"gene-drop",
"gene-burden",
"kinship",
"mapreduce"
],
"content": "Introduction\n\nSeqfam is a python package which is most useful in analysing next generation sequencing (NGS) DNA data from families with known pedigree information in order to identify rare variants that are potentially causal of a disease/trait. It was originally developed to analyse the whole exome sequencing data of a cohort of 200 families affected by a particular complex disease. Family-based study designs are often used to identify rare causal variants because such variants may be substantially more frequent in affected families than the general population (Auer & Lettre, 2015). Seqfam contains modules for Monte Carlo gene dropping (gene_drop.py) (MacCluer et al., 1986), flagging variants based on their pattern of occurrence within families (pof.py), verification of ascertained pedigrees via kinship coefficients (relatedness.py), regression-based gene burden testing (gene_burden.py), and an additional module that facilitates the creation of job submission scripts on a computer cluster (sge.py).\n\nFor a rare variant to be considered potentially causal of a particular trait/disease based on in silico analysis, it must satisfy various criteria, such as being biologically plausible and predicted to be pathogenic. The user can run analyses with the gene_drop.py and pof.py modules to acquire additional evidence. Given the structure of the families, Monte Carlo gene dropping can assess whether a variant is enriched in the cohort relative to the general population, and assuming the trait/disease is more prevalent in the cohort, such enrichment supports causality. The user can use the pof.py module to identify variants which are carried by most or all affected members of a family, or even which segregate between affected and unaffected members. The authors are unaware of existing software packages for performing these analyses in familial cohorts, so gene_drop.py and pof.py fulfil this need. The gene_drop.py module can be considered complementary to the RVsharing R package (Bureau et al., 2014), which calculates the probability of multiple affected relatives sharing a rare variant under the assumption of no disease association or linkage.\n\nThe gene_burden.py module implements the Combined Multivariate and Collapsing (CMC) burden test (Li & Leal, 2008) for detecting rare causal variants, where the multivariate test is a log-likelihood ratio test. The user can supply covariates to control for potential confounders such as divergent ancestry. This burden test should be applied to unrelated samples, and hence is of no use for cohorts containing few families. However, for cohorts containing a relatively large number of families, a sufficient number of unrelated cases can be extracted and combined with a separate set of unrelated controls. Burden tests aggregate rare variants in a gene or functional unit into a single score (Li & Leal, 2008; Madsen & Browning, 2009; Morris & Zeggini, 2010; Price et al., 2010), and are one broad class of statistical methods which combine the effects of rare variants in order to increase power over single marker approaches. Sequence kernel association testing (SKAT) (Wu et al., 2011) is another widely-used sub-category of such methods. In general, burden testing is more powerful than SKAT when a large proportion of variants are causal and are all deleterious/protective.\n\nStand-alone software exists for performing CMC testing with covariates e.g. RVTESTS (Zhan et al., 2016) and PLINK/SEQ, but gene_burden.py is an attractive alternative due to its use of the versatile Pandas library. The CMC test function takes malleable Pandas data frames as input/output, which gives the user great flexibility in pre/post-processing the data, and this in turn may reduce I/O overhead. For example, let us consider variant grouping (e.g. by gene) and aggregation. This is specified via columns in the data frame, so the user can experiment widely by modifying them in python code e.g. group variants by functional unit instead of gene, aggregate within alternative sets of population allele frequency ranges, include/exclude unaggregated variants, and even aggregate by another factor such as consequence. As well as this flexibility, the function also produces rich output, including information about the independent variables.\n\nThe potential for genetic discovery in DNA sequencing data is reduced when samples are mislabelled. Hence, necessary quality control steps include identifying duplicates, and in the case of familial samples, verifying the ascertained familial relationships described in the pedigrees. The relatedness.py module facilitates these quality control steps and is used in conjunction with KING software (Manichaikul et al., 2010). Given genotypes for relatively common variants, KING can efficiently calculate a kinship coefficient for each sample pair. The relatedness.py module can then map each kinship coefficient to a degree of relationship and check it corresponds with the pedigree. KING is often already part of NGS analysis pipelines, so incorporating relatedness.py is straightforward. Peddy (Pedersen & Quinlan, 2017) is an alternative which does not require KING.\n\nThe final module, sge.py, has general utility in running analyses of NGS data (and indeed any “big data”) on computer clusters. Many NGS data analyses can be cast as “embarrassingly parallel problems” and hence executed more efficiently on a computer cluster using a “MapReduce pattern”: the overall task is decomposed into independent sub-tasks (“map” tasks), then the map tasks run in parallel and after their completion, a “reduce” action merges/filters/summarises the results. For example, gene burden testing across the whole exome can be decomposed into independent sub-tasks by splitting the exome into sub-units e.g. chromosomes. Sun Grid Engine (SGE) is a widely used batch-queueing system, and analyses can be performed in a MapReduce pattern on SGE via so-called array jobs. The sge.py module can be used to automatically create the scripts required for submitting and running an array job.\n\n\nMethods\n\nThis section describes the functionality and methods employed by seqfam’s 5 modules, which are:\n\n1. gene_drop.py: Monte Carlo gene dropping;\n\n2. pof.py: variant pattern of occurrence in families;\n\n3. gene_burden.py: regression-based gene burden testing;\n\n4. relatedness.py: identification of duplicates and verification of ascertained pedigree information via kinship coefficients;\n\n5. sge.py: Sun Grid Engine (SGE) array job creation.\n\nFigure 1 provides a visual representation of modules 1–4.\n\nPanel A represents the Cohort.gene_drop method in the gene_drop.py module which performs Monte Carlo gene dropping. On a single iteration, for each family the algorithm seeds founder genotypes based on the variant population allele frequency and then gene drops via depth-first traversals. Having done this for all families, a simulated cohort allele frequency (AF) is calculated and following many iterations (e.g. 10,000), a p-value, the proportion of iterations where cohort AF < simulated cohort AF, is outputted. Panel B represents the Pof.get_family_pass_name_l method in the pof.py module. Prior to calling the method, each family is assigned a variant pattern of occurrence in family (POF) rule. The method then takes a variant’s genotypes and returns families whose POF rule is passed. Panel C represents the CMC.do_multivariate_tests method in the gene_burden.py module. This method takes sample affection status and variant genotypes across multiple genes, plus optionally covariates such as ancestry PCA coordinates. For each gene, the method aggregates the variants by allele frequency, constructs null and alternative hypothesis logit models which may include the covariates, and then performs a log-likelihood ratio test. Panel D represents the Relatedness.get_exp_obs_df method in the relatedness.py module. For input, this takes pedigree information and kinship coefficients from KING for each within-family sample pair. It maps these data to expected and observed degrees of relationship respectively, returning a data frame.\n\nGene dropping. By default, for each variant of interest, the gene_drop.py module performs 10,000 iterations of gene dropping in the familial cohort. In each iteration it gene drops in each family once, seeding the founder genotypes based on the population allele frequency. It then calculates the resulting simulated cohort allele frequency from samples specified by the user i.e. those which were sequenced. After completing all iterations of gene dropping, gene_drop.py outputs the proportion of iterations in which the true cohort allele frequency is less than or equal to the simulated cohort allele frequency: a low proportion, e.g. < 5%, is evidence of enrichment.\n\nThe module gene drops in a family in the following way. First, it assigns a genotype (number of copies of the mutant allele) to each founder using a Binomial distribution where the number of trials is 2 and the probability of success in each trial is the population allele frequency. Hence the founders are assumed to be unrelated. It then performs a depth-first traversal starting from each founder (1 per spousal pair), and for heterozygotes, uses a random number generator to determine which parental allele to pass onto the child. Thus, every individual in the family is assigned a genotype.\n\nVariant pattern of occurrence in families. For each family, the user can use the pof.py module to define a variant pattern of occurrence rule and check whether any supplied variants pass. The rule can specify a minimum value for the proportion of affected members (As) who are carriers (A.carrier.p), and/or a minimum difference between the proportion of As and unaffected members (Ns) who are carriers (AN.carrier.diff). Constraints for the number of genotyped As and Ns can also be added.\n\nAs an illustrative example, consider a cohort in which families can be categorised as follows based on their number of As and Ns:\n\n1. “A4N1”: ≥ 4 As and ≤ 1 N\n\n2. “A3N2”: ≥ 3 As and ≥ 2 Ns\n\nFor the A4N1 families, the user may be interested in variants carried by all As and so require A.carrier.p = 1, while for the A3N2 families, they may be interested in variants which are more prevalent in As than Ns and so require AN.carrier.diff ≥ 0.5.\n\nGene burden. To use the gene_burden.py module, the user must first read the various required data into Pandas data frames. These data include variant annotations by which to group (e.g. gene/functional unit) and aggregate (e.g. population allele frequency), and the genotypes, affection status and covariates for the unrelated samples. The user can specify multiple categories in which to aggregate variants (e.g. into population allele frequency ranges of 0–1% and 1–5%), and variants outside these categories (e.g. more common variants) remain unaggregated. An aggregated variant category takes the value 0 or 1. For each variant group, having aggregated the variants, gene_burden.py will perform a multivariate test, which is a log-likelihood ratio test based on Wilk’s theorem:\n\nΧ2=2(llh0 − llh1); df = dfh1 − dfh0\n\nwhere ll is log-likelihood, h1 is the alternative hypothesis, h0 is the null hypothesis and df is degrees of freedom. Specifically, it is a log-likelihood ratio test on null and alternative hypothesis logit models where the dependent variable is derived from affection status, the variant variables (aggregated and/or unaggregated) are independent variables in the alternative model and the covariates are independent variables in both. The logit models are fitted using the Broyden–Fletcher–Goldfarb–Shanno (BFGS) algorithm.\n\nSince aggregation by population allele frequency is common, the module provides a function to assign variants to allele frequency ranges based on multiple columns (derived from different population databases such as gnomAD (Lek et al., 2016)). The user specifies a preference order in case the variant is absent from the most preferred database(s).\n\nRelatedness. As input, the relatedness.py module requires pedigree information and a file containing kinship coefficients outputted by KING. For each sample pair, the module will map the pedigree information and kinship coefficient to an expected and observed degree of relationship respectively. The mapping from kinship coefficient to relationship is as specified in KING documentation: > 0.354 for duplicate samples/monozygotic twins, 0.177–0.354 for 1st degree relatives, 0.0884–0.177 for 2nd degree relatives, 0.0442–0.0884 for 3rd degree relatives, and < 0.0442 for unrelated. The user can change this mapping if they wish.\n\nComputer cluster array job creation. To use the sge.py module, the user must first create lists of map tasks, map tasks requiring execution and reduce tasks. The map tasks requiring execution are map tasks which have not previously completed successfully and hence need to run. Given these lists, the sge.py module can create all necessary scripts/files for submitting and running an array job. This includes scripts for all map tasks, a text file specifying that only the map tasks requiring execution should run, and a master executable submit script for submitting the array job to the job scheduler.\n\nSeqfam is compatible with Windows, Mac OS X and Linux operating systems. It is coded using Python 3.6, but can also be run by Python 2.7. It requires various data analysis libraries/packages, almost all of which can be acquired by downloading and installing the Anaconda python distribution. The StatsModels module, which is not in the Anaconda distribution, is also required. Having cloned the repository, the user should add the repository src directory to their environmental python path.\n\n\nUse cases\n\nThe repository contains additional scripts in src/examples which demonstrate the functionality of the modules on example data, including files in the data directory. The scripts are 1_test_gene_drop.py, 2_test_pof.py, 3_test_gene_burden.py, 4_test_relatedness.py, and 5_test_sge.py. The reader can also refer to Table 1 for a summary of the main user functions of the 5 seqfam modules, which includes their input/output. Data in the example data files are derived from the whole exome sequencing of a large cohort of over 200 families with inflammatory bowel disease (unpublished study1).\n\ntsv = tab-separated values; AF = allele frequency; POF = pattern of occurrence in family.\n\nThe only input file for 1_test_gene_drop.py is cohort.tsv, which contains the pedigree information for an example familial cohort. This cohort has 3,608 samples from 251 families, and the complexity of the families, calculated as 2n-f where n and f are the number of non-founders and founders respectively (Abecasis et al., 2002), has median 9 and range 0–103. The cohort.csv file is in fam file format (Purcell et al., 2007), meaning it has 1 row per individual and 6 columns for family ID, person ID, father, mother, sex and affection.\n\nThe 1_test_gene_drop.py script first creates a Cohort object from cohort.tsv, which stores each family tree, then calls the gene_drop method with the following arguments: pop_af and cohort_af are the allele frequency of a particular variant in the general population and cohort respectively, sample_genotyped_l is the list of cohort samples with a genotype, and gene_drop_n is the number of iterations of gene dropping to perform. Hence the samples in sample_genotyped_l are used by the user to calculate cohort_af, and by the method to calculate the simulated cohort allele frequencies. The method returns a p-value. The script calls the gene_drop method with ascending values for cohort_af, and so descending p-values are returned.\n\nIn benchmarking with the Unix time command on an Intel Xeon CPU E7-4830 v2 processor (20M Cache, 2.20 GHz), creating a cohort object and then executing a single call of the gene_drop method (gene_drop_n=1,000) required approximately 25 seconds of Computer Processing Unit (CPU) time. CPU time scales linearly with gene_drop_n and cohort size.\n\nThere are no input files for 2_test_pof.py. The example script first creates a Pof object which stores a couple of Family objects, each representing an example family and its variant pattern of occurrence rule. Next it calls the Pof object’s get_family_pass_name_l method with the argument genotypes_s, which is a Pandas Series containing sample genotypes for a particular variant. The method returns a list of families whose pattern of occurrence rule is passed by this variant.\n\nThe 3_test_gene_burden.py script uses the gene_burden.py module to perform CMC tests on example data: it performs 1 CMC test per gene where variants in the population allele frequency ranges of 0-1% and 1-5% are aggregated, and any variants ≥ 5% remain unaggregated. The input files are in the data/gene_burden directory: samples.csv, genotypes.csv and optionally covariates.csv. The samples.csv file contains the samples’ ID and affection status where 2 indicates a case and 1 a control, and genotypes.csv contains 1 row per variant with columns for the sample genotypes, and for variant grouping and aggregation e.g. gene and population allele frequency. A sample’s genotype is the number of alternate alleles which it carries (0-2). The covariates.csv file contains the covariates to control for, which in this case are ancestry PCA coordinates.\n\nThe script first reads samples.csv into a Pandas Series, and genotypes.csv and covariates.csv into Pandas DataFrames. These data frames are indexed by variant ID and covariate name respectively. Having created a CMC object, the script calls its assign_vars_to_pop_frq_cats method in order to map the variants to the desired population allele frequency ranges. Multiple population allele frequency columns (databases) are used here, ordered by descending preference. The mapping is stored in a new column in the genotypes data frame. Finally, the script calls the do_multivariate_tests method to perform the CMC tests, specifying the gene column for grouping the variants, and the new allele frequency range column for aggregation. The results are written to a CSV (comma-separated values) file and returned in a data frame. They include the number of variants in each aggregation category, the number of unaggregated variants (“unagg” column), the log-likelihood ratio test p-value with/without covariates (“llr_p” and “llr_cov_p”), and the coefficient/p-value for each aggregated variant variable (“_c” and “_p”).\n\nThe input files for 4_test_relatedness.py are cohort.tsv (as used in gene dropping), and data/relatedness/king.kinship.ibs which was outputted by KING and contains kinship coefficients for within-family sample pairs. The example script first creates a Relatedness object which stores the paths to these files, then calls the object’s find_duplicates and get_exp_obs_df methods. The former returns any within-family sample duplicates, and the latter returns a Pandas DataFrame containing the expected and observed degree of relationship for each within-family sample pair. Finally, the script prints the sample pairs which have a different expected and observed degree of relationship.\n\nThere are no input files for 5_test_sge.py. This script first makes lists of map tasks (map_task_l), map tasks to execute (map_task_exec_l), and reduce tasks (reduce_task_l). Here map_tasks_exec_l contains every other map task. Next, the script creates an SGE object which stores the directory where job scripts will be written (here data/sge). Finally, it calls the object’s make_map_reduce_jobs method with the following arguments: a prefix for all job script names (here “test”) and the above 3 lists. This writes the job scripts, and were they for a real array job (they are not), the user could then submit it to the job scheduler by running the master executable submit script data/sge/submit_map_reduce.sh. The test.map_task_exec.txt file specifies which map tasks to run i.e. the map tasks in the map_tasks_exec_l list.\n\n\nConclusions\n\nThis article has introduced seqfam, a python package, primarily designed for analysing NGS DNA data from families with known pedigree information in order to identify rare variants that are potentially causal of a disease/trait of interest. It currently includes modules for verification of pedigree information, gene dropping, applying variant pattern of occurrence rules in families, gene burden testing and job script generation on a computer cluster.\n\n\nData and software availability\n\nLatest source code and example files are available at: https://github.com/mframpton/seqfam\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.1173768 (Frampton, 2018)\n\nLicense: GNU General Public License v3.0\n\n\nFootnotes\n\n1Authors involved in the unpublished study: E. R. Schiffa, M. Framptona, F. Semplicia, N. Pontikosb, S. Bloomc, S. McCartneyc, R. Vegac, L. Lovatd, E. Woode, A. Hartf, D. Crespig, M. Furmang, S. Mannh, C. Murrayi, A. P. Levinea and A. W. Segala\n\naCentre for Molecular Medicine, Division of Medicine, University College London\n\nbInstitute of Ophthalmology, Moorfields Eye Hospital, University College London\n\ncDepartment of Gastroenterology. University College London Hospital\n\ndResearch Department of Tissue and Energy, Division of Surgery and Interventional Science, UCL, U.K.\n\neGastroenterology Department, Homerton University Hospital, London, U.K.\n\nfGastroenterology Department, St Marks Hospital, U.K.\n\ngCentre for Paediatric Gastroenterology, Royal Free Hospital, London, U.K.\n\nhGastroenterology Department, Barnet General Hospital, U.K.\n\niCentre for Gastroenterology, Royal Free Hospital, London, U.K.",
"appendix": "Competing interests\n\n\n\nThe authors declare no competing interests.\n\n\nGrant information\n\nThis work was funded by the Charles Wolfson Charitable Trust.\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 all study participants whose sequencing data and pedigree information were used in developing and testing seqfam, plus their referring clinicians.\n\n\nReferences\n\nAbecasis GR, Cherny SS, Cookson WO, et al.: Merlin--rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet. 2002; 30(1): 97–101. PubMed Abstract | Publisher Full Text\n\nAuer PL, Lettre G: Rare variant association studies: Considerations, challenges and opportunities. Genome Med. 2015; 7(1): 16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBureau A, Younkin SG, Parker MM, et al.: Inferring rare disease risk variants based on exact probabilities of sharing by multiple affected relatives. Bioinformatics. 2014; 30(15): 2189–2196. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrampton M: seqfam. Zenodo. 2018. Data Source\n\nLek M, Karczewski KJ, Minikel EV, et al.: Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016; 536(7616): 285–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi B, Leal SM: Methods for detecting associations with rare variants for common diseases: application to analysis of sequence data. Am J Hum Genet. 2008; 83(3): 311–321. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMacCluer JW, VandeBerg JL, Read B, et al.: Pedigree analysis by computer simulation. Zoo Biol. 1986; 5(2): 147–160. Publisher Full Text\n\nMadsen BE, Browning SR: A groupwise association test for rare mutations using a weighted sum statistic. PLoS Genet. 2009; 5(2). PubMed Abstract | Publisher Full Text | Free Full Text\n\nManichaikul A, Mychaleckyj JC, Rich SS, et al.: Robust relationship inference in genome-wide association studies. Bioinformatics. 2010; 26(22): 2867–2873. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorris AP, Zeggini E: An evaluation of statistical approaches to rare variant analysis in genetic association studies. Genet Epidemiol. 2010; 34(2): 188–193. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPedersen BS, Quinlan AR: Who’s Who? Detecting and Resolving Sample Anomalies in Human DNA Sequencing Studies with Peddy. Am J Hum Genet. 2017; 100(3): 406–413. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrice AL, Kryukov GV, de Bakker PI, et al.: Pooled Association Tests for Rare Variants in Exon-Resequencing Studies. Am J Hum Genet. 2010; 86(6): 832–838. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPurcell S, Neale B, Todd-Brown K, et al.: PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses. Am J Hum Genet. 2007; 81(3): 559–575. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu MC, Lee S, Cai T, et al.: Rare-variant association testing for sequencing data with the sequence kernel association test. Am J Hum Genet. 2011; 89(1): 82–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhan X, Hu Y, Li B, et al.: RVTESTS: An efficient and comprehensive tool for rare variant association analysis using sequence data. Bioinformatics. 2016; 32(9): 1423–1426. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "31578",
"date": "09 Mar 2018",
"name": "Brent S. Pedersen",
"expertise": [
"Reviewer Expertise Genomics",
"bioinformatics",
"algorithms"
],
"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\nFrampton et al describe seqfam, a set of tools to perform family-based analysis on sequence data. It does gene-dropping, burden-testing, relatedness evaluation, \"pattern of occurrence\", and contains a script for running cluster analysis.\nMy main critique is that this seems like a set of unrelated modules, some of which have (potentially) better alternatives. For example, the SKAT family of burden tests have been widely used in place of CMC implemented in this package and several implementations of CMC exist. Therefore, it's not clear from reading why this package is needed. In addition, as the authors note, this is only useful if there are sufficient unrelated controls. Another example is the relatedness module which relies on KING output. The additional utility provided in seqfam is a subset of the utility provided in peddy which does not rely on plink or KING. Again, it's not clear when the use of seqfam in this context would be preferable.\nThe parts that are more novel are the gene dropping and the \"POF\" scripts. Running the script currently requires setting the PYTHONPATH. The package should have a requirements.txt and a proper setup.py so it can be used in a more standard manner. I was able to run the example script for gene dropping and see sensible outputs. Other than this, there are no tests in the repository and nothing in the manuscript indicating any of the methods were evaluated for correctness or accuracy performance.\n\nThere is also very little documentation. For example,the gene_burden_test has:\n\ncmc_result_df = cmc.do_multivariate_tests(sample_s, geno_df, group_col=gene_col, agg_col=\"pop_frq_cat\", agg_val_l=[\"rare\",\"mod_rare\"], covar_df=covar_df, results_path=results_path)\n\nA user must read the code to see that agg_val_l is:\n\n`(list of strs): names of the aggregated categories.` but it's still not clear what that means or how to use it. For each module intended for use, there should be clear text and API documentation. Figure 1 and some of the manuscript would be useful as documentation as well.\nFigure 1B could be made clearer by adding a rule and using PASS/FAIL for how/if families meet that rule.\nThis is certainly just a matter of preference, but I found the manuscript hard to digest due to the layout. To get all the information on a particular tool, I have to keep 3 sections of the paper in my head, the intro, the methods, and then the use-cases. This would have been more readable to me with a short intro describing the use-case and more per-tool description in 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? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "31577",
"date": "22 Mar 2018",
"name": "Alexandre Bureau",
"expertise": [
"Reviewer Expertise Statistical genetics",
"genetic 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\nThe python package seqfam is a collection of genetic association analysis tools, some but not all tailored for familial data. The instructions provided by the authors allow for a successful installation of the seqfam package. Example scripts provided with the package perform the analyses described in the manuscript.\n\nThis manuscript, however, also raises concerns with these reviewers. Tools performing similar tasks already exist in other computing languages (PLINK/R) and are widely used. The gene dropping and filtering based on pattern of occurrence in families implemented in seqfam differ in some respects from approaches implemented in other packages, which have been thoroughly assessed for their statistical properties. As this is a software manuscript, an assessment of the statistical properties of the seqfam approaches is not required, but we note that it will be very hard to convince a practitioner to use this approach if no consideration of power for various settings is given. Moreover, we are also of the opinion that some of the seqfam tools should not be used as currently proposed.\n\nMajor concerns:\n\nReferring to gene dropping and the application of variant pattern of occurrence rules in families, the authors state in the abstract that they are \"unaware of other software which performs these tasks in familial cohorts\", but unfortunately missed a somewhat large body of literature. A reasonably thorough review of the literature is imperative for any manuscript.\nMost applicable in our opinion for testing rare variants are RareIBD (Sul et al. 20161) which uses the number of affected relatives carrying a rare allele similar to the allele frequency used in seqfam, and GESE (Qiao et al. 20172), based on the probability of multiple affected relatives sharing a rare variant, which we proposed in a recent manuscript that the authors do cite (Bureau et al. 20143). Of note, we have since expanded our approach to gene-based analyses, refined the rare variant definition based on haplotypes, and introduced a partial sharing test based on rare variant sharing probabilities for subsets of affected family members (Bureau et al. 20184, posted online after the seqfam manuscript was submitted, so the authors could not be aware of it). Another alternative is testing of co-segregation of variants with disease under a parametric linkage analysis as in the pedigree Variant Annotation, Analysis and Search Tool pVAAST (Hu et al. 20145).\nThere are also other implementations of variant filtering based on proportions of affected and unaffected carriers, e.g. the R package Mendelian of Broeckx et al. (2015)6.\n\nThe inference of the seqfam gene dropping test depends on knowledge of the variant allele frequencies in the study population. Methods that depend on such knowledge tend to be extremely sensitive to deviations from the truth (see for example the above mentioned Qiao et al 2017 and Bureau et al 2018 papers). In most instances we have only a very rough idea about exact allele frequencies (especially for rare variants) when big population-based studies of said population already have been carried out, or we have no knowledge at all if a new population is investigated. Thus, methods that do not depend on knowledge of allele frequencies are to be preferred, but at a minimum the authors must report the impact of allele frequency misspecification (as for example done in Bureau et al 2018), and the size of the control group from the appropriate population which is needed to obtain reliable allele frequencies for the proposed method (as for example done in the Qiao et al paper) to guide the user.\n\nThere is no notion of error control due to multiple comparisons in the manuscript. And with the number of variants present even in whole exome sequencing data, the recommended number of iterations of gene dropping (10,000) most likely will be inadequate to estimate the p-values with sufficient precision. For example, even if none of 10,000 iterations yields anything more extreme than observed in the actual data, the upper bound for the 95% confidence interval for the true p-value will be 0.00037 (for example type binom.test(0,10000)$conf in R). Thus, with a couple hundred variants assessed, the upper bound for the confidence interval would not beat the Bonferroni threshold to declare significance.\n\nThe inference based on so-called \"patterns of occurrence in families” (POFs) does not quantify the total evidence for linkage/association per se (see for example the RareIBD paper by Sul et al) but investigates user-chosen patterns instead. This could be powerful if one knew exactly what patterns to look for a priori, but we cannot imagine this ever being the case in practice, nor is any guidance on this offered in the manuscript. The example script involves specifying the pattern of occurrence rule with example families, but in practice family samples contain various family structures, so it is not clear how it generalizes. Why not simply input values for A.carrier.p and AN.carrier.diff? As it stands, it is left to the user to tinker with the vast number of possibilities. In that sense, inference based on these specific patterns should at best be considered secondary analysis.\n\nNo functionality is provided in seqfam to apply the gene burden test to familial samples. As the authors rightly point out, the test is valid with unrelated subjects. As it stands, users must extract unrelated subjects from familial cohorts outside of seqfam. This function should offer the option to perform selection of unrelated cases from familial cohorts, or otherwise it should be removed from seqfam.\n\nThe authors should also mention that in many family-based sequencing studies genotypes of founders are unavailable (as for example DNA is not available from family members several generations ago), and quite frequently only distantly related subjects are sequenced. That means that for these studies only a single unrelated subject can be extracted from each family, limiting the scope of the gene burden test substantially.\n\nWhen executing the test program, the p-value of the LR test is identical with or without covariates, so adjustment for covariates does not seem to do anything. The expressions “log-likelihood ratio test p-value with/without covariates” for llr_p and llr_cov_p are ambiguous. In the output, the llr_p test is missing when one of the variant classes is empty, while llr_cov_p is always reported. Is llr_cov_p a test of the covariates?\n\nThere are also some more minor concerns and questions:\n\nFor the gene dropping test it is not clear in the manuscript whether the variant frequency is computed only for affected family members or all family members together.\n\nThe gene_drop.py module would be more useful if it took genotype data as input like the gene_burden.py and pof.py modules and computed the cohort allele frequency for every variant. The authors state that the gene_drop.py module performs gene dropping “for each variant of interest”, but this is misleading, as the gene_drop method takes as argument the frequency of a single variant. Users must write a script to loop over variants in their genotype file (pre-filtered by population allele frequency and possibly other annotations), compute variant frequency in their cohort, and pass it to gene_drop. By contrast, competing packages GESE, RareIBD, pVAAST and our new Bioconductor RVS package process genotype data. They also offer the option to perform analyses at the gene level, while the gene_drop.py module is restricted to single variant analyses.\n\nFiltering of variants (e.g. those predicted to be pathogenic) is critical and relies heavily on available annotation. However, this information is largely available only for exomes. Filtering of variants by annotation should be discussed. Is seqfam foremost a package for targeted sequencing studies (i.e. exome sequencing)? If yes, this should be stated. Otherwise, some guidance on application to whole genome sequencing studies should be given.\n\nWhat exactly is the additional value of relatedness.py? For example, the GENESIS Bioconductor package allows for the fast calculation of genetic relatedness matrices (GRMs), which can easily be subsetted using the cut-offs suggested by the authors. It is unclear whether relatedness.py also updates the pedigrees accordingly (which can be done using kinship2 in R). Also, what happens if founders are found to be related? This should be part of the quality control and considered in the inference.\n\nThe relatedness.py module could be generalized by allowing input from other kinship computation packages than King.\n\nThe program for computer cluster job creation can be a useful utility program complementing the seqfam analysis tools, but it has no specific connection to family sequencing studies. It does not deserve to be featured as a main user function in the manuscript, and instead should be mentioned as a utility program.\n\nThere is no mention of dominant and recessive inheritance patterns, which are often encountered with rare causal variants. The gene dropping statistics is likely better suited for dominant variants, while different POF in families could be defined for dominant and recessive variants. This should be discussed.\n\nIs the rationale for developing the new software tool clearly explained? No\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? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No",
"responses": []
}
] | 1
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https://f1000research.com/articles/7-281
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https://f1000research.com/articles/6-1819/v1
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09 Oct 17
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{
"type": "Research Article",
"title": "A comparison of spatial heterogeneity with local cluster detection methods for chronic respiratory diseases in Thailand",
"authors": [
"Wongsa Laohasiriwong",
"Nattapong Puttanapong",
"Amornrat Luenam",
"Nattapong Puttanapong",
"Amornrat Luenam"
],
"abstract": "Background: The Centers for Disease Control and Prevention reported that deaths from chronic respiratory diseases (CRDs) in Thailand increased by almost 13% in 2010, along with an increased burden related to the disease. Evaluating the geographical heterogeneity of CRDs is important for surveillance. Previous studies have indicated that socioeconomic status has an effect on disease, and that this can be measured with variables such as night-time lights (NTLs) and industrial density (ID). However, there is no understanding of how NTLs and ID correlate with CRDs. We compared spatial heterogeneity obtained by using local cluster detection methods for CRDs and by correlating NTLs and ID with CRDs. Methods: We applied the spatial scan statistic in SaTScan, as well as local indices of spatial association (LISA), Getis and Ord’s local Gi*(d) statistic, and Pearson correlation. In our analysis, data were collected on gender, age, household income, education, family size, occupation, region, residential area, housing construction materials, cooking fuels, smoking status and previously diagnosed CRDs by a physician from the National Socioeconomic Survey, which is a cross-sectional study conducted by the National Statistical Office of Thailand in 2010. Results: According to our findings, the spatial scan statistic, LISA, and the local Gi*(d) statistic revealed similar results for areas with the highest clustering of CRDs. However, the hotspots for the spatial scan statistic covered a wider area than LISA and the local Gi*(d) statistic. In addition, there were persistent hotspots in Bangkok and the perimeter provinces. NTLs and ID have a positive correlation with CRDs. Conclusions: This study demonstrates that all the statistical methods used could detect spatial heterogeneity of CRDs. NTLs and ID can serve as new parameters for determining disease hotspots by representing the population and industrial boom that typically contributes to epidemics.",
"keywords": [
"chronic respiratory diseases",
"comparison",
"spatial heterogeneity",
"local cluster detection",
"methods",
"Thailand"
],
"content": "Introduction\n\nChronic respiratory diseases (CRDs) are a public health issue worldwide1. According to the World Health Organization, CRDs are the world’s leading cause of death. In Thailand, the Centers for Disease Control and Prevention reported that deaths from CRDs increased by almost 13% in 2010, and there was an increased burden resulting from these diseases2.\n\nSpatial clustering methods are important for statistical consideration, to develop models for prediction of disease risk positions. Disease risk positions are areas located near one another in statistically significant clusters. In this study, we indicate whether the geographical aggregation of CRDs is explained by chance or is statistically significant. It is likely that geographical areas share similar disease risk factors, since they are in a similar environment. Local spatial clustering methods are useful to identify the characteristics of clusters in terms of location, size, and prevalence of the disease4. These methods differ from standard methods3,4 used to identify the presence of spatial clustering in a whole study area. Local clustering methods in spatial epidemiology that are commonly applied to understanding spatial clustering (heterogeneity) are the Getis and Ord’s local Gi*(d) statistic, spatial scan statistics, and local indices of spatial association (LISA)5–8.\n\nAt present, there are no studies comparing the performance of these methods in epidemiological studies involving CRDs. In this study, we used local clustering methods to detect the spatial heterogeneity of CRDs and compared the performance of these local spatial clustering methods, including the local Gi*(d) statistic, the spatial scan statistic, and LISA. Spatial scan statistics performed in SaTScan circular version (version 9.4.2) were considered the best choice for detecting small, compact clusters9. A local version of Moran’s I was one of our selective cluster detection methods, due to its great performance on outlier detection, despite not being a good method for detecting large clusters3,10. The local Gi*(d) statistic was used to indicate locations surrounded by a cluster of high or low values. It shows areas where lower than average values tend to be found near each other, or areas where higher than average values tend to be found near each other, including the value at the location in which the spatial autocorrelation is being measured.\n\nIn a socioeconomic context, previous literature has recommended that night-time lights (NTLs) can substitute for other variables, such as urbanization, density, and economic growth. Data on NTLs have been used to indicate the actual scope of urban incorporations and appraise population scale in urban areas11–15 and population density16. These data have been employed to monitor the speed of urbanization17,18, as well as electricity used19. Moreover, NTLs have been utilized as a substitute for Gross Domestic Product (GDP), which works as an indirect indicator of administrative areas11,20–24. Remotely sensed NTL data can also be applied in epidemiological studies25,26. A 2011 study entitled “Explaining seasonal fluctuations of measles in Niger using night-time lights imagery” in Niger, by Bharti et al., found the concentration of the population affected by outbreaks of measles, and recommended that NTLs should be applied to public health studies27. Some studies in Israel have found that the incidence of breast and lung cancer in females is associated with NTL intensity in Israel28.\n\nIn Thailand, NTLs are used to estimate Gross Provincial Products (GPPs), because the luminosity of NTLs is normally dependent on the amount of economic activity in each area. The results showed that NTLs and GPP growth are significantly correlated and can represent the relationship between economic values and spatial inequality29. Industrial density (ID) may also be related to disease. However, previous studies have not investigated how NTLs and ID correlate with CRDs.\n\nThe government of Thailand has utilized various measures to attempt to prevent, diagnose, and treat CRDs. However, spatial clustering of diseases, particularly at a national level, has rarely been applied to examine characteristics of CRD clusters in terms of their location, size, and magnitude in Thailand. In addition, there has been limited use of socioeconomic indicators, such as NTLs and ID, in the epidemiology of CRDs.\n\nIn this study, commonly used methods for spatial clustering of CRDs were compared, including the use of the spatial scan statistic, LISA, and the local Gi*(d) statistic. We also analysed whether there was a correlation between NTLs and ID with CRDs. Our findings could help decision makers implement more relevant disease control policies using the performance of these local cluster detection methods, as well as the correlation of CRDs with NTLs and ID. The availability of a suitable strategy for spatial clustering of CRDs may allow for a better allocation of health care resources.\n\n\nMethods\n\nThailand occupies an area of 514,000 square kilometres, which consists of an area of 511,770 square kilometres of land and 2,230 square kilometres of water. Thailand shares a border with Myanmar, Cambodia, Laos and Malaysia. In 2010, there were 76 provinces, 878 districts (Amphoe), 7,225 sub-districts (Tambon), and 74,965 villages (Mooban) in Thailand.\n\nThe Ethical Committee of Khon Kaen University deemed this study to be exempt from requiring ethical approval (reference no. HE 582315).\n\nSpatial data. The geographical coordinates of administrative areas of Thailand were retrieved from DIVA-GIS online (http://www.diva-gis.org/gdata), which is publicly available. This database of provincial maps of Thailand was processed with Quantum GIS for further GIS based analysis. For GIS based analysis, we applied zonal statistics to calculate mean NTLs and ID. The spatial distribution of CRD prevalence was obtained from the province-level polygon map, which contains information regarding the latitudes and longitudes of each province. The calculated mean NTLs, ID and CRD prevalence were further visualized with province-level layers on the polygon map and labelled with the administrative code of each province.\n\nCRD, NTL and ID data. This study used data from the National Socioeconomics Survey (NSS), a cross-sectional study conducted by the National Statistical Office (NSO), Thailand, in 2010. Using stratified two-stage sampling, the survey selected a nationally representative sample to respond to a structured questionnaire. The questionnaires collected information on gender, age, household income, education, family size, occupation, region, residential area, housing construction materials, cooking fuels, smoking status and previous diagnosis of CRDs. A total of 17,040 individuals who met the inclusion criteria (aged between 18–59 years and diagnosed with having CRDs by a physician) were included in this analysis. The NSO administrative board officially allowed the research team to use the data (reference no.050601/1441).\n\nThe NTLs of Thailand used in this study were based on global stable lights imagery from 2010, acquired from the Operational Linescan System (OLS) sensor on-board satellite F18 under the Defense Meteorological Satellite Program (DMSP). All NTL data are publicly available (https://www.ngdc.noaa.gov/eog/dmsp/downloadV4composites.html). The ID data were calculated from GPPs, which represent the official statistics on provincial economic activity published by the National Economic and Social Development Board (NESDB). GPP data are also publicly available (http://www.nesdb.go.th/nesdb_en/more_news.php?cid=156&filename=index).\n\nThe Open GIS software Quantum GIS (version 2.8.5) was used to conduct exploratory spatial data analysis30. Spatial autocorrelation analysis was conducted using GeoDa (version 1.6.6) and the local Gi*(d) statistic31. Stata version 10.0 (StataCorp, CollegeStation, TX) was used to calculate CRD prevalence. SaTScan (version 9.0.1) was used to determine the presence of statistically significant CRD spatial clusters and identify their approximate locations.\n\nLocal indices of spatial association. The local Moran’s I investigates the local level of spatial autocorrelation of provinces with a high and low prevalence of CRDs32. Computation of LISA assesses the local version of Moran’s I for each location to determine the variation in spatial autocorrelation over the study area. Its significance is evaluated in five categories: High-High, Low-Low, Low-High, High-Low, and Not Significant. Spatial autocorrelation occurs when a high prevalence of CRDs correlates with a high prevalence in neighbouring areas (also known as hotspots), or when a low prevalence of CRDs correlates with a low prevalence in neighbouring areas (also known as cold spots)33. This study set the spatial weight matrix of 3 k-Nearest Neighbours around each province, meaning that the clustering relation was identified with three neighbouring provinces. The statistical significance level was 0.05, and the simulation used 999 permutations34 to evaluate the sensitivity of the results.\n\nGetis and Ord’s local Gi*(d) statistic. The local Gi*(d) statistic is used to test the statistical significance of local clusters and to determine the spatial dependence and relative magnitude between observations. In this study, the local Gi*(d) statistic was used to test statistically significant CRD prevalence and local autocorrelation, as well as to determine the dependence of neighbouring observation35 and local clustering. We used k-Nearest Neighbours, and contiguity was set as three provinces for a polygon contiguity spatial weight matrix, which was created depending on three provinces that share common boundaries and vertices. A 0.05 significance level and 999 permutations were used to identify significant clusters of local autocorrelation. A high value for the local Gi*(d) statistic indicates a hotspot, and a low value indicates a cold spot36,37.\n\nSpatial scan statistic. The spatial scan statistic, calculated in SaTScan38, was applied to analyse the geographic distribution of CRD prevalence in Thailand and determine whether there are any spatial geographical clusters of CRD prevalence as for high or low CRDs. We used the retrospective purely spatial Poisson model to identify spatially high or low clusters of CRDs, since it fit the assumption that the number of events in an area was Poisson distributed according to a known underlying population at risk. Purely spatial analysis, which ignores the time dimension of the cases, was performed to detect CRD clusters in the study areas. The purely spatial Poisson model imposed circular windows on the map and let the circle move over the area. Each circle moved until reaching a maximum population at risk. The radius of each circle increased continuously from to a maximum radius. Therefore, the window never encompassed more than 50% of the total population at risk, i.e., the maximum spatial cluster size, in this investigation. The likelihood function was maximized over all window positions and sizes, and the one with the maximum likelihood constituted the most likely cluster. For each window, we used a Monte Carlo simulation to test the null hypothesis that no significant clusters were created, supposing that the relative risk (RR) of CRDs was no different within the window compared to outside the window. The maximum number of random Monte Carlo replications was defined as 999. The likelihood function was maximized in the overall window locations and sizes39. The window with the maximum likelihood value of the spatial window was set to 50% of the population at risk.\n\n\nResults\n\nPearson correlation assessed the relationship between NTLs and ID with CRDs in all provinces. There was a moderate positive correlation of 0.34 (95% CI: 0.12-0.52; p-value 0.002) and 0.36 (95% CI: 0.15-0.54; p-value 0.001) with CRDs, which indicated that NTLs and ID had similar relationships to CRDs (Table 1 and Figure 1).\n\n(A) NTLs and (B) ID showed high values, representative of high population density.\n\nThe Univariate Moran’s I scatter of annual CRD prevalence among provinces in Thailand in 2010 showed a slightly positive spatial autocorrelation, as the Moran's I was 0.072, with statistical significance at 0.05. The Moran’s I indicated clustering patterns. There were hotspots in Bangkok and the perimeter provinces, namely, Samut Prakan, Nonthaburi, Pathum Thani and Nakhon Pathom. Cold spots and low prevalence areas, were located in the Nan and Phitsanulok provinces. Two high/low clusters were located in Trang, Songkhla, and part of the Phatthalung provinces, indicating that the distribution of the CRD in affected provinces was spatially autocorrelated (low clustering), even though the overall tendencies were not clear. This could be due to the nature of local Moran's I indices; the statistic cannot accommodate extreme values or outliers in the data set, such as the very low CRD prevalence in some provinces that were also included in the model. Therefore, its sensitivity in cluster detection may be reduced. Using results from other local spatial analysis methods could help explain the autocorrelations.\n\nConsidering NTLs and ID with CRDs in terms of bivariate Moran’s I scatter for annualized prevalence in 2010, the results showed a positive spatial autocorrelation, with Moran's I values of 0.345 and 0.374, respectively. These values were statistically significant, with a significance level of 0.05. Based on these results, hotspots were located in Bangkok and perimeter provinces, consisting of Samut Prakan, Nonthaburi, Pathum Thani and Nakhon Pathom. The results showed a high, significant correlation between the NTLs with CRDs, suggesting that brighter lights, together with high population densities of exclusively wealthier people, can be used to define the spatial extent of CRD development. Therefore, ID, which reflects urbanization, was correlated with CRDs (Figure 3 and Figure 4).\n\nFor the local Gi*(d) statistics, hotspots were located in Bangkok and perimeter provinces, consisting of Samut Prakan, Nonthaburi, Pathum Thani and Nakhon Pathom (Figure 5).\n\n(A) LISA (Univariate) cluster map, and (B) Moran scatter plot matrix of CRDs (p < 0.05) in 2010. There were 3 hotspots located in Bangkok and the perimeter provinces.\n\n(A) LISA (Bivariate; Night-time light with CRDs) cluster map, and (B) Moran scatter plot matrix of CRDs (p < 0.05) in 2010. We analysed NTLs with CRDs in terms of bivariate Moran’s I scatter for annualized prevalence. There were 5 hotspots located in Bangkok and the perimeter provinces.\n\n(A) LISA (bivariate; industrial density with CRDs) cluster map, and (B) Moran’s I scatter plot matrix of CRDs (p < 0.05) in 2010. We analysed ID with CRDs in terms of bivariate Moran’s I scatter for annualized prevalence. There were 5 hotspots located in Bangkok and the perimeter provinces.\n\nThere were 5 hotspots located in Bangkok and the perimeter provinces.\n\nThe spatial scan statistics revealed two spatial clusters. The primary cluster was located in 23 provinces. There were 157 cases compared to 213 expected cases. Thus, the ratio between observed and expected cases was 0.74. The p-value was smaller than 0.05, which indicated that the cluster was highly significant. The relative risk for the population inside the cluster compared to the population outside the cluster was 0.65, indicating that the risk of CRDs within this area was higher than locations outside it. This number is the estimated risk within the cluster, divided by the estimated risk outside the cluster. The analysis also revealed that a secondary cluster, which is the most likely cluster, was located in 32 provinces. The number of observed cases was 348, compared to 297 expected cases. Thus, the ratio between observed and expected cases was 1.17. According to these calculations, the relative risk inside the cluster is 1.37. The p-value was smaller than 0.05, indicating that the cluster was highly significant. The relative risk for the population inside the cluster compared to the population outside the cluster was 1.37, indicating that the risk of CRDs in this area was higher than locations outside it (Figure 6).\n\nThe spatial scan statistic, calculated in SaTScan, was used to detect spatial high or low CRD prevalence. We used the retrospective, purely spatial Poisson model. Based on these results, the spatial scan statistics revealed two spatial clusters.\n\nIn summary, as seen in Figure 1–Figure 6, spatial scan statistics, LISA, and local Gi*(d) statistics revealed similar results for areas with the highest clustering of CRDs. Nevertheless, the clusters for spatial scan statistics covered a wider area of the Bangkok, Central, Northeast and South clusters, while the clusters for LISA and the local Gi*(d) statistic were wider and more dispersed in Bangkok and the perimeter provinces.\n\n\nDiscussion\n\nIn this study, NTL and ID data were utilized to assess growth in provinces based on geography, economic growth, urbanization, or health metrics, as well as concentration of NTLs and ID. Areas with a high CRD prevalence also experienced high light growth and a high concentration of ID, and therefore NTLs and ID were probably induced CRDs. In other words, the results indicated that CRDs are affected by population density and industrial concentration. This finding was consistent with a previous study that stated that it is possible to use NTLs in public health studies26. The findings of this study suggest that NTLs and ID can substitute for some variables, such as urbanization, density, economic growth, and industrial concentration. NTLs and ID serve as a new tool for specifying disease hotspots by representing the population and industrial booms typically contributing to epidemics. In urban areas with migration, NTLs can indicate where populations are clustering, through the captured expansion and the increase brightness of lighted areas. The technique indicates fluctuations in population density, which affect epidemic risk and can replace current methods of outbreak surveillance26,40.\n\nLISA, spatial scan statistics, and local Gi*(d) statistics are common methods applied to investigate local clusters of diseases. These techniques help identify clusters and assess their statistical significance41. In this study, all of these methods provided comparable results in the detection of geographic areas of CRDs in both high and low- rate clusters. However, there were some inconsistencies. The identification of clusters by spatial scan statistics was more localized, compared to LISA and local Gi*(d) statistics, possibly due to their dissimilar role in defining clusters. LISA detected the neighbouring provinces with rates significantly correlated to each other, and areas with similar values were thus determined as a cluster32. In this study, LISA explored the correlation between the value of a certain area and the average value of neighbouring areas. Spatial scan statistic methods were used, and larger clusters were detected; the calculation of the maximum likelihood ratio of cases was made together with the underlying population in the area to investigate the cluster of provinces considered to have higher or lower rates. Using spatial scan statistics methods, larger clusters were detected, and the calculation of maximum likelihood ratio of cases was made simultaneously with the underlying population in the area to investigate the cluster of provinces and consider them for higher or lower rates7,42. Meanwhile, the local Gi*(d) statistic and LISA reported that the two methods shared similar results in terms of rising clusters (hotspots). Hanson and Wieczorek7 compared LISA to spatial scan statistics by exploring alcohol-related mortality in New York, USA (which identified some differences between two methods). It was concluded that the spatial scan statistic is the more sensitive method. A multi-method approach is adopted for cluster detection, since different methods tend to identify different characteristics in clusters, i.e., LISA identifies the core of the cluster, and the scan statistic identifies its extent. Furthermore, Jacquez and Greiling43 used LISA to analyse spatial clustering for diagnosis of breast, lung and colorectal cancers in Long Island, USA, and to identify significant spatial patterns for all of these diseases. Their analysis confirms the clustering of breast cancer mortality previously revealed by Kulldorff’s spatial scan statistic44, but they found that the two methods identified slightly different clusters. As a result, they recommended that a combination of statistics be used when studying local clustering to assure that different aspects of spatial patterns are fully identified and that the results from the suite of analyses are logically consistent.\n\nDue to its own principle for cluster detection, each method selected different geographic areas. The intersection of the selections indicated that these three methods resulted in different features in the same clusters, suggesting that various methods should be applied to identify clusters of CRDs at the provincial level in Thailand. These spatial cluster detection methods can be utilized collectively rather than individually. Furthermore, because each method has its own advantages and disadvantages, no single method is deemed the “gold standard” for cluster analysis45.\n\nThe strengths of this study included the methods used to analyse CRD cases, including spatial scan statistics, LISA, and local Gi*(d) statistics. All of these have been thoroughly investigated and compared to determine the appropriate methods for evaluating spatial clustering and cluster detection. NTLs and ID were included to investigate a correlation with CRDs. This study contained a large sample size that represents a nationally representative sample. Therefore, the results could represent the Thai population in general.\n\nAbbreviations: CRDs: Chronic respiratory diseases, NSO: National Statistical Office, NSS: National Socioeconomics Survey, LISA: Local Indices of Spatial Association, ID: Industrial density, NTLs: Night time lights.\n\n\nConclusions\n\nThis study utilized a geographic information system and spatial analyses, which can be applied to several epidemiological studies to investigate and clarify the spatial heterogeneity of CRDs in highly affected provinces in Thailand. LISA, spatial scan statistics, and local Gi*(d) statistics were calculated, with the aim of revealing the spatial characteristics of the CRDs. Spatial scan statistics performed much better in outlier detection in terms of power, compared to LISA and local Gi*(d) statistics. Based on our simulation, there was a large relative risk difference (two spatial clusters) and significant spatial heterogeneity. The findings suggested heterogeneity in the spatial pattern of CRDs in the study. Purely spatial retrospective analysis revealed persistence of CRD clusters in some geographical locations of the provinces on an annual basis. CRDs were affected by the concentration of NTLs and ID. We believe that NTLs and ID serve as a new tool for specifying disease hotspots by representing the population and industrial booms that typically contribute to epidemics. The results of the study provide helpful information on the common epidemiological situation of CRDs. Demonstrating the existence of CRDs hotspots in different provinces may enable the Ministry of Public Health or provincial health officers to launch remedial measures in the affected areas and formulate strategies for more effective handling of CRDs.\n\n\nData availability\n\nData used in this study were from the NSS; permission to use these data can be requested from the NSO. This study got approval from the NSS (reference no.050601/1441) to use the data on gender, age, household income, education, family size, occupation, region, residential area, housing construction materials, cooking fuels, smoking status and previously diagnosed CRDs by a physician. NTL data and ID data are publicly available.\n\nThe NTL data are from the Operational Linescan System (OLS) sensor on-board satellite F18 under the Defense Meteorological Satellite Program (DMSP) (https://www.ngdc.noaa.gov/eog/dmsp/downloadV4composites.html).\n\nThe ID data are from the National Economic and Social Development Board (NESDB) (http://www.nesdb.go.th/nesdb_en/more_news.php?cid=156&filename=index).\n\nDataset 1. Pearson correlation with CRDs dataset.\n\nDOI, 10.5256/f1000research.12128.d17870046\n\nDataset 2. NTLs and ID dataset.\n\nDOI, 10.5256/f1000research.12128.d17870147\n\nDataset 3. LISA (Univariate) cluster CRDs dataset.\n\nDOI, 10.5256/f1000research.12128.d17870248\n\nDataset 4. LISA (Bivariate; Night-Time Light with CRDs) cluster dataset.\n\nDOI, 10.5256/f1000research.12128.d17870349\n\nDataset 5. LISA (Bivariate; Industrial Density with CRDs) cluster dataset.\n\nDOI, 10.5256/f1000research.12128.d17870450\n\nDataset 6. Local Gi*(d) cluster CRDs dataset.\n\nDOI, 10.5256/f1000research.12128.d17870551\n\nDataset 7. Spatial scan statistics in SaTScan analysis of CRDs dataset.\n\nDOI, 10.5256/f1000research.12128.d17870652",
"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 are grateful to all contributors to this research, and especially to the NSO for the data.\n\n\nReferences\n\nWorld Health Organization: Global surveillance, prevention and control of chronic respiratory diseases: a comprehensive approach. GARD_Manual. 2007; World Health Organization: Geneva, Switzerland, 2007. Reference Source\n\nCenters for disease control and prevention Prevention: Global Health-Thailand. 2011, [cited 2017 Jan 26]. Reference Source\n\nWaller LA, Gotway CA: Applied spatial statistics for public health data. John Wiley & Sons; 2004. Publisher Full Text\n\nPfeiffer DU, Robinson TP, Stevenson M, et al.: Spatial Analysis in Epidemiology. Oxford University Press. 2008; 142. Publisher Full Text\n\nSong C, Kulldorff M: Power evaluation of disease clustering tests. Int J Health Geogr. 2003; 2(1): 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKulldorff M, Tango T, Park PJ: Power comparisons for disease clustering tests. Comput Stat Data Anal. 2003; 42(4): 665–84. Publisher Full Text\n\nHanson CE, Wieczorek WF: Alcohol mortality: a comparison of spatial clustering methods. Soc Sci Med. 2002; 55(5): 791–802. PubMed Abstract | Publisher Full Text\n\nFotheringham AS, Zhan FB: A comparison of three exploratory methods for cluster detection in spatial point patterns. Geogr Anal. 1996; 28(3): 200–18. Publisher Full Text\n\nKulldorff M, Huang L, Pickle L, et al.: An elliptic spatial scan statistic. Stat Med. 2006; 25(22): 3929–43. PubMed Abstract | Publisher Full Text\n\nKulldorff M: Tests of Spatial Randomness Adjusted for an Inhomogeneity. J Am Stat Assoc. 2006; 101(475): 1289–305. Publisher Full Text\n\nElvidge CD, Baugh KE, Kihn EA, et al.: Mapping city lights with nighttime data from the DMSP Operational Linescan System. Photogramm Eng Remote Sensing. 1997; 63(6): 727–34. Reference Source\n\nElvidge CD, Baugh KE, Kihn EA, et al.: Relation between satellite observed visible-near infrared emissions, population, economic activity and electric power consumption. Int J Remote Sens. 1997; 18(6): 1373–9. Publisher Full Text\n\nImhoff ML, Lawrence WT, Stutzer DC, et al.: A technique for using composite DMSP/OLS “city lights” satellite data to map urban area. Remote Sens Environ. 1997; 61(3): 361–70. Publisher Full Text\n\nElvidge CD, Baugh KE, Dietz JB, et al.: Radiance calibration of DMSP-OLS low-light imaging data of human settlements. Remote Sens Environ. 1999; 68(1): 77–88. Publisher Full Text\n\nSmall C, Elvidge CD, Balk D, et al.: Spatial scaling of stable night lights. Remote Sens Environ. 2011; 115(2): 269–80. Publisher Full Text\n\nZhuo L, Ichinose T, Zheng J, et al.: Modelling the population density of China at the pixel level based on DMSP/OLS non‐radiance‐calibrated night‐time light images. Int J Remote Sens. 2009; 30(4): 1003–18. Publisher Full Text\n\nZhang Q, Seto KC: Mapping urbanization dynamics at regional and global scales using multi-temporal DMSP/OLS nighttime light data. Remote Sens Environ. 2011; 115(9): 2320–9. Publisher Full Text\n\nElvidge CD, Baugh KE, Anderson SJ, et al.: The Night Light Development Index (NLDI): a spatially explicit measure of human development from satellite data. Soc Geogr. 2012; 7(1): 23–35. Publisher Full Text\n\nTownsend AC, Bruce DA: The use of night-time lights satellite imagery as a measure of Australia's regional electricity consumption and population distribution. Int J Remote Sens. 2010; 31(16): 4459–80. Publisher Full Text\n\nFlorida R, Gulden T, Mellander C: The rise of the mega-region. Econ Soc. 2008; 1(3): 459–76. Publisher Full Text\n\nHenderson JV, Storeygard A, Weil DN: A Bright Idea for Measuring Economic Growth. Am Econ Rev. 2011; 101(3): 194–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDoll CH, Muller JP, Elvidge CD: Night-time imagery as a tool for global mapping of socioeconomic parameters and greenhouse gas emissions. J Human Environ. 2000; 29(3): 157–62. Publisher Full Text\n\nSutton PC, Elvidge CD, Ghosh T: Estimation of gross domestic product at sub-national scales using nighttime satellite imagery. Int J Ecol Econ Stat. 2007; 8(S07): 5–21. Reference Source\n\nFlorida R, Mellander C, Gulden T: Global metropolis: assessing economic activity in urban centers based on nighttime satellite images. Prof Geogr. 2012; 64(2): 178–87. Publisher Full Text\n\nLi D, Zhao X, Li X: Remote sensing of human beings–a perspective from nighttime light. Geo Spat Inf Sci. 2016; 19(1): 69–79. Publisher Full Text\n\nHenderson JV, Storeygard A, Weil DN: Measuring Economic Growth From Outer Space. Am Econ Rev. 2012; 102(2): 994–1028. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBharti N, Tatem AJ, Ferrari MJ, et al.: Explaining seasonal fluctuations of measles in Niger using nighttime lights imagery. Science. 2011; 334(6061): 1424–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKloog I, Haim A, Stevens RG, et al.: Light at night co-distributes with incident breast but not lung cancer in the female population of Israel. Chronobiol Int. 2008; 25(1): 65–81. PubMed Abstract | Publisher Full Text\n\nChaiwat T: Night Lights, Economic Growth, and Spatial Inequality of Thailand. Puey Ungphakorn Institute for Economic Research. 2016. Reference Source\n\nSteiniger S, Hunter AJ: The 2012 free and open source GIS software map–A guide to facilitate research, development, and adoption. Comput Environ Urban Syst. 2013; 39: 136–50. Publisher Full Text\n\nAnselin L, Syabri I, Kho Y: GeoDa: an introduction to spatial data analysis. Geogr Anal. 2006; 38(1): 5–22. Publisher Full Text\n\nAnselin L: Local Indicators of Spatial Association—LISA. Geogr Anal. 1995; 27(2): 93–115. Publisher Full Text\n\nAnselin L, Syabri I, Kho Y: GeoDa: An introduction to spatial data analysis. 2004. Reference Source\n\nAnselin L: An introduction to EDA with GeoDa. Spatial Analysis Laboratory (SAL) Department of Agricultural and Consumer Economics, University of Illinois, Urbana-Champaign, IL. 2003. Reference Source\n\nHinman SE, Blackburn JK, Curtis A: Spatial and temporal structure of typhoid outbreaks in Washington, D.C., 1906–1909: evaluating local clustering with the Gi* statistic. Int J Health Geogr. 2006; 5(1): 13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGetis A, Ord JK: The analysis of spatial association by use of distance statistics. Geogr Anal. 1992; 24(3): 189–206. Publisher Full Text\n\nGetis A, Ord JK: Local spatial statistics: an overview. Spatial analysis: modelling in a GIS environment. 1996; 374. Reference Source\n\nKulldorff M: A spatial scan statistic. In: Communications in Statistics - Theory and Methods. 1997; 26(6): 1481–96. Publisher Full Text\n\nKulldorff M: SaTScanTM User Guide for version 8. 0. 2009.\n\nPrinceton University: Nighttime images help track disease from the sky. ScienceDaily. 2011, [cited 2017 Jan 30]. Reference Source\n\nAnselin L: Review of Cluster Analysis Software. The North American Association of Central Cancer Registries, Inc. 2004. Reference Source\n\nNødtvedt A, Guitian J, Egenvall A, et al.: The spatial distribution of atopic dermatitis cases in a population of insured Swedish dogs. Prev Vet Med. 2007; 78(3–4): 210–22. PubMed Abstract | Publisher Full Text\n\nJacquez GM, Greiling DA: Local clustering in breast, lung and colorectal cancer in Long Island, New York. Int J Health Geogr. 2003; 2(1): 3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKulldorff M, Feuer EJ, Miller BA, et al.: Breast cancer clusters in the northeast United States: a geographic analysis. Am J Epidemiol. 1997; 146(2): 161–70. PubMed Abstract | Publisher Full Text\n\nSasson C, Cudnik MT, Nassel A, et al.: Identifying High-risk Geographic Areas for Cardiac Arrest Using Three Methods for Cluster Analysis. Acad Emerg Med. 2012; 19(2): 139–46. PubMed Abstract | Publisher Full Text\n\nLaohasiriwong W, Puttanapong N, Luenam A: Dataset 1 in: A comparison of spatial heterogeneity with local cluster detection methods for chronic respiratory diseases in Thailand. F1000Research. 2017. Data Source\n\nLaohasiriwong W, Puttanapong N, Luenam A: Dataset 2 in: A comparison of spatial heterogeneity with local cluster detection methods for chronic respiratory diseases in Thailand. F1000Research. 2017. Data Source\n\nLaohasiriwong W, Puttanapong N, Luenam A: Dataset 3 in: A comparison of spatial heterogeneity with local cluster detection methods for chronic respiratory diseases in Thailand. F1000Research. 2017. Data Source\n\nLaohasiriwong W, Puttanapong N, Luenam A: Dataset 4 in: A comparison of spatial heterogeneity with local cluster detection methods for chronic respiratory diseases in Thailand. F1000Research. 2017. Data Source\n\nLaohasiriwong W, Puttanapong N, Luenam A: Dataset 5 in: A comparison of spatial heterogeneity with local cluster detection methods for chronic respiratory diseases in Thailand. F1000Research. 2017. Data Source\n\nLaohasiriwong W, Puttanapong N, Luenam A: Dataset 6 in: A comparison of spatial heterogeneity with local cluster detection methods for chronic respiratory diseases in Thailand. F1000Research. 2017. Data Source\n\nLaohasiriwong W, Puttanapong N, Luenam A: Dataset 7 in: A comparison of spatial heterogeneity with local cluster detection methods for chronic respiratory diseases in Thailand. F1000Research. 2017. Data Source"
}
|
[
{
"id": "28274",
"date": "18 Dec 2017",
"name": "Mathuros Tipayamongkholgul",
"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 methods, the author used SatScan and used 50% of population at risk to be a radius at the province level. Using such technique at the provincial level (large geographic area) may lead to inaccurate cluster as you can see in the result that one cluster cover the whole region.\n\nIn the result, Moran’ I present very low spatial correlation (0.07), although it presented statistically significant. The correlational value cannot accept the spatial correlation that exists, and therefore all following spatial data analysis may not be necessary.\n\nLISA and Local G were used for different purpose, I don’t think comparison between this two is needed.\n\nThe number of CRD may relate with the accessibility to healthcare service in Bangkok metropolitan and some surrounding area. The author may explain whether this point may affect the study 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? 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": [
{
"c_id": "3468",
"date": "06 Mar 2018",
"name": "Amornrat Luenam",
"role": "Author Response",
"response": "Explanation to question 1.We used the SaTScan, using the Poisson probability model and constrained to clusters no larger than 50% of the population at risk and 50% of this study to locate potential circular cluster areas based on the recommendation of Kulldorff M. Kulldorff M (2016) suggested that 50 percent is the default maximum, a smaller maximum may be requested. However, the recommended value is 50 percent (Kulldorff M. 2018, Kulldorff M, Nagarwalla.1995) which reported that a window sized up to 50% of the population at risk can generally reduce negative clusters. Whereas 90%, is more appropriately interpreted as a lower disease rate in the 10% of the area outside the ‘cluster’ rather than as an excess disease rate covering almost the whole study region, therefore, it did not appropriate with our study.References:Abdurrob A., Kulldorff M. SaTScan Tutoria # 3 Advanced Options. 2016. Available: https://www.satscan.org/tutorials/advancedoptions/SaTScanTutorialAdvancedOptions.pdfKulldorff M, 2018. SaTScanTM User Guide for version 9.5 Kulldorff M, Nagarwalla N. Spatial disease clusters: detection and inference. Statistics in medicine. 1995;14(8):799–810.Explanation to question 2 and 3 We conducted Global Moran I, G* test and LISA based on contents and suggestions introduced by Anselin (1995). Specifically, all three tests were undertaken because of the following reasons as clarified in Anselin (1995). (1) Global Moran I has a limitation. The statistical insignificance of Global Moran I might not indicate that the local association does not exist (Getis and Ord 1992, p. 201). Therefore, the methods of G* and LISA have been developed in order to overcome this incomplete capability of Global Moran I, and both are capable of identifying the local associations.(2) Based on statistical inference, G* can identify only two cases of localized associations, which are hotspots of high-high and cold spots of low-low. However, LISA has extended this capability, and it can indicate four categories of hotspots, which are high-high, high-low, low-high and low-low. Specifically, LISA can identify both cases of positive spatial correlation (e.g. high-high and low-low) and those of negative spatial association (e.g. high-low and low-high) Based on these clarifications, this paper included results obtained from Global Moran I, G* test, and LISA in order to generate the robust results, statically examining spatial associations for both aspects - the global vs. local and univariate vs. bivariate. References: Anselin, L. Local Indicators of Spatial Association—LISA. Geographical Analysis. 1995; 27: 93–115.Getis, A., and K. Ord. ‘The Analysis of Spatial Association by Use of Distance Statistics.” Geographical Analysis. 1992; 24:189-206.Explanation to question 4We study the CRDs morbidity which is mainly linked to the epidemic of tobacco exposure and indoor and outdoor air pollution in Asian countries (Tan WC, Ng TP, 2008), whereas the mortality may relate with the accessibility to healthcare service in Bangkok metropolitan and some surrounding area.References:Tan WC, Ng TP. COPD in Asia: where East meets West. Chest. 2008; 133(2):517-527."
}
]
},
{
"id": "29646",
"date": "29 Jan 2018",
"name": "Edson Zangiacomi Martinez",
"expertise": [
"Reviewer Expertise Epidemiology",
"biostatistics",
"Bayesian models"
],
"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 interesting data about the possible associations between socioeconomic status (night-time lights and industrial density) and chronic respiratory diseases. The manuscript is well written, the methods and the results sections are adequate, and should be of great interest to the readers. Overall, it is an important study, but their limitations are not mentioned. For example, ecological studies do not permit cause-and-effect conclusions or evidence that risk-modifying factors are related to risk of the disease, since they do not link individual exposure histories to individual outcome events. Thus, I believe the authors can provide a paragraph on the limitations 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": "3467",
"date": "06 Mar 2018",
"name": "Amornrat Luenam",
"role": "Author Response",
"response": "There are two aspects of limitations. The first is based on the availability of data. Therefore, there are limited resolutions in both spatial and time dimensions of data. The second limitation is caused by the characteristic of data and analytical method that do not permit the cause-and-effect conclusion, particularly the connection between individual exposure histories and individual outcome events."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1819
|
https://f1000research.com/articles/6-1863/v1
|
20 Oct 17
|
{
"type": "Research Article",
"title": "Evaluation of predicted Medfly (Ceratitis capitata) quarantine length in the United States utilizing degree-day and agent-based models",
"authors": [
"Travis Collier",
"Nicholas Manoukis",
"Nicholas Manoukis"
],
"abstract": "Invasions by pest insects pose a significant threat to agriculture worldwide. In the case of Ceratitis capitata incursions on the US mainland, where it is not officially established, repeated detections are followed by quarantines and treatments to eliminate the invading population. However, it is difficult to accurately set quarantine duration because non-detection may not mean the pest is eliminated. Most programs extend quarantine lengths past the last fly detection by calculating the amount of time required for 3 generations to elapse under a thermal unit accumulation development model (“degree day”). A newer approach is to use an Agent-Based Simulation (ABS) to explicitly simulate population demographics and elimination. Here, predicted quarantine lengths for 11 sites in the continental United States are evaluated using both approaches. Results indicate a strong seasonality in quarantine length, with longer predictions in the second half of the year compared with the first; this pattern is more extreme in degree day predictions compared with ABS. Geographically, quarantine lengths increased with latitude, though this was less pronounced under the ABS. Variation in quarantine lengths for particular times and places was dramatically larger for degree day than ABS, generally spiking in the middle of the year for degree day and peaking in second half of the year for ABS. Analysis of 34 C. capitata quarantines from 1975 to 2017 in California shows that, for all but two, quarantines were started in the second half of the year, when degree day quarantine lengths are longest and have the highest uncertainty. For a set of hypothetical outbreaks based on these historical quarantines, the ABS produced significantly shorter quarantines than degree day calculations. Overall, ABS quarantine lengths were more consistent than degree day predictions, avoided unrealistically long values, and captured effects of rare events such as cold snaps.",
"keywords": [
"biosecurity",
"Mediterranean fruit fly",
"eradication",
"invasive pest",
"agriculture"
],
"content": "Introduction\n\nInvasions by insects, pathogens and pests are increasingly a defining challenge of the 21st century, facilitated by global connectivity, climatic shifts, and other factors1,2, with a particularly severe impact on agriculture3. Invasions by insects that do not become established have a lower public profile than those that are “successful” from the point of view of the insect. However, there is a greater chance that cases of invasion followed by elimination will be detected and studied when the invading species is of environmental, human health, or economic concern4. Eradicating local populations of such insects can be desirable and feasible5 depending on several factors.\n\nOne factor determining the feasibility of elimination is if the new environment is only marginally or seasonally suitable to the invading insect, facilitating its eradication. Another is when the high cost of allowing establishment leads to extensive efforts for eradication. The invasion of the malaria mosquito species Anopheles gambiae into Northeastern Brazil in the 1930’s6 is one example of an invasive insect that was successfully eradicated primarily due to the second of these factors7,8.\n\nIn the case of An. gambiae there have been no reports of reinvasion, but there are examples of insects that recurrently invade areas outside their native range and are recurrently eliminated within relatively few generations. The Gypsy moth Lymantria dispar in Canada9 is one such species. Arguably, another example is the screwworm Cochlyomyia hominivorax along the current northernmost edge of its range in Panama10 and more recently in Florida11.\n\nOne of the most important instances of repeated invasion and elimination by an economically important insect pest is that of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Medfly) in California. The last four decades have seen a repeated pattern of invasion, detection, and response interspersed by periods of no detections12,13. While it has been suggested that this pattern is the result of cryptic establishment14, the majority view is that Medfly in California is an example of a “metainvasion”, consisting of multiple sequential or overlapping introductions15 and repeated eradication16. Still other researchers point to the possibility of different situations in different regions of the state17,18. Medfly is occasionally found in other parts of the mainland US such as Florida19, and in other countries or areas that are considered free of the pest including Eastern Australia, Mexico and Chile20.\n\nThe response plan to Medfly in California and the other “free” regions mentioned above is extensive and costly, including a quarantine when detections exceed an established standard (more than a male or unmated female fly is detected)21. A practical and important problem is how long to maintain the countermeasures and quarantine after flies are no longer detected. Predicting the likely duration of this ‘post last detection’ quarantine period (hereafter just called quarantine length) would help with management decision-making and planning, and could allow potential cost savings by having sufficient but not excessive resources available.\n\nCurrently, most programs extend quarantine periods past when the last fly is found, by calculating the amount of time required for a given number of generations (usually three) to elapse under a thermal unit accumulation (“degree day”) physiological development model. Degree day based quarantine lengths have been codified in some legal regulations, including United States Federal code22, California23, and Florida. However, the procedure prescribed only defines when the end of a quarantine period has been reached after the fact.\n\nFor planning and resource allocation, policy makers and managers typically attempt to predict the quarantine lengths by using normal temperatures for forward projection. Although it frequently works fairly well, this approach is mathematically flawed and also provides no indication as to the variance or uncertainty of those predictions. Even a more rigorous treatment of degree day based values from historical temperature data can still produce highly variable results depending on relatively small changes in temperatures or details of the model formulation24, in addition to neglecting important aspects of the biology.\n\nRecently, another approach to determining effective quarantine durations against Medfly via Agent-Based Simulations (ABS)25 was introduced. The MED-FOES system simulates a population of individual Medflies under inundative sterile insect technique (SIT) and other controls, explicitly modeling elimination as opposed to the degree day approach, which only determines the time for a specific number of generations to elapse to estimate quarantine duration. MED-FOES also allows for the sampling of parameter space (temperature dependent mortality for each stage, fecundity, etc.), producing a distribution of possible outcomes. While an ABS can be arbitrarily complex, MED-FOES is parameterized in such a way that it can model a ‘typical’ or hypothetical outbreak from only hourly temperature data, and is therefore similar to degree day methods in its input data requirements. It is also possible to vary the initial population to model a specific outbreak.\n\nIn this paper, predicted quarantine length (PQL) for 11 sites in the continental United States were analyzed (Figure 1 and Table 1) based on both the standard thermal accumulation degree day method26 as well as the MED-FOES ABS27. Seasonal variation dominates quarantine duration, so we aggregated the PQL values for each day of the year (Jan. 1, Jan. 2, etc.) across a large number of years (65 for most locations) to produce normals. This approach enables comparison of the standard degree day method to the ABS, but more importantly provides insight into seasonal and spatial variations, prediction uncertainties, and model reliability.\n\nLabels correspond to last three letters of the weather station call-signs in Table 1.\n\n\nMethods\n\nHourly air temperature data for 11 sites was downloaded from NOAA’s publicly available Integrated Surface Database (ISD) dataset28,29.\n\nThe airport sites shown in Figure 1 were chosen for their biological relevance and availability of high quality hourly data over a long time frame.\n\nSites are referred to here by the last three letters of the callsign shown in Table 1. For 8 sites (SFO, FAT, LAX, RIV, SAN, JAX, TPA, and MIA), temperature data starting on 1950-01-01 was used. The 3 other sites contained large (>14 days) gaps or other problems in the early years of their data, so data starting on 1970-01-01 for IAH and 1973-01-01 for BUR and MCO was used. For all sites, temperature data from the start date through 2017-05-15 was used to generate PQLs for dates ranging from the start date to 2016-01-01.\n\nData was fetched and parsed using the Fetching and parsing ISH.ipynb program. Records for the same station callsign were merged, since identification, format, and precise location of stations has changed over time. The data was then cleaned using the Cleaning temperatures.ipynb by removing outliers, identifying large gaps (> 3 hours), resampling to every hour on the hour using linear interpolation, and filling the large gaps using day-over-day linear interpolation (interpolating using values for the same hour of day from previous and following days). The resulting temperature datasets are available.\n\nDegree-days were computed by the single-sine method26, using a base development temperature of 12.39°C (53.3°F) and 345.56 degree-days Celsius (DDc; 622 DDf) per generation following the standard set by California Department of Food and Agriculture regulation 3406(b)23,30. Since hourly temperature data are available, we also calculated degree-days by simple summation for comparison24. For each date, the number of days required for 3 generations of degree-day based life cycles was computed. These calculations are implemented in Temperature functions.ipynb.\n\nMED-FOES25,27 is an agent-based simulation explicitly modeling the eradication of a population of Medflies under inundative sterile male releases (sterile insect technique or SIT) and other interventions, such as increased trapping and foliar sprays. A MED-FOES simulation models a single non-spatial population, starting from a given population size and age distribution, tracking the number of individuals through time until the last fly (Agent) dies and the population is eliminated. In addition to hourly temperatures, simulation parameters include: the initial population, additional mortality induced by control efforts, the effectiveness of SIT, and a large number of biological parameters for which ranges are known from the literature including temperature-dependent development and mortality. The simulations were performed using the same hourly time series of temperature values used for degree-day calculations.\n\nDue to the fact that many of the parameters are only known to within a range, 2500 individual MED-FOES simulations were run for each start date at each site, evenly sampling different regions of parameter-space via the Latin Hypercube Sampling31 procedure. This set of simulations, encompassing a range of possible elimination outcomes, is referred to as a ‘run’. The number of days from the start date required for 95% of the simulations in a run to be eliminated is taken as a conservative prediction of needed quarantine length and referred to as ABS PQL25.\n\nVarying the start date for different simulations was achieved by simply starting at different points in the input temperature file; for this study a run was started every 7 days over the range of dates available for each site. Each set of runs for a single site over a range of starting dates is referred to as a ‘runset’. All runsets were conducted with the same input parameters aside from temperature. Initial population numbers were chosen as a “standard outbreak” based on seven real outbreaks modeled previously25. The 7 day interval ABS PQL values were upsampled to daily values using linear interpolation to allow day-of-year aggregations across years and comparisons with daily degree day based PQLs.\n\nMED-FOES version 0.6.2 was run under Open Grid Scheduler/Grid Engine 2011.11 on a CentOS 6.6 HPC cluster. The MED-FOES code, configuration files, helper scripts, and raw results are available. Overall, we created 11 runsets (one for each site). Each runset contained runs starting every 7 days over the input temperature data range for that site, and each run contained 2500 individual simulations sampling different regions of biologically plausible parameter space. This sums to a total of approximately 86×106 simulations.\n\nThe main results reported here are ‘normals’ in a meteorological sense of the term, but without the typical running mean smoothing which would complicate interpretation. For a variable of interest (eg. temperature or PQL), all values for the same calendar day irrespective of year (eg. 20-July) are aggregated, and summary statistics such as mean, minimum, maximum, and standard deviation are computed for each aggregation. Temperature functions.ipynb contains the code used to perform normal calculations, and the code generating figures as well as all statistical analysis is Summary Figures.ipynb (Jupyter Notebook32, module and version information documented in the file).\n\nThe results reported here are the normals of PQL, computed using the full temperature time series as opposed to computing PQL from the normal of the temperature time series. While the latter is fairly common practice, it is not mathematically proper since, as with means, the normal of a function of X is not generally equal to the function applied to the normal of X. Additionally, by computing the normals of the predicted quarantine durations, we can investigate properties of the distribution of values as shown in Figure 3 and Figure 4 and the “supernorm” supplementary figure S1, supplementary figure S2, and supplementary figure S3.\n\nFigure 2 shows the mean of the normal PQL based on 3 generation degree day accumulation and MED-FOES 95% elimination along with the minimum and maximum of the normals for temperatures. Figure 3 and Figure 4 show the standard deviations (σ) of the normals for the degree day and ABS based PQL.\n\nYear range of input temperature data used is inclusive. All panels have identical limits except SFO quarantine.\n\nThere is significant variation in PQL across both time and location. The temporal variation in PQL is dominated by a yearly cycle, characterized by the normal values shown in Figure 2. Table 2 shows the percentage of variance in quarantine length predictions captured by the mean of the normal yearly cycle (R2) for each site. At all but one site, greater than 75% of the variance in both degree day and ABS based PQLs is accounted for by the mean normal, and the majority exceed 90%. SFO is an exception to this common trend, with the mean normal accounting for only 9.1% of the variation in degree day based PQL and 28.0% of the ABS based PQL. This is also reflected in supplementary figure S2 and supplementary figure S3.\n\nSeasonal variation, evidenced by the general shape of the curves shown in Figure 2, is doubtless familiar to anyone engaged in Medfly pest management. Outbreaks starting in the late summer, autumn, or early winter will extend through relatively cold periods, when thermal dependent development will be slow and therefore extend the duration of quarantine required for 3 generations of degree days to accumulate (referred to as DD PQL hereafter). Similarly, outbreaks starting in the spring or early summer often lead to short quarantines due to the relatively high temperatures.\n\nThis familiar pattern is also seen in the ABS PQLs despite it being quite different in nature from simple degree day accumulation. However, the ABS predictions show a smaller seasonal swing. The ABS generally produces a smaller overall range of PQLs, with longer quarantines than DD PQL for spring and early summer outbreaks, and shorter quarantines for late summer through early winter in almost all cases.\n\nA particular feature of interest, shown most dramatically at FAT in Figure 2, is that ABS PQL often flattens out or even dips for quarantines starting in the late autumn or early winter. This can be due to relatively rare and brief cold-snaps, normally lasting only a few hours, which increase mortality. Since DD PQL does not account for mortality, it misses the effect of cold-snaps entirely. This effect is most clearly seen at more northern and inland sites where cold-snaps are more likely: particularly FAT and RIV, but also BUR, LAX, JAX, and IAH.\n\nPQL generally shows a positive correlation with latitude, and sites are ordered by latitude in the figures and tables here. As seen in Figure 2, higher latitude sites tend to have longer PQLs as well as larger seasonal swings for both degree day and ABS based predictions.\n\nFigure 5 shows the relationship between PQL and latitude. An ordinary least squares fit to the median PQL at each site shows a significant slope for both DD PQL (F =14.08, p=0.005) and ABS PQL (F =10.55, p=0.010), but the degree day based predictions are more sensitive to latitude than the ABS (coefficients of 17.39 and 4.78 respectively). Additionally, the ABS predictions are more stable for SFO, and to a lesser extent FAT, where the degree day model for Medfly produced PQLs that appear either unrealistically long (SFO) or are subject to rapid and extreme seasonal variation in the mid year (FAT).\n\nFor each site, the mean, median, and inter-quartile range are shown, similar to a boxplot. An ordinary least-squares linear fit to the median values is shown by the green lines. The left panel is for single sine degree day predictions, and MED-FOES ABS based predictions are in the right panel.\n\nIn addition to the variation associated with latitude, large differences in PQLs computed for the same start date can exist between even relatively nearby sites. For example, the differences in both degree day and ABS PQLs for the three sites in the Los Angeles region (LAX, BUR, RIV) (shown in the supplementary figure S4) display a strong seasonal component with a spike in July and/or August. The difference in DD PQL between LAX and BUR is normally about a month (overall median=35 days; overall 25% & 75% quantiles are 28 & 45 days), but the median difference of the normal exceeds 75 days in August with some PQL differences up to 142 days. Differences in ABS PQLs are more seasonally stable, with the LAX minus BUR difference not exceeding 42 days for any start date in the 43 years analyzed here.\n\nFigure 3 and Figure 4 report the standard deviation (σ) of the normal for DD PQL and the MED-FOES ABS PQL respectively. These indicate the year to year variability of the PQL for outbreaks starting at a given time of the year and can be used to gauge the uncertainty of predictions based on past PQLs relative to the actual quarantine length which will be required. Similar information is represented by the inter-quartile ranges shown in Figure 5 and supplementary figure S2 and supplementary figure S3. The distributions of PQL values for a site and day-of-year (aggregating across years) are generally not highly skewed, making σ a relatively easy to interpret measure of uncertainty.\n\nExcluding SFO, the mean normal is a good predictor of DD PQL with σ values below 20 days except for the late summer and early autumn, where variance increases due to quarantines extending through the cold season. FAT and, to a lesser extent, RIV show this increase more dramatically, presumably due to their more arid/inland climates where both daily and seasonal temperature ranges are larger (also see Figure 2). The standard deviation generally decreases with decreasing latitude, together with reduced means. The standard deviation in DD PQL for SFO shows an inversion of the seasonal trend other sites exhibit. This is due to the colder temperatures leading to extremely long DD PQLs, frequently extending across two winter seasons.\n\nThe standard deviations of the ABS PQL normals shown in Figure 4 are generally about ½ as large as for DD PQL. This indicates that the ABS PQL not only shows less dramatic seasonal swings, but is also produces more consistent predictions across years. Values again generally decrease with latitude, but less consistently than DD PQL σ of normals. Also, unlike with the DD PQL, the results for SFO appear consistent with other sites.\n\nA notable feature is that BUR, LAX, and SAN all show an increase in the year to year variation in ABS PQLs starting in July and extending through November, while that increase for all other sites starts in July or August but extends to January or February. Additionally, results for FAT show a sharp increase in uncertainty starting in September, fitting with the more arid/inland climate. RIV shows a significant but more gradual increase.\n\nThirty-four Medfly quarantines in CA dating from 1975 to early 2017 were analyzed (supplementary table S1). The start of all but two of these quarantines was in the latter half of the year (July through December), when DD PQLs are typically relatively long, with 68% (23/34) occurring in September through October, when DD PQLs are longest. August, the month where uncertainty in DD PQL often spikes (see Figure 3), accounts for 30% (7/34) of historic quarantines.\n\nFor each historic quarantine start date, the DD PQL and ABS PQL for the closest of the 11 sites analyzed above (see Figure 1 and Table 1) to the actual outbreak location was determined (see supplementary table S1). For this set of hypothetical quarantines, the ABS produced significantly shorter quarantines (mean=169.7 days, σ=21.8 days) than simple 3 generation degree day accumulation (mean=234.2 days, σ=79.2 days) (df =33, t=6.01, p<10−5). Additionally, the variance in the difference between quarantine lengths using a specific date and the mean of the normal PQL for that day of year was smaller for the ABS (σ=8.2 days) than with degree day (σ=25.9 days) (df =33, F =9.92, p<10−8).\n\n\nDiscussion\n\nThe principal contributions of this work can be broken down into three categories:\n\n1) Comparison of PQLs as determined by the degree day and ABS methods.\n\n2) Variation in average PQLs across time of year and space; and\n\n3) Variation in PQLs within a time of year and location.\n\nConsideration of all three of these by program managers, planners and other decision makers is likely to improve management of Medfly outbreaks by informing resource allocation ahead of outbreaks, reducing quarantine costs in some cases, and reducing risk from premature quarantine suspension in others. The results presented cover most of the latitudinal range of Medfly suitability within the United States, as well as many sites of probable introduction, and will hopefully find use as a general guide.\n\nEradication models are extremely difficult to test for accuracy given the impracticality of experimental introductions and the sparse and idiosyncratic nature of historic outbreaks. However, analyzing the timing and locations of historic outbreaks suggests that quarantine lengths would generally be more consistent and shorter on average in California if estimated by ABS compared with degree day.\n\nRequiring a fixed number of generations (typically 3) of degree days to pass is a “tried and true\" method, but not explicitly an extirpation model. It may overestimate required quarantine length through cold weather25 and may underestimate length when growth conditions are very favorable, which somewhat paradoxically leads to shorter degree day based quarantine periods after the last fly detection since generation times are shorter. However, the simplicity of the degree day calculation is a point in its favor, together with its record of generally avoiding subsequent detections after eradication measures and quarantine establishment20.\n\nABS results may be used to inform and modulate responses and treatments such as delimination trapping, fruit sampling, and eradication measures which are under the some discretion of managers. In situations where DD PQL greatly exceed those from the ABS, it is likely that degree day is missing important effects, such as cold snaps, which may justify shortening quarantine periods. On the other hand, in cases where the ABS predicts longer times to elimination, it is plausible that the degree day indicated quarantine is optimistically short, and eradication treatments and SIT releases should be conducted even more aggressively than normal to ensure eradication is achieved within the degree day based period.\n\nA few specific results arising from overall comparisons of different locations are worth highlighting. In general, DD PQLs for Medfly generated from San Francisco International Airport temperature data are almost certainly too long for the entire year. The ABS PQLs are flatter and seem more realistic at around 200 days for San Francisco compared with the 400–550 days of DD PQLs. For several other California locations (typified by Fresno and Riverside) DD PQLs are in close alignment with those from the ABS for the first half of the year but go significantly longer in the cooler months. For three of the four Florida locations analyzed, DD PQLs are significantly shorter than the ABS results (Miami, Tampa, and Orlando). The extent of the difference in those Florida locations is smaller in the later months of the year, but the generality of this pattern suggests that the margin of safety for quarantines as calculated by degree day in those locations may be smaller than expected.\n\nThere is significant variation in PQL depending on the location of the outbreak, with the extremes in our study sites represented by Miami and San Francisco. These geographic results could be compared to previous efforts to model climatic suitability of different parts of the US. One of the early studies on the subject focused on Medfly found higher climatic suitability in Florida locations (Fort Pierce and Orlando) compared with California sites33. Within California, however, those authors found a higher number of suitable months in coastal areas such as Oceanside compared with Riverside and Fresno, roughly paralleling our findings (compare Los Angeles or San Diego with Fresno or Riverside). A more recent analysis of climatic suitability likewise concludes that coastal S. California is the most favorable area of the state for Medfly, but favorability drops inland in the south due to desert conditions. Suitability in central and northern California is limited by cold temperatures and freezes34.\n\nAn important aspect of ABS PQLs is variation within particular times of years and locations. Rare events like cold snaps can increase mortality in the ABS, and thereby lead to shorter PQLs than expected based on historical averages, or DD PQLs. The specificity of the ABS is helpful for determining when quarantines might be safely suspended due to a rare event, something that might not be captured by the degree day model. The degree day model includes only development for generating PQLs, and development is halted at low temperatures, extending quarantine lengths. The ABS, however, also includes mortality for generating PQLs, which means that low temperatures can significantly reduce estimates.\n\nHistorically in California, quarantines have most frequently occurred at times of year when degree day based quarantines are drawn out by cold weather and the MED-FOES ABS model predicts significantly shorter durations. Furthermore, 30% of those historic quarantines happened in August where there is a great deal of uncertainty in forward predictions of degree day quarantine durations based on normal values. If we assume those historic CA quarantines are a guide, the ABS model would very likely produced more predictable and shorter quarantine durations for future outbreaks.\n\nCombination of the two methods analyzed here could leverage the best aspects of both methods for determining optimal quarantine length. The initial quarantine length estimate could be quickly produced via degree-day calculation or the ABS based on the distribution of PQL values generated using historical temperatures. This would generate not just a single “typical” value as the current method of projecting using historical average/normal temperatures does, but a range of outcomes. The median “most likely” value may be used for official estimates, while the variance and extremes would provide managers and affected parties additional information vital for planning.\n\nOnce the three generation period has started after the last fly find, weekly ABS simulations could indicate the likelihood that the pest has been successfully eliminated. If a threshold of 95% of simulations show elimination, the decision to end quarantine early could be made, or in the case where the ABS has not reached the 95% threshold at the end of the DD PQL additional measures could be considered to reduce the risk of re-detection.\n\n\nData and software availability\n\nAll data, non-standard programs, and scripts used are available in the GitHub repository: https://github.com/travc/paper-Predicted-MF-Quarantine-Length-Data-and-Code, archived source code as at the time of publication is available at https://doi.org/10.5281/zenodo.100669835. Files are documented in the repository’s README, and the analysis scripts (.ipynb files) are viewable online at GitHub. Efforts were made to make the code understandable. It is our intent that someone with a reasonable level of programming knowledge will be able to not only replicate our analysis, but also use portions of the provided code as a basis for their own analysis.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was funded by USDA-ARS, project number 2040-22430-025-00D, and by the Headquarters Research Associate program (TCC).\n\n\nAcknowledgements\n\nWe thank J. Hendrichs (FAO/IAEA), S. Gieb (USDA-ARS), S. Sim (USDA-ARS), and T. Fezza (USDA-APHIS) for comments on an early draft of this paper, and N. Mullaly (USDA-APHIS) for data on historical outbreaks. This work was supported by the US Department of Agriculture, Agricultural Research Service. Opinions, findings, conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the USDA. USDA is an equal opportunity provider and employer.\n\n\nSupplementary material\n\nFigure S1: Daily normal of hourly temperatures. Hourly temperature data aggregated by day of year.\n\nClick here to access the data\n\nFigure S2: Degree day based PQL supernorm. Single sine degree day based predicted quarantine lengths aggregated by day of year.\n\nClick here to access the data\n\nFigure S3: MED-FOES PQL based supernorm. MED-FOES based predicted quarantine lengths aggregated by day of year.\n\nClick here to access the data\n\nFigure S4: Difference in PQL at nearby sites (LA basin example). The difference in PQL values for the same start date (including year) between sites in the LA basin. For each date over the range of available data (1973 through 2015 for LAX-BUR and RIV-BUR, 1950 through 2015 for LAX-RIV), PQL values for each site are computed and the differences taken. The resulting differences are then aggregated by day-of-year. Lines show the median while the shaded region is the 25% to 75% quantile range for each day-of-year aggregation of differences.\n\nClick here to access the data\n\nTable S1: Historical quarantines. Historical quarantines in California.\n\nClick here to access the data\n\n\nReferences\n\nSimberloff D, Martin JL, Genovesi P, et al.: Impacts of biological invasions: what’s what and the way forward. Trends Ecol Evol. 2013; 28(1): 58–66. PubMed Abstract | Publisher Full Text\n\nPimentel D, Pimentel M, Wilson A: Plant, Animal, and Microbe Invasive Species in the United States and World. Biological Invasions. Springer Berlin Heidelberg, Berlin, Heidelberg. 2007; 193: 315–330. Publisher Full Text\n\nPaini DR, Sheppard AW, Cook DC, et al.: Global threat to agriculture from invasive species. Proc Natl Acad Sci U S A. 2016; 113(27): 7575–7579. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiebhold AM, Tobin PC: Population Ecology of Insect Invasions and Their Management. Annu Rev Entomol. 2008; 53(1): 387–408. PubMed Abstract | Publisher Full Text\n\nMyers JH, Simberloff D, Kuris AM, et al.: Eradication revisited: dealing with exotic species. Trends Ecol Evol. 2000; 15(8): 316–320. PubMed Abstract | Publisher Full Text\n\nSoper FL, Wilson DB: Anopheles gambiae in Brazil, 1930 to 1940. The Rockefeller Foundation, New York, 1943. Reference Source\n\nCausey OR, Deane LM, Deane MP: Ecology of Anopheles gambiae in Brazil. Am J Trop Med. 1943; s1-23(1): 73–94. Publisher Full Text\n\nKilleen GF, Fillinger U, Kiche I, et al.: Eradication of Anopheles gambiae from Brazil: lessons for malaria control in Africa? Lancet Infect Dis. 2002; 2(10): 618–627. PubMed Abstract | Publisher Full Text\n\nGray DR: Hitchhikers on trade routes: A phenology model estimates the probabilities of gypsy moth introduction and establishment. Ecol Appl. 2010; 20(8): 2300–2309. PubMed Abstract | Publisher Full Text\n\nRobinson AS, Vreysen MJ, Hendrichs J, et al.: Enabling technologies to improve area-wide integrated pest management programmes for the control of screwworms. Med Vet Entomol. 2009; 23 Suppl 1: 1–7. PubMed Abstract | Publisher Full Text\n\nMatthews J, Caudell JN: In the news spring 2017. Hum Wildl Interact. 2017; 11(1): 3. Reference Source\n\nCarey JR: Establishment of the Mediterranean fruit fly in California. Science. 1991; 253(5026): 1369–1373. PubMed Abstract | Publisher Full Text\n\nPapadopoulos NT, Plant RE, Carey JR: From trickle to flood: the large-scale, cryptic invasion of California by tropical fruit flies. Proc Biol Sci. 2013; 280(1768): 20131466. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarey JR, Papadopoulos N, Plant R: The 30-year debate on a multi-billion-dollar threat: Tephritid fruit fly establishment in California. Am Entomol. 2017; 63(2): 100–113. Publisher Full Text\n\nDavies N, Villablanca FX, Roderick GK: Bioinvasions of the Medfly Ceratitis capitata: Source estimation using DNA sequences at multiple intron loci. Genetics. 1999; 153(1): 351–360. PubMed Abstract | Free Full Text\n\nHaymer DS, He M, McInnis DO: Genetic marker analysis of spatial and temporal relationships among existing populations and new infestations of the Mediterranean fruit fly (Ceratitis capitata). Heredity. 1997; 79(3): 302–309. Publisher Full Text\n\nBonizzoni M, Zheng L, Guglielmino CR, et al.: Microsatellite analysis of medfly bioinfestations in California. Mol Ecol. 2001; 10(10): 2515–2524. PubMed Abstract | Publisher Full Text\n\nGasperi G, Bonizzoni M, Gomulski LM, et al.: Genetic Differentiation, Gene Flow and the Origin of Infestations of the Medfly, Ceratitis Capitata. Genetica. 2002; 116(1): 125–135. PubMed Abstract | Publisher Full Text\n\nSzyniszewska AM, Leppla NC, Huang Z, et al.: Analysis of seasonal risk for importation of the mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), via air passenger traffic arriving in Florida and California. J Econ Entomol. 2016; 109(6): 2317–2328. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcinnis DO, Hendrichs J, Shelly T, et al.: Can polyphagous invasive tephritid pest populations escape detection for years under favorable climatic and host conditions. Am Entomol. 2017; 63(2): 89–99. Publisher Full Text\n\nGilbert AJ, Bingham RR, Nicolas MA, et al.: Insect trapping guide. 13th edition. CDFA., Sacramento CA, 2013. Reference Source\n\nUnited States Code of Federal Regulations: Title 7 Subtitle B Chapter III Part 301.32-10 Treatments. 73 FR 32432, June 9, 2008, as amended at 75 FR 4240, Jan. 26, 2010. 2017. Reference Source\n\nCaliforina Code of Regulations: Mediterranean Fruit Fly Interior Quarantine. Title 3, Section 3406. Last vistited 2017-07-17. Reference Source\n\nRoltsch WJ, Zalom FG, Strawn AJ, et al.: Evaluation of several degree-day estimation methods in California climates. Int J Biometeorol. 1999; 42(4): 169–176. Publisher Full Text\n\nManoukis NC, Hoffman K: An agent-based simulation of extirpation of Ceratitis capitata applied to invasions in California. J Pest Sci (2004). 2014; 87(1): 39–51. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaskerville GL, Emin P: Rapid estimation of heat accumulation from maximum and minimum temperatures. Ecology. 1969; 50(3): 514–517. Publisher Full Text\n\nManoukis NC, Hall B, Geib SM: A computer model of insect traps in a landscape. Sci Rep. 2014; 4: 7015, WOS:000344760700005. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith A, Lott N, Vose R: The Integrated Surface Database: Recent Developments and Partnerships. Bull Am Meteorol Soc. 2011; 92(6): 704–708. Publisher Full Text\n\nIntegrated Surface Database (ISD). National Centers for Environmental Information (NCEI) formerly known as National Climatic Data Center (NCDC). Last visited 2017-07-05. Reference Source\n\nMediterranean fruit fly: Regulation and quarantine boundaries. Last vistited 2017-07-17. Reference Source\n\nBlower SM, Dowlatabadi H: Sensitivity and uncertainty analysis of complex models of disease transmission: An hiv model, as an example. Int Stat Rev. 1994; 62(2): 229–243. Publisher Full Text\n\nPérez F, Granger BE, et al.: IPython: a system for interactive scientific computing. Comput Sci Eng. 2007; 9(3): 21–29. Publisher Full Text\n\nMessenger PS, Flitters NE: Bioclimatic Studies of Three Species of Fruit Flies in Hawaii. J Econ Entomol. 1954; 47(5): 756–765. Publisher Full Text\n\nGutierrez AP, Ponti L: Assessing the invasive potential of the Mediterranean fruit fly in California and Italy. Biol Invasion. 2011; 13(12): 2661–2676. Publisher Full Text\n\nCollier T, Manoukis N: travc/paper-Predicted-MF-Quarantine-Length-Data-and-Code: Initial submission [Data set]. Zenodo. 2017. Data Source"
}
|
[
{
"id": "27623",
"date": "21 Nov 2017",
"name": "Teresa Vera",
"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 explains the significance of using two different simulation models to calculate quarantine length after repeated pest invasions, particularly from Ceratitis capitate. Quarantine length is a key issue: non- detection doesn’t mean the pest invasion has been eliminated. Therefore, authors have selected eleven sites in the USA to compare quarantine lengths estimated with the usual simulation model –called degree-day model- and a newer one –called Agent-based simulation (ABS) in order to compare both. The first approach calculates quarantine lengths taking into account the time needed to pass 3 pest generations and a thermal approach. The second approach calculates the lengths considering population and elimination. I think it’s a very interesting paper showing how important is the approach selected in order to determine quarantine lengths. The comparison of both approaches gives some useful information about which approach could suit better in function of season, latitude and longitude etc. When both methods are combined, managers can select the best aspects of each one to optimize quarantine lengths. As authors remark at the end of Discussion, using this combination, vital information for planning is provided to managers and affected parts. Only a few recommendations: In my opinion, selection of sites is not well explained. Authors mention the sites “were chosen for their biological relevance and availability of high quality data”, the last is easy to understand; however the first is not so obvious. I’d recommend a brief explanation of why airports have a biological relevance. Regarding Statistical analysis, I recommended a longer explanation: it seems like the only parameter compared is temperature, what about ABS inputs (as initial population, mortality and so on)? I like very much the detailed information provided in the Results section. I’d recommend some changes in the Discussion section since discussion of the results is put together with conclusions. It could be interesting to add a Conclusion section after Discussion, and then the article structure would be clearer. Finally, reference sources tend to be quite old; I’d recommend authors to update them, at least some of them.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3277",
"date": "18 Dec 2017",
"name": "Travis Collier",
"role": "Author Response",
"response": "Thank you for the review and useful recommendations. Apologies for the long delay in responding. We are planning to address your review in detail along with an additional review and posting a revised paper, but that second review is taking a long time to come in. I don't want your contribution to go too long without being acknowledged though. Thank you"
}
]
},
{
"id": "29994",
"date": "18 Jan 2018",
"name": "Daniel M. Borchert",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research was well done and provides valuable analysis and insight into an ongoing challenge that is faced in monitoring fruit fly quarantines.\n\nOne point of note is the base developmental threshold reported for the Medfly DD model of 53.3 F should be 54.3 F. I am not sure if this was a typo in the manuscript or if it was carried through the calculations. This would likely impact the length of the PQL for the degree day models in both the lower and higher latitude areas, but I do not think that the results would be dramatically different. I recommend that this be checked and revised if necessary.\n\nIn my opinion, the ABS models do provide an improvement to the estimations of Predicted Quarantine Length and can be a beneficial tool in the regulatory area.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "3360",
"date": "19 Jan 2018",
"name": "Travis Collier",
"role": "Author Response",
"response": "Good catch on base temp... It is fortunately just a typo. The code used to do all the calculations, which is posted on Github, has the correct 54.3'C value.We will correct that and address Teresa Vera's comments in a revision shortly.Thank youTravis"
},
{
"c_id": "3361",
"date": "19 Jan 2018",
"name": "Travis Collier",
"role": "Author Response",
"response": "I meant 54.3'F, not 'C in the above comment. Sorry For the record, the degree-day model used is: Base temp = 54.3'F (12.3889'C) DD per generation = 622 DDf (345.5556 DDc) from \"Ceratitis capitata- Review of Degree Day Models\" (2011): USDA APHIS Medfly Action Plan also in CDFA regs sec 3406: https://www.cdfa.ca.gov/plant/medfly/docs/regs/3406-TXT-medfly.pdf The original source appears to be: Tassan, R. L., K. S. Hagen, A. Cheng, T. K. Palmer, G. Feliciano and T. L. Bough. 1982. Mediterranean fruit fly life cycle estimations for the California eradication program. CEC/IOBC Symposium Athens November 1982. pp. 564-570 Of possible note: An alternative model used by Chile and I suspect other places, which might be more applicable to coastal central CA is from Grout and Stoltz (2007): Base temp = 9.9'C DD per generation = 338 DDc"
}
]
},
{
"id": "29759",
"date": "24 Jan 2018",
"name": "John M. Kean",
"expertise": [
"Reviewer Expertise entomology",
"biosecurity",
"surveillance",
"population dynamics",
"modelling"
],
"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 thoroughly support the intent of this paper: to bring additional accuracy and rigour to biosecurity decision making. Specifically, the question of “when is eradication achieved” is an interesting and important one. It is not easy to address, however, but this paper makes an important contribution.\n\nBiosecurity aware economies have recognised the need for science-based reform of international practices around quarantines1. Current practice has largely withstood the test of time, but is based on simplistic assumptions. As this paper points out, better decisions might be made with the better biological data and population dynamic tools now available.\n\nThis paper describes a well-designed comparison between an existing “rule of thumb” approach and a more biologically savvy modelling approach. The strengths and weaknesses of each method are alluded to and the reasons for misalignment explored. The authors suggest a balanced approach to utilise the strengths and minimise the weaknesses of each. On the whole, I support the study and its findings, with two caveats.\n\nFirst, some additional details are needed to fully understand what the agent-based simulations (ABS) are doing. Critical information is lacking on the temperature relationships and simulated management in the model. While this information would be available from the computer code provided, I am loathe to search through 423 MB of source code to find it. Without these details I cannot make a confident assessment of the study.\n\nSecond, the study highlights the importance of low temperatures in fruit fly phenology, but glosses over some of the biologically relevant complications. More detailed comments on the paper sections are given below.\n\nIntroduction\n\nThe examples given for eradications are all historical, including the cited review paper2, which is not very optimistic about eradication as an effective tool for managing invasions. The science of eradication has advanced considerably in recent years and better understanding of invasive ecology, improved surveillance and control tools, and important advances in understanding and managing social expectations around eradication programmes mean that biosecurity agencies can now conduct such operations with much greater certainty, efficacy and efficiency. I suggest replacing the cited review paper2 with Liebhold et al. (2016)3 which gives a more up-to-date review of eradication science.\n\nI note that the quarantine guideline of three generations is not universally used. For example, Australia uses one generation plus thirty days.\n\nI am intrigued by the statement that predicting generation times into the future using normal temperatures is “mathematically flawed” and would like further clarification about what the authors mean.\n\nMethods\n\nIt would be very useful to specify the developmental parameters used by the ABS, especially if they differ from those used for the day-degree approach. I note that the day degree parameters used in California differ considerably from other published values, which find a base development temperature of 8 to 10°C 4.\n\nNo details are given about the assumed starting populations and age structures for ABS runs. This seems a critical detail. Also, what were the assumed management conditions? Without management the simulated populations would presumably increase (on average) and the fact that eradication was achieved in the simulations suggests that some sort of management must have been in place. Details of this are critical for understanding what the simulation results mean and how applicable they may be to different cases.\n\nIn addition, I feel an important aspect of quarantine has been completely ignored – that of surveillance efficacy. The relevant international guidelines (ISPM 6, 9 and 26) specify that surveillance and monitoring are a key part of any programme aiming to prove freedom from a pest. For fruit flies, surveillance trapping is used to monitor eradications, with the quarantine period applying from the time of the last confirmed detection. Therefore surveillance effort is a critical factor in determining an appropriate quarantine length. If surveillance were perfect then no quarantine period would be needed because we would know that nothing is there. Conversely, if surveillance is poor then a large population might conceivably persist undetected for a very long time, necessitating a very long quarantine period indeed. I assume that the ABS simulated the surveillance practices used in California, but some details are needed. I believe that fruit fly surveillance practices differ between different parts of California – could this explain part of the differences in results between sites (Figure 2)?\n\nResults\n\nThe ABS results were tallied as the simulated times after which 95% of simulated populations were eradicated. Depending on the situation, regulator might prefer greater (e.g. 99%) or lesser certainty. It would be useful to see an example of a population survival curve to understand how quarantine duration affects the risk of non-eradication. I suspect that such information would be very useful for biosecurity authorities in their decision-making.\n\nFigure 2 has a spelling mistake – “extripation”.\n\nConclusions\n\nA key point in the paper is the hypothesis that cold snaps may help to knock out fruit fly populations more quickly than expected by the simple day-degree method. The implication is that the ABS method may enable significant cost savings in such situations by allowing a substantially shorter quarantine period. This is a valuable insight, but should be tempered by the fact that fruit flies may have special tricks to enable them to survive cold snaps. The torpor and cold survival thresholds for Queensland fruit fly, for example, are conditioned by previous exposure to low temperatures56, an effect that I suspect is not captured in the ABS. Therefore, I would urge some caution in applying the ABS recommendations in such situations.\n\nOver all, Medfly development and survival at relatively low temperatures seems to be a key factor in setting quarantine periods that start in late summer/autumn, and in understanding the different predictions from the day-degree and ABS methods. However linear day degree models, as used in both approaches, are known to have questionable validity near the predicted developmental threshold. Also (as noted by the authors) small changes in the threshold temperature parameter might cause relatively large changes in the predicted quarantine times. This is not a particular issue for this study, but is a common problem in the simulation of insect phenology, especially in temperate climates. Given the economic importance of fruit flies, better data and models for behavioural and physiological thresholds could be very useful for making better biosecurity decisions.\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": []
}
] | 1
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https://f1000research.com/articles/6-1863
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https://f1000research.com/articles/7-276/v1
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05 Mar 18
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{
"type": "Systematic Review",
"title": "The effectiveness of exercise-based vestibular rehabilitation in adult patients with chronic dizziness: A systematic review",
"authors": [
"Burak Kundakci",
"Anjum Sultana",
"Alan J Taylor",
"Mansour Abdullah Alshehri",
"Anjum Sultana",
"Alan J Taylor",
"Mansour Abdullah Alshehri"
],
"abstract": "Background: Dizziness is a non-specific term used by patients to describe several symptoms ranging from true vertigo, light headedness, disorientation or sense of imbalance. Vestibular rehabilitation (VR) is a specific form of exercise-based therapy programme aimed at alleviating the primary and secondary problems of a vestibular pathology. The aim of this study was to investigate the effectiveness of exercise-based vestibular rehabilitation in adult patients with chronic dizziness. Methods: The following five databases were searched: the Cochrane Central Register of Controlled Trials (CENTRAL, the Cochrane Library), MEDLINE, PubMed, the Physiotherapy Evidence Database (PEDro) and Scopus (Elsevier). Two investigators independently reviewed all articles and a systematic review of literature was performed using the PRISMA guidelines (Preferred Reporting Items for Systematic Reviews and Meta-Analyses). The articles were included if they met the following inclusion criteria: (1) randomised controlled trial, (2) people with chronic dizziness, (3) adults aged 18 or over, (4) exercise-based VR, (5) VR exercises compared with sham or usual care, non-treatment or placebo and (6) only studies published full text in English. Results: The initial search identified 304 articles, four of which met the criteria for analysis. All studies involved some form of vestibular rehabilitation, including vestibular compensation, vestibular adaptation and substitution exercises. These exercises were compared with usual medical care (three studies) or placebo eye exercise (one study). The Vertigo Symptom Scale was the most commonly used outcome measure to assess subjective perception of symptoms of dizziness (three studies). According to the PEDro scale, three studies were considered to be of high quality, and one was rated as fair. Conclusions: This review suggests that exercise-based vestibular rehabilitation shows benefits for adult patients with chronic dizziness with regard to improvement in the vertigo symptom scale, fall risk, balance and emotional status.",
"keywords": [
"Vestibular Rehabilitation",
"Exercise",
"Physiotherapy",
"Chronic Dizziness",
"Vertigo",
"Balance"
],
"content": "Introduction\n\nDizziness is a non-specific term used by patients to describe several symptoms ranging from true vertigo, light headedness, disorientation or sense of imbalance1. It is a frequent complaint, especially in elderly patients, with a reported prevalence of dizziness of approximately 30% of older adults, affecting more women (36%) than men (22%). Importantly, its prevalence rises with age2. Consequently, dizziness leads to a significant personal and health care burden. It is associated with a risk of falls, an increased level of fear and anxiety, and decreased independence in older people. In a recent study, Tinetti et al.3 reported adverse health, functional and psychological consequences due to chronic dizziness in patients aged 72 years or older. It was concluded over one year of follow-up that chronic dizziness was associated with increased risk of falling, worsened depressive symptoms, self-reported health and decreased confidence in performing social activities.\n\nDizziness may be classified into four categories: vertigo, pre-syncope, disequilibrium and psycho-physiological1. Vertigo means a rotatory movement either of the patient or of their surroundings. It is generally provoked by head movements, such as movements in bed like turning over or looking upwards. Vertigo suggests disturbances of the vestibular system4. Pre-syncope is a medical term defining an imminent loss of consciousness. Patients may feel that they are about to faint, often accompanied by nausea or sweating4. Disequilibrium refers to a sense of imbalance or unsteadiness, and being in a dark room or closing eyes generally deteriorates symptoms. It is attributed to somatosensory abnormality, visual illusions or cerebellar disorders4. Psycho-physiological symptoms are a combination of sensations such as swimming, floating and rocking or a feeling of being removed from one’s body. This symptom can be created by anxiety and central disorders4.\n\nDizziness may occur due to numerous diseases or disorders. The causes of dizziness can be grouped into otological, neurological and medical disorders. Otological causes consist of middle ear disease, unilateral peripheral vestibular dysfunction, benign paroxysmal positional vertigo (BPPV), bilateral vestibular failure, Ménière’s syndrome, and benign recurrent vertigo. Neurological causes include the 7th cranial nerve, cranio-cervical junction, cerebellum, and cortex problems. Furthermore, dizziness is associated with general medical issues such as orthostatic hypotension, low cardiac output, hyperventilation, hypoglycaemia, anaemia and psychological extremes1. Patients benefit from a full neuro-otological examination, explanation of their symptoms and appropriate reassurance. Even though medical management of dizziness depends on the diagnosis, numerous treatments such as vestibular rehabilitation (VR)4, medication5, cognitive behavioural therapy (CBT)6, Tai Chi exercises7 and surgery5 have been advocated as management strategies.\n\nVestibular rehabilitation is a specific form of exercise-based therapy programme aimed at alleviating the primary and secondary problems of a vestibular pathology. The original protocols date back to 1946, when Cawthorne and Cooksey defined hierarchical group activities according to their difficulties to challenge the central nervous system. These exercises are mainly based on graded activities including eye, head and body movements stimulating the vestibular system8. Today, specific exercises have been improved and further described in the VR therapy, with each exercise, such as habituation, adaptation and substitution, based on different physiological and behavioural rationales. Habituation exercises, also called compensatory exercises, use repetitive movements or provoking stimuli. The movements that provoke patients’ symptoms are identified and the patient performs these exercises until they no longer respond adversely to the stimuli. Adaptation exercises are repeated head and eye movements which aid the central nervous system by adapting to a loss or an alteration in vestibular system input. The head is moved to the left and right side, while the eyes are kept fixed on a stationary target. Substitution exercises organise the use of the remaining sensory inputs to aid postural control. It is aimed at the patient using an intact sensory system instead of a sensory system providing no input or error message for the central nervous system4.\n\nThe management of dizziness is important issue in primary care at the systemic level9. The occurrence of dizziness, in addition to impacting directly on patient health and quality of life, is associated with a large economic burden on the health care system. The total national cost of patients with dizziness visiting the emergency department (ED) in the US exceeds $4 billion annually, representing approximately 4% of total ED costs10. In spite of the increase in use of VR, currently, there is a lack of literature investigating the effectiveness of exercise-based VR in adult patients with chronic dizziness. Therefore, the aim of this systematic review is to appraise the efficacy of exercise-based VR in adult patients with chronic dizziness. Previous reviews attempted to investigate either the effectiveness of VR on all types of chronic vestibular disorders11 or general management of the patient with chronic dizziness12. This systematic review (SR) aims to provide a summary of the evidence on the effectiveness of specifically exercise-based VR in adult patients with chronic dizziness.\n\n\nMethods\n\nThe literature search was performed by two independent researchers (B.K. and A.S.) for articles published in English. For the identification of relevant studies, the following databases were used from their establishment date as databases until January 2017: the Cochrane Central Register of Controlled Trials (CENTRAL, Cochrane Library), MEDLINE, PubMed, the Physiotherapy Evidence Database (PEDro) and Scopus (Elsevier). The key search terms used were “dizziness”, “chronic dizziness”, “vestibular rehabilitation”, “habituation exercise” and “adaptation exercise”, modified in terms of the glossary of each database and combined using Boolean operators. Table 1 shows the search strategy for one of the selected databases (PubMed). The titles and abstracts were examined according to the inclusion and exclusion criteria separately by two authors (B.K. and A.S.). In case of any doubt, the full text of an article was read to determine its relevance. Furthermore, reference lists of all identified articles were screened for additional relevant literature. After that, if there was a disagreement between the two authors (B.K. and A.S.), a third author (A.J.T.) was consulted.\n\nThe articles were included if they were based on the following inclusion criteria: (1) randomised controlled trials (RCTs), (2) people with chronic dizziness, whereby chronicity means longer than three months, or studies determined the case as “chronic” in the title, (3) adults aged 18 or over, (4) exercise-based VR, (5) VR exercises compared with sham or usual care, non-treatment or placebo and (6) only studies published full text in English. Articles with the following criteria were excluded: (1) non-randomised studies or non-controlled studies, (2) patients who have chronic dizziness due to only specific diagnosed causes, such as BPPV, migraine-associated vertigo or Ménière's disease, (3) studies including patients younger than 18, (4) passive VR (Canalith repositioning manoeuvres), (5) studies comparing the effectiveness of two different VR exercises and (6) non-English language studies.\n\nThe following key data were independently extracted from the included articles by the two review authors (B.K. and A.S.). The data extracted included participant characteristics (population, number of participants, age and gender), types of intervention (intervention group and control group), types of outcome measures (primary and secondary outcomes) and the main findings. The methodological quality of the included studies and risk of bias were assessed according to the PEDro scale, which is commonly used in SRs13. It was reported that the PEDro scale has valid and reliable criteria to measure the study quality of RCTs14. The PEDro scale consists of a checklist of 11 questions regarding validity and interpretation of results of controlled trials and it is rated by assigning one point according to the positive answers, with the total number of positive answers scored out of 1015. The first question is not used to calculate the PEDro score as it shows the sample eligibility criteria. Higher scores on the PEDro scale demonstrates a more appropriate study design. Articles were scored independently by the two authors (B.K. and A.S.) and any discrepancies between the authors in the early stages of selection and assessment of risk of bias of the articles were resolved by consensus or if required, consultation with the third author (A.J.T.). The quality of studies was rated in accordance with the following scoring system, taken from prior SRs16,17, ≤3 low quality, 4-5 fair quality and ≥6 high quality.\n\n\nResults\n\nA total of 376 electronic publication records were retrieved from the systematic database search strategy. After 206 duplicates were removed, the remaining 170 articles were investigated through the title and abstract assessment and 16 records were identified as potentially eligible. After full text analysis, 12 of the remaining full text articles did not meet the inclusion criteria and were excluded because of the reasons such as language, chronicity, specific diagnosed dizziness, and lack of randomisation. As a result, a total of four articles18–21 met the inclusion criteria. The flow of studies and the reasons for exclusion are demonstrated in a PRISMA diagram (Figure 1).\n\nThree of the included articles originated from the UK and one was from the US. All the included studies were RCTs. A total of 687 participants were included, predominantly females (n=503). In general, the diagnosis of dizziness was non-specific, however a few causes reported included Ménière's disease, BPPV and labyrinthitis. In three studies18,20,21, exercises were delivered under the supervision of trained nurses, and in one study19, booklet-based exercises were used. All forms of VR which are compensatory, adaptation or substitution exercises were selected as the main intervention. These exercises were compared with usual medical care in three studies19–21, while they were evaluated versus placebo eye exercises in one study18. The most common outcome measure was self-reported dizziness measured by the Vertigo Symptom Scale, Visual Analogue Scale or Dizziness Handicap Inventory. Other outcome measures included gait speed (Dynamic Gait Index (DGI)), balance (Postural Stability Eyes Open and Closed, Romberg Test), anxiety and depression (Hospital Anxiety and Depression Scale), quality of life (Short Form-36) and other tests (computerised dynamic posturography, customised computerised software). Table 2 describes the main study characteristics of the included studies.\n\nIG - Intervention Group; CG - Control Group; n - Number; DGI - Dynamic Gait Index; VR - Vestibular Rehabilitation; QALY - Quality Adjusted Life Year.\n\nAs mentioned before, the methodological quality of the included studies and risk of bias were evaluated by using the PEDro criteria, whereby the greater the PEDro score, the more convenient the study design. Consequently, according to the PEDro assessment conducted by the authors (B.K. and A.S.), the results demonstrated that three studies were rated as high quality, and one was fair quality. Table 3 shows the quality appraisal of included studies in detail. A lower scoring criteria amongst studies also indicates more risk of bias. The common criteria that the included articles failed to meet were allocation concealment, blinding of participants’ therapists or outcome assessors in accordance with the PEDro scale. Hence, the highest risk of bias for these articles might be associated with selection and detection.\n\nAll studies in this review differ in outcome measures used, comparator interventions or delivery method. Even though a meta-analysis was planned, it was not possible to perform it due to heterogeneity. Hence a descriptive, narrative synthesis of the main findings and quality of the included articles will be presented in this SR.\n\nThe main findings of each study can be found in Table 2. Only one study18 did not present confidence intervals (CI) for between group differences. The other three studies19–21 reported confidence intervals for the within group change scores.\n\nAll the included studies18–21 assessed whether there was an improvement in dizziness symptoms, as measured by the Vertigo Symptom Scale19–21, the Visual Analogue Scale (VAS)18 or the Disability Rating Scale18. Three studies19–21 reported that participants treated with VR demonstrated statistically significant improvement compared with participants treated with usual medical care. However, the study of Hall et al.18 did not detect any significant change in the dizziness VAS score.; the magnitude of change in the dizziness VAS score was small for the gaze stabilisation group, and moderate for the placebo eye exercise group (standardised response mean (SRM) = 0.4 and 0.7, respectively).\n\nBalance and risk of falls were investigated by the Romberg Test19–21, Vertigo Balance Scale19, Dynamic Gait Index18 and computerised dynamic posturography18. All studies found significant differences in balance and risk of falls, with the improvement in all balance and risk of falls tests being significantly greater in the VR group compared to the routine medical care.\n\nAnxiety and depression were evaluated using Anxiety and Depression Scale19–21, Geriatric Depression Scale or State-Trait Anxiety Inventory18. While three studies19–21 demonstrated significant improvement in anxiety and depression, Hall et al.18 could not find any significant difference.\n\n\nDiscussion\n\nThe use of exercise-based VR has been investigated in terms of its ability to manage adult patients with chronic dizziness. The literature search confirmed the dearth of RCTs evaluating the efficacy of VR. However, despite the scarcity of studies, the articles included in this SR suggested that VR might be an area for future research with regard to severity of dizziness, fall risk, handicap, postural control and emotional status. However, owing to the differences in the methodologies of included studies, it was not possible to determine the most effective VR protocol, or the ideal frequency and intensity of the interventions.\n\nIn this context, the databases mentioned previously were scrutinised to identify relevant literature. Four studies18–21 were found to be eligible in terms of participant characteristics, study design, interventions and comparators. Three of these studies were rated as high quality, whereas one was rated as fair quality in accordance with the PEDro scale (Table 3). These studies commonly failed to meet criteria regarding allocation concealment, blinding of participants, therapists or assessors. Therefore, even though these studies were determined as adequate quality, there was a risk of selection as well as detection bias, which may lead to significant alteration in the findings.\n\nOther limitation of these studies was that they most commonly used subjective outcome measures such as Vertigo Symptom Scale, VAS, and Short Form-36. Nonetheless, the objective outcome measures were significant to ensure reliable rationale for treatment. Furthermore, they may demonstrate significant limitations quantifying objectively. On the other hand, subjective outcome measures are based on the perception of the individual with regards to the symptoms. The Hall et al.18 study contained a very small cohort (n = 37), which makes it difficult to generalise the results to a wide range of dizzy patients.\n\nThe effectiveness of VR in dizziness has been currently evaluated by several studies11,22,23. However, previous systematic reviews evaluated the effect of VR very generally11,23 or focused on only passive repositioning manoeuvres22. For instance, a SR conducted by Kendall et al.23 evaluated all non-pharmacological interventions and did not focus on only VR, while Ricci et al.11 studied the effectiveness of VR for all vestibular problems and dizziness in general. Given the lack of targeted studies in this area, this SR aimed to critically appraise the existing evidence and provide further information regarding the effectiveness of exercise-based VR to tackle chronic dizziness in adult patients. To the authors’ knowledge, this study is the first SR investigating the effectiveness of exercise-based VR for chronic dizziness in adult patients.\n\nThe authors’ acknowledge several weaknesses which exist within this SR. Firstly, five databases were investigated to identify relevant literature regarding VR and the research process did not include grey literature. Therefore, it is possible some relevant studies were overlooked. Secondly, studies published in languages other than English were excluded, but there might have been some relevant and valuable studies in those non-English articles (language bias). Finally, studies in this SR generally compared the effectiveness of VR interventions that were nurse-delivered or booklet-based, without supervision, other types of VR protocols may not have been represented in the literature. It may have been preferable to compare the same delivery method or similar exercise protocols. Consequently, there is a need for future studies, including homogenous samples.\n\nThe subjective measurement of symptoms was the most commonly used outcome measure in all the included studies. Three of the studies19–21 used the Vertigo Symptom Scale, whereas one study18 assessed the alteration in symptoms using the Disability Rating Scale and the VAS. Yardley et al.21 reported that the relative risk of the improvement in symptoms of the VR group versus the usual medical care group was 1.78 (95% CI, 1.31 to 2.42), while 56 out of 83 participants (67%) in the VR group reported easing in symptoms, in the usual medical care group it was only reported by 33 of 87 patients (38%) at the three-month follow-up.\n\nHall et al.18 were unable to find significant difference between the gaze stabilisation exercise group and control group to whom placebo eye exercises were applied, except for fall risk measured by Dynamic Gait Index (DGI) (p = 0.026). However, in this study, the paucity of the number of participants (n = 37) and the compliance of patients make the findings questionable. Firstly, the number of patients was limited in this study. Secondly, patients in the control group were compliant with the placebo eye exercises eighty percent of the time, while in the gaze stabilisation group, the compliance of the patients was sixty percent of the time (p = 0.04). This result is consistent with the study of Shumway-Cook et al.24, which concluded that patients who are compliant with the therapy sessions less than 75% of the time demonstrated less improvement compared the patients who are fully adherent to therapy. Last, this difference in the findings might arise due to the duration of experiment. Participants were assessed at baseline and at discharge, which was only 2–3 weeks for many participants. Likewise, another included study by Yardley et al.19 failed to reach significance at 12 weeks, while it found a clear improvement compared to the routine care group at one year follow-up. Therefore, long-term outcomes might be more effective than short-term ones.\n\nAnother outcome measure was the postural control evaluated by using static (Romberg Test)20,21 and dynamic (DGI)18 balance tests and computerised dynamic posturography18. Although the static balance tests are easy to apply, they are deficient in the evaluation of functional aspects of balance and mobility. Functional balance tests may complete these aspects providing information regarding characteristics, gait and balance. However, it is also limited in determining muscle tightness or weakness and motor coordination problems, which are important aspects of an individualised treatment plan. Conversely, computerised dynamic posturography might assess all of these signs and is useful for determining the diagnosis of vestibular disorder and an appropriate treatment plan to complete other conventional balance tests.\n\n\nConclusion\n\nThis review suggests that exercise-based VR shows benefits for adult patients with chronic dizziness in terms of improvement in the vertigo symptom scale, fall risk, balance and emotional status. Three out of four studies demonstrated a significant difference in the VR group compared to the usual medical care group. Furthermore, it was also suggested that VR is cost-effective, either by direct delivery in primary care units or via a booklet-based delivery. However, this study could not determine the most effective VR protocol or the optimal treatment frequency and intensity, so further studies are required to elucidate the optimal methods of delivery and dosages.\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\nSupplementary material\n\nSupplementary File 1: Completed PRISMA checklist.\n\nClick here to access the data.\n\n\nReferences\n\nMurdin L, Davies R: Dizziness. Medicine. 2008; 36(10): 535–539. Publisher Full Text\n\nLee R, Elder A: Dizziness in older adults. Medicine. 2013; 41(1): 16–19. Publisher Full Text\n\nTinetti ME, Williams CS, Gill TM: Health, functional, and psychological outcomes among older persons with chronic dizziness. J Am Geriatr Soc. 2000; 48(4): 417–421. PubMed Abstract | Publisher Full Text\n\nAlrwaily M, Whitney SL: Vestibular rehabilitation of older adults with dizziness. Otolaryngol Clin North Am. 2011; 44(2): 473–496. PubMed Abstract | Publisher Full Text\n\nDerebery MJ: The diagnosis and treatment of dizziness. Med Clin North Am. 1999; 83(1): 163–177. 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Acad Emerg Med. 2013; 20(7): 689–696. PubMed Abstract | Publisher Full Text\n\nRicci NA, Aratani MC, Doná F, et al.: A systematic review about the effects of the vestibular rehabilitation in middle-age and older adults. Rev Bras Fisioter. 2010; 14(5): 361–371. PubMed Abstract | Publisher Full Text\n\nBronstein AM, Lempert T: Management of the patient with chronic dizziness. Restor Neurol Neurosci. 2010; 28(1): 83–90. PubMed Abstract | Publisher Full Text\n\nda Costa BR, Hilfiker R, Egger M: PEDro's bias: summary quality scores should not be used in meta-analysis. J Clin Epidemiol. 2013; 66(1): 75–77. PubMed Abstract | Publisher Full Text\n\nde Morton NA: The PEDro scale is a valid measure of the methodological quality of clinical trials: a demographic study. Aust J Physiother. 2009; 55(2): 129–133. PubMed Abstract | Publisher Full Text\n\nYamato TP, Maher C, Koes B, et al.: The PEDro scale had acceptably high convergent validity, construct validity, and interrater reliability in evaluating methodological quality of pharmaceutical trials. J Clin Epidemiol. 2017; 86: 176–181. PubMed Abstract | Publisher Full Text\n\nHerd CR, Meserve BB: A systematic review of the effectiveness of manipulative therapy in treating lateral epicondylalgia. J Man Manip Ther. 2008; 16(4): 225–237. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMay S, Comer C: Is surgery more effective than non-surgical treatment for spinal stenosis, and which non-surgical treatment is more effective? A systematic review. Physiotherapy. 2013; 99(1): 12–20. PubMed Abstract | Publisher Full Text\n\nHall CD, Heusel-Gillig L, Tusa RJ, et al.: Efficacy of gaze stability exercises in older adults with dizziness. J Neurol Phys Ther. 2010; 34(2): 64–69. PubMed Abstract | Publisher Full Text\n\nYardley L, Barker F, Muller I, et al.: Clinical and cost effectiveness of booklet based vestibular rehabilitation for chronic dizziness in primary care: single blind, parallel group, pragmatic, randomised controlled trial. BMJ. 2012; 344: e2237. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYardley L, Beech S, Zander L, et al.: A randomized controlled trial of exercise therapy for dizziness and vertigo in primary care. Br J Gen Pract. 1998; 48(429): 1136–1140. PubMed Abstract | Free Full Text\n\nYardley L, Donovan-Hall M, Smith HE, et al.: Effectiveness of primary care–based vestibular rehabilitation for chronic dizziness. Ann Intern Med. 2004; 141(8): 598–605. PubMed Abstract | Publisher Full Text\n\nHelminski JO, Zee DS, Janssen I, et al.: Effectiveness of particle repositioning maneuvers in the treatment of benign paroxysmal positional vertigo: a systematic review. Phys Ther. 2010; 90(5): 663–678. 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}
|
[
{
"id": "31549",
"date": "16 Mar 2018",
"name": "Shamekh Mohamed El-Shamy",
"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 the invitation to review this paper.\n\nGeneral comment:\nThis paper aimed ‘to investigate the effectiveness of exercise-based vestibular rehabilitation in adult patients with chronic dizziness.' Generally, the manuscript content was well-written. The introduction section was concise and clear. The methods and results sections were well-structured. The discussion section was well-explained.\nSpecific comments: Strengths:\nIntroduction section: Generally, the introduction section covered all necessary data, including: definition of dizziness, epidemiology, impact of dizziness on society, classifications of dizziness and their definitions, etiologies of dizziness and its symptoms, brief summary about available interventions, vestibular rehabilitation, the importance of this review and the aim. Methods section: Reporting the data sources, an example of search strategy in detail that was presented in table 1 which showed the specific terms that used in the search process, inclusion and exclusion criteria, data extraction, risk of bias assessment, data synthesis and analysis. Result section: Study selection and characteristics were reported clearly, risk of bias assessment, and reporting the results and divided them based on the outcome measures which was easy to read and follow. Discussion section: interpretation of the results and reporting some of the limitations of the study.\n\nWeaknesses:\nGrey literature was not included in the search process. The authors did not report the effect size for each study to determine the effectiveness level of the intervention. The authors assessed the risk of bias for each included study, but they did not report the overall quality of a body of evidence. The discussion section did not include enough supporting evidence.\n\nGenerally, although the existence of these weaknesses, there are no necessary changes to the content of the manuscript.\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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
},
{
"id": "33648",
"date": "14 May 2018",
"name": "Alia Alghwiri",
"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 systematic review is an interesting paper that highlights the effectiveness of exercise-based vestibular rehabilitation in adult patients with chronic dizziness.\nThe rationale behind conducting this paper is clear.\nThe methods section has sufficient and clear details.\nThe results of the study was stated clearly and a summary of the findings was provided in a good way.\nThe discussion and conclusion provide adequate information that is supported by the results.\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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-276
|
https://f1000research.com/articles/7-275/v1
|
05 Mar 18
|
{
"type": "Research Article",
"title": "Significance of intra-operative blood pressure data resolution: A retrospective, observational study",
"authors": [
"Senthil Packiasabapathy",
"Ammu T. Susheela",
"Fernando Mujica",
"Balachundhar Subramaniam",
"Senthil Packiasabapathy",
"Ammu T. Susheela",
"Fernando Mujica"
],
"abstract": "Background: With evolving techniques for analysis of blood pressure (BP) variability, the importance of sampling resolution for intra-operative BP still remains to be examined. This study aims at comparing BP data with beat-by-beat vs. 15 second resolution. Methods: This is a retrospective analysis of intra-arterial BP data obtained from cardiac surgical patients from the intra-operative period. Data was collected from two sources for each patient, one with beat-by-beat frequency, other at a frequency of once every 15 seconds. The fraction of time and area under the curve beyond systolic BP thresholds of 95 – 135 mmHg were calculated using data from both sources, for each patient. These were compared using Wilcoxon ranked sum test for paired samples using R-statistics version 3.4.3. Results: There was a statistically significant difference (P < 0.001) between the parameters from the two sources. This was especially true for parameters below and outside the thresholds. Only time fraction showed significant difference above the 135 mmHg threshold. Conclusion: Our preliminary analysis shows a definitive difference between BP descriptors, depending on sampling resolution. But the impact of this difference on the outcome predicting models of the parameters stands to be ascertained. Future larger studies, powered to examine the impact of sampling resolution on outcome predictive ability of BP descriptors, with special emphasis on dynamic markers of complexity are warranted.",
"keywords": [
"Blood pressure",
"Beat by beat sampling",
"Cardiac surgery",
"Cardiopulmonary bypass"
],
"content": "Introduction\n\nIntra-operative blood pressure (BP) analysis has gained popularity not only because it forms a target for intervention and optimization, but also because of its predictive ability for major post-operative adverse events1. Technology has contributed to the improved accuracy of invasive real-time BP monitoring, with high temporal resolution. But we do not yet know how much temporal resolution is an adequate resolution, for various BP analyses. This observational study was designed to address the above-said issue.\n\nMany analytical techniques have been explored to decipher the information provided by the BP waveform. The journey began from absolute BP cut-offs to population thresholds, percentage changes from baseline1,2 to static3,4 and dynamic properties5 of the BP waveform. For this study, we computed the duration and area under the curve (AUC) beyond a certain BP threshold, as described by Aronson et al.3 They used invasive systolic BP (SBP) data, sampled every 30 seconds. The threshold used for final analysis was 95 and 135 mmHg of SBP. BP excursions beyond these thresholds were analysed in terms of the number of excursions and duration and magnitude of excursions, which were used to calculate the AUC above and below the thresholds. They found that the duration of excursion showed the most significant association with 30-day mortality. For every minute of excursion outside the threshold, the odds ratio for an increase in mortality was shown to be 1.03, using a multiple logistic regression model.\n\nBeat-by-beat (BBB) sampling provides a voluminous quantity of data to store and analyse. But we do not know if this would significantly improve the outputs of various analytical methods. We sought to clarify the importance of the sampling resolution in BP variability analyses. We hypothesized that SBP parameters (time and AUC outside the range of 95–135 mmHg) computed from BBB BP data would be significantly different from the same descriptors derived from low-resolution BP data, sampled every 15 seconds.\n\n\nMethods\n\nThis is a retrospective, observational, single center, exploratory analysis conducted at Beth Israel Deaconess Medical Center, after the approval of the Institutional Review Board (approval number: 2008P000478). BP data was collected from 200 patients who underwent cardiac surgery between January 2008 and June 2014, from an ongoing NIH funded study (RO1GM098406), after verbal informed consent.\n\nThis manuscript adheres to the STROBE statement guidelines.\n\nAll patients over 18 years of age, undergoing elective cardiac surgery under cardiopulmonary bypass were considered eligible for BP data collection. This cohort was chosen because of the highly protocolized nature and the relatively tighter peri-operative BP control employed in the setting of a cardiac surgery. Baseline parameters such as age, gender, comorbidities, medications, nature of surgery, and risk scores were also collected.\n\nAll the patients had a radial arterial catheter inserted during the pre-operative period. BBB waveforms were securely exported from OR Philips monitors (Philips Medical, Andover, MA). BP data sampled every 15 seconds was also collected for all the patients via the institution’s Anesthesia Information Management System (AIMS) (CompuRecord, Philips Healthcare, Andover, MA, USA). The BP data was pre-processed to remove artefacts. After excluding BP data of insufficient quality and length, 193 datasets were included in the final analysis.\n\nWe calculated the duration and AUC (magnitude times duration of excursions) of SBP outside 95–135 mmHg range. This included duration and AUC above, below and outside the SBP range. These parameters were calculated both from BBB and AIMS data for each patient. To standardize for the differences in the duration of data acquisition between BBB and AIMS, time fraction outside the thresholds was used for final analysis, rather than the absolute duration.\n\nStatistical testing was performed using R version 3.4.3. Data are presented as median (interquartile range) or n (%) depending upon the variable. Shapiro Wilk Test was used to test for normality. Wilcoxon Rank-Sum test for paired samples was used to compare BP descriptors between BBB and AIMS data.\n\n\nResults\n\nThe baseline characteristics of the patient cohort are described in Table 1. We were able to find a statistically significant difference in AUC and time fraction between BBB and AIMS data, below (< 95 mmHg) and outside the range (P < 0.001; Table 2). A similar significant difference was also found with the fraction of time above the threshold (P =0.03; Table 2). The AUC above the range (>135 mmHg) was similar between BBB and AIMS data. In general, both the descriptors had lower values when calculated from BBB data, compared to AIMS data.\n\n* Variables are presented as N (%), mean ± SD, or median (IQR) based on the type and distribution.\n\nBP, blood pressure; BBB, beat-by-beat; AIMS, Anesthesia Information Management System; AUC, area under the curve.\n\n* Statistical significance P < 0.05.\n\n** In mmHg per minute.\n\nAll values are median (IQR).\n\n\nDiscussion\n\nThe time fraction and AUC of SBP excursions were significantly lower with the BBB data when compared to the AIMS data. This could be explained by the fact that the data points in the AIMS data represent values averaged over 15 seconds. The 15 seconds duration between subsequent data points in AIMS data would accommodate approximately 15 or more BBB data points depending on the heart rate and this extrapolation could account for the relatively larger time fraction and AUC obtained from AIMS data. AUC below and outside the range showed significant difference (P < 0.001) between the two sources. Fraction of time above, below and outside the range showed significant difference (Table 2).\n\nThe descriptors used in this study are static parameters and do not take into account the temporal structure of the BP waveform. Hence intuitively, the impact of sampling frequency should be relatively less. But when we consider measures such as the multi-scale entropy5,6, non-linearity7, which measure the temporal dynamics and complexity of the BP waveform, BBB data would prove to be significantly more accurate compared to lower resolution data. This could be a topic for future studies.\n\nOur study is limited by the sample size included in the analysis. For the same reason, it was underpowered to analyse outcome correlation of the parameters studied. Also, only two BP parameters were used in the study among a number of other parameters described in the literature.\n\n\nConclusions\n\nWe were able to show that the BP parameters differed significantly depending on the frequency of source data acquisition. Future directions include studies that are adequately powered to test the impact of sampling resolution on the ability of the parameters to predict outcomes, as well as analyzing the significance of data resolution in computing other BP parameters of variability and complexity.\n\n\nData availability\n\nDataset 1: Variables collected throughout this study for the 193 participants. 10.5256/f1000research.13810.d1961068",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nBalachundhar Subramaniam, MD, MPH, is supported by the National Institutes of Health, Research Project Grant GM 098406.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nMonk TG, Bronsert MR, Henderson WG, et al.: Association between Intraoperative Hypotension and Hypertension and 30-day Postoperative Mortality in Noncardiac Surgery. Anesthesiology. 2015; 123(2): 307–19. PubMed Abstract | Publisher Full Text\n\nSalmasi V, Maheshwari K, Yang D, et al.: Relationship between Intraoperative Hypotension, Defined by Either Reduction from Baseline or Absolute Thresholds, and Acute Kidney and Myocardial Injury after Noncardiac Surgery: A Retrospective Cohort Analysis. Anesthesiology. 2017; 126(1): 47–65. PubMed Abstract | Publisher Full Text\n\nAronson S, Stafford-Smith M, Phillips-Bute B, et al.: Intraoperative systolic blood pressure variability predicts 30-day mortality in aortocoronary bypass surgery patients. Anesthesiology. 2010; 113(2): 305–12. PubMed Abstract | Publisher Full Text\n\nLevin MA, Fischer GW, Lin HM, et al.: Intraoperative arterial blood pressure lability is associated with improved 30 day survival. Br J Anaesth. 2015; 115(5): 716–26. PubMed Abstract | Publisher Full Text\n\nSubramaniam B, Khabbaz KR, Heldt T, et al.: Blood Pressure Variability: Can Nonlinear Dynamics Enhance Risk Assessment During Cardiovascular Surgery? J Cardiothorac Vasc Anesth. 2014; 28(2): 392–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCosta M, Goldberger AL, Peng CK: Multiscale entropy analysis of complex physiologic time series. Phys Rev Lett. 2002; 89(6): 068102. PubMed Abstract | Publisher Full Text\n\nGoldberger AL: Giles f. Filley Lecture. Complex Systems. Proc Am Thorac Soc. 2006; 3(6): 467–471. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPackiasabapathy S, Susheela AT, Mujica F, et al.: Dataset 1 in: Significance of intra-operative blood pressure data resolution: A retrospective, observational study. F1000Research. 2018. Data Source"
}
|
[
{
"id": "31519",
"date": "15 Mar 2018",
"name": "Janet M.C. Ngu",
"expertise": [
"Reviewer Expertise Cardiac Anesthesiology",
"Hemodynamic monitoring",
"Epidemiology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis well written manuscript compared blood pressure data collected using two sampling resolutions: beat-by-beat contours vs. numeric data captured q15 seconds. This study represents an important first step to validating and comparing hemodynamic data obtained using different sampling methods and proposes the concept of time fractions as a standardizing method to account for case duration. The authors provided a thoughtful discussion of main findings. As the authors suggested, future studies of the association between different sampling methods and outcomes would be very interesting and a welcomed addition to the literature\n\nWe have only minor suggestions:\nCan the authors briefly explain what constituted artefacts and how artefacts were removed from BBB tracings and from the AIM data? Also did these algorithms produce consistent removal of artefacts in both BP recording modalities? The authors are to be commended for their use of time fraction. Can the authors clarify the method of calculation for time fraction (i.e., was case duration used as the denominator)? Looks like although the median AUC values were very different between the BBB and AIMS groups, their corresponding time fractions were not very different (despite statistical significance).\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": "3532",
"date": "22 Mar 2018",
"name": "Senthil Packiasabapathy",
"role": "Author Response",
"response": "Thank you reviewers, for your approval and kind comments!1. Can the authors briefly explain what constituted artefacts and how artefacts were removed from BBB tracings and from the AIM data? Also did these algorithms produce consistent removal of artefacts in both BP recording modalities?An automated algorithm was used to eliminate artifacts. It excluded SBP values < 50 mm Hg and > 250 mm Hg; DBP values < 20 mm Hg and > 150 mm Hg; DBP ≥ SBP, and SBP – DBP < 10 mm Hg. Yes this was used for both BP sources.2. The authors are to be commended for their use of time fraction. Can the authors clarify the method of calculation for time fraction (i.e., was case duration used as the denominator)?Thank you, Time fraction was used because the total duration of BP data acquisition could be different for BBB and AIMS. The total duration for which BP data was acquired was used as the denominator.3. Looks like although the median AUC values were very different between the BBB and AIMS groups, their corresponding time fractions were not very different (despite statistical significance).Yes, agreed. AUC takes into account both the magnitude and duration of the BP excursions. As we see there is not a lot of difference in the time fraction (despite statistical significance), it is possible that the difference in the magnitude between BBB and AIMS has contributed to the differences in AUC."
}
]
},
{
"id": "34645",
"date": "15 Jun 2018",
"name": "Goverdhan Dutt Puri",
"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 have only following suggestions/questions\nThough the absolute difference in fraction of time for the two methods of data collection is very small (Table2) but the difference in AUC beyond specified thresholds is almost double. Is it due to difference in the valid time period of analysis by two different methods i.e. BBB and AIMS? In that case it would be better that the time period analyzed in each patient by two different methods be also depicted - the explanation given by authors is not satisfactory. Looking at the time period of analysis by two methods may clarify it. Is the Philips monitor sending the data for AIMS every 15 second or every 5 seconds? Is the data averaged after collection at server or before being sent from peripheral monitor to central server? How is area under curve calculated for AIMS data? If the peak of wave was SBP, what was time dimension – is it the total systolic time or total epoch time of 15 sec? As no waveform was available in this case. Or was it the SBP above the threshold multiplied by the time till next SBP value comes within the threshold? Why was MAP not chosen?\n\nOtherwise it’s a well written manuscript with great future implications.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3746",
"date": "20 Jun 2018",
"name": "Senthil Packiasabapathy",
"role": "Author Response",
"response": "Thank you for your approval and kind comments! Please find the response to your queries below. 1. Though the absolute difference in the fraction of time for the two methods of data collection is very small (Table2) but the difference in AUC beyond specified thresholds is almost double. Is it due to the difference in the valid time period of analysis by two different methods i.e. BBB and AIMS? In that case, it would be better than the time period analyzed in each patient by two different methods be also depicted - the explanation given by authors is not satisfactory. Looking at the time period of analysis by two methods may clarify it.Thank you for your question. Yes. The area under the curve values is almost double due to the difference in valid time period analysis. The various values of the fraction of time as a part of time period analysis is given under the data sheet in excel in the article. 2. Is the Philips monitor sending the data for AIMS every 15 second or every 5 seconds? Is the data averaged after collection at server or before being sent from peripheral monitor to central server?Thank you. The OR Philips monitor secured the beat by beat data while the Anesthesia Information Management System (AIMS) collected the blood pressure data every 15 seconds. 3. How is area under curve calculated for AIMS data? If the peak of wave was SBP, what was time dimension – is it the total systolic time or total epoch time of 15 sec? As no waveform was available in this case. Or was it the SBP above the threshold multiplied by the time till next SBP value comes within the threshold?Thank you. Area under curve (AUC) was analyzed as the summation of the integrated SBP-time curve excursions, capturing the product of magnitude (mm Hg) and duration (min) of BP outside the predefined SBP ranges. So it was total epoch time of 15 seconds. 4. Why was MAP not chosen?Thank you. We chose intraoperative systolic blood pressure over mean arterial pressure following the methodology mentioned in the article by Aronson et al.1Reference: “Aronson S, Stafford-Smith M, Phillips-Bute B, et al.: Intraoperative systolic blood pressure variability predicts 30-day mortality in aortocoronary bypass surgery patients. Anesthesiology. 2010; 113(2): 305–12.”"
}
]
}
] | 1
|
https://f1000research.com/articles/7-275
|
https://f1000research.com/articles/7-273/v1
|
05 Mar 18
|
{
"type": "Case Report",
"title": "Case Report: Complex ureteral stenosis treated with ileal substitution",
"authors": [
"Raquel Catarino",
"André Cardoso",
"Carlos Ferreira",
"Diogo Pereira",
"Tiago Correia",
"Manuel Cerqueira",
"Frederico Carmo Reis",
"Rui Prisco",
"André Cardoso",
"Carlos Ferreira",
"Diogo Pereira",
"Tiago Correia",
"Manuel Cerqueira",
"Frederico Carmo Reis",
"Rui Prisco"
],
"abstract": "Ileal substitution of the ureter is a complex procedure, considered a surgery of the last resort in ureteral repair and is useful in the presence of an extensive ureteral stricture. It is indicated in cases of long or multiple ureteral stenosis. There are few large studies in the literature reporting the outcome of this procedure. We present a case report of a patient with long ureteral stenosis surgically treated with ileal substitution of the right ureter, with an isoperistaltic ileal segment of 22 cm, with no detubularization. The patient had no perioperative complications and presented normal renal function. Currently, after 20 months of follow-up, the patient is asymptomatic, presents no urinary infections, no relapse of stenosis and has preserved renal function. In conclusion, ileal substitution of the ureter is a surgical technique that should be considered in cases of long, proximal or multiple ureteral stenosis, when there is no other surgical option.",
"keywords": [
"Ureteral stenosis",
"Ileal ureteral substitution",
"Bowel ureteral replacement",
"reconstructive urology."
],
"content": "Introduction\n\nUreteral stenosis is associated with ischemia, iatrogenic lesions, periureteral fibrosis, malignancy, congenital factors, radiation, urinary lithiasis, infections (namely tuberculosis and schistosomiasis), and idiopathic conditions. After the diagnosis of a ureteral stricture, surgical treatment is indicated if the patient presents clinical symptoms and a functionally significant obstruction1.\n\nThere are several options for the surgical treatment of ureteral stricture disease. Endourological interventions can be performed in short ureteral strictures and include ureteral stent placement, balloon dilation and endoureterotomy. Surgical repair options are more complex and need a careful evaluation of the nature, location and length of the stricture, in order to plan the appropriate surgical procedure. Surgical repair options include ureteroureterostomy, ureteroneocystostomy, Psoas Hitch procedure, Boari flap, downward nephropexy, transureteroureterostomy, bowel substitution and autotransplantation.\n\nIn case of long, proximal or multiple ureteral stenosis, the defect can be bridged with non-urothelial tissues, including bowel substitution1–3. Ileal substitution of the ureter is a complex procedure, considered a surgery of the last resort in ureteral repair and is useful in the presence of an extensive ureteral stricture.\n\nThere are few large studies in the literature reporting the outcome of this procedure2,3. We present a case report of a patient with long ureteral stenosis surgically treated with ileal substitution of the right ureter.\n\n\nCase description\n\nA woman of 43 years, with no relevant past medical history, presented lumbar pain, oliguria and bilateral peripheral oedema. She presented renal dysfunction, with serum creatinine of 6 mg/dl [normal range 0.5-1.2mg/dl] and renal echography demonstrated right ureterohydronephrosis and left renal atrophy.\n\nThe patient was submitted to ureteral stent placement with rapid improvement of renal function after one week. She removed the catheter for a CT scan after one month, which revealed circumferential thickening of the right ureter and caudally a complete obliteration of the lumen for 4 cm (Figure 1).\n\nWith ureterorenoscopy, we visualized multiple concentric ureteral stenosis, and a biopsy revealed no malignancy. Mycobacteriologic and parasitologic urine tests were negative.\n\nSince the patient later presented acute obstructive renal dysfunction after 2 days without urinary diversion, a ureteral stent was placed, but in spite of the ureteral diversion, the patient presented further deterioration of renal function (assessed using serum creatinine levels) after one month, so a right nephrostomy was placed, with complete consequent restoration of renal function.\n\nWe proposed a curative surgical procedure, which the patient agreed to undergo17 months after the initial diagnosis, and the patient was submitted to ileal substitution of the right ureter, with an isoperistaltic ileal segment of 22 cm, with no detubularization.\n\nThe procedure was done using a midline incision, followed by medial mobilization of the right colon, isolation of the right ureter and section of the pieloureteral junction. We performed an enterectomy of 22cm of ileum (15cm away of the ileocecal valve), followed by a terminoterminal enteral anastomosis. Next, we reflected the cecum and ascending colon superiorly and placed the ileal segment in the retroperitoneum. Finally, we checked the orientation of the ileal segment and performed the anastomoses of the intact, isoperistaltic ileal segment at the level of the renal pelvis and at the bladder (Figure 2 and Figure 3).\n\nThe patient was discharged from the hospital with no medication. Total hospital stay of the patient was 14 days. The patient removed the urethral catheter after 4 weeks and the nephrostomy catheter after 6 weeks.\n\nThere were no perioperative complications and renal function was unaltered, with serum creatinine of 1.2 mg/dl. Pathological study of the ureter demonstrated chronic inflammation and fibrosis, with no malignant cells.\n\nAfter 20 months of follow-up, the patient is asymptomatic, presents no urinary tract infections, no relapse of stenosis and has preserved renal function. She is now medicated with oral sodium bicarbonate 6 mg per day in order to correct metabolic acidosis.\n\n\nDiscussion and conclusions\n\nIleal substitution of the ureter was described in 1959 by Goodwin and co-workers4. Historically, it has been applied in ureteral stenosis associated with tuberculosis infection. However, it has been used more frequently with the new onset of ureteral stenosis associated with endoscopic procedures, as well as post radiation stenosis.\n\nIleal substitution of the ureter is a complex procedure, considered a surgery of the last resort in ureteral repair and is useful in the presence of an extensive ureteral stricture. It is indicated in cases of long or multiple ureteral stenosis. The ileum is considered the best ureteral segment for ureteral substitution because of its peristalsis, good vascular supply and absorptive properties.\n\nThe Yang Monti principle consists of the interposition of reconfigured bowel segments, where a small segment of bowel is isolated, detubularized and retubularized in the opposite axis, forming a longer tube. The procedure can be performed using two or more reconstructed bowel segments to obtain a longer graft. This technique has the advantage of using a shorter bowel length, leading to a smaller intestinal absorptive surface and decreased mucus production5,6.\n\nKnown contraindications of bowel substitution include renal dysfunction (serum creatinine above 2 mg/dl), bladder outlet obstruction, inflammatory bowel disease and radiation enteritis2,3,7,8. Common complications include ileal and anastomotic stenosis, mucus obstruction, urinary infection, renal dysfunction, metabolic acidosis and rare cases of malignancy. However, the risk of metabolic acidosis and renal dysfunction are rare in patients with no renal impairment pre-operatively2,3,7–9.\n\nThere are few large studies in the literature reporting the outcome of ileal substitution of the ureter; however, the published series demonstrate very good results2,3,7,8. The largest reported series, published by Armatys and co-workers2, includes the study of 91 patients submitted to ileal ureter replacement, with a mean follow-up of 36 months. They reported anastomotic stricture in 3.3% of the patients and fistula in 6.6%. Serum creatinine remained stable or decreased in the majority of the patients (74%) and hyperchloremic acidosis was reported in 3.2% of the patients.\n\nIn conclusion, we present a case report of a patient with long and multiple ureteral stenosis successfully treated with ileal substitution. Ileal substitution of the ureter is a surgical technique that should be considered in cases of long, proximal or multiple ureteral stenosis, that present no other surgical option. This procedure is certainly a better alternative to a nephrectomy, permanent ureteral stenting or permanent nephrostomy tube.\n\n\nConsent\n\nWritten informed consent for the publication of the patient’s 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\nKnight RB, Hudak SJ, Morey AF: Strategies for open reconstruction of upper ureteral strictures. Urol Clin North Am. 2013; 40(3): 351–361. PubMed Abstract | Publisher Full Text\n\nArmatys SA, Mellon MJ, Beck SD, et al.: Use of ileum as ureteral replacement in urological reconstruction. J Urol. 2009; 181(1): 177–181. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChung BI, Hamawy KJ, Zinman LN, et al.: The use of bowel for ureteral replacement for complex ureteral reconstruction: long-term results. J Urol. 2006; 175(1): 179–183; discussion 183–174. PubMed Abstract | Publisher Full Text\n\nGoodwin WE, Winter CC, Turner RD: Replacement of the ureter by small intestine: clinical application and results of the ileal ureter. J Urol. 1959; 81(3): 406–418. PubMed Abstract | Publisher Full Text\n\nYang WH: Yang needle tunneling technique in creating antireflux and continent mechanisms. J Urol. 1993; 150(3): 830–834. PubMed Abstract | Publisher Full Text\n\nMonti PR, Lara RC, Dutra MA, et al.: New techniques for construction of efferent conduits based on the Mitrofanoff principle. Urology. 1997; 49(1): 112–115. PubMed Abstract | Publisher Full Text\n\nMatlaga BR, Shah OD, Hart LJ, et al.: Ileal ureter substitution: a contemporary series. Urology. 2003; 62(6): 998–1001. PubMed Abstract | Publisher Full Text\n\nVerduyckt FJ, Heesakkers JP, Debruyne FM: Long-term results of ileum interposition for ureteral obstruction. Eur Urol. 2002; 42(2): 181–187. PubMed Abstract | Publisher Full Text\n\nAusten M, Kalble T: Secondary malignancies in different forms of urinary diversion using isolated gut. J Urol. 2004; 172(3): 831–838. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "31508",
"date": "12 Mar 2018",
"name": "Arnaldo Figueiredo",
"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 report illustrates the use of a bowel segment to restore the urine flow from the kidney to the bladder. The authors appropriately consider that as the \"last resort\" option for long ureteral strictures. From the description of the case it looks like that the renal pelvis and the proximal ureter were salvageable (in fact, the anastomosis was performed to the pelvis), making the reader wonder whether alternatives such as a auto-transplantation to the iliac fossae could be implemented, avoiding the need for interposing bowel. Using 22cm of bowel was probably excessive, given the anticipated lenghtening that always occurs (as illustrated in the post-operative reconstructed images from a CT). Also, the authors could include tappering of the ureter as an option to reduce the risks of urine reabsortion.\nFinally, it would be interesting to know the final diagnosis of the pathology behind the ureteral stricture, to further support the therapy chosen.\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": "186664",
"date": "15 Aug 2023",
"name": "Tzu-Ping Lin",
"expertise": [
"Reviewer Expertise Urological reconstructive surgery",
"endourology",
"cancer 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 author presents a case who received ileal ureter substitution due to right ureter obstruction. It would need some revisions to consider approval.\nWhy the stricture sites that the ureterorenoscope can pass still need bypass surgery?\n\nPlease discuss more why this patient needs a long term supplement of oral sodium bicarbonate for metabolic acidosis. We think this is the main finding of this case report.\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": []
}
] | 1
|
https://f1000research.com/articles/7-273
|
https://f1000research.com/articles/7-120/v1
|
29 Jan 18
|
{
"type": "Research Article",
"title": "City-based action to reduce harmful alcohol use: review of reviews",
"authors": [
"Peter Anderson",
"Eva Jané-Llopis",
"Omer Syed Muhammad Hasan",
"Jürgen Rehm",
"Eva Jané-Llopis",
"Omer Syed Muhammad Hasan",
"Jürgen Rehm"
],
"abstract": "Background: The World Health Organization global strategy on alcohol called for municipal policies to reduce the harmful use of alcohol. Yet, there is limited evidence that documents the impact of city-level alcohol policies. Methods: Review of reviews for all years to July 2017. Searches on OVID Medline, Healthstar, Embase, PsycINFO, AMED, Social Work Abstracts, CAB Abstracts, Mental Measurements Yearbook, Health and Psychosocial Instruments, International Pharmaceutical Abstracts, International Political Science Abstracts, NASW Clinical Register, and Epub Ahead of Print databases. All reviews that address adults, without language or date restrictions resulting from combining the terms (“review” or “literature review” or “review literature” or “data pooling” or “comparative study” or “systematic review” or “meta-analysis” or “pooled analysis”), and “alcohol”, and “intervention” and (“municipal” or “city” or “community”). Results: Five relevant reviews were identified. Studies in the reviews were all from high income countries and focussed on the acute consequences of drinking, usually with one target intervention, commonly bars, media, or drink-driving. No studies in the reviews reported the impact of comprehensive city-based action. One community cluster randomized controlled trial in Australia, published after the reviews, failed to find convincing evidence of an impact of community-based interventions in reducing adult harmful use of alcohol.\n\nConclusions: To date, with one exception, the impact of adult-oriented comprehensive community and municipal action to reduce the harmful use of alcohol has not been studied. The one exception failed to find a convincing effect. We conclude with recommendations for closing this evidence gap.",
"keywords": [
"Cities",
"Municipalities",
"communities",
"harmful use of alcohol"
],
"content": "Introduction\n\nIn response to the 2011 United Nations declaration on non-communicable diseases (NCDs) (United Nations General Assembly, 2011; United Nations, 2014), the World Health Organization proposed a target to reduce the harmful use of alcohol by 10% between the years 2010 and 2025 (World Health Organization, 2014a), based on three possible indicators, adult per capita alcohol consumption, prevalence of heavy episodic drinking, and measures of alcohol-related morbidity and mortality (World Health Organization, 2014b).\n\nThe bulk of alcohol-related severe health problems, including mortality, occurs in middle age (Office for National Statistics, 2015), and, it is amongst this age group that policy and programme interventions are likely to bring the greatest health and productivity gains (Organisation for Economic Co-operation and Development, 2015). Heavy drinkers are responsible for the majority all alcohol-related harm (Rehm et al., 2013). It is also amongst this group, compared with lighter drinkers, that disproportionally greater health gains can be made for the same absolute reduction in alcohol consumption (Rehm & Roerecke, 2013). Thus, if alcohol policy is to be most efficient in reducing the harmful use of alcohol, it should preferentially address adult drinkers, and, in particular, those who drink heavily.\n\nDriving the NCD alcohol target are WHO’s Global Strategy to reduce the harmful use of alcohol (World Health Organization, 2010), and WHO’s three ‘best buys’ (World Economic Forum & World Health Organization, 2011; World Health Organization, 2013). One of the ten target areas within the global strategy is community action, with a specific call to: “strengthen capacity of local authorities to encourage and coordinate concerted community action by supporting and promoting the development of municipal policies to reduce harmful use of alcohol, as well as their capacity to enhance partnerships and networks of community institutions and nongovernmental Organizations” (World Health Organization, 2010).\n\nCities are a potentially important setting and jurisdictional level for reducing NCDs (De Leeuw et al., 2015; Farrington et al., 2015). There is a range of evidence-based interventions that fall within municipal jurisdictional responsibility and which could be implemented at city level to reduce the harmful use of alcohol (Anderson & Baumberg, 2006; Anderson et al., 2009; Anderson et al., 2012; Burton et al., 2017; Fitzgerald et al., 2016; Martineau et al., 2013). Despite a long history of calls for city-based policies and action plans (Mathrani & Anderson, 1998; Ritson, 1995; World Health Organization, 1998), and an equally long history of research endeavour (Giesbrecht et al., 1990), a systematic review of the impact of alcohol policies, undertaken prior to the launch of the WHO global strategy, was unable to include community actions within its cost-effectiveness estimates, due to insufficient evidence of impact (Anderson et al., 2009).\n\nSpurred by a target of a global beer producer to reduce the harmful use of alcohol by 10% over the five-year period 2016–2020 in pilot cities in at least nine different middle- and high-income countries (ABInBev, 2016), we have undertaken a review of reviews to investigate the potential impact of city-based action to reduce the harmful use of alcohol. In our review, we have focused on reviews that summarize the literature of comprehensive community and municipal action, often based on a municipal comprehensive strategy and action plan, as put forward by the World Health Organization (Anderson, 1991; Ritson, 1995). Because we anticipated very few such reviews, we have supplemented our findings with an overview of what could be implemented within a comprehensive municipal strategy to reduce the harmful use of alcohol, based on the published evidence base (Anderson & Baumberg, 2006; Anderson et al., 2009; Anderson et al., 2012; Burton et al., 2017; Fitzgerald et al., 2016; Martineau et al., 2013).\n\n\nMethods\n\nDuring July 2017, we conducted a systematic literature search on OVID Medline, Healthstar, Embase, PsycINFO, AMED, Social Work Abstracts, CAB Abstracts, Mental Measurements Yearbook, Health and Psychosocial Instruments, International Pharmaceutical Abstracts, International Political Science Abstracts, NASW Clinical Register, and Epub Ahead of Print databases to identify reviews that addressed community and municipal alcohol interventions. With no language or date restrictions, the search used the following combination of terms: (“review” or “literature review” or “review literature” or “data pooling” or “comparative study” or “systematic review” or “meta-analysis” or “pooled analysis”), and “alcohol” and “intervention” and (“municipal” or “city” or “community”), supplemented with hand searches of included reviews.\n\nOur inclusion criteria were reviews and overviews (whether or not systematic) that discussed the implementation of comprehensive policies and programmes to reduce the harmful use of alcohol at the community or municipal level. We excluded reviews of specific alcohol policy interventions, for example restrictions on hours and days of sale, that may or may not have been part of a city action plan. Two authors (OSMH and PA) independently reviewed titles and abstracts for selecting papers for full text review and selecting papers to include. Discrepancies, which only related to whether or not the publication addressed comprehensive approaches, were resolved with discussion. The result of the search, analysed during July 2017, is summarized in Figure 1. As only five relevant reviews were identified, three of which were by the same author, we did not attempt to analyze them for their quality, but rather describe their methods and findings.\n\n\nResults\n\nOnly five relevant reviews were identified (Giesbrecht et al., 2014; Giesbrecht & Greenfield, 2003; Giesbrecht & Hayden, 2006; Gorman & Speer, 1996; Toomey & Lenk, 2001). Three included the same first author, and the most recent publication (Giesbrecht et al., 2014) included all publications of, and reached similar conclusions to, the previous four reviews. None of the reviews were systematic and none adhered to standard guidelines. Subsequent to the publication dates of the reviews, our search identified one further large randomized study of the effectiveness of community action (Shakeshaft et al., 2014).\n\nOf the 23 individual studies mentioned in the 2014 review (Giesbrecht et al., 2014), twelve included adults, mostly with a focus on younger adults. Six were from North America, two were from Nordic countries, and four were from Australia/New Zealand. Four of the 12 studies targeted bars, three media campaigns, two drink-driving, one overall access, one a specific location (a beach), and one primary health care-based brief advice programmes. No studies reported comprehensive community or municipal interventions. The four bar studies found an effect in reducing violence, which tapered off over time. The three media studies found no impact. The two drink-driving studies led to reduced alcohol-involved traffic crashes. The access and location study led to reductions in the harmful use of alcohol, but the brief advice initiative did not.\n\nThe large study, outside of the reviews, was a randomized trial comprising 20 communities in Australia that each had populations of 5,000–20,000 inhabitants (Shakeshaft et al., 2014). Communities were pair-matched, and one member of each pair was randomly allocated to the experimental group. Thirteen interventions were implemented in the experimental communities from 2005 to 2009: community engagement; general practitioner training in alcohol screening and brief intervention (SBI); feedback to key stakeholders; media campaign; workplace policies/practices training; school-based intervention; general practitioner feedback on their prescribing of alcohol medications; community pharmacy-based SBI; web-based SBI; Aboriginal Community Controlled Health Services support for SBI; Good Sports program for sports clubs; identifying and targeting high-risk weekends; and hospital emergency department–based SBI. The study failed to detect an impact of the interventions in reducing the harmful use of alcohol in routine data, but found a significant reduction of average alcohol use in surveys. Unfortunately, the study appeared under powered for the routine data, and had low response rates to measurement surveys. The community interventions were not able to be fully comprehensive and were not able to include a range of potentially impactful interventions, including sales taxes and restrictions on availability. Further, no process evaluation was reported; thus, whether or not the included interventions were implemented as planned, or the extent to which they were implemented is not reported.\n\nOne of the five reviews that we identified included a summary of feasible intervention options at the local and municipal level (Giesbrecht & Haydon, 2006). We have included and expanded on these in Table 1, adding an overview of the evidence base (Anderson & Baumberg, 2006; Anderson et al., 2009; Anderson et al., 2012; Burton et al., 2017; Fitzgerald et al., 2016; Martineau et al., 2013), with implementation illustrations at the city level. Dependent on jurisdictional responsibilities, city-level interventions that might have a meaningful contribution to reducing the harmful use of alcohol include sales taxes, restrictions on density of outlets and days and hours of sale, drink-drive restrictions, and scale-up of individual level advice and treatment programmes (Organisation for Economic Co-operation and Development, 2015).\n\n\nDiscussion\n\nCities can be natural units for promoting health (de Leeuw et al., 2015; Farrington et al., 2015), including reducing the harmful use of alcohol (Mathrani & Anderson, 1998). Although not necessarily having the full jurisdictional responsibilities of national governments for all health policy issues, they often have greater flexibility and are an important site for innovative environmental measures that make healthier choices easier choices, shifting social norms in the process (Reeve et al., 2015). Cities are members of many networks, including Healthy Cities networks, which are natural vehicles for deployment to full scale globally.\n\nThis review has identified a suite of evidence-based policies and programmes that could, dependent on jurisdictional responsibilities, be implemented at the city level to reduce the harmful use of alcohol. Indeed, some of the studies identified by the reviews that focused on one particular programme area, for example enforcement of bar regulations, stepped up drink-driving activities, and strengthened access regulations, found some impacts in reducing the harmful use of alcohol. However, only one study evaluated a suite of interventions, and found no convincing evidence of impact. This study, though, was unable to include a range of potential impactful interventions (price and availability), and did not report implementation fidelity of the included interventions Shakeshaft et al., 2012).\n\nWe have not been able to find evidence for the effective impact of comprehensive municipal action plans in reducing the harmful use of alcohol. There is, thus, dissonance between calls for action and evidence. This contrasts with other topic areas, such as smoking (Moreland-Russell et al., 2016; Perlman et al., 2016), obesity (Reeve et al., 2015; Sisnowski et al., 2016), and physical activity (Giles-Corti et al., 2016; Sallis et al., 2016a; Sallis et al., 2016b; Stevenson et al., 2016), where there is experience of coordinated city action and a supporting evidence base.\n\nWe conclude our paper by discussing how this dissonance might be reduced. First, there are needs for multi-city studies that test the impact of developed and implemented municipal action plans in reducing the harmful use of alcohol. To match the WHO target of a 10% reduction in the harmful use of alcohol over a 15-year time frame, recognizing the importance of addressing adults, and, in particular heavy drinkers, the evidence base indicates that municipal action plans need to include, where jurisdictional authority allows, all of: sales taxes to increase the price of alcohol; reductions in the availability of alcohol through restrictions on outlet density and days and hours of sale; intensive implementation of drink drive restrictions through sobriety checkpoints and/or unrestrictive (random) breath testing; and, widespread deployment and scale-up of health care based screening and brief advice and treatment programmes, see Table 1. Municipal action plans should not be based on public education or mass media programmes alone, as these have been found to be ineffective in reducing the harmful use of alcohol (Martineau et al., 2013).\n\nIdeally, multi-city studies should be set up as randomized trials with control cities in different sites with sufficient sample size with respect to both cities and individuals within cities to test whether the municipal action plans work. While this may be difficult to realize, given that such randomized trials would have very high costs, and there are not many examples of sufficiently powered community trials in the literature, a different design in combining national aggregate and individual level data could be used to achieve better control (Gmel et al., 2004). Longitudinal individual level data could be collected by drawing representative samples from the adult population in the cities, with an oversampling of heavier drinkers. This would allow an analysis of how the city actions affect individual drinking trajectories, as recommended by a number of authors (Fitterer et al., 2015; Holmes et al., 2015). The evaluation should triangulate the individual cohort data with the aggregate-level data, and with other routinely collected data, such as alcohol-attributable hospitalizations (Shakeshaft et al., 2014) to test if the municipal actions lead to a reduction in the harmful use of alcohol. Wastewater-based epidemiology can be used to contrast alcohol consumption between implementation cities and comparator cities (Ryu et al., 2016). Logic models should be developed to undertake process evaluation (Moore et al., 2015) to ascertain implementation fidelity and to identify and empirically demonstrate effective best practices to reduce harmful consumption of alcohol that could be adopted for scale-up in other cities and countries (Barker et al., 2016).",
"appendix": "Competing interests\n\n\n\nJR, EJL and OSMH report no conflict of interest. PA has received fees from AB InBev Foundation.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nABInBev: Our Global Smart Drinking Goals. (acessed 26 July 2017). Reference Source\n\nAnderson P: The Management of Drinking Problems. WHO Regional Publications, European series No. 32, Copenhagen, World Health Organization Regional Office for Europe. (In English, French, German, Russian and Hungarian), 1991.\n\nAnderson P, Baumberg B: Alcohol in Europe: A public health perspective. London: Institute of Alcohol Studies, 2006. Reference Source\n\nAnderson P, Chisholm D, Fuhr DC: Effectiveness and cost-effectiveness of policies and programmes to reduce the harm caused by alcohol. Lancet. 2009; 373(9682): 2234–46. 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}
|
[
{
"id": "30366",
"date": "05 Feb 2018",
"name": "Joan Ramón Villalbí",
"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 relevant paper providing needed perspectives on interventions to reduce the harm caused by alcohol. It is presented as a review of reviews, but in fact it goes a bit beyond it, as the contents of table 1 are in themselves a valuable side-product. I think the paper is acceptable to be indexed in its current form, and may be useful to many practitioners.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "31220",
"date": "26 Feb 2018",
"name": "R.H.L.M Bovens",
"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 think this paper contributes very much to the discussion what to do about reducing alcohol problems in society among adult people. It shows in the world there is little effort to focus these problems in an integrative way, using evidence based interventions as the ‘three best buys’: reducing advertisement, availability and raising taxes. Authors did their job accurate.\nI just have one comment: The paper focuses on adult people. In the research were, as far as I know only reviews that address adults. Why do the authors do not discuss the gap between the policy used focussing youth and the policy focussing adults? For example the research carried out by Karen Schelleman at your own university shows small evidence for effect of city-based actions in the Netherlands (Karen Schelleman-Offermans, R. Knibbe, E. Kuntsche: Preventing Adolescent Alcohol Use: Effects of a Two-Year Quasi-Experimental Community Intervention Intensifying Formal and Informal Control. Journal of Adolescent Health 54(3) November 2013; Karen Schelleman-Offermans, R. Knibbe, M. Derickx & H. van de Mheen: A Process Evaluation of a Community Intervention to Reduce Youth Drinking. Sucht 59(5):249-259 · January 2013). The thing is many countries are focusing only on youth, I think that is the main reason there are so few researches about community interventions in the adult world. This is the reason why I chose ‘Partly’ in the first question: accurate: Yes, but in my view not complete. Paying attention to the gap between the policy regarding youth and regarding adults would improve the paper seriously.\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": "3474",
"date": "05 Mar 2018",
"name": "Peter Anderson",
"role": "Author Response",
"response": "Author response: Thank you for this important comment. We have added a paragraph to the discussion that stresses, from a health perspective, the importance of addressing adults as opposed to adolescents. We noted that there is considerably more research that focuses on community-based action to reduce the harmful use of alcohol among adolescents rather than among adults and suggested that this is due to an undue focus of existing alcohol policy on youth-oriented actions, that have tended to strengthen during the first decade of the 21st century, at the expense of policies that would address adults."
}
]
}
] | 1
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https://f1000research.com/articles/7-120
|
https://f1000research.com/articles/7-157/v1
|
07 Feb 18
|
{
"type": "Research Note",
"title": "Reflections on the use of the World Health Organization’s (WHO) OneHealth Tool: Implications for health planning in low and middle income countries (LMICs)",
"authors": [
"John Q. Wong",
"Nel Jason Haw",
"Jhanna Uy",
"Diana Beatriz Bayani",
"John Q. Wong",
"Jhanna Uy",
"Diana Beatriz Bayani"
],
"abstract": "The World Health Organization (WHO) launched the OneHealth Tool (OHT) to help low and middle income countries to develop their capacities for sector-wide priority setting. In 2016, we sought to use the OHT to aid the Philippine Health Insurance Corporation (PHIC), the national health insurer of the Philippines, in decisions to expand benefit packages using cost-effectiveness analyses. With technical support from the WHO, we convened health planning officers from the Philippine Department of Health (DOH) and the Philippine Health Insurance Corporation (PHIC) conduct generalized cost-effective analyses (GCEA) of selected un-financed noncommunicable disease interventions using OHT. We collected epidemiological and cost data through health facility surveys, review of literature such as cost libraries and clinical practice guidelines, and expert consultations.\nAlthough we were unable to use GCEA results directly to set policy, we learnt important policy lessons which we outline here that might help inform other countries looking to inform service coverage decisions. Additionally, the entire process and GCEA visualizations helped high-level policymakers in the health sector, who have traditionally relied on ad hoc decision making, to realize the need for a systematic and transparent priority-setting process that can continuously provide the evidence needed to inform service coverage decisions.",
"keywords": [
"priority-setting",
"low and middle income countries",
"WHO CHOICE",
"generalized cost-effectiveness analysis",
"OneHealth Tool",
"sector-wide planning"
],
"content": "Introduction\n\nStriving for universal health coverage requires priority setting to maximize health gains from limited resources (Chalkidou et al., 2016). Countries like the Philippines struggle with building technical and institutional capacity for health technology assessment (HTA).\n\nIn 2015, the national insurer Philippine Health Insurance Corporation (PHIC) wanted to redefine their health benefits package. We developed a list of priority conditions based on disease burden and a corresponding list of cost-effective interventions (Wong et al., 2018). This paper documents our subsequent attempt on a more definitive list of interventions using cost-effectiveness analyses (CEA) in the Philippine setting. At that time, however, we had insufficient technical capacity to conduct the volume of necessary primary CEAs within the timeframe of our policy window. We sought the help of the World Health Organization (WHO) to conduct generalized cost-effectiveness analysis (GCEA) using their OneHealth Tool (OHT) software.\n\nThe appeal of GCEA and OHT was its perceived feasibility for our purposes. OHT had been used successfully in 20 low and middle income countries (LMICs) for sector-wide planning (Avenir Health, 2017), and WHO-CHOICE had conducted regional-level GCEA for a good range of diseases in our priority list of conditions ((World Health Organization, 2017). Because GCEA uses a “do nothing” comparator, we would be able to simultaneously analyze multiple diseases and new and existing alternatives within and outside the health sector to determine the most efficient mix of interventions to fund at the national-level (Hutubessy et al., 2003). Lastly, GCEA could be done through the user-friendly OHT software with its built-in health impact models, templates for costing and epidemiological inputs, and data visualization functions.\n\nWe hope that our reflections on our experience with GCEA using OHT will aid other LMICs looking to scale up their priority setting efforts.\n\n\nMethods\n\nBased on the available models in the OHT at that time (April 2016), twelve interventions across three diseases - cardiovascular disease, diabetes, chronic obstructive pulmonary disorder - were chosen for analysis. OHT has global or regional defaults for all of the values in the models, and we attempted to replace all of the defaults with the best available local data. To do this, we collected epidemiological and cost data from health facility surveys, desk reviews, and clinical experts. A summary of data sources for each input is detailed in Table 1.\n\nNote: A full documentation of the changes, together with the references and notes, are found in Supplementary File 1.\n\nWe invited WHO-Geneva to conduct a five-day workshop on the use of OHT, with DOH and PHIC planning officers, and some senior officials as participants. Over the course of the workshop, we used the default parameters of the model as a starting point of discussion. We used a consensus approach in modifying these defaults. We first consulted available data we had gathered, then all workshop participants discussed iteratively which data inputs and resource requirements were appropriate in the Philippine context until a consensus was reached.\n\nOn the last day, we plotted the cost-effectiveness results on an isoquant graph. Each intervention represents a point, and the graph is to be interpreted diagonally. The graph (Figure 1) shows the cost of the intervention on the horizontal axis, and effectiveness on the vertical axis. We have also added price tags to each data point to indicate the five-year budget impact.\n\nCalculations made using OneHealth Tool.\n\n\nLessons learned\n\nOur study raised awareness among policy makers that cost-effectiveness can be used for making decisions on service coverage. They found the GCEA results straightforward and understandable. However, for the policy question, GCEA methods were deemed insufficient to satisfy policy makers’ demands. GCEA was geared for choosing a complement of mutually compatible interventions based on a ‘do-nothing’ scenario. The results ended up validating the programs already covered, but failed to address the question of expanding service coverage. We realized that a CEA with current practice as the comparator would be more appropriate.\n\nAnother limitation of GCEA is the limited availability of models, making it difficult to conduct a sector-wide exercise that completely incorporates the Philippine baseline context. While more models have been added since our last use, such as modules on neglected tropical diseases, it will take time before the full breadth of interventions will be covered. Most of the disease packages available were based on WHO’s list of global priorities, which were already covered in the Philippines. In addition, the process of modifying defaults in the OHT was more difficult than expected. Expert opinion and clinical practice guidelines varied widely, which made it challenging to reconcile the input values, especially in the absence of a local methods guideline for economic evaluation.\n\nThe undertaking also led to the policy makers’ realization that no systematic process existed within PHIC for developing benefit packages. The failure in providing an answer to the question of “Which intervention to cover next?” through the GCEA pushed all stakeholders to consider setting up a process and value framework for priority setting alongside an institution that will facilitate such processes. While PHIC and DOH are not completely new to HTA, there was a need to establish a single, harmonized priority setting process for the health sector that would enable these two institutions to make better policy decisions based the questions they are currently faced with. This envisioned an HTA system, characterized by societal principles, with specific criteria and steps and supporting legal framework that would respond to the next level of questions such as which drug to reimburse, given that both institutions are flooded by demands from various stakeholders to cover a wide range of interventions.\n\nThis whole exercise re-emphasizes that defining a health benefits package cannot be a one-off exercise. It is influenced by a myriad of factors, and entails much stakeholder consultation and awareness of societal and institutional values. We recognize that the next step for the Philippines is to embed in the health system a process of both evidence generation and use for making investment decisions in the public sector. Developing capacity at both technical and sectoral dimensions must also be prioritized. Despite the limitations of the GCEA, it was a practical exercise to demonstrate the importance of evidence use in supporting policy surrounding service coverage decisions.\n\n\nData and software availability\n\nThe OneHealth Tool (OHT) may be downloaded freely at from Avenir Health. At the time this study was conducted, the Spectrum version of the OHT was 5.42. The results may be recreated by following the detailed documentation of changes to the OHT defaults found in Supplementary File 1.\n\nData entered into OHT was obtained from sources listed in Table 1",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe project was funded jointly by the United Nations Children’s Fund (UNICEF) and the Philippine Health Insurance Corporation (PHIC).\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 other members of the project team who helped us with data collection and analysis - John Valdes, Kathy Chavez, Deo Estanislao, Ingrid Espinosa, Michael Mo, Katherine Reyes, and Christian Nuevo. We would also like to thank planning staff and senior officers of the Philippine Department of Health (DOH) and the Philippine Health Insurance Corporation (PHIC) for attending the workshop and contributing to the analysis of the results. Finally, we would like to thank the World Health Organization (WHO) Geneva, specifically the Department of Health Financing and Governance (HGF) for providing the necessary technical assistance to conduct the analysis.\n\n\nSupplementary material\n\nSupplemental File 1. Documentation of changes to WHO OneHealth Tool (OHT). Input-by-input changes to the interventions considered to recreate the results of the study.\n\nClick here to access the data.\n\n\nReferences\n\nAvenir Health: Avenir Health: OneHealth Tool Country Applications.2017. Reference Source\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. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHutubessy R, Chisholm D, Edejer TT, et al.: Generalized cost-effectiveness analysis for national-level priority-setting in the health sector. Cost Eff Resour Alloc. 2003; 1(1): 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO:World Health Organization: WHO-CHOICE Interventions. 2017. Reference Source\n\nWong JQ, Uy J, Haw NJ, et al.: Priority Setting for Health Service Coverage Decisions Supported by Public Spending: Experience from the Philippines. Health Systems and Reform. 2018; 4(1): 19–29. Publisher Full Text"
}
|
[
{
"id": "30630",
"date": "08 Feb 2018",
"name": "Kalipso Chalkidou",
"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 valuable and original contribution describing the analytical methods, data sources and overall policy approach to reviewing and attempting to adjust the health benefits package in the Philippines (based on a selection of mostly NCD interventions) using WHO’s OneHealth tool. A number of middle income countries are currently in a similar position, en route to Universal Healthcare Coverage and making a systematic attempt at revising their Benefits Package as part of the national or social health insurance funds and not many resources exist on how such a process can be carried out (e.g. see https://www.cgdev.org/publication/whats-in-whats-out-designing-benefits-universal-health-coverage for the Centre for Global Development guide to designing benefits packages).\n\nThe authors succinctly summarise the challenges they faced including the limited breadth of the OneHealth models available to cover the whole spectrum of diseases the country faces. They also highlight the problematic assumption of the “null comparator”. The latter is a core element of the WHO approach to cost effectiveness analysis and makes structuring the decision problem in a way that reflects the reality policy makers are faced with, where the option of doing nothing is neither realistic nor ethical, impossible.\n\nThe authors also flag up as major weaknesses the lack of national guidelines on the methods and processes for revising the package. Again there is limited global guidance as to best practice in the methods (for one example see the iDSI Reference Case to economic evaluation here http://www.idsihealth.org/resource-items/idsi-reference-case-for-economic-evaluation/) and processes for managing such a methodologically and politically challenging exercise.\n\nWe need more such examples to be publicised and for a dialogue to start within and between countries as to how best to support policy makers achieve UHC in a fair and sustainable way. The authors may not have the answers but pose important questions for driving discussion as well as 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? 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "30629",
"date": "01 Mar 2018",
"name": "Lotte M.G. Steuten",
"expertise": [
"Reviewer Expertise Health Technology Assessment",
"priority setting",
"comparative effectiveness",
"cost-effectiveness analyses"
],
"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 described their reflections on the experience with Generalized Cost Effectiveness Analysis (GCEA) using the WHO One Health Tool (OHT), to aid other Low-Middle Income Countries (LMICs) looking to scale up their priority setting efforts.\nThey clearly describe the rationale and aim of using this tool to support redefining the benefits package of the national insurer of the Philippines. Most importantly, they identify key limitations on various levels: 1) limitations of the OHT itself; 2) limitations in availability of models and data specific to the Philippine context; and 3) the unmet need to establish a single, harmonized priority setting process for the health sector that would enable multiple stakeholders (in this case the Department of Health and the Philippine Health Insurance Corporation) to make better policy decisions based on the questions they are currently faced with. Based on these limitations and their experiences the authors provide general recommendations that are relevant to the Philippines and to other LMICs.\nThe paper is valuable and sound, given that its aims are quite humbly to share reflections on the use of the OHT for a priority setting exercise. However, these reflections –however well described and sensible- should not be read as (and are not intended to be) the result of a systematic evaluation of experiences based on all stakeholders inputs. A degree of subjectivity should be acknowledged and the paper would have benefit from more specific or detailed suggestions to improve the priority setting exercise and experience, from the perspective of the different participants. That said, this paper is a valuable contribution to the literature and more examples such as these should be welcomed and shared to drive the HTA field forward, globally as well as locally.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-157
|
https://f1000research.com/articles/7-261/v1
|
02 Mar 18
|
{
"type": "Method Article",
"title": "Gene Unprediction with Spurio: A tool to identify spurious protein sequences",
"authors": [
"Wolfram Höps",
"Matt Jeffryes",
"Alex Bateman",
"Wolfram Höps",
"Matt Jeffryes"
],
"abstract": "We now have access to the sequences of tens of millions of proteins. These protein sequences are essential for modern molecular biology and computational biology. The vast majority of protein sequences are derived from gene prediction tools and have no experimental supporting evidence for their translation. Despite the increasing accuracy of gene prediction tools there likely exists a large number of spurious protein predictions in the sequence databases. We have developed the Spurio tool to help identify spurious protein predictions in prokaryotes. Spurio searches the query protein sequence against a prokaryotic nucleotide database using tblastn and identifies homologous sequences. The tblastn matches are used to score the query sequence’s likelihood of being a spurious protein prediction using a Gaussian process model. The most informative feature is the appearance of stop codons within the presumed translation of homologous DNA sequences. Benchmarking shows that the Spurio tool is able to distinguish spurious from true proteins. However, transposon proteins are prone to be predicted as spurious because of the frequency of degraded homologs found in the DNA sequence databases. Our initial experiments suggest that less than 1% of the proteins in the UniProtKB sequence database are likely to be spurious and that Spurio is able to identify over 60 times more spurious proteins than the AntiFam resource. The Spurio software and source code is available under an MIT license at the following URL: https://bitbucket.org/bateman-group/spurio",
"keywords": [
"Gene prediction",
"machine learning",
"gaussian process model",
"bioinformatics",
"sequence analysis"
],
"content": "Introduction\n\nSequencing of genomes has now become routine with the DNA archives containing the sequences of over 100,000 complete genomes, while the direct sequencing of proteins is still low throughput and not a routine technique. Fortunately, computational methods exist to predict the protein sequence of genes from genomic DNA sequence. At least for bacterial DNA, these methods are fast and accurate. Existing tools for bacterial gene prediction claim accuracy figures of over 99% suggesting that almost all known genes in well annotated genomes are identified by these methods1. However, many extra genes are predicted, some of which may be real and some of which may be false. Even if the false positive rate of the methods is only 0.1%, then within a database of 100 million proteins like UniProt we would still expect to find 100,000 spurious protein predictions. Given the widely varying quality of gene prediction pipelines still in use2, we expect that the actual number of spurious proteins is likely to be much higher. An important question to address is what fraction of sequence databases are spurious gene predictions. In this paper we begin to address this problem by creating a generic tool to identify spurious proteins.\n\nWe term the task of identifying and deleting spurious gene predictions as gene unprediction. Gene unprediction would allow for the quality control and refinement of existing genomic annotation as well as helping to identify shortcomings in existing gene prediction pipelines. One existing tool that can aid in gene unprediction is the AntiFam database3. AntiFam is a collection of profile-HMM models that can be used to identify members of potentially spurious protein families. AntiFam release 4.0 contains 65 entries that identify a range of spurious proteins. Some of these models were families initially built and included into the Pfam database (RRID:SCR_004726)4, but later removed when it was pointed out they contained only spurious proteins. Many more AntiFam entries were constructed to model shadow ORFs which appear on the opposite strand of well-known genes, such as the 23S rRNA5. However, the AntiFam approach does not scale well. Each family requires the effort of a curator to build it and verify its status as spurious. Many spurious proteins may be singletons, appearing only once in the sequence database and so could not form a family of spurious proteins to be included in AntiFam.\n\n\nMethods and results\n\nOur approach to identifying spurious genes is to identify stop codons in homologous genomic DNA sequences. If we see many stop codons falling within what would be the homologous protein sequence from related organisms then we will infer that this DNA region is unlikely to be under selection at the protein level and is likely to be a spurious gene prediction. Still we must expect to find stop codons in homologous DNA sequences that are not indicative of incorrect gene prediction. Firstly the homologous DNA sequence may have sequencing errors leading to erroneous stop codons. A second reason is that stop codons are sometimes recoded for amino acids. The most prevalent examples include recoding of UGA codons as tryptophan in members of Entomoplasmatales and Mycoplasmatales6, and more widely, UGA can also be interpreted as selenocysteine7, as well as UAG which can be recoded as pyrrolysine in archaebacteria8. Pseudogenization is a real process and so we must expect some level of stop codons to be found in homologous regions of known genes. Certain organisms have a high level of pseudogenization, in particular obligate intracellular pathogens such as buchnera species may contain up to 50% of pseudogenes9.\n\nHere we describe two examples that illustrate the concept of identifying spurious proteins by inspecting homologous DNA sequence. The first example is from a known spurious protein identified by the AntiFam resource. This protein is an uncharacterized protein from the microbe Acinetobacter bereziniae (UniProt accession: N8YUQ2) which was revealed to be a translated CRISPR YPRES repeat sequence. In Figure 1A below we show a summary visualization of the tblastn output, with each line representing a similar DNA sequence. Stop codons are identified with white pixels and give the appearance of snow falling, hence we call these blizzard plots. This is a clear case where almost every homologous DNA sequence contains stop codons throughout the alignment.\n\n(A) A blizzard plot of Acinetobacter bereziniae protein F963_00691 (UniProt accession: N8YUQ2). (B) Apolipoprotein N-acyltransferase from Mycobacterium smegmatis (UniProt: A0QZ13). Each row on the plot shows the alignment region of potential protein from the tblastn search. Stop codons are shown as white pixels and methionine codons are shown as red pixels. The significance level of the match is indicated by the rainbow colour scale on the right.\n\nThe second example (Figure 1B) shows an example protein from UniProtKB/Swiss-Prot (Apolipoprotein N-acyltransferase from Mycobacterium smegmatis (UniProt: A0QZ13). The plot is almost totally devoid of stop codons within the aligned regions. The single example stop codon is very close to the C-terminus of the protein meaning it is likely a benign change. It is interesting to see that there are black dots also within the similar sequences which represent deletions in the homologous sequence that occur in the multiple of three bases. This represents an additional line of evidence for the coding potential of the query sequence.\n\nThe Spurio tool is based on running the tblastn software (RRID:SCR_011822) (we have used BLAST version 2.7.1+) using the query protein to search against a collection of microbial genome sequences. The tblastn output is parsed to include only matches more significant than the threshold E-value. We explored a range of E-values in the benchmarking and identified 10 to be a good balance between precision and recall. For the genome collection, we chose a non-redundant set of 1,507 full genomes of bacteria and archaea provided by the ENA genome database10. As we mentioned earlier, Entomoplasmatales and Mycoplasmatales use an alternative genetic code, in which the UGA codon is interpreted as tryptophan6. To account for this, these bacteria are processed in a separate homology search where the correct genetic code is used.\n\nOur tool proceeds to transform the results of the homology search, which can be visualized as a blizzard plot, into a probability estimate for the underlying sequence to be spurious. To perform this classification, Spurio extracts three features from the set of homologous sequences. The central one, describing the relative amount of stop codons, is given in the equation F1 below. The '+1' pseudocount is a compromise for the logarithm to be applicable even if zero stop codons are found. Note also that stop codons are only counted if they fall within the region of similarity reported by tblastn. Finally, because homologous over-extension of alignments11 can cause pairwise alignments to extend into non-homologous regions, we only count stop codons if they fall within the body of the tblastn matching region (Not within the first or last 10 amino acids of match positions). Additionally, Spurio uses the logarithmized number of homologous sequence hits (Equation F2) and the protein sequence length (Equation F3) as features, which together describe the dimensions of the corresponding blizzard plot.\n\n\n\n\n\n\n\nHaving extracted and preprocessed features, we use a probabilistic Gaussian process classifier12 to estimate the probability of a protein to be spurious. As a supervised learning technique, the Gaussian process classifier is dependent on training samples to infer the underlying feature distribution. For this, we created a balanced sample set of protein sequences. The positive set is composed of 3,107 likely spurious proteins derived from the AntiFam resource (version 4.0) (See Supplementary File 1). The negative control set of 3,107 proteins that are genuinely translated were randomly selected from UniProtKB/Swiss-Prot (RRID:SCR_002380) (See Supplementary File 1). The distribution of these sample sequences after preprocessing suggests that the feature space is adequate for the separation of real and spurious sequences (see Figure 2).\n\nProtein sequences were sampled from either Swiss-Prot (3,107 sequences shown in blue) or AntiFam (3,107 spurious sequence shown in orange). After preprocessing, every protein sequence is represented by a single dot in three-dimensional space. This dataset was later used for the training and testing a probabilistic classifier. (A) Shows the log length versus the normalised log of the stop codons per aligned position. (B) Shows the log number of tblastn hits versus the normalised log of the stop codons per aligned position. The raw data set can be found associated with this paper.\n\nOn this set of sample data, we trained a Gaussian process model with a radial basis function kernel implemented in the python package scikit-learn13. Figure 3 shows the model after training on all samples, overlaid with 500 test samples. The performance for the whole approach is reviewed in the following section.\n\nSequences classified as spurious are coloured blue and non-spurious proteins are coloured orange. The classification is performed in three dimensions. Shown above are cross-sections along the sequence length dimension. 500 test data samples are projected to the nearest layer in this plot. 8-fold cross validation suggests a mean prediction accuracy of 96.8%.\n\nThe Spurio software (version 1.0) was tested using 8-fold cross validation on the previously described set of 3,107 samples per class. This led to 8 iterations of 5,438 training- and 776 test samples each. Based on this procedure, we report a mean accuracy of 96.8% (training: 97.0%) and area under the curve of 0.991 (training: 0.992). The results are summarized in Figure 4.\n\nThe transparent lines represent the individual results of each cross-validation run, the mean over all runs is shown in blue. (A) Receiver Operator Characteristic curve. (B) Precision-Recall curve. (C) Calibration plot showing the reliability of the model versus a perfectly calibrated model.\n\nTo further understand the performance of Spurio we ran it on 100,000 random bacterial proteins (See Supplementary File 2) from UniProtKB/TrEMBL version 2017_12 in order to estimate the number of spurious proteins (See Supplementary File 3). 5,392 Sequences did not yield any homologous sequences and were excluded. How the remaining proteins are distributed in the probability space of the Gaussian process classifier is shown in Figure 5. We see that the large majority of spurious proteins are found to be in the shorter length ranges of 30–150 amino acids as we might expect from incorrect gene predictions. As expected, we identify many more real than spurious proteins.\n\nThe Spurio output data can be found associated with the paper.\n\nTo illustrate the predictions by Spurio we have selected a representative example, the AZOBR_140218 protein from Azospirillum brasilense (UniProt: G8AMM6). This protein is 648 amino acids long and so would appear to be very likely a true protein coding gene. However, Spurio gives it a probability score of 0.979 indicating it is very likely to be Spurious. Inspection of the Blizzard plot (Figure 6) shows that the DNA homologues of this sequence have a large number of stop codons. Further investigation shows that this protein is on the opposite strand to the translational GTPase TypA (UniProt: A0A060DFP7) which strongly suggests that the AZOBR_140218 protein is indeed spurious and is a shadow ORF. Interestingly searching this spurious protein for homologues identifies many proteins including some that are erroneously annotated as the enzyme 1-deoxy-D-xylulose 5-phosphate reductoisomerase (see UniProt: R5CSG3 as an example).\n\nSee Figure 1 for a description of the features of the blizzard plot.\n\nIf we select an arbitrary threshold of 0.8 or greater to represent a spurious protein then 0.82% of the 100,000 sample of proteins are predicted to be spurious. Of these 26% have matches to Pfam which is somewhat surprising (see Table 1). However, if we consider proteins with no Pfam match we find that 3.8% of them have a Spurio score > 0.8 compared to just 0.25% of proteins with a Pfam match. Thus proteins with no Pfam match are 15 times more likely to be predicted as spurious than those with a Pfam match. If we search the sample of 100,000 proteins with AntiFam we find it identifies only 12 that are spurious (see Supplementary File 4). Therefore, Spurio is able to identify 62 times more spurious proteins than AntiFam. Of the 12 AntiFam matched proteins, 9 had Spurio scores of 0.97 or greater. The results of the AntiFam search can be found in Supplementary materials.\n\nIt is interesting to highlight an example where Spurio does did not match a protein that AntiFam did. If we take the example ALP79_101044 (UniProt: A0A0W8HJ99) we find that it has a Spurio score of 0.14 and has a strong Pfam match to the FAD_binding_3 family (Pfam: PF01494). The blizzard plot (Figure 7) shows that there is very little similarity detected to other organisms in the N-terminal 100 amino acids. It has an AntiFam match at the N-terminus of the protein from residues 1-25 to a translation of a tRNA. It seems likely that the protein should start at the methionine which is at position 31 of the existing sequence in UniProt.\n\nSee Figure 1 for a description of the features of the blizzard plot. The Pfam domain architecture of this protein has been added at the top of the figure.\n\nWe continued to investigate whether sequences predicted as spurious are less likely to be members of existing protein families in Pfam than those sequences predicted to be true proteins. We would expect that spurious proteins would be unlikely to fall into Pfam families and so in a perfect world we would see the expected number of Pfam matches at a Spurio score of 0 and see no Pfam matches at a Spurio score approaching 1. Figure 8 shows that in the 100,000 sequences from TrEMBL this is the case for predicted values from zero up to 0.6. But above that value we see an excess of matches to Pfam. To understand what is causing this excess of matches to families we created a list of the top ten most frequently occurring Pfam families, shown in Table 2. Inspection shows that eight out of the top ten Pfam families are related to transposon function. It is known that there can be many copies of degraded transposons within a genome. The larger than normal number of these degraded copies compared to proteins with normal cellular functions makes them appear to be spurious proteins.\n\nPfam accessions for the families that are likely to be transposon associated are underlined.\n\n5,392 Sequences did not yield any homologous sequences and were excluded. Another 4,363 samples were processed by spurio, but were released later than the current version in InterPro and were thus excluded. The plot shows the remaining 90,245 sequences.\n\nWe expected that selenoproteins may present problems for the Spurio method. To examine this we took an example selenoprotein GrdA from Carboxydothermus hydrogenoformans (UniProt: Q3A9J5) and ran Spurio on it. We found that indeed it was scored as 0.891 probability to be spurious (see Figure 9). One can clearly see in the blizzard plot the conserved selenocysteine position as a column of stop codons. It is interesting to note that selenoproteins that have been mispredicted to contain premature stop codons are unlikely to be predicted as spurious.\n\nSee Figure 1 for a description of the features of the blizzard plot.\n\n\nDiscussion\n\nThe identification of spurious genes is an area of genomic annotation that has received very little attention. This is partly due to the difficulty of proving that a gene is not expressed in any condition. We have made a generic tool to discover spuriously predicted proteins from bacterial genome sequences. Our attempt is reasonably successful, but we find that while we can indicate likely spurious genes, there are some failure modes that mean that the Spurio results should be considered indicative and that they will require inspection for some applications. For example, transposon related genes are apt to be predicted as spurious because they have many pseudogenized homologues. It may be possible that this could be turned into a positive attribute to help identify regions of a genome with high predicted spuriousity that may be transposons.\n\nIn order to improve the accuracy of Spurio we recommend that users focus on proteins that do not fall into known Pfam families as well as short proteins less than 150 amino acids in length. A use case where Spurio may be particularly appropriate is in the case of overlapping genes. If genes are called on opposite strands then Spurio could be used to detect if either or both the genes may be due to spurious gene prediction. A preliminary study of 21,452 genes in overlapping pairs (>50 nucleotide overlap) showed that 8.7% (1,867) of them had a Spurio score of 0.8 or higher (See Supplementary File 5).\n\nSpurio could be further developed by the addition of new features for training the model. Possible features could include the fraction of residues covered by Pfam domains. We would expect that spuriousness would negatively correlate with this feature. Also the number or proportion of insertions or deletions may carry useful information to discriminate real from spurious genes. It is worth noting that Pearson showed that protein sequences are essentially random and so features based on protein sequence or composition may not be informative14. Because we have found that transposons have a propensity to be predicted as spurious it may be beneficial to have a feature that measures how many times a protein matches within a particular genome, i.e. the average copy number. Transposons are often found in multiple copies per genome. We might expect this to be higher for transposon proteins.\n\nAlthough we did not see amino acid recoding to be an important factor in testing Spurio, it would be possible to attempt to make an ab initio prediction of recoding of stop codons. For example if we saw a TGA stop codon was consistently aligned to cysteine residues in the tblastn output we could predict that stop codon as a selenocysteine position. This may make an incremental enhancement of prediction accuracy.\n\nWith a method to assess the level of spurious proteins in hand we can assess the quality of a variety of protein sequence datasets. One future avenue to explore, would be to use Spurio as a quality control metric for complete proteomes. By looking at the fraction of predicted spurious proteins on a per proteome basis one could assign a quality index. In addition, we could also investigate how the quality of protein datasets has changed over time. It has been suggested that the quality of databases and their annotations may degenerate over time due to new protein sequences being based on previous erroneous protein sequences. Spurio gives us an initial estimate of 0.82% of TrEMBL proteins being spurious. Depending on your perspective this might be considered reassuringly low, or alarmingly high. Whatever your perspective, we believe that Spurio gives us a new and important tool to address issues of gene misprediction and we hope this will motivate further work in the area of gene unprediction.\n\n\nOperation\n\nTo run Spurio, blast15 and bedtools16 must first be installed. Spurio has several Python dependencies, which are listed in the requirements.txt file. Spurio requires Python 3.\n\n\nSoftware availability\n\nSpurio software and source code is available at: https://bitbucket.org/bateman-group/spurio\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.118443717\n\nLicense: MIT",
"appendix": "Competing interests\n\n\n\nThe authors have no competing interests.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nSupplementary File 1: Training data for the Spurio classifier. This file contains the 3,107 positive AntiFam proteins and the negative set of 3,107 UniProtKB/Swiss-Prot proteins. As well as the UniProtKB identifier we include the feature values used by the classifier.\n\nClick here to access the data.\n\nSupplementary File 2: List of the 100,000 protein sample from UniProtKB/TrEMBL. List accession numbers of the 100,000 bacterial protein sample randomly taken from UniProtKB/TrEMBL from UniProtKB version 2017_12.\n\nClick here to access the data.\n\nSupplementary File 3: Spurio matches to the 100,000 sample of UniProtKB/TrEMBL. This file contains the Spurio match data for the 94,602 proteins which could be scored by Spurio from the 100,000 random TrEMBL sample sequences. Column 1 contains the TrEMBL accession, column 2 contains the Spurio score, Columns 3 to 5 contain the feature values used by the Spurio classifier.\n\nClick here to access the data.\n\nSupplementary File 4: AntiFam matches to the 100,000 sample of UniProtKB/TrEMBL. This tab delimited file describes the 12 AntiFam matches found in the 100,000 random TrEMBL sample sequences. The Spurio scores for each are included for reference.\n\nClick here to access the data.\n\nSupplementary File 5: Spurio matches to the 21,452 overlapping proteins from UniProtKB/TrEMBL. This file contains the Spurio match data for the 21,452 overlapping proteins sampled from UniProtKB/TrEMBL. Column 1 contains the TrEMBL accession, column 2 contains the Spurio score, Columns 3 to 5 contain the feature values used by the Spurio classifier.\n\nClick here to access the data.\n\n\nReferences\n\nDelcher AL, Bratke KA, Powers EC, et al.: Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics. 2007; 23(6): 673–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWood DE, Lin H, Levy-Moonshine A, et al.: Thousands of missed genes found in bacterial genomes and their analysis with COMBREX. Biol Direct. 2012; 7: 37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEberhardt RY, Haft DH, Punta M, et al.: AntiFam: a tool to help identify spurious ORFs in protein annotation. Database (Oxford). 2012; 2012: bas003. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFinn RD, Coggill P, Eberhardt RY, et al.: The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016; 44(D1): D279–85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTripp HJ, Hewson I, Boyarsky S, et al.: Misannotations of rRNA can now generate 90% false positive protein matches in metatranscriptomic studies. Nucleic Acids Res. 2011; 39(20): 8792–802. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBové JM: Molecular Features of Mollicutes. Clin Infect Dis. 1993; 17(Suppl 1): S10–31. PubMed Abstract | Publisher Full Text\n\nZinoni F, Birkmann A, Stadtman TC, et al.: Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli. Proc Natl Acad Sci U S A. 1986; 83(13): 4650–4. PubMed Abstract | Free Full Text\n\nSrinivasan G, James CM, Krzycki JA: Pyrrolysine Encoded by UAG in Archaea: Charging of a UAG-Decoding Specialized tRNA. Science. 2002; 296(5572): 1459–62. PubMed Abstract | Publisher Full Text\n\nLiu Y, Harrison PM, Kunin V, et al.: Comprehensive analysis of pseudogenes in prokaryotes: widespread gene decay and failure of putative horizontally transferred genes. Genome Biol. 2004; 5(9): R64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSilvester N, Alako B, Amid C, et al.: The European Nucleotide Archive in 2017. Nucleic Acids Res. 2018; 46(D1): D36–D40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPearson WR: Selecting the Right Similarity-Scoring Matrix. Curr Protoc Bioinformatics. 2013; 43: 3.5.1–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeeger M: Gaussian processes for machine learning. Int J Neural Syst. 2004; 14(2): 69–106. PubMed Abstract | Publisher Full Text\n\nGarreta R, Moncecchi G: Learning scikit-learn: Machine Learning in Python. Packt Publishing Ltd; 2013; 100. Reference Source\n\nLavelle DT, Pearson WR: Globally, unrelated protein sequences appear random. Bioinformatics. 2010; 26(3): 310–8. 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\nQuinlan AR, Hall IM: BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–842. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHöps W, Jeffryes M, Bateman A: Spurio (Version v1.0). Zenodo. 2018. Data Source"
}
|
[
{
"id": "31447",
"date": "13 Mar 2018",
"name": "Arne Elofsson",
"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 report a novel tool to detect \"spurious\" hits in gene assignments. The methodology is based on the assumption that \"homologous gene sequences\" that contain a substantial amount of stop codons indicate that a gene is \"not under selective pressure.\n\nThe tool seems to be superior to the earlier tool (antifam) and is therefore a useful tool for automatic annotations of genomes.\nAlthough this assumption most likely is correct in most cases - it also might miss non-spurious genes, in particular orphan genes. It is generally accepted that there exist a birth and death process where novel (orphan) genes can occur from non-coding regions and also that existing genes turns into pseudo-genes. This could be discussed further.\nThere exist quite good set of orphans in drosophila and yeast - it would be interesting to see how spurio would rank these. (I am aware that this tool is mainly for bacteria - but at least in yeast most of these orphan genes are single exon so it should be possible to run it I think).\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? Partly",
"responses": []
},
{
"id": "31445",
"date": "12 Apr 2018",
"name": "Daniel H. Haft",
"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\nProkaryotic structural and functional annotation improves over time as growing resources, such as Pfam or CDD, add to the collections of rules that automated annotation pipelines can call on for genome analysis. A considerable amount of genomic “dark matter” remains in the form of proteins not currently reached by any annotation rule.\n\nMost large clusters in the dark matter really do represent real proteins in need of characterization and a name. But some merely appear to be real, and to be suitable for the invention of new “domain of unknown function” protein families, when actually they reflect a long legacy of false-positive errors in the prediction of protein-coding regions. The authors here introduce Spurio, a tool that finds suspicious proteins whose would-be homologs from related DNA show a statistically damning “blizzard” of stop codons spread across their sequence alignments.\n\nAs the authors make clear, Spurio does not provide a clear yes/no decision for which proteins are real. It provides merely a list of proteins that is highly enriched in false predictions, vs. those lacking evidence of falseness. Some protein families, encoded by selfish genetic elements such as transposons, have members decay into pseudogenes so frequently a blizzard of stop codons can mislead. What Spurio actually offers is a new analytical metric that can integrate into workflows for building new protein families, or for deprecating old ones, or for culling bad data from large databases such as UniProt and RefSeq.\n\nSome human review, or use in combination with other indicators, may be necessary for most uses.\n\nSpurio is likely to find its most enthusiastic users among the biocurators and bioinformaticians who build new protein family definitions such as the HMMs of Pfam, and the developers of prokaryotic annotation pipelines such as RAST or PGAP.\n\nBecause so many researchers in the biology and biochemistry of bacteria and archaea depend on these resources, as they try to make better sense of genomic and metagenomic “dark matter,” Spurio may contribute positively to the infrastructure of bioinformatics, with most beneficiaries unaware of its theory and its role.\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/7-261
|
https://f1000research.com/articles/6-1991/v1
|
13 Nov 17
|
{
"type": "Research Note",
"title": "Multi-species consumer jams and the fall of guarded corals to crown-of-thorns seastar outbreaks",
"authors": [
"Mohsen Kayal",
"Jane Ballard",
"Mehdi Adjeroud",
"Jane Ballard",
"Mehdi Adjeroud"
],
"abstract": "Outbreaks of predatory crown-of-thorns seastars (COTS) can devastate coral reef ecosystems, yet some corals possess mutualistic guardian crabs that defend against COTS attacks. However, guarded corals do not always survive COTS outbreaks, with the ecological mechanisms sealing the fate of these corals during COTS infestations remaining unknown. In August 2008 in Moorea (17.539° S, 149.830° W), French Polynesia, an unusually dense multi-species aggregation of predators was observed feeding upon guarded corals following widespread coral decline due to COTS predation. Concurrent assaults from these amplified, mixed-species predator guilds likely overwhelm mutualistic crab defense, ultimately leading to the fall of guarded corals. Our observations indicate that guarded corals can sustain devastating COTS attacks for an extended duration, but eventually concede to intensifying assaults from diverse predators that aggregate in high numbers as alternative prey decays. The fall of guarded corals is therefore suggested to be ultimately driven by an indirect trophic cascade that leads to amplified attacks from diverse starving predators following prey decline, rather than COTS assaults alone.",
"keywords": [
"Predator outbreak",
"Acanthaster",
"Mutualistic defense",
"Guardian crab",
"Trapezia",
"Mixed-species predator guild",
"Trophic cascade",
"Density dependence."
],
"content": "Introduction\n\nIdentifying ecological processes that drive species trajectories is a prerequisite for ecosystem management. However, community dynamics are sometimes governed by unexpected, indirect interactions and complex emergent properties that can cause runaway responses and abrupt ecological shifts (Silliman et al., 2013; Terborgh & Estes, 2010). Outbreaks of the coral predator crown-of-thorns seastar (COTS) cause widespread coral mortality across the Indo-Pacific Ocean (Pratchett et al., 2014) with often drastic impacts on diverse reef communities (Kayal et al., 2012). However, some coral species possess mutualistic allies that can deter COTS predation. In particular, trapeziid crabs inhabiting large pocilloporids are known for their ability to effectively defend their host corals from COTS assaults (Glynn, 2013; McKeon & Moore, 2014), although guarded pocilloporids do not always survive COTS outbreaks (Leray et al., 2012; see Figure 1). Despite increasing understanding of factors determining coral susceptibility to COTS predation (Glynn, 1976; Kayal et al., 2011; Kayal & Kayal, 2017; Pratchett, 2001; Rouzé et al., 2014), the processes sealing the fate of guarded corals during outbreaks have remained unknown. Here we provide insights into the ecological mechanisms underlying the fall of guarded corals during predatory COTS outbreaks.\n\nPictures were taken at 6 m depth on Tiahura reef in Moorea, French Polynesia, before (a) and after (b) this location was invaded by crown-of-thorns seastar (COTS) swarms. White feeding scars characteristic of recent COTS predation can be seen on several of the guarded coral colonies (Pocillopora eydouxi) in b.\n\n\nMethods\n\nOur observations were performed at the peak of an intense crown-of-thorns seastar (COTS) outbreak that decimated coral communities around the island of Moorea (17.539° S, 149.830° W), French Polynesia, between 2003 and 2010. General patterns in propagation of COTS swarms around the island, and impacts on corals and other reef communities were described by Kayal et al. (2012); Kayal et al. (2017). Here, we provide complementary observations that unveil processes leading to the fall of large pocilloporid assemblages that benefit from “anti-COTS” mutualistic defense, the so-called guarded corals. In Moorea, these assemblages are dominated by Pocilloporida eydouxi, a species that hosts trapeziid crabs able to deter COTS predation (Leray et al., 2012; McKeon & Moore, 2014; Figure 1). Our observations were performed using SCUBA on the outer reef slope at Tiahura where the COTS outbreaks in Moorea were initiated and had particularly detrimental impacts (Kayal et al., 2012).\n\n\nResults and discussion\n\nIn August 2008 at 12 m depth on Tiahura reef, we observed an unusually dense aggregation of coral-eating butterflyfishes jamming around guarded pocilloporids, the last coral bastions that had yet resisted swarms of the predatory seastar (Figure 2, Supplementary Image 1). Widespread coral decline had previously wiped out much of resident populations of coral-feeding butterflyfishes (Kayal et al., 2012), pushing starving survivors to aggregate around the guarded corals. The aggregation of 9 butterflyfishes within a single square-meter (9 fish.m-2), as captured in Figure 2, was particularly surprising, as density of the coral-feeding butterflyfish assemblage on this reef location had dropped to the much lower average value of 4.3±0.9 SE fish.200m-2 (surveyed in June 2008, equivalent to 0.02 fish.m-2). The observed aggregation thus represented a more than 400-times concentration of the predation pressure exerted by the butterflyfishes, and was targeting a guarded pocilloporid that was already under attack by COTS (Figure 2, Supplementary Image 1).\n\nThis aggregation was observed following widespread coral decline (note the absence of live coral in the background) in August 2008 at 12 m depth on Tiahura reef in Moorea, French Polynesia. The predator guild was composed of a crown-of-thorns seastar (COTS) and nine butterflyfishes from species Chaetodon ornatissimus, C. pelewensis, C. quadrimaculatus, C. reticulatus. White feeding scars characteristic of recent COTS predation can be seen on the guarded coral (Pocillopora eydouxi).\n\nGuarded pocilloporids in Moorea have shown the ability to resist devastating COTS predation for several years (McKeon & Moore, 2014; Figure 1). However, concurrent assaults from such locally amplified, mixed-species predatory guilds likely overwhelm the ability of trapeziid crabs and other exo-symbionts to defend host pocilloporids, ultimately causing the fall of guarded corals. Indeed, coral occupation by mutualist communities is determined by strict rules of territoriality and competition (Glynn, 2013; Leray et al., 2012), which limits the abundance of inhabiting guardians in host colonies, and therefore their ability to sustain predatory assaults. Coral decline has already been identified as an engine of COTS movements and prey selection during outbreaks (Kayal et al., 2011; Kayal et al., 2012; Silliman et al., 2013). Our observations suggest that further cascading effects include aggregating diverse predators in numbers surpassing mutualistic defenses, eventually leading to the collapse of guarded corals. We therefore advocate the importance of controlling COTS outbreaks at the earliest stages, before trophic cascades could lead to a runaway collapse of coral communities.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMohsen Kayal’s Ph.D. studies were supported by grants from Polynésienne des Eaux and Planète Urgence.\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 Image 1. Additional photographs capturing the predatory guild aggregating around a guarded coral following widespread coral decline. Pictures (a, b) were taken in August 2008 at 12 m depth on Tiahura reef in Moorea, French Polynesia, at the peak of an intense outbreak of the coral-eating crown-of-thorns seastar (COTS). The observed macro-predator aggregation was composed of one COTS and nine individuals from resident butterflyfish species Chaetodon ornatissimus, C. pelewensis, C. quadrimaculatus, C. reticulatus (see Figure 2). White feeding scars characteristic of recent COTS predation can be seen on the guarded coral (Pocillopora eydouxi).\n\nClick here to access the data.\n\n\nReferences\n\nGlynn PW: Some physical and biological determinants of coral community structure in the eastern Pacific. Ecol Monogr. 1976; 46(4): 431–456. Publisher Full Text\n\nGlynn PW: Fine-scale interspecific interactions on coral reefs: functional roles of small and cryptic metazoans. Smithson Contrib Mar Sci. 2013; 39: 229–248. Reference Source\n\nKayal M, Bosserelle P, Adjeroud M: Bias associated with the detectability of the coral-eating pest crown-of-thorns seastar and implications for reef management. R Soc Open Sci. 2017; 4(8): 170396. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKayal M, Kayal E: Colonies of the fire coral Millepora platyphylla constitute scleractinian survival oases during Acanthaster outbreaks in French Polynesia. Mar Biodivers. 2017; 47(1): 255–258. Publisher Full Text\n\nKayal M, Lenihan HS, Pau C, et al.: Associational refuges among corals mediate impacts of a crown-of-thorns starfish Acanthaster planci outbreak. Coral Reefs. 2011; 30(3): 827–837. Publisher Full Text\n\nKayal M, Vercelloni J, Lison de Loma T, et al.: Predator crown-of-thorns starfish (Acanthaster planci) outbreak, mass mortality of corals, and cascading effects on reef fish and benthic communities. PLoS One. 2012; 7(10): e47363. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeray M, Béraud M, Anker A, et al.: Acanthaster planci outbreak: decline in coral health, coral size structure modification and consequences for obligate decapod assemblages. PLoS One. 2012; 7(4): e35456. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcKeon CS, Moore JM: Species and size diversity in protective services offered by coral guard-crabs. PeerJ. 2014; 2: e574. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPratchett MS: Influence of coral symbionts on feeding preferences of crown-of-thorns starfish Acanthaster planci in the western Pacific. Mar Ecol Prog Ser. 2001; 214: 111−119. Publisher Full Text\n\nPratchett MS, Caballes CF, Rivera-Posada JA, et al.: Limits to understanding and managing outbreaks of crown-of thorns starfish (Acanthaster spp.). Oceanogr Mar Biol Annu Rev. 2014; 52: 133–200. Reference Source\n\nRouzé H, Lecellier G, Mills SC, et al.: Juvenile Trapezia spp. crabs can increase juvenile host coral survival by protection from predation. Mar Ecol Prog Ser. 2014; 515: 151–159. Publisher Full Text\n\nSilliman BR, McCoy MW, Angelini C, et al.: Consumer fronts, global change, and runaway collapse in ecosystems. Annu Rev Ecol Evol Syst. 2013; 44: 503–538. Publisher Full Text\n\nTerborgh J, Estes JA: Trophic cascades: predators, prey, and the changing dynamics of nature. Island Press, Washington DC. 2010. Reference Source"
}
|
[
{
"id": "27894",
"date": "12 Dec 2017",
"name": "Peter W. Glynn",
"expertise": [
"Reviewer Expertise Reef coral biology and ecology."
],
"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 alpheid shrimp guard, Alpheus lottini, also should be noted as defending pocilloporid corals from COTS attacks. This shrimp guard occurs world-wide on pocilloporid corals.\n\nIt would also be worth noting the defensive behaviour, if any, of the crustacean guards toward the fish corallivores.\n\n‘White feeding scars’ are referred to in Fig. 1 and Fig. 2 (supplementary image). These are difficult to make out in the photographs. I suggest adding arrows to make these easier to see. Also, it would be useful to know the approximate diameters of the P. eydouxi colonies.\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\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "30512",
"date": "07 Feb 2018",
"name": "Ciemon Frank Caballes",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting observation of a relatively high concentration of a multi-species predator guild on corals that were initially spared from crown-of-thorns seastar (COTS) predation. The ultimate demise of \"guarded\" Pocilloporids may have been due to a high density of starving COTS (at the peak of an outbreak) feeding on whatever coral was left and overwhelming mutualistic crabs in the process. The overall impact of the butterflyfish, in terms of coral mortality, was most likely lower compared to COTS.\nIt is unclear whether this was a widespread occurrence or a one-time observation. A brief description of the feeding behaviour of COTS and butterflyfishes (relative contribution to coral mortality), as well as the defensive behaviour of Trapeziid crabs will be useful.\nMETHODS: Change Pocilloporida eydouxi to Pocillopora eydouxi.\nSUPPLEMENTARY IMAGE 1: Feeding scars are not clear in the pictures.\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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1991
|
https://f1000research.com/articles/7-259/v1
|
02 Mar 18
|
{
"type": "Research Article",
"title": "Reliability of unconventional torso anthropometry using a three-dimensional scanner in Peruvian children and adolescents",
"authors": [
"Carlos Alberto Delgado",
"Roberto Shimabuku",
"Erika Alarcón",
"Luis Huicho",
"Augusto Cesar Ferreira De Moraes",
"SAYCARE Study Group",
"Roberto Shimabuku",
"Erika Alarcón",
"Luis Huicho",
"Augusto Cesar Ferreira De Moraes"
],
"abstract": "Background: Three-dimensional (3D) scanners have made it possible to measure and display body surface and shape with high precision. These are fast measurements with minimum discomfort, which is especially useful when children are involved. The objective was to assess the reliability and validity of a 3D-scanner for measuring unconventional torso parameters in children and adolescents. Methods: This is a sub-sample of the SAYCARE study, an observational multicentre research effort being conducted in six South American countries, aimed at developing methods to collect data on cardiovascular health biomarkers, lifestyles, and environmental, social and family risk factors. Images were captured using a portable scanner (iSense, Cubify, USA) attached to a Tablet 128Gb with OSX (Ipad-Air Apple, USA). Images were reshaped to exclude head, hair, arms and legs; area and volume were measured using 3D design software ((Rhinoceros for OSX, v5.02, USA). Results: The sub-sample for our study comprised 54 girls and 46 boys, aged 6 to 17 years old, from two private schools in Lima, Peru. Out of 100 participants, 82 were scanned twice. There was strong reliability (rho_c> 0.80) between first and second measurements of area and volume in boys of every age group. In girls, the reliability coefficient was moderate (rho_c> 0.70) only for area comparison in adolescents older than 10 years of age. The mean torso area was 0.55 m2 (SD 0.08) in girls and 0.63 m2 (SD 0.13) in boys. The overall mean torso volume was 24.4 l (SD 5.33) in girls and 31.47 l (SD 10.14) in boys. Area under ROC curve oscillates between 0.5707 and 0.6383 when volume/area ratio was compared to the selected “gold standard” (waist to height ratio > 0.5). Conclusion: Use of portable and low cost 3D-scanners provides a reliable but inaccurate alternative for area and volume torso measurements in children and adolescents.",
"keywords": [
"Reliability",
"accuracy",
"three dimensional",
"3D",
"scanner",
"anthropometry",
"torso",
"children",
"adolescents"
],
"content": "Introduction\n\nThree-dimensional (3D) scanning is a time saving procedure and due to minimum discomfort it has become acceptable to use in children when body components and disease risk are being studied1,2. Moreover, body mass index or head circumference may also be evaluated accurately through 3D scanners that capture three-dimensional images3–5.\n\nScanning devices that scan the body surface generating 3D images originated in the garment industry6. When adapted for computers or personal devices, 3D scanners can measure and display with precision the size and shape of a person's body and the surface of the skin, and offer great potential for medical applications7,8.\n\nCurrently, there are several safe, accurate and reliable portable devices that perform their function in a few seconds4,9. However, despite technological advances, there are few studies using 3D images in children. Pfeiffer et al.10 reported in 2006 the first study of prevalence of flatfoot in children using 3D measurements. Prieto et al.11 reported a study measuring burnt skin area on different ages and Djordjevic et al.12 reported on facial symmetry in adolescents.\n\nTorso 3D measurements are reported in relation to breast position assessment for plastic surgery13 and scoliosis follow-up without x-ray exposure14,15. Clinical or public health relevance for torso 3D measurements in relation to the obesity pandemic still requires additional research to define its usefulness.\n\n\nMethods\n\nThis is a sub-sample of a larger project called South American Youth/Child cARdiovascular and Environmental Study (SAYCARE), an observational multicentre feasibility study based at public and private schools, aimed at developing methods for collecting reliable, comparable and validated data on cardiovascular health biomarkers, lifestyles, and environmental, social and family risk factors in children and adolescents. A detailed description of the SAYCARE sampling and recruitment methodology, data collection and quality control activities has been published elsewhere16.\n\nSample size calculation was performed considering a comparison between observed body surface area (BSA) mean obtained by a 3D scanner (2,139; SD=224) and a calculated BSA mean value using a mathematical formula (2,225), as reported by Schloesser et al.17. We included a type I error α of 0.05 and a type II error β of 0.95. The estimated sample size was 72, and was increased to 86 allowing an anticipated loss up to 20%. Thirty-six female and 46 male participants were recruited. There were 66 adolescents (29 girls and 37 boys) and 16 children under 10 years old (7 girls and 9 boys).\n\nFor data collection, the schools were initially contacted and received a formal invitation with detailed information about the study. The schools were selected for their proximity to the institute and researchers in charge of the study, for being public or private, and because they had students in the required age groups. For the schools that agreed to participate, an information letter and a verbal explanation were provided to the potential participants and their parents or legal guardians.\n\nImages were captured using a portable scanner (iSense, Cubify, USA) attached to a Tablet 128Gb with OSX (Ipad-Air Apple, USA). Special training was not required to use these devices. The training in the use of the scanner was carried out with the support of the local dealer technician during one morning. The training included information on safety, assembly, calibration and how to scan and export images with the scanner attached to the tablet. The scanner was operated by two authors (CD and EA). It allows scanning objects from 30cm to 3 meters in size. Images were capturing by rotating around the subject with the device focused towards the centre. Some training practical sessions by scanning objects were conducted before actually scanning people. The acquired images were rebuilt as objects without texture and processed with software ad-hoc for analysis and processing of digital images. The images were manually reshaped using the 3D design software Rhinoceros for OSX, v5.3.2 (Robert McNeel & Associates, USA) in order to exclude hair in girls or arms in boys and girls, retaining only the torso (Figure 1). Area and volume were measured using 3D design software (Rhinoceros for OSX, v5.3.2).\n\nBody surface scanning was performed in a room with daylight, and with doors and windows closed. Girls were evaluated in a standing position, with their arms over their heads, holding their hair. Boys were evaluated in a standing position with the arms at the sides and the palms forward.\n\nA: Waist to Height Ratio=0.4; B Waist to Height Ratio=0.6. In addition a 3D scan example is presented for Figure 1 B - Torso 3D scan: Model ID 3DPX-008635. Available from https://3dprint.nih.gov/discover/3dpx-008635.\n\nDescriptive analysis included mean, standard deviation and coefficient of variation. Reliability for area in m2 and volume in litres1 was made comparing the first and the second measurement, through the concordance correlation coefficient (rho_c). A new variable was constructed by dividing volume over area in order to apply a curve ROC analysis and estimate the accuracy, sensitivity and specificity for certain values of this method. The Waist-to-Height Ratio (WHtR) was considered as a “gold-standard” to measure obesity, considering a 0.5 as the cut-off point for abdominal obesity. We used a WHtR > 0.5 as gold standard for obesity classification, because this ratio has been reported as accurate in cross-sectional studies for children and adults18. Waist and height measurements were obtained by conventional anthropometric measurements during fieldwork. Expert anthropometrist hired for fieldwork took the anthropometric measures. The size was measured with a stadiometer with the feet not raised from the ground and with the head in the Frankfort plane. The waist was measured with a non-elastic and flexible tape measure, at the midpoint between the last rib and the iliac crest.\n\nThe statistical analyses were performed using the Stata software version 12.1 (StataCorp, College Station, Texas) and were stratified by sex and age group.\n\n\nResults\n\nTable 1 shows the descriptive values for first measurements of area, volume, waist, height and waist to height ratio (WHtR). The original sample comprised 54 girls and 46 boys. Images from 18 participants were excluded because they were incomplete or scanning could not be repeated twice. We obtained complete images for analysis from 36 girls and 46 boys. All 82 children studied were from two private schools in Lima.\n\nN=Number; SD=Standard Deviation; CV=Coefficient of Variation (SD/Mean); W/H ratio=Waist to Height ratio.\n\nTable 2 shows a descriptive analysis that includes the mean values for torso scanned area and volume measurements. It also shows the reliability coefficients for first and second area and volume measurement by sex and age group. In boys the reliability coefficients were strong (rho_c > 0.80) in every comparison between first and second area and volume measurements at any age group. In girls, this coefficient was moderate (rho_c > 0.70) only for area comparison in adolescents older than 10 years of age.\n\nArea-1=First area measurement, Area-2=Second area measurement, Volume-1=First volume measurement, Volume-2=Second volume measurement. rho_c= Concordance correlation coefficient, p=p-value\n\nTable 3 shows mean values for torso scanned area and volume measurements classified by WHtR and age group. It also shows the reliability coefficients for first and second area and volume measurement. The reliability coefficients for obesity were strong (rho_c> 0.80) in every comparison between first and second area and volume measurements at any age group.\n\nArea-1=First area measurement, Area-2=Second area measurement, Volume-1=First volume measurement, Volume-2=Second volume measurement. rho_c= Concordance correlation coefficient, p=p-value\n\nFigure 1 shows examples of 3D scanned images of the torso captured from two male teenagers with opposite values of WHtR. Both adolescents shown in Figure 1 have similar age (both are 16 years-old) and height (both had a height around 1.7 m), but dissimilar WHtR (Image A=0.4 and Image B=0.6).\n\nFigure 2 shows that the first and second three-dimensional torso area and volume measurements have a monotonic correlation.\n\nA: Area measurements; B: Volume measurements. Syntaxes used by STATA program included the command: concord to graph area and volume concordance.\n\nFigure 3 shows the ROC curve for different cut-off points for volume/area ratios of 44 and 48. The area under the ROC curve ranged between 0.5707 and 0.6383 when volume/area ratio was compared to the cut-off point used as compared to the “gold standard” (WHtR > 0.5). In order to measure accuracy volume (in l)/area (in m2) ratio selected cut-off were 44 and 48. Sensitivity was higher (75%) than specificity (39%) when using the volume/area ratio = 44. Specificity was higher (80%) than sensitivity (47%) when using volume/area ratio = 48.\n\nA: Volume to Area ratio = 44; Sensitivity 75%; Specificity 39%. B: Volume to Area ratio = 48; Sensitivity 47%; Specificity 80%. Syntaxes used by STATA program included the command: roctab to graph area and volume area under the curve.\n\n\nDiscussion\n\nPortable scanners are reliable, time-saving devices, and are applicable in childhood nutritional research. Torso 3D measurements obtained by using low cost, portable scanners may increase unconventional anthropometric assessment in children and adolescents.\n\nIn this study, we showed strong reliability of 3D scanning images for torso area and volume measurements, particularly in boys and obese children. Our results are in line with previous reports showing that 3D scanner devices are reliable for different anthropometric measurements2,17,19, and pave the way for further studies with larger numbers of participants.\n\nTo the best of our knowledge, we did not find published papers that used the iSense hand-held technology. However, Knoops et al.20 compared four 3D scanning systems for describing facial form, including the Structure Sensor (Occipital Inc., San Francisco, CA, USA) that is similar to the scanning device used in our study. Those authors found that Structure Sensor performance was within a clinically acceptable range of 2 mm, showing fair agreement with systems more than tenfold its cost, therefore being of great promise for clinical use. For our study, we invested less than $2,000 USD for each scanner attached to a tablet when purchased from local dealers. Portability and user-friendly performance are also important assets for fieldwork.\n\nOf note, the reliability was higher in obese children and adolescents than in non-obese children and girls. As mentioned above in the Methods, images were manually reshaped to exclude head, hair, arms and legs, and then area and volume were measured using 3D software. It may be possible that the reshaping done after the capture of images introduced a bias, which can be more evident when dealing with smaller images.\n\nSeveral authors assessed various 3D scanners in order to understand its usability for unconventional anthropometry.1,17. Santos et al.1 studied 3350 Brazilian children at 6 years old with the aim to describe variation in childhood body shape and size by using three-dimensional photonic scanner using TC2 Three-Dimensional Photonic Scanner (TC2, Cary, NC, USA; www.tc2.com), traditional anthropometry and dual X-ray absorptiometry. These authors found that the component termed corpulence showed strong correlations with traditional anthropometric and body composition measures. Schloesser et al.17 determined the body surface area (BSA) in healthy term and near-term neonates by 3D scanning and compared their results with those from five mathematical formulae for each subject. These authors found that scanned BSA for a full-term new-born was slightly lower than that calculated by mathematical formulae.\n\nIn our study, Area and Volume 3D measurements have strong reliability, but Area to Volume ratio, which was tested as an empirical approach to a 3D diagnostic tool for obesity shows low accuracy. A possible explanation is related to bias linked to manually reshaping of images. Area and volume are not directly measurable by conventional anthropometry but could be well-calculated using reconstruction algorithms from 3D surface imaging systems to assess obesity21. Obesity in children is a tractable condition, and if it is labelled as an epidemiologic pandemic22 or part of a bigger picture23, highly sensitive tools including automatic processing for early diagnosis are required.\n\nThe capture of three-dimensional images using a low-cost portable scanner can be done in approximately 100 seconds with high reliability between measurements. However the scanner is extremely sensitive to movements, and if this happens, it is necessary to repeat the whole procedure. In addition, when scanning people with comparison purposes, it was observed that some uniformity is required in the amount of clothes that can be used to perform the body surface scans.\n\nA major strength in our study is that we were able to assess the performance of a portable, low cost device to evaluate unconventional torso anthropometry in youths in a middle-income country, so we are adding to scientific literature with results from people and places not well studied.\n\n\nConclusions\n\nThe use of portable and low cost 3D scanners provides a reliable but inaccurate alternative for area and volume as unconventional anthropometric torso measurements in children and adolescents.\n\n\nData availability\n\nDataset 1: 3D-Scanner-Unconventional-Anthropometry_Database.\n\nAvailable from: https://dataverse.harvard.edu Dataset Persistent ID doi:10.7910/DVN/BLH6BS\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nEthics and consent\n\nInstitutional Research Ethic Committees approved the study protocol in each center where the SAYCARE team collected data: University of Buenos Aires (Argentina), National Institute for Child’s Health at Lima (Peru), University of Antioquia (Colombia), Catholic University of Uruguay (Uruguay), University of Talca (Chile), University of Sao Paulo (Brazil), and Federal University of Piauí (Brazil).\n\nFor those who agreed to participate, informed written consent had to be signed by the parent. This had to be signed by a parent or legal guardian and by adolescent participants, before the enrolment. Adolescents, under 18 years-old are not legally able to consent alone. In addition, in Lima, children over 8 years of age were also asked to give their consent.\n\nIn Lima - Peru, the study was approved by the Review Board of the National Institute for Child’s Health (Document number 00222-CEI-INSN-2015, February 25th, 2015). The informed consent was obtained from all participants and guardians of the children, clarifying doubts when needed, through written communications, telephone or face-to-face conversations.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe SAYCARE Study was supported mainly by Brazilian Government from National Counsel of Technological and Scientific Development (CNPq; proc. 471266/2013-2) and São Paulo State Government from São Paulo Research Foundation (FAPESP; proc. 2014/11468-6). The SAYCARE Study has also been co-funded by other agencies in the other countries: (i) Collaborative Projects Fund (R.D. N°501-2015-INSN-DG-OEA) granted by the Instituto Nacional de Salud del Niño, Lima, Perú; (ii) Sustainability Strategy at the University of Antioquia 2014-2015, Research group of social and economic determinants of health and nutrition, and Demography and Health Research Group at the University of Antioquia, Medellin, Colombia, and Interuniversity Services Corporation (CIS) from UdeA; (iii) Secretary of University Extension and Student Welfare, University of Buenos Aires; (iv) European Regional Development Fund (MICINN-FEDER) to GENUD Research Group.\n\nDr. Augusto César F. de Moraes was given a post-doctoral scholarship from São Paulo Research Foundation — FAPESP (proc. 2014/13367-2 and 2015/14319-4).\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 acknowledge school principals, teachers and other staff who provided authorization and support in the following schools: “Salesianos”, “María Auxiliadora”, “Mariano Melgar”, “Marianitos” and “Cuna Jardín”. We also thank children, adolescents and their parents for their willing participation. We acknowledge Mr. Andrew Mello Silva skillful management of SAYCARE database.\n\nWe thank the SAYCARE Study Group for its contribution to our research.\n\n\nReferences\n\nSantos LP, Ong KK, Day F, et al.: Body shape and size in 6-year old children: assessment by three-dimensional photonic scanning. Int J Obes (Lond). 2016; 40(6): 1012–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWells JC, Stocks J, Bonner R, et al.: Acceptability, Precision and Accuracy of 3D Photonic Scanning for Measurement of Body Shape in a Multi-Ethnic Sample of Children Aged 5-11 Years: The SLIC Study. PLoS One. 2015; 10(4): e0124193. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWells JC, Treleaven P, Cole TJ: BMI compared with 3-dimensional body shape: the UK National Sizing Survey. Am J Clin Nutr. 2007; 85(2): 419–25. PubMed Abstract | Publisher Full Text\n\nGeil MD, Smith A: Accuracy and reliability of a system for the digital capture of infant head shapes in the treatment of cranial deformities. JPO: Journal of Prosthetics and Orthotics. 2008; 20(2): 35–8. Publisher Full Text\n\nIfflaender S, Rüdiger M, Koch A, et al.: Three-Dimensional Digital Capture of Head Size in Neonates – A Method Evaluation. PLoS One. 2013; 8(4): e61274. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTreleaven P, Wells J: 3D body scanning and healthcare applications. Computer. 2007; 40(7): 28–34. Publisher Full Text\n\nHoffmann J, Westendorff C, Leitner C, et al.: Validation of 3D-laser surface registration for image-guided cranio-maxillofacial surgery. J Craniomaxillofac Surg. 2005; 33(1): 13–8. PubMed Abstract | Publisher Full Text\n\nWang J, Gallagher D, Thornton JC, et al.: Validation of a 3-dimensional photonic scanner for the measurement of body volumes, dimensions, and percentage body fat. Am J Clin Nutr. 2006; 83(4): 809–16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEtxaniz O, Solaberrieta E, Mínguez R, et al.: Digital modelling of a human skull. Journal of Achievements in Materials and Manufacturing Engineering. 2008; 27(1): 55–8. Reference Source\n\nPfeiffer M, Kotz R, Ledl T, et al.: Prevalence of Flat Foot in Preschool-Aged Children. Pediatrics. 2006; 118(2): 634–9. PubMed Abstract | Publisher Full Text\n\nPrieto MF, Acha B, Gómez-Cía T, et al.: A system for 3D representation of burns and calculation of burnt skin area. Burns. 2011; 37: 1233–40. PubMed Abstract | Publisher Full Text\n\nDjordjevic J, Toma AM, Zhurov AI, et al.: Three-dimensional quantification of facial symmetry in adolescents using laser surface scanning. Eur J Orthod. 2014; 36(2): 125–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi D, Cheong A, Reece GP, et al.: Computation of breast ptosis from 3D surface scans of the female torso. Comput Biol Med. 2016; 78: 18–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGorton GE 3rd, Young ML, Masso PD: Accuracy, reliability, and validity of a 3-dimensional scanner for assessing torso shape in idiopathic scoliosis. Spine (Phila Pa 1976). 2012; 37(11): 957–65. PubMed Abstract | Publisher Full Text\n\nRamsay J, Seoud L, Barchi S, et al.: Assessment of Breast Asymmetry in Adolescent Idiopathic Scoliosis Using an Automated 3D Body Surface Measurement Technique. Spine deform. 2017; 5(3): 152–8. PubMed Abstract | Publisher Full Text\n\nCarvalho HB, Moreno LA, Silva AM, et al.: Design and Objectives of the South American Youth/Child cARdiovascular and Environmental (SAYCARE) Study. Obesity (Silver Spring). 2018; 26 Suppl 1: S5–S13. PubMed Abstract | Publisher Full Text\n\nSchloesser RL, Lauff M, Buxmann H, et al.: Three-dimensional body scanning: a new method to estimate body surface area in neonates. Neonatology. 2011; 100(3): 260–4. PubMed Abstract | Publisher Full Text\n\nBrowning LM, Hsieh SD, Ashwell M: A systematic review of waist-to-height ratio as a screening tool for the prediction of cardiovascular disease and diabetes: 0·5 could be a suitable global boundary value. Nutr Res Rev. 2010; 23(2): 247–69. PubMed Abstract | Publisher Full Text\n\nPepper MR, Freeland-Graves JH, Yu W, et al.: Validation of a 3-dimensional laser body scanner for assessment of waist and hip circumference. J Am Coll Nutr. 2010; 29(3): 179–88. PubMed Abstract | Publisher Full Text\n\nKnoops PG, Beaumont CA, Borghi A, et al.: Comparison of three-dimensional scanner systems for craniomaxillofacial imaging. J Plast Reconstr Aesthet Surg. 2017; 70(4): 441–9. PubMed Abstract | Publisher Full Text\n\nXu B, Yu W, Yao M, et al.: Three-dimensional surface imaging system for assessing human obesity. Opt Eng. 2009; 48(10): nihpa156427. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDietz WH: The response of the US Centers for Disease Control and Prevention to the obesity epidemic. Annu Rev Public Health. 2015; 36: 575–96. PubMed Abstract | Publisher Full Text\n\nLobstein T, Jackson-Leach R, Moodie ML, et al.: Child and adolescent obesity: part of a bigger picture. Lancet. 2015; 385(9986): 2510–20. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "32551",
"date": "04 May 2018",
"name": "Lina Jaeschke",
"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\nSummary The manuscript by Delgado et al. describes the application of a 3D scanner to assess body surface area and body volume in Peruvian children and adolescents in the age of 6 to 17 years. The study included 82 children and adolescents and authors used concordance correlation coefficients to assess ‘reliability’ of body surface area and body volume estimation, and ROC analyses to assess ‘validity’ of the volume-to-area ratio as compared to the manually measures waist-to-height ratio. Authors conclude that the 3D scanner ‘provides a reliable but inaccurate alternative for area and volume torso measurement’.\nWith regard to the growing interest in finding new technologies and novel markers to assess the individual anthropometry as potential risk factor, the study deals with an interesting topic and promising measurement technique. However, there are major concerns about the study protocol used and the presentation of the study in the manuscript, especially in terms of the methods.\nPlease see detailed comments below.\n\nMajor comments Introduction\n1. Although touching an interesting field, i.e., the assessment of 3D body volume and body surface area, I think that the scientific impact and predictive value of assessing body surface area and volume, and, thus, the rational for the study and the potential big advantage that 3D body scanners may possess, was not make clear. One decisive point is only mentioned shortly in the discussion section: the potential of 3D scanners to be a ‘diagnostic tool for obesity’. In this respect, also the ‘unconventionality’ (see title) of the measures ‘surface area’ and ‘volume’ was unfortunately not pointed out, although this would clearly highlight the scientific relevance of the manuscript. Further, a short overview over current methods to assess body surface area and volume, and their shortcomings would underline the value of 3D body scanners.\n2. The scanner technique used by the authors is different from many of the currently used stationary 3D body scanners (e.g., the Vitus Smart XXL), not only due to the portability of the scanner but also due to the technique underlying the scanner. However, in the introduction and discussion section, the authors cite studies using such other stationary scanning devices to point at the ‘accuracy’ and ‘precision’ of 3D scanners, and authors further compared their findings with other stationary scanning devices. Discussing differences between (and limitations of) the current (portable) and other (stationary) scanning techniques would help to point at the ‘novelty’ of the technology and further to meaningfully interpret the current findings.\nMethods 3. Overall, the statistical methods used, are reported insufficiently, so that I believe that analyses presented are not reproducible by others.\n4. Further, the scan protocol is not made clear. Persons were scanned twice – what was the time interval between measures? Some more information about the scanning technique is needed to understand the procedure and study (e.g. What kind of image is created? A whole body image? Which/how many anthropometric measures are determined? The authors mentioned using a 3D design software – is it specific for the body scanner? The authors report that scanning device captures pictures by ‘rotating around the subject’ – was rotating based on a manual movement by the study personnel or was it performed automatically by the device?).\n5. Further, could the authors please explain how they defined reliability (short-term vs. long-term reliability, i.e., technical reliability of the device vs. true variability/stability of the measure) and validity? This is currently not made clear to the reader and, thus, the aim and methods are not really clear.\n6. Why did the authors use rho_c to assess reliability and ROC analyses to investigate the agreement of the volume-to-area and the waist-to-height ratio (which is their concept to proof validity)? Did the authors also thought about using ICC and Bland Altman plots instead?\n7. Typically, persons being investigated are scanned with only wearing underwear and a bathing cap. In the present study, children and adolescents were scanned wearing loose clothing as presented in Figure 1. I am not sure if this makes sense, when aiming to investigate reliability and validity of the torso in terms of body surface area and volume. The authors themselves discuss that for ‘comparison purposes [...] some uniformity is required in the amount of clothes’. Uniformity, however, was not fulfilled in the present study.\n8. There were huge differences in the scanning procedures between sexes: boys were scanned ‘in a standing position with the arms at the sides and the palms forward’, while girls were scanned ‘in a standing position, with their arms over their heads, holding their hair’. However, it is important to consider that previous studies indicate the significant impact of the arm position on abdominal measures assessed by 3D body scanners.\n9. It is not clear to me, why captured 3D images were edited so extensively (‘The acquired images were rebuilt as objects without texture [...] images were manually reshaped [...] in order to exclude hair in girls or arms in boys and girls, retaining only the torso’). Typically, researchers using 3D scanning techniques evaluate whole body images. Could the authors please explain the rational/need for this editing? Why was it not possible to evaluate the whole body image? Further, was there any standard operating procedure defining the ‘manual’ editing to make the process less subjectively and, therefore, reproducible?\n10. Similarly, the differences in editing 3D images between sexes seem to be problematic in my point of view (‘images were manually reshaped [...] in order to exclude hair in girls or arms in boys and girls’). Probably, authors wanted to account for (typically) longer/more voluminous hair in girls than in boys. However, did the authors consider excluding hair in both sexes or using bathing caps to reduce bias due to sex differences in data handling?\n11. Regarding editing of images, in the methods the authors report that ‘images were manually reshaped [...] in order to exclude hair in girls or arms in boys and girls’, while in the abstract and discussion they say that ‘images were manually reshaped to exclude head, hair, arms, and legs’ – how was reshaping done? If heads were excluded, exclusion of hair (and the description of exclusion of hair in girls in the methods) seems to be unnecessary.\n12. I am not sure about using the waist-to-height ratio as standard reference to assess validity of the 3D scanner-based volume-to-area ratio, since both ratios reflect different anthropometric information on body shape. However, what is the predictive value of the waist-to-height ratio in terms of health risk? Pointing at this issue may support the authors’ use of this ratio to assess validity. Nevertheless, in my point of view, the concept used here may only assess validity indirectly, since the waist-to-height ratio is not a commonly used standard reference to assess body surface area or volume (for the latter, air displacement plethysmography may be the better ‘gold standard’). Pointing at this limitation would be beneficial for the manuscript.\n13. The ROC analysis is not quite clear to me. Figures show three dots for each curve only – could the authors please be more precise about the analyses, underlying data, and choosing cut points. What is meant by ‘certain values of this method’? Typically, the ROC shows sensitivity and 1-specifity over a range of cut points. However, in the results, figures suggest that the ROC is only shown for the cut points 44 and 48 for the volume-to-area ratio – or how can the figures be interpreted? Further, calculation of AUC is not reported in the methods section.\nResults 14. Results are partly described sparsely and do not always represent the ‘whole picture’ of results shown in the tables (see minor comments for details).\n15. The AUC observed is 0.5707 and 0.6383. An acceptable discrimination is typically considered for AUC >0.7. Could the authors provide an evaluation and interpretation of the observed AUC and an underlying definition for such evaluation?\nDiscussion 16. The discussion section would strongly improve, when authors may more discuss their own results. Currently, their own results are hardly discussed.\n17. The authors did several sub-group analyses but did not really discuss differences found between measures, sexes, age groups, and ‘obesity’ groups, or probable reasons for different findings.\n18. Strengths and limitations should be addressed in more detail, also with regard to the comments made on the scanning protocol and data handling.\n19. Finally, I am not sure about the conclusion the authors made. It is concluded that 3D scanners provide “a reliable but inaccurate alternative” – Is it really an alternative if the measurement is inaccurate? Further, if at all, what it is an alternative for (see also comment #1)?\nAbstract 20. In the background section, authors mention that 3D scanners allow measuring and displaying body surface and shape with ‘high precision’. However, is investigating the ‘precision’ not one of the aims of the authors’ study (in terms of reliability)?\n21. In the background section, authors state that investigating ‘unconventional’ torso measures was one of the current aims? For me, it is not clear what is meant by ‘unconventional’. Probably, body area and volume are meant. However, both are first mentioned in the methods section. I would further recommend to shortly make clear the predictive value of measuring body surface area and volume, and, thus, the potential big advantage of 3D body scanners.\n22. In the methods section, there is no information about statistics. Thus, the following results are hardly to follow and to interpret. E.g. What scan protocol was applied (obviously authors scanned persons twice – what was the time interval between measures)? How did the authors define reliability (short-term vs. long-term reliability, technical reliability of the device vs. true variability/stability of the measure) and how was it analyzed (obviously by using rho)? How was validity defined and analyzed (obviously by using ROC analyses with the waist-to-height ratio as standard reference)?\n23. In the methods section, there is no information about age range and sample size included.\n24. In the results section, I do not think that it is useful to report mean body surface area and volume as averaged over all ages, as ages included ranged from 6 to 17 years (as mentioned inthe main text), therefore, encompassing a wide range of body weight, height, and shape.\n25. Please refer to major comment #19.\nMinor comments Introduction 1. In the introduction section, authors say that 3D scanning is a ‘time saving procedure’ but in the discussion section they say that scanning requires around 100 seconds, with the scanner being ‘extremely sensitive to movements’. In my point of view, 100 seconds is quite a long time for standing motionlessly, especially for children. Further, ‘time saving’ would only make sense for me if knowing the number of measures assessed in this time period and if knowing the time required for alternative measurements.\n2. First paragraph, second sentence and elsewhere: ‘3D’ instead of ‘three-dimensional’\n3. Second paragraph, first sentence: please check wording.\nMethods 4. Second paragraph, first sentence: What does the numbers in parenthesis stand for? What data was the basis for the sample size calculation?\n5. Second paragraph: Please check the numbers on participants recruited. In the abstract and results section, authors report to have recruited 100 participants (54 females and 46 males), while here, authors report to have recruited 82 participants (36 females and 46 males). Obviously, the difference may be explained by the 18 participants excluded due to incomplete pictures as mentioned in the results section. However, please align numbers on recruited (vs. included/assessed) participants.\n6. Did the authors consider a selection bias due to the recruitment of participants from only 2 private schools being proximate to the institute/researchers in charge?\n7. Fourth paragraph: I find it a bit confusing that authors say that training in usage of the scanner was not required, while reporting the way of training done in the present study at the same time.\n8. Sixth paragraph: ‘gold standard’ instead of ‘gold-standard’\n9. How did the authors define ‘accuracy’?\n10. Was there any definition for interpreting rho_c, e.g., as ‘poor’ or ‘good’?\n11. Last paragraph: Analyses were also performed stratified by waist-to-height ratio.\nResults 12. First paragraph: ‘not be repeated’ instead of ‘not be repeated twice’\n13. Obviously, only girls were excluded due to incomplete scanning – was there any reason for that?\n14. Second paragraph, last sentence: Maybe you want to add the information that for other comparisons (volume and younger ages) in girls, coefficients were weak.\n15. Third paragraph: ‘the reliability coefficient for area and volume’ instead of ‘the reliability coefficient for obesity’ (since the term ‘obesity’ is used by the authors with regard to waist-to-height ratio)\n16. Third paragraph, last sentence: Authors say that rho_c was ‘>0.8 in every comparison [...] at any age’. However, this is not true for several sub-group analyses, e.g., in children for area and waist-to-height ratio ≤0.5, rho_c was 0.6000.\n17. Fourth paragraph: This finding is interesting but it is not further discussed. What is the implication of this result?\n18. Table 1: Here, waist-to-height ratio is abbreviated by using ‘W/H’, while in the text, authors use ‘WHtR’. Please align.\n19. Tables 1 to 3: the highest age included is 17 years; thus, I would recommend changing age group ‘15-18 years’ to ’15-17 years’.\n20. Tables 1 to 3: What do the p-values stand for?\n21. In addition to figure 3, a table showing sensitivity, specificity and AUC for all assessed cut points for the volume-to-area ratio would help to interpret results.\n22. Figures 2 and 3: Since all other analyses were made separately for sexes, age groups, and waist-to-height groups, I think that providing stratified figures for the different groups would be more in line with the other results and would, thus, help to interpret results as a whole.\nDiscussion 23. First paragraph: Does these statements are based on the authors’ results or based on previous research?\n24. Fourth paragraph: I would recommend to use ‘waist-to-height ratio <0.5’ or ‘waist-to-height ratio ≤0.5’ instead of ‘obese’ and ‘non-obese’, respectively, to facilitate following the discussion with regard to the results.\n25. I do not understand the explanation that reshaping ‘smaller images’ may be more bias-sensitive and, thus, may have caused the finding that rho_c was larger for waist-to-height ratios >0.5 than for ratios ≤0.5. The editing of images was not made in the torso area and the differences in the size of the images may not have been that huge to fully explain differences in rho_c between waist-to-height ratios >0.5 vs. ≤0.5. Did the author thought about other reasons, why rho_c was larger for waist-to-height ratios >0.5 than for ratios ≤0.5?\n26. Did the authors thought about the generalizability of their findings, keeping in mind that the study population was recruited from 2 private schools in Lima?\n27. Fifth paragraph: I do not think that this paragraph is that relevant for the discussion without putting these previous findings in context with the current findings; see major comment 2.\n28. Sixth paragraph: ‘Strong reliability’ does not hold true for all sub-groups assessed in the current study, e.g., rho_c was mainly weak in girls and for waist-to-height ratios ≤0.5.\n29. Sixth paragraph, third sentence onwards: I would recommend moving this part into the background section, since it is the basic topic of the current study and highlights its scientific relevance.\n30. Eighth paragraph: I do not think that this is a strength of the current study, since it was its aim.\nConclusion 31. First sentence: ‘provide’ instead of ‘provides’\nAbstract 32. In the methods section, last sentence, please delete one of the two left parentheses.\n33. In the method section, please introduce the abbreviations rho_c, SD, and ROC.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "32195",
"date": "08 May 2018",
"name": "Markus Scholz",
"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\nMajor issues:\nAbstract: The authors stated that the scanner is “inaccurate”. To my opinion this conclusion is far too general and not supported by the presented data. The research question is not sufficiently motivated. Why are the authors interested in the torso? Existing literature is not sufficiently discussed. Please consider Glock F et al. Pediatr Res. 2017 and Kuehnapfel et al. Sci Rep. 2016 Sample Size calculation: What is the null-hypothesis used for the sample size calculation and how does it relate to the research question? From the torso picture (figure 1), I conclude that the subjects are not undressed. This is unusual in anthropometric research. How does clothing impact your results? I have concerns that scanning posture is different for girls and boys. What is the impact of scanning posture on results? Could this be a reason for the different reliability results in girls and boys? The measure used for obesity is unusual to my opinion and should be better justified. WHtR is not a gold standard for defining obesity at least to my knowledge. Why not using BMISDS or WHR? Figure 2: Should be replaced by Bland-Altman plots which are more informative. Table 2: Confidence intervals of rho_c values are more relevant regarding reliability than p-values. Please replace. Figure 3: ROC analysis is not carried out correctly. Please provide the complete ROC curve and its corresponding area. Alternatively, I would recommend removing the analysis since WHtR is not a gold standard of obesity diagnosis as far as I know. Language polishing is required throughout the paper.\nMinor issues:\nAbstract: “Area under ROC curve oscillates…” This cannot be true. The area is fixed. Methods page 3, col 1: SD=224 can hardly be true Please provide times between first and second measurement.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
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https://f1000research.com/articles/7-259
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https://f1000research.com/articles/7-258/v1
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02 Mar 18
|
{
"type": "Brief Report",
"title": "Comparing organic versus conventional soil management on soil respiration",
"authors": [
"Bence Mátyás",
"Maritza Elizabeth Chiluisa Andrade",
"Nora Carmen Yandun Chida",
"Carina Maribel Taipe Velasco",
"Denisse Estefania Gavilanes Morales",
"Gisella Nicole Miño Montero",
"Lenin Javier Ramirez Cando",
"Ronnie Xavier Lizano Acevedo",
"Maritza Elizabeth Chiluisa Andrade",
"Nora Carmen Yandun Chida",
"Carina Maribel Taipe Velasco",
"Denisse Estefania Gavilanes Morales",
"Gisella Nicole Miño Montero",
"Lenin Javier Ramirez Cando",
"Ronnie Xavier Lizano Acevedo"
],
"abstract": "Soil management has great potential to affect soil respiration. In this study, we investigated the effects of organic versus conventional soil management on soil respiration. We measured the main soil physical-chemical properties from conventional and organic managed soil in Ecuador. Soil respiration was determined using alkaline absorption according to Witkamp. Soil properties such as organic matter, nitrogen, and humidity, were comparable between conventional and organic soils in the present study, and in a further analysis there was no statically significant correlation with soil respiration. Therefore, even though organic farmers tend to apply more organic material to their fields, but this did not result in a significantly higher CO2 production in their soils in the present study.",
"keywords": [
"soil respiration",
"conventional soil management",
"organic soil management"
],
"content": "Introduction\n\nResearch related to the benefits of organic management1 has become increasingly important in sustainable agriculture. Organic soil management can contribute to meaningful socio-economic and ecologically sustainable development. Kilcher states that \"Organic agriculture reduces the risk of yield failure, stabilizes returns and improves the quality of life of small farmers’ families\"2. Soil management has great potential to affect soil respiration, which is an important qualitative indicator of soil microbial activity3. Soil respiration is released as a result of soil organic matter decomposition. The present study aims to investigate the effects of organic versus conventional management on CO2 production of some Northern Ecuadorian agricultural soils. Our hypothesis was that major soil respiration will be observed in soils under organic management due to the increased amount of applied organic materials.\n\n\nMethods\n\nSoil samples from 23 organic farms and conventionally managed neighbouring farms were analyzed. In total, 17 sampling sites were located in organic farms, while 6 sampling sites were located in chemical fertilizer-treated areas. The sampling sites were chosen according to proximity of organic and conventionally managed farms in which the same crops are produced. Further details about each of the sampling sites can be found in Table 1. Approximately 1000 g of soil samples of 0–20 cm depth were taken. The following crops were produced in the examined areas: broccoli, potato, tomato and carrot.\n\nVariables are follows: areas of examined lands (m2), Name of crops, soil management (Organic/Conventional), Total crop production (kg), Applied fertilizer (kg), Type of fertilizers, Concentration of NPK, Concentration of NPK, Amount of NPK (Kg), GPS coordinates of the examined lands.\n\nSoil moisture content was determined gravimetrically, drying the soil at 105°C for 24 hours according to Fernández et al. (2008)4. Soil texture was measured using sodium hexametaphosphate ((NaPO3)6) according to Bouyoucos (1962)5. To measure the soil chemical properties, the samples were sieved through a 2mm mesh and pre-incubated at 25° for 72 hours. Soil pH in distilled water (soil/water, 1/2.5, w/w) was determined according to Karkanis (1991)6. In addition, we measured the electrical conductivity (EC) using a glass electrode according to Karkanis (1991)6. Cylinder volume was determined according to Agostini et al. (2014)7. Soil organic matter was determined according to Walkley and Black (1934)8. We measured the phosphorous content according to Olsen (1954)9. The Sand/Silt/Clay ratio was determined by Bouyoucos’s method (1936)10, while the cation exchange capacity was determined according to ISO 11260 (1994)11 protocol.\n\nThe experiment was applied at 25°C. 0, 1M NaOH (10ml) was placed in laboratory bottles (250ml), a sterile gauze pad were filled with 10 g of soil sample according to Witkamp (1966)12. After 10 days, the amount of CO2 was subsequently measured by standardized titration against 0.1N HCl using firstly phenolphthalein and then methyl orange indicator according to Witkamp (1966)12.\n\nThe below formula was applied to calculate soil respiration:\n\nm(CO2) = VxNx22 CO2\n\nAnd CO2 production (for 10 days):\n\nmg(CO2) * 100g – 1 * 10 day – 1 = methyl orange factor * HCI – phenolphthaleinloss) * NAOH factor * 2, 2 * Moisture multiplication factor\n\nwhere\n\nMoisture multiplication factor=(moisturecontent%+100)100\n\nWe determined the volume of the examined soils (counting with 0 – 20 cm depth) using topsoil calculator tool (https://www.tillersturf.co.uk/topsoil-calculator). The results of soil respiration was then estimated in kg(CO2)/ha/day.\n\nTo evaluate the behavior within results, two types of test were performed: i) Student’s t-test for comparing means between conventional and organic crop systems in terms of soil respiration (kg/CO2/ha/day), organic matter (%) and nitrogen (%). Furthermore, Person’s and Spearman’s correlation were fixed in order to test data covariation and correlation. ii) ANOVA was used to compare conventional and organic crop system and the type of crop harvested in the sampling site.\n\n\nResults\n\nThe results of soil respiration from areas of organic and conventional soil management are comparable (Dataset 1).\n\nFor soil respiration, conventional soil mean was 88.50 and organic mean was 98.64, showing and increment around 10%. However, there were no statistically significant differences between group means as determined by one-way ANOVA (p =0.15), comparing conventional and organic systems. Pearson‘s and Kendell‘s tests have showed no correlation. Soil respiration correlation coefficient with organic matter was lower than 0.05 and with nitrogen content was lower than 0.12. This analysis did not consider the differences between conventional and organic systems (Figure 1).\n\nThere were statistically significant differences between group means as determined by one-way ANOVA (p < 0.05), comparing crop types. Furthermore, a post hoc test (Duncan) was fixed. There was only one crop (carrot) in conventional system (odds lower than 0.05) that differs drastically from the others, as pointed out in (Figure 2).\n\nConsidering soil characteristics (pH, CIC, K, and Electric conductivity), Student’s t-test was applied to identify differences between conventional and organic systems. Only the characteristics from carrot crop systems (conventional or organic) have shown differences in terms of means (p < 0.05). Furthermore, the mean of conventional crop system was lower in every characteristic evaluated. Besides, these results were in congruence with Figure 2, leading us to believe that the cropping system has no influence on soil respiration, which is in contrast to the influence that soil characteristics have over soil respiration in this study.\n\n\nConclusions\n\nOrganic farmers tend to apply more organic material to their fields, but this did not result in a significantly higher CO2 production in their soils. The difference between organic and conventional soils (10% in mean) is not enough to conclude that the soil respiration under these two systems was different, considering the analysis of their variance.\n\nSoil properties like organic matter, nitrogen, and humidity, were comparable between conventional and organic soils in the present study, and in a further analysis there was no statically significant correlation with soil respiration. However, biological significance should be investigated in a posteriori research including microbial community profile of the soil and specific interactions in highlands (over 2500 m.a.s.l.).\n\n\nEthics\n\nOral consent was obtained from the farmers for the collection of soil samples from their land. Their only request was to inform them about the results of the soil characteristics, that we have already done personally on 9 November, 2017.\n\n\nData availability\n\nDataset 1: Raw data for various parameters calculated in conventional and organic managed soils. Parameters as follows: pH, Organic material (percentage), Total Nitrogen (percentage), Match (mg/kg), Potassium (cmol/kg), Electrical conductivity (dS/m), CIC (cmol/kg), Soil moisture content (percentage), Sand (percentage), Silt-limo (percentage), Clay (percentage), Texture (class), Soil respiration (kg/CO2/ha/day). DOI, 10.5256/f1000research.13852.d19552913",
"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\nvan Diepeningena AD, de Vosa OJ, Korthalsb GW, et al.: Effects of organic versus conventional management on chemical and biological parameters in agricultural soils. Appl Soil Ecol. 2006; 31(1–2): 120–135. Publisher Full Text\n\nKilcher L: How organic agriculture contributes to sustainable development. Kassel University Press, University of Kassel at Witzenhausen JARTS, 2007; (Supplement 89): 31–49. Reference Source\n\nBaldock JA, Wheeler I, McKenzie N, et al.: Soils and climate change: Potential impacts on carbon stocks and greenhouse gas emissions, and future research for Australian agriculture. Crop Pasture Sci. 2012; 63(3): 269–283. Publisher Full Text\n\nFernández L, Roldán T, Zegarra H, et al.: Manual de técnicas de análisis de suelos aplicadas a la remediación de sitios contaminados. Can Agr Eng, Mexico. 2008. Reference Source\n\nBouyoucos GJ: Hydrometer method improved for making particle size analyses of soils. Agron J. 1962; 54(5): 464–465. Publisher Full Text\n\nKarkanis PG, Au K, Schaalje GB: Comparison of 4 Measurement Schedules for Determination of Soil Particle-Size Distribution by the Hydrometer Method. Can Agr Eng. 1991; 33(2): 211–215. Reference Source\n\nde los Ángeles Agostini M, Monterubbianesi MG, Studdert GA, et al.: Un método simple y práctico para la determinación de densidad aparente. Ciencia Del Suelo. 2014; 32(2): 171–176. Reference Source\n\nWalkley A, Black IA: An examination of the Degtjareff method for determining organic carbon in soils: Effect of variations in digestion conditions and of inorganic soil constituents. Soil Sci. 1934; 63: 251–263.\n\nOlsen SR, Cole CV, Watanabe FS, et al.: Estimation of available phosphorus in soils by extraction with sodium bicarbonate. USDA, Washington, DC., 1954. Reference Source\n\nBouyoucos GJ: Directions for making mechanical analysis of soils by the hydrometer method. Soil Science. 1936; 42(3): 225–230. Publisher Full Text\n\nISO 11260: Soil quality -Determination of effective cation exchange capacity and base saturation level using barium chloride solution. 1994.\n\nWitkamp M: Decomposition of Leaf Litter in Relation to Environment, Microflora, and Microbial Respiration. Ecology. 1966; 47: 194–201. Publisher Full Text\n\nMátyás B, Chiluisa Andrade ME, Yandun Chida NC, et al.: Dataset 1 in: Comparing organic versus conventional soil management on soil respiration. F1000Research. 2018. Data Source"
}
|
[
{
"id": "31652",
"date": "15 Mar 2018",
"name": "Anita Jakab",
"expertise": [
"Reviewer Expertise Agricultural environmental management",
"soil management",
"agricultural soil science"
],
"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 worked at the differences between organic and conventional soil management. This research examined an important and topical issue especially the soil respiration under changing plant and soil conditions.\n\nIntroduction and methods The research investigated 23 soil samples in Ecuador. The samples were located from organic (17 samples) and conventionally managed neighboring farms (6 samples). In the research trials broccoli, potato, tomato and carrot were applied as test plant. Soil properties were measured after 1000 g soil samples of 0-20 cm depths of soil were taken in every picked area. The soil moisture, texture, pH, electrical conductivity, cylinder volume, organic matter, phosphorus content, sand/silt/clay ratio and cation exchange capacity, and the soil respiration were analyzed in laboratory. The values of the soil parameters are presented in a dataset, which inform about the important soil parameters especially the calculated soil respiration in kg (CO2)/ha/day). The protocols (description of the tests) are clear and traceable, especially the formula to calculate soil respiration. The study describes the applied type of fertilizers especially the concentration of NPK fertilizers.\n\nComment on the Methods - The sampling time and vegetation status are important for the evaluation, this information is missing in the study. If it’s possible, describe the followings: When the soil sampling happened? What was the state of the vegetation of test plants? - A bit more detail of the soil properties inform us about the actual soil status. The studied soils are classified as sandy textured soil, according to the soil classification (Franco Arenoso). The most typical parameters of the samples are the following: high sandy texture, neutral pH, good/very good organic matter-nitrogen and phosphorus content, 10-20% moisture content. I suggest describing it in the Methods.\n\nResults The results of the study are described with sufficient statistical analysis. It also describes the statistically significant/not significant results. There were solely statistically significant differences between crop types (for soil respiration by one-way ANOVA correlation test). - The Figure 1 contains a typographical error (Orgacin matter instead of Organic matter). - It may be more informative, if you use a line diagram instead of dot diagrams in the first figure. - The Figure 2 include the soil respiration values in kg CO2/ha /day, which would be more clear with the average values.\n\nConclusion The results have briefly evaluated and conclusions straightforward formulated. I quite agree with observations of the study that emphasizes the importance of further microbiological studies.",
"responses": []
},
{
"id": "31654",
"date": "19 Mar 2018",
"name": "Ankit Singla",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe submitted manuscript by Bence et al. is good work which is suitable for publication in F1000 research. Authors have compared the organic practices and conventional practices, and compared their effects on soil respiration which is very important aspect. Standard methodologies were followed which ensures reproducibility of the results. The findings were subjected to the statistical analysis and conclusion drawn nicely.\nHowever, I have below suggestions for improvement which may be considered as minor revisions:\n\"Physical-chemical\" could be replaced by \"Physico-chemical\" throughout the manuscript. In abstract, word \"statically\" should be replaced by \"statistically\" In result, \"showing and increment around 10%.\" should be \"showing an increment around 10%.\" The discussion could be added more so that the findings of the study will become stronger.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-258
|
https://f1000research.com/articles/7-55/v1
|
15 Jan 18
|
{
"type": "Opinion Article",
"title": "Response heterogeneity: Challenges for personalised medicine and big data approaches in psychiatry and chronic pain",
"authors": [
"Agnes Norbury",
"Ben Seymour",
"Ben Seymour"
],
"abstract": "Response rates to available treatments for psychological and chronic pain disorders are poor, and there is a considerable burden of suffering and disability for patients, who often cycle through several rounds of ineffective treatment. As individuals presenting to the clinic with symptoms of these disorders are likely to be heterogeneous, there is considerable interest in the possibility that different constellations of signs could be used to identify subgroups of patients that might preferentially benefit from particular kinds of treatment. To this end, there has been a recent focus on the application of machine learning methods to attempt to identify sets of predictor variables (demographic, genetic, etc.) that could be used to target individuals towards treatments that are more likely to work for them in the first instance. Importantly, the training of such models generally relies on datasets where groups of individual predictor variables are labelled with a binary outcome category − usually ‘responder’ or ‘non-responder’ (to a particular treatment). However, as previously highlighted in other areas of medicine, there is a basic statistical problem in classifying individuals as ‘responding’ to a particular treatment on the basis of data from conventional randomized controlled trials. Specifically, insufficient information on the partition of variance components in individual symptom changes mean that it is inappropriate to consider data from the active treatment arm alone in this way. This may be particularly problematic in the case of psychiatric and chronic pain symptom data, where both within-subject variability and measurement error are likely to be high. Here, we outline some possible solutions to this problem in terms of dataset design and machine learning methodology, and conclude that it is important to carefully consider the kind of inferences that particular training data are able to afford, especially in arenas where the potential clinical benefit is so large.",
"keywords": [
"personalised medicine",
"big data",
"machine learning",
"psychiatry",
"chronic pain",
"individual differences",
"response heterogeneity",
"clinical trial design"
],
"content": "Introduction\n\nThe proportion of patients who respond to available treatments for psychological and chronic pain disorders is often low. For example, in major depression, roughly 40% of individuals experience a ‘clinically significant’ response (decrease in symptom severity score above some minimum value) over the course of treatment (e.g. 1,2). Similarly, a recent meta-analysis of available pharmacotherapies for neuropathic pain found estimates of ‘number needed to treat’ (number of patients needed to be treated to prevent one additional adverse clinical outcome) for effective treatments ranged from 4–10, indicating poor response rates3. For patients, this often means a lengthy process of cycling through different treatment options, in a sequence that may be significantly influenced by non-clinical concerns (e.g. relative drug cost, therapist availability, local health authority guidelines), and where there may be inadequate data on the safety and effectiveness of switching regimes (e.g. 4). For psychological conditions, this process can be particularly lengthy, given the significant period of time before common pharmacological treatments are expected to take effect (e.g. 4–6 weeks to conclude a particular drug treatment is ineffective,4). Together, this results in a substantial burden of suffering and disability to individuals with a diagnosis of these disorders, before (if) an effective treatment option can be found.\n\nIt is generally assumed that differential response to a particular treatment across individuals can be at least partially explained by patient heterogeneity within a certain diagnostic category – i.e. that individuals who present to the clinic with similar sets of symptoms may have different underlying pathologies. This seems a particularly reasonable assumption in the case of both mental health disorders and chronic pain, as diagnosis is often made purely on the basis of self-reported symptom checklists, and our lack of knowledge into the aetiology of these conditions means we have little opportunity for differential diagnosis. Indeed, in the case of psychiatric disorders, such as depression, diagnosis can often be made on the basis of directly contradictory symptom reports (e.g. sleeping too much vs sleeping too little), and there may be many different ways to meet diagnostic criteria (e.g. 227 possible symptom combinations for major depressive disorder, according to DSM-IV5). Similarly, even patients with a diagnosis of a particular pain condition are likely to have distinct patterns of nervous system damage, involving multiple pathways (e.g. 6), and definitions of chronic pain itself can vary dramatically across research groups and clinical centres7.\n\nEven if we lack insight into pathological mechanisms, it seems likely that if we are able to use some kind of predictive method to direct individuals towards treatments that are likely to be more effective for them – then even a small increase in the resulting response rate could potentially have a large effect on disease burden for individual patients. There has therefore recently been great interest in doing just this for psychiatric data, via application of supervised learning methods to large datasets of individual clinical predictors and treatment response data (see 8 for an excellent recent review of potential clinical advantages and best methodological practice in this area).\n\nThe current gold standard approach is firstly to define a set of features and targets for various machine learning algorithms to train on. In this context, features are individual difference variables that may potentially relate to future treatment outcome (clinical, demographic, physiological, genetic, behavioural, etc. information). The target variable (that the algorithm must learn to predict) is usually a binary category label, such as ‘responder’ or ‘non-responder’ (whether or not an individual has exhibited symptom improvement above some threshold level, following a particular course of treatment). Various supervised learning algorithms can then be trained on this labelled dataset (ideally using a rigorous cross-validated approach), and assessed in terms of their predictive accuracy on independent ‘unseen’ (during model training) data. Finally, the best model can be brought forward to a randomised controlled trial framework, where treatment allocation by current clinical guidelines could be compared to algorithm-assisted treatment assignment8.\n\nThis approach is highly attractive, as the potential clinical gains from even a small increase in likelihood of treatment response for a particular individual are large. However, across the field of medicine in general, attempts to make such clinical gains via a personalised medicine approach have not often fulfilled their initial promise – with relatively few reaching the clinic (e.g. 9). Here, we explore a basic statistical issue that may limit the effectiveness of this process – i.e. the reliability of distinguishing between treatment ‘responders’ and ‘non-responders’ in the first place. We further discuss the reasons why this problem may be particularly acute in the case of available data regarding psychiatric disorders and chronic pain conditions, and some potential solutions.\n\n\nThe problem of response heterogeneity\n\nThe problem of properly identifying response heterogeneity, or, more simply, reliably distinguishing between responders and non-responders to a particular treatment, on the basis of randomised controlled trial (RCT) data, has previously been highlighted across various fields of medicine10–12. If not properly addressed, this constitutes an absolute limit on the effectiveness of predictive models at the level of input or training data, thereby limiting their future clinical usefulness.\n\nThe issue is best illustrated by considering the nature of data collected during RCTs, and the kind of inferences this process affords. The foundation of an RCT is that the mean effect of an intervention (e.g. active drug treatment) is derived by comparing what happened, on average, to the (randomly allocated) participants in the intervention group to what happened, on average, to participants in the control (e.g. placebo) arm. The random allocation of participants to the intervention vs control arms allows the control group to function as an illustration of what we might have expected to occur in the intervention group, had they not received the active treatment – in turn allowing us to draw conclusions about the overall (average) effects of the treatment itself12. Crucially, we can only draw this inference by direct comparison to the control arm data.\n\nThis basis of an RCT means that we cannot identify responders and non-responders by considering individuals in the intervention group alone. In other words, we cannot legitimately label an individual who received a particular active treatment as a ‘responder’ (or not) because we do not know what would have happened to that particular individual if they had been in the comparator (or placebo) arm10. This kind of information is very hard to obtain at the individual (cf the group) level, as there is no good way to obtain a control observation. Formally, to properly infer whether a particular participant responded or didn’t respond to a particular treatment, we would require knowledge of what would have happened if a key event (treatment administration) both did and did not occur (a form of counterfactual reasoning), which is not possible in the real world11.\n\nVariability of change (e.g. t2 – t1 symptom score) in the intervention arm is not a true estimate of variability in treatment response, because it includes components of within-subject variation and measurement error10. Even if measurement error is small (i.e. we can precisely measure the outcome variable of interest), for many medical interventions, the outcome variable will depend on a complex interplay of biological factors (e.g. time of day, stress level, etc.), and so within-subject variability will be relatively high. This means that the reliability of within-subject measurements across time points can be somewhat poor, and large variation in changes between study time points may be evident − even where there is no true individual difference in treatment response.\n\nUnfortunately, for psychiatric and chronic pain symptom data, both measurement error and within-subject variation are likely to be high. Measurement error may be higher than other areas of medicine, as the main tools used to assess clinical outcomes are patient or clinician-completed questionnaire measures, which are relatively low precision tools. Further, although self-reported symptom levels are considered the gold standard outcome measure for both psychiatric disorders and chronic pain conditions13, reliability is limited by factors such as cognitive capacity and level of insight for patient-rated measures (e.g. 14), and by interviewer skill and inter-rater agreement for clinician-rated measures (e.g. 15–17). Finally, these classes of disorders represent episodic, chronically relapsing conditions, which will likely contribute to large within-subject variation, particularly at typical RCT follow-up timescales (often around 6 months–1 year; cf e.g. median duration of a depressive episode of ~20 weeks,18). If the variation in outcome due to these sources is greater than that due to any true individual differences in treatment response, it will be very hard to detect the latter under a conventional RCT framework.\n\nA further problem in predicting true response heterogeneity is susceptibility of symptom change data to regression to the mean and mathematical coupling artefacts19,20. Regression to the mean refers to the phenomenon whereby if an individual is selected on the basis of having an extreme measurement value at time point one, their second measurement value will, on average, be closer to the mean of the population distribution (due to the influences of measurement error and normal within-subject variation). A corollary of this effect is that t1 severity is often a significant covariate of change in symptom score between t1 and t2, – meaning that individuals with higher initial scores may appear to show the greatest improvement in symptom levels at follow-up, even when the true magnitude of change does not vary across individuals (see 10 for a worked example). The fact the t1 score is used to calculate both quantities (i.e. they are mathematically coupled) results in further inflation of this relationship (see 20). Care should therefore be taken when key predictors in response algorithms closely index t1 severity, as this may result in a poorly generalising model. However, in previous studies in psychiatric datasets, baseline severity score is usually included among the features used to train response prediction algorithms (e.g. 21–23).\n\nThese factors may help explain why previous attempts to apply machine learning approaches to outcome prediction in psychiatric datasets have thus far had limited success in terms of out-of-sample (unseen data) classification. For example, a recent methodologically rigorous trial aiming to predict significant response (remission) following treatment with a particular antidepressant achieved only ~60% classification accuracy when the model was applied in external validation datasets23. However, as previously noted, tools with only modest true predictive value may still have reasonably high clinical utility compared to current best practice8; therefore this is still an approach very much worth pursuing.\n\n\nPotential solutions\n\nThe problem of identifying true response heterogeneity is a problem of appropriately partitioning variance components in observed outcomes11. The ability to properly identify differential response to a particular treatment in different individuals requires replication at the level at which the differential response is claimed (i.e., that particular treatment in that particular individual). Differential treatment response (i.e. identification of patient by treatment interactions) can therefore be identified by use of repeated period cross-over designs – a form of trial where each participant receives both placebo and active treatments more than once11. However, in practice, these designs are rare, as they are likely to be impractical (prohibitively lengthy and expensive) and/or unethical. This kind of design also assumes that treatments wash out fully between administrations, which might not be reasonable for some interventions (e.g. psychological therapies)24.\n\nAn alternative approach is to improve the way data from existing RCTs is used to train predictive models. For example, it has been suggested that the uncertainty in each individual’s ‘response’ (change in symptom score in the active treatment group) could be expressed as a confidence interval by reference to the standard deviation of the change scores in the control (placebo) group multiplied by the appropriate value from the t distribution (e.g. individual change score ± 1.96*SD of control arm changes for a 95% CI, see 24). The probability that any given individual in the intervention group is a true responder (true change score is greater than the minimum clinically significant change) can then be derived from individual CIs using a Bayesian approach10. Appropriate supervised learning algorithms could then be trained to predict (continuous) treatment response probability, as opposed to dividing individuals into binary response categories (e.g. using Gaussian process regression,25).\n\nIt also may be important to think carefully about the nature of the predictors (features) included in supervised learning model training data – as those that reference initial clinical severity may be vulnerable to regression to the mean-related artefacts. There are statistical methods that have proposed to correct for regression to the mean when correlating t2-t1 symptom changes with initial severity level (see 20). However, these may require additional measurements (e.g. multiple estimates of t1 value, in order to control for effects of measurement error).\n\nWhen a particular experiment is not feasible, one alternative is to train models from observational (non-experimental) data that are able to make counterfactual predictions – i.e. of the outcomes that would have been observed, had we run that particular experiment. For example, Saria and colleagues have recently developed a counterfactual Gaussian process (CGP) approach to modelling clinical outcome data26. The CGP is trained on observational (non-experimental) time series data, in order to form a model of clinical outcomes under a series of treatments in continuous time. Crucially, the CGP is trained using a joint maximum likelihood objective, which parses dependencies between observed actions (e.g. treatments) and outcomes in the data. This feature allows prediction of how future trajectories (symptom levels) may change in response to different treatment interventions, and has previously been shown to successfully predict real clinical data (renal health markers following different kinds of dialysis,26,27).\n\nThis modelling approach requires datasets with semi-continuous measurement of the relevant clinical outcome (both pre- and post- intervention), in order to generate hypothetical treatment response traces – a kind of data that is not usually available from existing RCTs. Given sufficient attention to patient confidentiality and other ethical concerns, it may be possible to obtain appropriate training data from health service clinical records; however, frequency and consistency of symptom reporting may pose analytical problems (e.g. 27). The use of personal devices such as smartphones or other wearable technology to regularly self-record symptom levels may be a potential source of this kind of data in the future, given sufficient insight and patient compliance (e.g. 28). The CGP approach also rests on two key mathematical assumptions: that there will be a consistency of outcomes between training observations and future outcomes, given a particular treatment; and that there are no important confounding variables missing from the dataset26. It may require careful consideration as to whether these are reasonable assumptions for modelling psychological and chronic pain symptomatology.\n\n\nConclusions\n\nThe issues discussed above underline the importance of focusing on where data comes from when considering strategies for personalised medicine. In particular, it is problematic to designate individual data points from a conventional RCT design as ‘responders’ or ‘non-responders’ to a particular treatment, as this is in effect a single-arm (no control) study not adjusted for other important sources of response variation. This might be particularly important when considering patients with episodic, chronically-relapsing disorders as control variability is likely to be high (and symptom measurement itself is often imprecise). One solution to this problem is to use data derived from repeated cross-over design clinical trials, although in practice these can be prohibitively difficult and/or ethically problematic. It may be possible to alleviate these issues with careful model design, but this may still require changes to the way data is collected and monitored in the future in order to maximise potential clinical utility.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAN and BS are supported by the Wellcome Trust (grant number 097490/Z/11/A to BS).\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\nRush AJ, Trivedi MH, Wisniewski SR, et al.: Acute and longer-term outcomes in depressed outpatients requiring one or several treatment steps: a STAR*D report. Am J Psychiatry. 2006; 163(11): 1905–1917. PubMed Abstract | Publisher Full Text\n\nPigott HE, Leventhal AM, Alter GS, et al.: Efficacy and effectiveness of antidepressants: current status of research. Psychother Psychosom. 2010; 79(5): 267–279. PubMed Abstract | Publisher Full Text\n\nFinnerup NB, Attal N, Haroutounian S, et al.: Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. Lancet Neurol. 2015; 14(2): 162–173. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCleare A, Pariante CM, Young AH, et al.: Evidence-based guidelines for treating depressive disorders with antidepressants: A revision of the 2008 British Association for Psychopharmacology guidelines. J Psychopharmacol. 2015; 29(5): 459–525. PubMed Abstract | Publisher Full Text\n\nZimmerman M, Ellison W, Young D, et al.: How many different ways do patients meet the diagnostic criteria for major depressive disorder? Compr Psychiatry. 2015; 56: 29–34. PubMed Abstract | Publisher Full Text\n\nSmith SM, Dworkin RH, Turk DC, et al.: The Potential Role of Sensory Testing, Skin Biopsy, and Functional Brain Imaging as Biomarkers in Chronic Pain Clinical Trials: IMMPACT Considerations. J Pain. 2017; 18(7): 757–777. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSteingrímsdóttir ÓA, Landmark T, Macfarlane GJ, et al.: Defining chronic pain in epidemiological studies: a systematic review and meta-analysis. Pain. 2017; 158(11): 2092–2107. PubMed Abstract | Publisher Full Text\n\nGillan CM, Whelan R: What big data can do for treatment in psychiatry. Curr Opin Behav Sci. 2017; 18: 34–42. Publisher Full Text\n\nDrucker E, Krapfenbauer K: Pitfalls and limitations in translation from biomarker discovery to clinical utility in predictive and personalised medicine. EPMA J. 2013; 4(1): 7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAtkinson G, Batterham AM: True and false interindividual differences in the physiological response to an intervention. Exp Physiol. 2015; 100(6): 577–588. PubMed Abstract | Publisher Full Text\n\nSenn S: Mastering variation: variance components and personalised medicine. Stat Med. 2016; 35(7): 966–977. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDahly D: Response Heterogeneity. 2017. Reference Source\n\nDworkin RH, Turk DC, Farrar JT, et al.: Core outcome measures for chronic pain clinical trials: IMMPACT recommendations. Pain. 2005; 113(1–2): 9–19. PubMed Abstract | Publisher Full Text\n\nAlwin DF, Krosnik JA: The Reliability of Survey Attitude Measurement: The Influence of Question and Respondent Attributes. Sociol Methods Res. 1991; 20: 139–181. Publisher Full Text\n\nKobak KA, Feiger AD, Lipsitz JD: Interview quality and signal detection in clinical trials. Am J Psychiatry. 2005; 162: 628. PubMed Abstract | Publisher Full Text\n\nEngelhardt N, Feiger AD, Cogger KO, et al.: Rating the raters: assessing the quality of Hamilton rating scale for depression clinical interviews in two industry-sponsored clinical drug trials. J Clin Psychopharmacol. 2006; 26(1): 71–74. PubMed Abstract | Publisher Full Text\n\nRothman B, Yavorsky C, De Fries A, et al.: P02-88 - Quantifying rater drift on the HAM-D in a sample of standardized rater training events: Implications for reliability and sample size calculations. Eur Psychiatry. 2011; 26: 683. Publisher Full Text\n\nSolomon DA, Keller MB, Leon AC, et al.: Recovery from major depression. A 10-year prospective follow-up across multiple episodes. Arch Gen Psychiatry. 1997; 54(11): 1001–1006. PubMed Abstract | Publisher Full Text\n\nOldham PD: A note on the analysis of repeated measurements of the same subjects. J Chronic Dis. 1962; 15(10): 969–977. PubMed Abstract | Publisher Full Text\n\nTu YK, Gilthorpe MS: Revisiting the relation between change and initial value: a review and evaluation. Stat Med. 2007; 26(2): 443–457. PubMed Abstract | Publisher Full Text\n\nRiedel M, Möller HJ, Obermeier M, et al.: Clinical predictors of response and remission in inpatients with depressive syndromes. J Affect Disord. 2011; 133(1–2): 137–149. PubMed Abstract | Publisher Full Text\n\nKessler RC, van Loo HM, Wardenaar KJ, et al.: Testing a machine-learning algorithm to predict the persistence and severity of major depressive disorder from baseline self-reports. Mol Psychiatry. 2016; 21(10): 1366–1371. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChekroud AM, Zotti RJ, Shehzad Z, et al.: Cross-trial prediction of treatment outcome in depression: a machine learning approach. Lancet Psychiatry. 2016; 3(3): 243–250. PubMed Abstract | Publisher Full Text\n\nHopkins WG: Individual responses made easy. J Appl Physiol (1985). 2015; 118(12): 1444–1446. PubMed Abstract | Publisher Full Text\n\nRasmussen CE, Williams KI: Regression. In Gaussian Processes for Machine Learning. The MIT Press; 2006. Reference Source\n\nSchulam P, Saria S: What-If Reasoning with Counterfactual Gaussian Processes. ArXiv170310651 Cs Stat. 2017. Reference Source\n\nSoleimani H, Subbaswamy A, Saria S: Treatment-Response Models for Counterfactual Reasoning with Continuous-time, Continuous-valued Interventions. ArXiv170402038 Cs Stat. 2017. Reference Source\n\nFaurholt-Jepsen M, Vinberg M, Christensen EM, et al.: Daily electronic self-monitoring of subjective and objective symptoms in bipolar disorder--the MONARCA trial protocol (MONitoring, treAtment and pRediCtion of bipolAr disorder episodes): a randomised controlled single-blind trial. BMJ Open. 2013; 3(7): pii: e003353. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "29856",
"date": "18 Jan 2018",
"name": "Greg Atkinson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, the authors discussed the concept of inter-individual differences in response to treatment interventions, particularly those focussed on psychological-related outcomes. The consideration of inter-individual responses is an important issue and the authors provide further insights previously not considered in detail within the domain of psychology. The topic is generally discussed accurately in the context of the current literature, statements are generally correct and supported by relevant citations. I have thoroughly read and considered the manuscript, which was interesting in content and constructed with a logical flow. I have only minor comments for the authors’ consideration.\n\nThe article focuses on the prediction of response heterogeneity, especially prediction of responders/non-responders. Have you considered the roadmap that has been suggested1 to actually confirm whether the general amount of true heterogeneity is clinically important or not BEFORE we might explore for predictors of that heterogeneity?\n\nPage 3, Right hand Column, Lines 27 onwards– whilst discussing RCT trial design and highlighting that responders/non-responders cannot be identified through the analysis of intervention sample data alone, perhaps it might be appropriate to address any research making similar claims even in the total absence of any control sample data.\n\nPage 4, Left hand column, Line 8 and conclusion. The arguments that you make on this point, particularly in the conclusion are, at present, unsupported by scientific literature and require justification. You allude to the fact that a lack of ‘true’ counterfactual information makes an RCT in effect a single-arm (no control study). It is agreed that one cannot say with 100% certainty whether the intervention group as a whole or any specific individual in the intervention group is a positive responder, as what would have happened to that person if they had been in the control group is of course unknown. This is the fundamental counterfactual basis of the RCT. Nevertheless, as the control group variability over the same time period as the intervention effectively provides our best guess of the counterfactual (what would have happened to individuals in the intervention group if they had been in the control arm), I feel that this applies to changes at both the group mean and the individual level, and that disregarding RCTs as ‘single-arm studies’ is unsupported. According to the previously-mentioned “roadmap” that has been presented, the analysis of the control group changes (specifically the comparison of change variance between treatment and control) can provide information as to what the general clinical importance is of “true” individual response heterogeneity. By “true”, one knows from this comparison whether the overall amount of heterogeneity in changes surpasses the overall amount of random within-subject heterogeneity of changes in the control group. If heterogeneity of change is similar between treatment and control, it could be argued that moving on to attempts to predict treatment response variability is a somewhat meaningless exercise.\n\nPage 4, Left hand column, Lines 43 – 54. Whilst discussing regression to the mean and the mathematical coupling of pre- to post change scores, the use of covariates (especially baseline values of the study outcome) in the statistical model (ANCOVA) could be suggested as a potential solution to this – a notable absence in many studies’ data analyses.\n\nPages 4 – 5. You make a number of pertinent suggestions for potential solutions to the problem, and briefly allude to the methods recently suggested (1,3). We have suggested how this might be approached in RCTs and tied to an appropriate anchor usually a minimal clinically important difference or smallest worthwhile change (1,2). Addressing these issues may assist the reader in applying this methodology in their applied practice and/or research environments.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": [
{
"c_id": "3464",
"date": "01 Mar 2018",
"name": "Agnes Norbury",
"role": "Author Response",
"response": "Thank you for taking the time to review this manuscript. Please see below for responses to your main comments, which are addressed in the revised manuscript.1. We have added reference to the proposed ‘roadmap’ to determine whether clinically important response heterogeneity exists prior to quantifying it. 2. Regarding the reference to ‘single arm’ studies, we have made it more explicit that although the underlying RCTs (in order to determine treatment effectiveness as the group level) are not single arm, machine learning algorithms concerned with predicting individual differences in treatment response are usually only trained on the active treatment arm data (please see below):“Although the underlying RCT datasets almost always consist of at least two arms (e.g. active treatment vs placebo), machine learning algorithms employed to predict psychiatric treatment response are usually trained on active treatment arm data alone – without reference to control arm symptom changes (e.g. [13,14]). Unless sufficient care is taken, these kinds of predictive models may therefore be the inferential equivalent of single arm trials, and the resultant categorisation of symptom change scores may be unduly influenced by sources of variance causally unrelated to true treatment response.”3. We have added reference to the use of ANCOVA under a traditional statistical framework as a way of guarding against regression to the mean and mathematical coupling artefacts – with discussion of equivalent techniques (or their absence) in the machine learning literature (please see below):“It also may be important to think carefully about the nature of the predictors (features) included in supervised learning model training data – as those that reference initial clinical severity may be vulnerable to regression to the mean-related artefacts. Under a traditional statistical framework, an effective way of dealing with these artefacts is to include baseline scores as covariates in models of symptom change data (i.e., conduct an ANCOVA). An interesting issue is that in machine learning, there is not really an equivalent concept to covariates of no interest – rather, model features are selected purely on the basis of their predictive capacity. One recent paper that explicitly addresses this issue comes from Rao and colleagues, who propose a method for removing known confounds from predictive models based on functional imaging data. Rao et al. suggest that a potential solution is to first fit linear models to each image feature using the confound variables as predictors, then consider the residuals of this model to be ‘adjusted’ data − suitable to be used as input features for a confound-controlled predictive model [26]."
}
]
},
{
"id": "29962",
"date": "02 Feb 2018",
"name": "William G. Hopkins",
"expertise": [
"Reviewer Expertise Research design and analysis",
"with special reference to physical activity",
"sport and lifestyle."
],
"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 represents a valuable contribution to the developing literature on quantification of individual responses to treatments and identification of patients who respond positively to treatments. Perhaps there should be some attention to the issue of identifying negative responders, since it is more important to avoid harming individual patients than to miss out on benefitting them. By \"harm\" I don't mean side effects. I also point out in my review that prediction models could be developed from identified modifiers of the treatment effect in controlled trials, something that you should also mention. I also have a strong view about repeatability of individual responses to treatments, something that you will need to address by refuting my claim or accommodating it. Otherwise there are only minor points for you to consider. These were all made on a first and only read-through, so although you explain some points further on, it is important to avoid any confusion in the first place.\n\n\"This may be particularly problematic in the case of psychiatric and chronic pain symptom data, where both within-subject variability and measurement error are likely to be high.\" I don't understand inclusion of \"within-subject variability\"? Are you referring to real changes of individual subjects pre to post the treatment that occur even in the absence of an active treatment? I normally think about that as another kind of measurement error. Perhaps you need to clarify by stating \"both within-subject variability over the period of the treatment and short-term measurement error \" Also, solitary \"this\" is a grammatical error known as an ambiguous antecedent. There are a few other instances in the manuscript that need to be fixed.\n\n\"especially in arenas where the potential clinical benefit is so large\" I don't understand the use of \"so\". Are you referring to the arenas of psych disorders and chronic pain? Are the \"potential clinical benefits\" in these arenas any larger than in any other arenas of pathology? And you haven't established (in the Abstract, anyway) that there is the potential for large benefit when individual responders have been characterised. Maybe something along the lines of \"trustworthy identification of characteristics of positive responders to treatments could result in substantial clinical benefit in psychological disorders, chronic pain, and other pathologies.\"\n\nIn the Introduction, you make it clear–at some length–that non-responders to one kind of treatment could be responders to another, and it may therefore be possible to improve the health of the majority of patients by targeting specific patients with specific treatments, where the pathology has a range of underlying causes and a range of treatments is available. Maybe you need to make that clearer in the Abstract.\n\n\"variables that may potentially relate\" is a \"double doubtful.\" Remove either \"may\" or \"potentially\".\n\n\"i.e. the reliability of distinguishing between treatment ‘responders’ and ‘non-responders’\". I think validity would be a better word than reliability. Also no need for quote marks.\n\ne.g. and i.e. normally have commas after them and are used only in parentheses.\n\n\"Crucially, we can only draw this inference by direct comparison…\" Make it \"Crucially, we can draw this inference only by direct comparison…\" Check for any other instances of this misplaced modifier.\n\nYou tend to use too many parenthetical asides. Remove parentheses from as many as you can.\n\n\"Formally, to properly infer whether a particular participant responded or didn’t respond to a particular treatment, we would require knowledge of what would have happened if a key event (treatment administration) both did and did not occur (a form of counterfactual reasoning), which is not possible in the real world.\" I found this sentence confusing. What is counterfactual reasoning? If I understand this sentence correctly, I disagree with it. In crossovers it is possible to determine the outcome with an individual who received the active and the control treatment. You go on to state that yourself.\n\n\"(e.g. time of day, stress level, etc.)\" Either e.g. or etc., but not both!\n\n\"Measurement error may be higher than [in] other areas of medicine, as the main tools used to assess clinical outcomes are patient or clinician-completed questionnaire measures, which are relatively low[-]precision tools.\" I think this statement is false, so you'd better support it with references. The square root of the alpha reliability (which provides an upper limit to the criterion validity correlation of multi-item instruments) and short-term retest reliability ICC could well be high enough to reasonably identify responders to short-term treatments. Even VASs have high short-term ICCs. The ICCs over periods for long-term treatments are likely to be a different story, as you point out.\n\n\"If the variation in outcome due to these sources is greater than that due to any true individual differences in treatment response, it will be very hard to detect the latter under a conventional RCT framework.\" It depends what you mean by \"detect\". To be on the safe side, perhaps you should state \"The greater the variation in outcome due to these sources compared with that of true individual responses, the harder it will be to characterize the latter in a controlled trial.\" Note that I have removed superfluous words. Bottom line is that you can make up for the short- and log-term errors with a big-enough sample size, at least for characterizing the mean effect and its modifiers.\n\n\"The fact the t1 score is used to calculate both quantities…\" I fully understand regression to the mean, but I re-read this paragraph and still don't know what \"both quantities\" refers to.\n\n\"The ability to properly identify differential response to a particular treatment in different individuals requires replication at the level at which the differential response is claimed (i.e., that particular treatment in that particular individual).\" This rather obscurely worded claim has been made by others, but it is false. You can have a patient who responds individually to a treatment on one administration of the treatment. Whether that patient would respond similarly again following washout and reapplication of the treatment is irrelevant. What matters is that the patient has obtained benefit from the treatment when it was applied the first time. Period. Whether you can adequately quantify the extent of an individual patient's response to the (first) application of the treatment depends on short- and long-term errors of measurement and on the magnitude of the response in that patient, but regardless, you can certainly characterise modifiers of the treatment effect with realistic sample sizes: 4x the sample size required to characterize the mean effect (Hopkins, 2006). The identified modifiers could then be used to build courses-for-horses (treatments-for-patients) prediction models, something you haven't considered. Anyway, there is no need to confuse everyone by raising the spectre of repeatability of the treatment effect in individuals.\n\n\"However, these may require additional measurements (e.g. multiple estimates of t1 value, in order to control for effects of measurement error).\" No, repeating the t1 assessment reduces but does not eliminate the effect of regression to the mean. What you need for the adjustment is the reliability ICC over the time-frame of the treatment. In fact, the control group effectively provides that: when you predict the likelihood of an individual's response in a controlled trial using a mixed model in which the pre-test (t1) is included as a modifying covariate, you have controlled for (adjusted away the effect of) regression to the mean.\n\nI don't understand the paragraph headed Counterfactual probabilistic modelling. You will have to explain what is going on here without the jargon, for my benefit and for those who are even less statistically savvy. I see the word \"trajectory\" in there, which suggests to me that you are talking about clinical trials with multiple repeated measurements during the course of the treatment. That's a luxury that may not be available in many settings, and in any case, it requires an appropriate model for the time course, which is bound to be non-linear. You still need a control group, if you want to eliminate the contribution of the placebo effect.\n\nAh, I see you provide some explanation in the next paragraph. Please make the preceding paragraph clearer.\n\n\"The CGP approach also rests on two key mathematical assumptions: that there will be a consistency of outcomes between training observations and future outcomes, given a particular treatment…\" No. See above.\n\n\"and that there are no important confounding variables missing from the dataset.\" Be more explicit. If it's a properly balanced controlled trial (or randomized with a sufficiently large sample size), what's the problem?\n\n\"One solution to this problem is to use data derived from repeated cross-over design clinical trials…\" Well, no, because it introduces what I called above the spectre of repeatability.\n\n\"It may be possible to alleviate these issues with careful model design…\" Explain \"careful\". You will need to have identified the point(s) explaining \"careful\" previously, as this sentence is in the Conclusions.\n\nHopkins, WG. Estimating sample size for magnitude-based inferences. Sportscience 10, 63-70 (http://www.sportsci.org/2006/wghss.htm)\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": [
{
"c_id": "3463",
"date": "01 Mar 2018",
"name": "Agnes Norbury",
"role": "Author Response",
"response": "Thank you for taking the time to review this manuscript. Please see below for responses to your main comments, which are addressed in the revised manuscript. 1. We have added reference to the possibility that appropriately formulated data-driven models could be used to predict probability of harm (symptom increase above a clinically significant threshold), as well as probability of successful treatment response (symptom decrease above some clinically significant threshold). 2. We have added reference to methods for the identification of treatment ‘modifiers’, whilst controlling for other artefacts, under a traditional statistical approach – plus a discussion of an analogous approach from the field of machine learning (please see below):“It also may be important to think carefully about the nature of the predictors (features) included in supervised learning model training data – as those that reference initial clinical severity may be vulnerable to regression to the mean-related artefacts. Under a traditional statistical framework, an effective way of dealing with these artefacts is to include baseline scores as covariates in models of symptom change data (i.e., conduct an ANCOVA). This approach can then be used to test if a given between-subjects variable is a significant modifier of treatment effect by adding it to the model (providing measurement error is sufficiently low, [24]). An interesting issue is that in machine learning, there is not really an equivalent concept to ‘covariates of no interest’ – rather, model features are usually selected purely on the basis of their predictive capacity. One recent paper that explicitly addresses this problem comes from Rao and colleagues, who propose a method for removing known confounds from predictive models based on functional imaging data. Rao et al. suggest that one solution is to first fit linear models to each image feature using the confound variables as predictors, then consider the residuals of this model to be ‘adjusted’ data − suitable to be used as input features for a confound-controlled predictive model [26].” 3. Regarding the issue of ‘repeatability’, we would argue that since this perspective deals with the ability to predict the responses of future patients (i.e., individuals with similar characteristics to past ‘responders’), then repeatability of these responses is not only relevant, but vital. Although we would not deny the real benefit to an individual patient of any significant improvement in clinical outcome, if this benefit is not ‘repeatable’ in that it is reliably casually related to treatment administration (e.g. score improvement is largely due to fluctuations in symptoms that would have occurred otherwise), then it should not be taken as evidence for use of that treatment in future (similar) individuals. 4. Some changes to grammar have also been made throughout the manuscript in order to increase clarity."
}
]
},
{
"id": "30529",
"date": "20 Feb 2018",
"name": "Ricardo Silva",
"expertise": [
"Reviewer Expertise 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\nI would like to thank the authors for the informative review of the many difficulties pertinent to modeling the treatment of psychiatric disorders. My main take will be on the statistical and machine learning aspects, causal inference in particular, as I do not have a clinical background.\nWith the availability of larger sources of data, it is reasonable to ask what can be leveraged in order to better predict how individual patients will respond to particular treatments. This raises questions of causal inference, as treatments are meant to be interventions that will (or are expected to) change the condition of a patient. The article properly addresses factors that make this challenging, including measurement error. I do not have much to comment on measurement error as this goes into the specifics of the domain, but I would like to second the authors' warning on how important this issue is. Even different ways of executing the data collection protocol in different environments (e.g., the way questionnaires are applied) can have an influence on what response distribution we obtain in the end.\nI will focus instead on what it means to perform counterfactual reasoning, and which challenges in performing it are warranted. I share much of the view of Dawid 1, that many problems of causal inference are decision-theoretical rather than genuinely counterfactual. In fact, counterfactuals are incompatible with out-of-sample problems, if only because it is impossible to be \"contrary to a fact\" that has not happened yet and can still happen. In the classical statistical approach for causal inference, the motivation is intrinsically an in-sample problem, where the randomness is all in the treatment assignment: we are dealing with what would have happened to a particular group of people, at a particular time, at a particular environment, had treatment assignments been different 2. In the questions raised by the authors, we are interested in what will happen to a patient given a treatment coming from a set of two or more choices (to follow one psychiatric treatment, or an alternative, or none etc.), not what would have happened had things been different. This is a predictive policy question, not unlike what is found in contextual bandits (but notice that, unlike contextual bandits, we are not necessarily interested in exploration-exploitation trade-offs, but on the assessment of a policy that maps features of an individual to an action). This boils down to evaluating hypothetical actions, and picking the most beneficial one according to some risk/utility function. This is still a causal inference question as it concerns the effects of actions, but it does not need to be framed as counterfactual reasoning as the notion of rewinding the clock to apply a different treatment to an individual is never on the cards when assessing out-of-sample treatment recommendation. Put simply, the estimand does not involve counterfactuals.\nHow to obtain such a model is however a nontrivial question and requires much work, which I briefly discuss below in the context of the article. As a final remark before continuing, I will say that, unlike Dawid, I have no issues on using counterfactual models to address hypothetical questions. By the end of the day, there are many equivalent languages to express causal assumptions, and we should be free to choose our \"syntactic sugar\" as it is seen fit, as long as we know what the limitations of our models are. The following is agnostic to whether counterfactuals have motivated the model or some other predictive counterfactual-free causal approach has been used instead.\nSo, what are the challenges of causal inference for heterogeneous effects? It is still the case that RCTs are much limited in this scenario: it is one thing to use a RCT so show that there exists a group of people which at particular time and at a particular environment would have shown different responses had treatment assignment been different. It is a different ballgame to extract meaningful predictive power of a dataset if the sample is not representative of the target population. This sample selection bias, where volunteers of a RCT are very likely not to be representative, is pervasive in many sciences. To the best of my understanding, this is also the case in psychiatric research. Some mitigation can be done to transfer some conclusions to the test set of interest 3 by tapping into some aspects of the process that remain invariant out-of-sample. Unfortunately, this may not be enough, and attending to the needs of many individuals of interest may require unwarranted extrapolations from the training set. Although RCTs are extremely desirable for causal inference, as a well-designed trial will remove unmeasured confounding, the selection bias that is natural in studies with human subjects may be too strong for its conclusions to be applicable to a large fraction of out-of-sample personalised treatments. I'm particularly skeptical of putting any effort on cross-over designs: those have the shortcomings of RCTs (sample not being representative), while adding assumptions such as \"treatments wash out fully between administrations\" which seem unbelievable to me in the context of psychiatric research, while the motivation (estimating counterfactuals) is unnecessary for the ultimate goal of deciding among the initial treatment options for out-of-sample patients.\nA promising venue is to exploit observational data, under the provision that we understand its many limitations. We need causal assumptions about which sources of confounding exist and how to remove them, and to which extent a RCT may provide information on the degree of confounding for patient profiles observed in the general population (via the observational data), but which are far from those found in the RCT sample. While the machine learning literature has done much in terms of providing ways of combining data from a set of experiments and observations 4, 5, they put much emphasis on finding causal networks as opposed to reliably estimating heterogeneous effects. This remains open, and with the added difficulty of dealing with assumptions about measurement error. Hopefully we will see this raising research questions for those up to the challenge, and with the motivation of improving the lives of the many who rely on our better understanding of psychiatric treatments and their effectiveness.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": [
{
"c_id": "3462",
"date": "01 Mar 2018",
"name": "Agnes Norbury",
"role": "Author Response",
"response": "Thank you for taking the time to review this manuscript, and for providing a thoughtful meditation on individual response prediction from a casual inference perspective. We have updated the manuscript to touch upon some of the issues you have raised (please see below): “Counterfactual probabilistic modelling and other causal inference methodsWhen a particular experiment is not feasible, an alternative is to train models on observational (non-experimental) data that are able to make counterfactual predictions – i.e. of the outcomes that would have been observed, had we run that particular experiment. For example, Saria and colleagues have recently developed a counterfactual Gaussian process (CGP) approach to modelling clinical outcome data 27 . The CGP is trained on observational symptom trajectory data to form a model of clinical outcomes under a series of treatments in continuous time. Crucially, the CGP is trained using a joint maximum likelihood objective, which parses dependencies between observed actions (e.g. treatments) and outcomes in order to infer the existence of causal relationships between the two. This feature allows the prediction of how future trajectories (symptom levels) may change in response to different treatment interventions, and has previously been shown to successfully predict real clinical data (renal health markers following different kinds of dialysis, 27, 28 ).Thus far, the CGP has only been empirically tested as a clinical decision support tool on the same subjects from whom model training data was derived [27]. However, it can be argued that prediction of the response of future patients to particular treatment options is an inference problem that does not necessarily involve counterfactual reasoning. Under these circumstances, we require a model that can infer causes that are likely to be active for out-of-sample data (individuals with certain features, who may not have received any treatment yet), as opposed to in-sample data (individuals whose clinical data a particular model was trained on, who might have showed a different response to different treatment strategies) [29].This perspective raises the issue of how representative the individuals who make up a particular training dataset are of the general population (from whom future patients will be drawn). Importantly, concerns have previously been raised as to the effects of various sources of selection bias on the representativeness of participants in RCTs compared to the population at large [30]. Specific forms of selection bias that have been identified in RCTs for psychological disorders include exclusion of individuals with comorbidities (which for many some conditions may be more common than ‘pure’ presentation), selection of less severe cases (e.g. in psychotic disorders, where ability to consent and treatment compliance may be of heightened concern), or, conversely, application of minimum severity thresholds (e.g. in mood disorders, to reduce the likelihood of spontaneous remission over the trial period) [31,32]. Further, methods of recruitment to RCTs (particularly the requirement to self-select into trials) may influence the distribution of various psychological traits in trial participants, prior to any further eligibility criteria being applied (e.g. [33]). Although there are methods designed to mitigate the effects of sample selection bias when transferring predictive models to a different test set (see [34]), it remains an open question as to whether these are sufficiently robust for successful out-of-sample treatment prediction at the individual level.The success of causal inference modelling approaches to response prediction may therefore depend upon availability of different kinds of data to that derived from traditional RCTs – involving semi-continuous measurement of the relevant clinical outcome (both pre- and post- intervention), and gathered from more representative sources than some previous RCT datasets. Given sufficient attention to patient confidentiality and other ethical concerns, it may be possible to obtain appropriate training data from health service clinical records; however, frequency and consistency of symptom reporting may pose analytical problems (e.g. 28). The use of personal devices such as smartphones or other wearable technology to regularly self-record symptom levels may be a potential source of this kind of data in the future, given sufficient insight and patient compliance (e.g. 35).A further important feature of predictive models derived from observational data is that they depend on explicit assumptions about the existence and causal consequences of any confounding variables present in the dataset. For example, the CGP approach requires both that there will be a consistency of outcomes between training observations and future outcomes, given a particular treatment, and that there are no important confounding variables missing from the dataset [27]. It will therefore be necessary to carefully consider how well such assumptions are met when considering applying these kinds of models to psychological and chronic pain symptom data.”"
}
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https://f1000research.com/articles/7-55
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https://f1000research.com/articles/7-256/v1
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01 Mar 18
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{
"type": "Research Article",
"title": "Evaluation of portal pressure by doppler ultrasound in patients with cirrhosis before and after simvastatin administration – a randomized controlled trial",
"authors": [
"Nadia Elwan",
"Raafat Salah",
"Manal Hamisa",
"Ebtsam Shady",
"Nehad Hawash",
"Sherief Abd-Elsalam",
"Nadia Elwan",
"Raafat Salah",
"Manal Hamisa",
"Ebtsam Shady",
"Nehad Hawash"
],
"abstract": "Background: Portal hypertension is one of the most frequent complications of cirrhosis. β-adrenergic blockers, with or without organic nitrates, are currently used as hypotensive agents. Statins such as simvastatin seem to be safe for patients with chronic liver diseases and exert multiple pleiotropic actions. This study aimed to assess PTH using Doppler ultrasound in patients with cirrhosis before and after simvastatin administration. Methods: This randomized controlled clinical trial was conducted on 40 patients with cirrhosis who were randomized into 2 groups: group I included 20 patients with cirrhosis who were administered 20 mg of simvastatin daily for 2 weeks and then 40 mg daily for another 2 weeks, and group II included 20 patients with cirrhosis who did not receive simvastatin as a control group. All patients underwent full clinical examination, laboratory investigations, and abdominal Doppler ultrasound at baseline and after 30 days to evaluate portal vein diameter, blood flow volume, direction and velocity of portal vein blood flow, hepatic artery resistance and pulsatility indices, splenic artery resistance index, portal hypertension index (PHI), liver vascular index, and modified liver vascular index (MLVI). Results: There was a highly significant decrease in the hepatic artery resistance index in group I, from 0.785 ± 0.088 to 0.717 ± 0.086 (P < 0.001). There was a significant decrease in the PHI in group I , from 3.915 ± 0.973 m/sec to 3.605 ± 1.168 m/sec (P = 0.024). Additionally, there was a significant increase in the MLVI in group I from 11.540 ± 3.266 cm/sec to 13.305 ± 3.222 cm/sec, an increase of 15.3% from baseline (P = 0.009). No significant adverse effects were detected. Conclusions: Simvastatin is safe and effective in lowering portal hypertension. [ClinicalTrials.gov Identifier: NCT02994485]",
"keywords": [
"Simvastatin",
"Portal hypertension",
"Cirrhosis",
"Doppler",
"Ultrasound."
],
"content": "Introduction\n\nLiver cirrhosis is considered to be the most common cause of portal hypertension (PHT)1. Increased portal inflow and increased outflow resistance are associated with the development of PHT2. Liver transplantation is indicated in patients with advanced cirrhosis complicated by PHT; furthermore, morbidity and mortality are increased in these patients3,4.\n\nThe ideal hypotensive drug for PHT should decrease portal pressure by lowering intrahepatic vascular resistance while maintaining or increasing hepatic blood flow3. Moreover, it should improve liver function through its antifibrotic effects, and it should be able to increase nitric oxide bioavailability in the liver to help fulfill many of these requirements3,5–8.\n\nCurrently, the available therapies for PHT are based on the use of β-adrenergic blockers, with or without organic nitrates, and allow achievement of the target hemodynamic response in less than half of patients. Moreover, about 30% of patients may have contraindications or may not tolerate β-blockers9.\n\nStatins such as simvastatin are used mainly for cardiovascular diseases and metabolic syndrome. They exert multiple pleiotropic effects and can be used safely in patients with chronic liver diseases10. They decrease Rho-kinase activity in activated hepatic stellate cells11. In addition, statins have anti-inflammatory, immunomodulatory, and antioxidant properties12. Simvastatin is also known to induce Krüppel-like factor 2, which improves liver fibrosis and PHT by increasing nitric oxide bioavailability13.\n\nColor Doppler ultrasound is an important non-invasive tool that can be used to record portal venous system blood flow14. This study aimed to evaluate PHT by Doppler ultrasound in patients with cirrhosis before and after simvastatin administration.\n\n\nPatients and Methods\n\nThis randomized controlled study was conducted in the Department of Tropical Medicine at Tanta University Hospital. Forty patients with cirrhosis and PHT were enrolled from April to November 2016. All patients provided written informed consent, and the study was approved by the Ethics Committee of the Faculty of Medicine at Tanta University. All patients had code numbers to ensure anonymity. The study was registered on www.clinicaltrials.gov (ClinicalTrials.gov Identifier: NCT02994485).\n\nPatients diagnosed with liver cirrhosis by ultrasound (a coarse echogenic pattern, surface irregularity, attenuated hepatic veins, and a bulky caudate lobe) who also had clinical signs of PHT (esophageal varices, splenomegaly, ascites, and grade I-II hepatic encephalopathy) were enrolled in the study.\n\nThe exclusion criteria for this study were pregnancy, hepatocellular carcinoma, portal vein thrombosis, grade III-IV hepatic encephalopathy, a history of treatment with calcium channel blockers, statin use during the previous 3 months, and a known allergy to any statin.\n\nThe patients in the study were randomized to either receive or not receive simvastatin. group I included 20 patients with cirrhosis who were administered 20 mg of simvastatin daily for 2 weeks followed by 40 mg of simvastatin daily for another 2 weeks, and group II included 20 patients who did not receive simvastatin as a control group. The included patients received their routine treatments of diuretics, liver support, and anti-diabetic or anti-hypertensive therapies during the study.\n\nAll patients provided detailed history including age, sex, residence, job, marital status, special habits, history of diabetes mellitus or anti-diabetic therapy, hypertension, anti-hypertension therapy, history of surgical shunts, history of gastrointestinal bleeding, history of upper endoscopy, and bleeding varices. A thorough clinical examination was conducted to assess for liver or spleen ascites, lower limb edema, jaundice, and hepatic encephalopathy.\n\nAll patients underwent routine laboratory investigations in our Tropical Medicine Clinic, including complete blood count (hemoglobin level, platelet count, and white blood count), kidney function tests (blood urea and serum creatinine), liver function profile (liver enzyme and total serum bilirubin levels, which were also measured 2 and 4 weeks after the beginning of the study to exclude any increase from baseline in group I), coagulation profile (international normalization ratio [INR], prothrombin time [PT], and prothrombin activity), random blood sugar (this was also measured 2 and 4 weeks after the beginning of the study to exclude any increase from baseline in group I), Child–Pugh score (assessed in all studied patients), and serum alpha-fetoprotein level.\n\nAbdominal ultrasound was performed on all patients in the study to assess the liver (echogenicity, surface, edge, and size), attenuation of hepatic veins, spleen size, and presence of ascites.\n\nDoppler ultrasound was performed on all patients to measure the portal vein diameter (PVD), portal vein velocity (PVV), portal vein blood flow (PVBF), portal vein flow direction, hepatic artery resistance index and hepatic artery pulsatility index (HARI and HAPI, respectively), splenic artery resistance index (SARI), portal hypertension index (PHI), and modified liver vascular index and liver vascular index (MLVI and LVI, respectively).\n\nDoppler analysis was performed during quiet respiration or while the patients held their breath15. All parameters were measured twice, at the beginning and at the end of the study. We placed the Doppler gate in the hilum of the spleen and in the porta hepatis of the liver. The same observer usually unified the method for measuring each index to avoid interobserver variability and calculated the mean of 3 consecutive measurements.\n\nPVD was measured from the hilar segment when crossing the inferior vena cava while the patient was in the recumbent supine position. It was recorded in millimeters.\n\nPVBF was calculated automatically after recording the peak, lowest, and mean venous velocity of the flow and the measurement of a cross-sectional area of the vessel lumen in a transverse plane. It was recorded in liters per minute (L/min). Portal vein flow direction: the direction of portal blood flow was shown by color Doppler, indicating if it was toward (hepatopetal) or away from the liver (hepatofugal).\n\nPVV was calculated automatically after measuring (Vmax) and (Vmin). It was recorded in centimeters per second (cm/sec).\n\nHARI: The hepatic artery was evaluated by demonstrating the artery proper while crossing the portal vein. HARI was calculated automatically after measuring the hepatic artery peak velocity and end diastolic velocity measured in meters per second (m/sec) at the porta hepatis. The resistance index was calculated using the following equation: [peak systolic velocity (V max) - end diastolic velocity/peak systolic velocity (V min)/mean velocity]16.\n\nHAPI was calculated automatically using the following equation: [(V max) - (V min)/mean velocity]16.\n\nThe resistance index and wave form of the right hepatic vein was measured as the maximum negative velocity - minimum negative velocity (or positive velocity in case of triphasic flow signal)/maximum negative velocity.\n\nHepatic vein waveforms were described as triphasic, biphasic, monophasic, or not assessed because of severe attenuation.\n\nSARI: Color Doppler allowed identification of the main branches of the splenic artery by placing the transducer below the left costal margin17. SARI was measured automatically after measuring (Vmax) and (Vmin), which were measured in meters per second (m/sec) by putting the cursor in the main branches of the splenic artery at the splenic hilum at the left intercostal space18.\n\nThe resistance index was calculated using the following equation: [(Vmax) - (Vmin)]/peak systolic velocity]16.\n\nPHI was calculated as (HARI × 0.69) × (SARI × 0.87)/PVV19. It was recorded in m/sec.\n\nLVI was calculated as PVV/HAPI20,21. It was recorded in cm/sec.\n\nMLVI was calculated as PVV/HARI20,21. It was recorded in cm/sec.\n\nAll abdominal ultrasound and Doppler ultrasound assessments were performed with a Toshiba Nemio XG apparatus (Toshiba, Japan) by using a 3.5 MHz convex probe with B-mode and color Doppler ultrasound in the Tropical Medicine Department. Before evaluation, the patients fasted for at least 6 hours. During the evaluation, the patients were in the supine position.\n\nThe Statistical Package for the Social Sciences software (version 19, IBM Corp., Armonk, NY) was used for statistical analysis after organization and tabulation of our data. The mean and standard deviation were used for numerical variables. Additionally, the t-test and paired t-test were used for comparison of mean values between groups. Differences in mean values between the four variables studied were tested using analysis of variance (ANOVA). When the value of ANOVA (F) was significant, Tukey’s test was used. Percentages, numbers, and the chi square test were used for categorical variables. The P value was considered non-significant if it was > 0.05, significant if it was < 0.05, and highly significant if it was < 0.001).\n\n\nResults\n\nForty patients with cirrhosis and PHT were enrolled from April to November 2016 (38 were hepatitis C positive, 1 was hepatitis B positive, and 1 had combined hepatitis B and C infection). There was no statistically significant difference between the two groups with regard to age, sex, clinical features, medical history (hypertension, diabetes mellitus, and history of diuretics therapy), or Child–Pugh classification (Table 1, Table 2).\n\nDM: Diabetes mellitus, GI: Gastrointestinal, HTN: Hypertension, LL: Lower leg\n\nRegarding laboratory investigations, there was no significant difference between the two groups.\n\nALT: Alanine transferase, AST: Aspartate aminotransferase, Hb: Hemoglobin, WBCs: White blood cells\n\nRegarding the Doppler parameters of the studied groups (Table 3), there was a significant difference in PVD between groups I and II at baseline (13.210 ± 2.353 mm vs. 14.805 ± 2.528 mm; P = 0.046). There was no difference between PVD at baseline and PVD after 30 days in both groups.\n\nPVD: Portal vein diameter, PVV: Portal vein velocity, PVFV, HARI: Hepatic artery resistance index, HAPI: Hepatic artery pulsatility index, PHI: Portal hypertension index, LVI: Liver vascular index, MLVI: Modified liver vascular index, SARI: Splenic artery resistance index\n\nThere was no significant difference in the baseline mean values of PVV, PVBF, and HAPI between groups I and II (P = 0.881, 0.930, and 0.894, respectively). No difference was detected between baseline measurements and the measurements taken after simvastatin administration for 30 days in group I (P values: 0.358, 0.180, and 0.064, respectively).\n\nThere was no significant difference in HARI at baseline between groups I and II (0.785 ± 0.088 vs. 0.739 ± 0.079; P = 0.088). There was a highly significant decrease in HARI after simvastatin administration for 30 days in group I, from 0.785 ± 0.088 to 0.717 ± 0.086 (P < 0.001), an estimated 8.7% decrease from baseline.\n\nPHI was significantly higher in group I than in group II at baseline (3.915 ± 0.973 m/sec vs. 3.080 ± 0.610 m/sec; P = 0.007). PHI was significantly decreased in group I after simvastatin administration for 30 days, from 3.915 ± 0.973 m/se to 3.605 ± 1.168 m/sec (P = 0.024), a 7.9% decrease from baseline.\n\nThe mean LVI at baseline was 6.420 ± 2.561 cm/sec in group I and 6.140 ± 2.011 cm/sec in group II with no significant difference (P = 0.703). LVI was 6.420 ± 2.561 cm/sec at baseline and 7.094 ± 2.135 cm/sec after 30 days of simvastatin administration with no difference (P = 0.188).\n\nThe mean MLVI at baseline was 11.540 ± 3.266 cm/sec and 12.170 ± 3.506 cm/sec in groups I and II, respectively, with no significant difference (P = 0.560). MLVI was significantly increased in group I after simvastatin administration for 30 days, from 11.540 ± 3.266 cm/sec to 13.305 ± 3.222 cm/sec (P = 0.009), an increase of 15.3% from baseline. The MLVI was significantly higher in group I, after simvastatin administration for 30 days, than in group II (13.305 ± 3.222 cm/sec vs. 11.000 ± 2.968 cm/sec; P = 0.024).\n\nThe mean SARI value was 0.697 ± 0.073 and 0.609 ± 0.101 in groups I and II, respectively, with a significant difference between them (P = 0.003). There was no significant difference between SARI at baseline and after simvastatin administration for 30 days in group I (0.697 ± 0.073 vs. 0.668 ± 0.083; P = 0.23).\n\nPHI decreased significantly in group I after simvastatin administration for 30 days, from 3.915 ± 0.973 m/sec to 3.605 ± 1.168 m/sec (P = 0.024), a decrease of 7.9% from baseline (Figure 1).\n\nNo significant difference was found in the adverse effects noted during the study between the 2 groups in terms of myalgia, muscle pain, diarrhea, or worsening of ascites. The incidence of myalgia or muscle pain was reported to be 10% in group I and 20% group II (P = 0.376). The incidence of diarrhea was reported to be 10% in group I and 5% in group II (P = 0.548). The incidence of worsening ascites was reported to be 15% in group I and 25% in group II (P = 0.429) (Table 4).\n\n\nDiscussion\n\nPHT is considered to be an inevitable outcome of liver cirrhosis1. We conducted a randomized controlled clinical trial to study PHT by Doppler ultrasound in patients with cirrhosis prior to and after receiving simvastatin, and we studied other Doppler parameters reflecting hepatic fibrosis in patients with cirrhosis.\n\nThere was no significant difference regarding age, sex, or Child–Pugh score between the two studied groups, but there was a significant difference between the two groups with regard to INR, PT, and prothrombin activity at baseline. INR and PT were significantly higher, while prothrombin activity was significantly lower in group II than in group I. This may be because 50% of the patients in group II had Child–Pugh class C with advanced cirrhosis, and 45% had Child–Pugh class B cirrhosis. This was in accordance with the findings of Schuppan and Afdhal22, who reported that PT is increased in patients with advanced cirrhosis, as those patients have significantly impaired synthetic function.\n\nBefore simvastatin treatment, PVD was significantly higher in group II than in group I, which might be related to the severity of liver disease. This was in accordance with the findings of Shateria et al.23. In contrast, Ong and Tan24 reported that PVD does not correlate with high portal pressure or cirrhosis severity, while Lafortune et al.25 concluded that PVD might even decrease with an increase in PHT severity.\n\nWe also found that the mean PVV and mean PVBF were lower than normal in both groups. This was similar to the findings of Al-Nakshabandi26, who reported that a flow velocity of <16 cm/sec is a diagnostic feature of PHT in patients with cirrhosis. This decrease in PVV may be due to the presence of PHT, which results in increasing resistance to portal blood flow27. This was in accordance with the findings of Achim et al.28. They compared the mean PVBF and PVV between patients with cirrhosis and a healthy control group and found that these parameters were significantly lower in patients with cirrhosis, and this decrease was more notable in patients with Child–Pugh class B and C.\n\nFurthermore, HARI and HAPI were higher than normal. This may be due to the increase in hepatic arterial vascular resistance parallel to the rise in the portal pressure29. Additionally, it may be explained by the hepatic artery buffer response mechanism30.\n\nThe results of this study were similar to those of other studies28,31, which reported that HARI was higher in patients with cirrhosis and PHT than in control subjects, and the findings of Zhang et al.32, who concluded that HAPI was higher in patients than in controls and that portal pressure was significantly positively correlated with HAPI.\n\n\nConclusions\n\nIn conclusion, simvastatin significantly decreased PHI and HARI in patients with cirrhosis and PHT. Moreover, simvastatin significantly improved liver perfusion, as shown by the increased MLVI in patients with cirrhosis and PHT. These effects were achieved with or without the administration of β-adrenergic blockers. Therefore, simvastatin could be a valuable therapy for PHT, as simvastatin administration was associated with lowered hepatic resistance without harmful effects on systemic circulation. Additionally, simvastatin is relatively safe for patients with cirrhosis and PHT whether compensated or not.\n\n\nData Availability\n\nDataset 1: Dataset for Groups 1 and 2 of the study 10.5256/f1000research.13915.d19599733\n\n• N: Number\n\n• Group: 1, 2\n\n• Sex: •Male: 1 •Female: 2\n\n• Previous portal hypertension-related GIT bleeding: •Yes: 1 •No: 0\n\n• Endoscopy: •Yes: 1 •No: 0\n\n• Varices and portal gastropathy: •Yes: 1 •No: 0\n\n• History of β-blocker use: •Yes: 1 •No: 0\n\n• Hypertension (HTN): •Yes: 1 •No: 0\n\n• History of diuretic use: •Yes: 1 •No: 0\n\n• Diabetes mellitus (DM): •Yes: 1 •No: 0\n\n• Hepatic encephalopathy: •Yes: 1 •No: 0\n\n• Hepatitis B virus (HBV): •Yes: 1 •No: 0\n\n• Hepatitis C virus (HCV): • Yes: 1• NO: 0\n\n• Jaundice: •Yes: 1 •No: 0\n\n• Lower limb (LL) edema: •Yes: 1 •NO: 0\n\n• Ascites: •Yes: 1 No: 0\n\n• Child–Pugh class: •Child–Pugh class A: 1 •Child–Pugh class B: 2. •Child–Pugh class C: 3\n\n• Myalgia: •Yes: 1 •No: 0\n\n• Diarrhea: •Yes: 1 •No: 0\n\n• Worsening of ascites: •Yes: 1 •No: 0\n\n• Observed improvement in muscle cramps: •Yes: 1 •No: 0\n\n• ALT Alanine transferase\n\n• AST Aspartate aminotransferase\n\n• HAPI Hepatic artery pulsatility index\n\n• HARI Hepatic artery resistance index\n\n• Hb Hemoglobin\n\n• LVI Liver vascular index\n\n• MLVI Modified liver vascular index\n\n• PHI Portal hypertension index\n\n• PVD Portal vein diameter\n\n• PVBF Portal vein blood flow\n\n• PVV Portal vein velocity\n\n• SARI Splenic artery resistance index\n\n• RBCs Red blood cells\n\n• WBCs White blood cells\n\n\nEthics and consent\n\nAll patients provided written informed consent, and the study was approved by the Ethics Committee of the Faculty of Medicine, Tanta University. All patients had code numbers to ensure patient anonymity.\n\n\nList of abbreviations\n\n",
"appendix": "Competing interests\n\n\n\nThe authors declare that they do not have any conflicts of interest regarding simvastatin or any of its manufacturers. All the authors confirm that they do not have any financial or non-financial competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\nAll costs associated with this study were personally covered by the authors.\n\n\nSupplementary Material\n\nSupplementary file 1: CONSORT flow diagram\n\nClick here to access the data.\n\nSupplementary file 1: CONSORT check list\n\nClick here to access the data.\n\nSupplementary file 1: Study protocol\n\nClick here to access the data.\n\n\nReferences\n\nBosch J, Abraldes JG, Berzigotti A, et al.: The clinical use of HVPG measurements in chronic liver disease. Nat Rev Gastroenterol Hepatol. 2009; 6(10): 573–582. PubMed Abstract | Publisher Full Text\n\nSanyal AJ, Bosch J, Blei A, et al.: Portal hypertension and its complications. Gastroenterology. 2008; 134(6): 1715–1728. PubMed Abstract | Publisher Full Text\n\nBosch J, Abraldes, JG, Groszmann R: Current management of portal hypertension. J Hepatol. 2003; 38(Suppl 1): S54–S68. PubMed Abstract | Publisher Full Text\n\nVargas V, Rimola A, Casanovas T, et al.: Applicability of liver transplantation in Catalonia at the end of the millennium (A prospective study of adult patient selection for liver transplantation). Transpl Int. 2003; 16(4): 270–275. PubMed Abstract\n\nFailli P, DeFranco RM, Caligiuri A, et al.: Nitrovasodilators inhibit platelet-derived growth factor-induced proliferation and migration of activated human hepatic stellate cells. Gastroenterology. 2000; 119(2): 479–492. PubMed Abstract | Publisher Full Text\n\nYu Q, Shao R, Qian HS, et al.: Gene transfer of the neuronal NO synthase isoform to cirrhotic rat liver ameliorates portal hypertension. J Clin Invest. 2000; 105(6): 741–748. 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PubMed Abstract | Publisher Full Text\n\nTrebicka J, Hennenberg M, Laleman W, et al.: Atorvastatin lowers portal pressure in cirrhotic rats by inhibition of RhoA/Rho-kinase and activation of endothelial nitric oxide synthase. Hepatology. 2007; 46(1): 242–253. PubMed Abstract | Publisher Full Text\n\nVioli F, Calvieri C, Ferro D, et al.: Statins as antithrombotic drugs. Circulation. 2013; 127(2): 251–257. PubMed Abstract | Publisher Full Text\n\nZafra C, Abraldes JG, Turnes J, et al.: Simvastatin enhances hepatic nitric oxide production and decreases the hepatic vascular tone in patients with cirrhosis. Gastroenterology. 2004; 126(3): 749–755. PubMed Abstract | Publisher Full Text\n\nBerzigotti A, Piscaglia F; EFSUMB Education and Professional Standards Committee: Ultrasound in portal hypertension--part 2--and EFSUMB recommendations for the performance and reporting of ultrasound examinations in portal hypertension. Ultraschall Med. 2012; 33(1): 8–32; quiz 30-1. PubMed Abstract | Publisher Full Text\n\nScheinfeld MH, Bilali A, Koenigsberg M: Understanding the spectral Doppler waveform of the hepatic veins in health and disease. Radiographics. 2009; 29(7): 2081–2098. PubMed Abstract | Publisher Full Text\n\nMcNaughton DA, Abu-Yousef MM: Doppler US of the liver made simple. Radio Graphics. 2011; 31(1): 161–188. PubMed Abstract | Publisher Full Text\n\nBolognesi M, Sacerdoti D, Merkel C, et al.: Splenic Doppler impedance indices: Influence of different portal hemodynamic conditions. Hepatology. 1996; 23(5): 1035–1040. PubMed Abstract | Publisher Full Text\n\nSacerdoti D, Gaiani S, Buonamico P, et al.: Interobserver and interequipment variability of hepatic, splenic, and renal arterial Doppler resistance indices in normal subjects and patients with cirrhosis. J Hepatol. 1997; 27(6): 986–992. PubMed Abstract | Publisher Full Text\n\nPiscaglia F, Donati G, Serra C, et al.: Value of splanchnic Doppler ultrasound in the diagnosis of portal hypertension. Ultrasound Med Biol. 2001a; 27(7): 893–899. PubMed Abstract | Publisher Full Text\n\nHaktanir A, Cihan BS, Celenk C, et al.: Value of Doppler sonography in assessing the progression of chronic viral hepatitis and in the diagnosis and grading of cirrhosis. J Ultrasound Med. 2005; 24(3): 311–321. PubMed Abstract | Publisher Full Text\n\nZhang L, Duan YY, Li JM, et al.: Hemodynamic features of Doppler ultrasonography in patients with portal hypertension: intraoperative direct measurement of portal pressure in the portal venous system. J Ultrasound Med. 2007; 26(12): 1689–1696. PubMed Abstract | Publisher Full Text\n\nSchuppan D, Afdhal NH: Liver cirrhosis. Lancet. 2008; 371(9615): 838–851. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShateria K, Mohammadib A, Moloudic F, et al.: Correlation between sonographic portal vein diameter and flow velocity with the clinical scoring systems MELD and CTP in cirrhotic patients: is there a relationship? Gastroenterology Research. 2012; 5(3): 112–119. Publisher Full Text\n\nOng TZ, Tan HJ: Ultrasonography is not reliable in diagnosing liver cirrhosis in clinical practice. Singapore Med J. 2003; 44(6): 293–295. PubMed Abstract\n\nLafortune M, Marleau D, Breton G, et al.: Portal venous system measurements in portal hypertension. Radiology. 1984; 151(1): 27–30. PubMed Abstract | Publisher Full Text\n\nAl-Nakshabandi NA: The role of ultrasonography in portal hypertension. Saudi J Gastroenterol. 2006; 12(3): 111–117. PubMed Abstract | Publisher Full Text\n\nBerzigotti A, Seijo S, Reverter E, et al.: Assessing portal hypertension in liver diseases. Expert Rev Gastroenterol Hepatol. 2013; 7(2): 141–155. PubMed Abstract | Publisher Full Text\n\nAchim CA, Bordei P, Dumitru E: The role of ultrasonography in the evaluation of portal hemodynamics in healthy adults and pathologic conditions. ARS Medica Tomitana. 2016; 22(2): 128–134. Publisher Full Text\n\nHarkanyi Z: Doppler Ultrasound signs of portal hypertension in cirrhosis. Ultrasound Clin. 2006; 1(3): 443–455.\n\nGülberg V, Haag K, Rössle M, et al.: Hepatic arterial buffer response in patients with advanced cirrhosis. Hepatology. 2002; 35(3): 630–634. PubMed Abstract | Publisher Full Text\n\nYu Ya F, Ya EV: Possibilities of Doppler ultrasonography in valuing of morfofunctional condition of liver for patients with viral hepatitis and hepatocirrhosis. Intercollegas. 2014; 1(1): 65–72. Reference Source\n\nZhang L, Duan YY, Li JM, et al.: Hemodynamic features of Doppler ultrasonography in patients with portal hypertension: intraoperative direct measurement of portal pressure in the portal venous system. J Ultrasound Med. 2007; 26(12): 1689–1696. PubMed Abstract | Publisher Full Text\n\nElwan N, Salah R, Hamisa M, et al.: Dataset 1 in: Evaluation of portal pressure by doppler ultrasound in patients with cirrhosis before and after simvastatin administration – a randomized controlled trial. F1000Research. 2018. Data Source"
}
|
[
{
"id": "31377",
"date": "07 Mar 2018",
"name": "William Beaubien-Souligny",
"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 had the opportunity to review the manuscript entitled “Evaluation of portal pressure by Doppler ultrasound in patients with cirrhosis before and after simvastatin administration – a randomized controlled trial.” In this project, the authors performed ultrasound assessment to determine whether treatment with simvastatin resulted in findings suggestive of a decrease in portal vein pressure compared to placebo.\nThe subject is of interest for clinicians involved in the care of cirrhosis patients, particularly as the studied agent is already widely available. While these data are clinically relevant and must be reported, I have numerous concerns about data reporting in this work. I will list the main issues that must be addressed in my opinion, followed by other minor points.\n\nMain concerns:\nAs a randomized controlled trial, the comparator is the placebo group (as it was written in the Clinicaltrials.gov entry). It is clear, just by reading the abstract of this article, that the results are not adequately reported. Group 1 and group 2 (placebo) are not compared in the result section of the abstract: the main result reported is a pre-post analysis without comparison with the control group. The main analysis of your study should be as described in your clinicaltrials.gov entry: comparing the intervention and the placebo arm on the primary outcome (The number of patients with reduced portal pressure after intervention). It is also important clearly determine what was the primary outcome of the study in the method section (what ultrasound parameter is the most clinically important for the evaluation of portal hypertension). Other less important echographic parameters should be considered secondary outcomes.\n\nIn order for the readers to adequately assess the risk of bias in this study, I think it is important for the authors to give information about the following points which are included in the Cochrane Risk of Bias assessment tool:\nPlease describe the method of randomization Please describe the method of allocation concealment (if any) Please describe if there was blinding during outcome assessment (does the ultrasonographer knew patient allocation to one arm or the other) There was no study protocol available for review. The file identified as the study protocol was a copy of the NCT entry and thus was insufficient. I think points a and b are especially important as baseline differences were seen between the intervention and the placebo arm (more severe cirrhosis in the intervention arm)\n\nThe discussion section should be heavily modified. The authors do not discuss previous clinical trials of the subject. There is at least 4 previous trials on the subject. (PMID: 29099421, 26321186, 26774179, 19208350) This is a vital part of the discussion section that must highlight how this work compares with previous studies. Additionally, the limitations of the study are not discussed which is usually an integral part of the discussion section.\n\nOther specific comments:\nAbstract: Please see main concern no 1 Methods\nPlease clarify the inclusion criteria for portal hypertension. Were all the features required to be present or only one of them was sufficient? How were patients approached for this study? On an outpatient basis or during hospitalisation? Please make sure to indicate that this is a open-label study both in the abstract and in the method. Please describe how you determined that the distribution for the continuous variables was normal Please describe how you determined sample size\n\nResults:\nToo much emphasis is put on the baseline values. I don’t think it is necessary to repeat what is found in the Tables except for clinically or statistically significant differences between groups. Focus on the comparison of outcome between the intervention and the control group. Pre-post analysis should be secondary in a randomized clinical trial. How many patients were on nadolol. Were the dose modified during the treatment period?\n\nDiscussion\nPlease see main concern no 3 Please provide a small summary of the main findings at the beginning. The discussion about echographic parameters is of limited interest in its present form. I would suggest to focus of the clinical implications of these findings. Surely, you think that some of those markers represent good surrogates for the severity of portal hypertension and may be used as a pharmacologic treatment target? This should represent an opportunity to describe what is the best marker (primary outcome) and if it was modified by statin therapy. It should be discussed in the results that the baseline imbalance in PHI between the intervention and control group is problematic in this study and probably affect the lack of difference between the two groups at 30 days. Was this imbalance due to chance? Giving more information (see major point 2) would help the reader to understand the possible explanations.\n\nI will be available to reconsider this manuscript after these issues are addressed. Thank you for the opportunity to review this work.\n\nSincerely\n\nWilliam Beaubien-Souligny MD FRCPC\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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "31373",
"date": "16 Mar 2018",
"name": "Mohamed Ahmed Samy Kohla",
"expertise": [
"Reviewer Expertise Viral hepatitis",
"hepatocellular carcinoma",
"liver cirrhosis",
"portal hypertension"
],
"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 entitled: Evaluation of portal pressure by doppler ultrasound in patients with cirrhosis before and after simvastatin administration – a randomized controlled trial, is quite interesting and of clinical significance which could have a good impact on treatment of portal hypertension with Simvastatin, which is a safe and well tolerated drug.\nHowever, I have few comments:\nI think this study actually aims at evaluation of portal hemodynamics rather than portal pressure, because Doppler U/S can asses portal hemodynamics rather than measurement of portal pressure,therefore, i suggest a minor modification of the title: Evaluation of portal hemodynamics by doppler ultrasound in patients with cirrhosis before and after simvastatin administration – a randomized controlled trial\n\nData on MELD score should be provided, we can compare the mean MELD score in the 2 groups.\n\nDid all patients in the 2 groups had an upper GI endoscopy before enrollment? If yes, the endoscopic findings should be shown to asses varices and portal hypertensive gastropathy.\n\nIn the method section, the authors mentioned measurement of Random blood sugar and AFP but this was not shown in the tables.\n\nWere patients on beta blockers excluded from the study? Was there a drug free interval before enrollment?\n\nWhy was the dose of Simvastatin escalated from 20 mg to 40 mg per day after 2 weeks?\n\nMy recommendation is that, this manuscript is suitable for publication (accepted with minor changes).\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": []
},
{
"id": "31479",
"date": "03 Apr 2018",
"name": "Abidullah Khan",
"expertise": [
"Reviewer Expertise General internal medicine",
"medical education",
"endocrine and rheumatology"
],
"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 congratulate Elwan N et al for their efforts. The research ideas being brought forward in this manuscript are worth appreciation. However, the authors need to address the following deficiencies before any consideration for final publication is made.\n1) TITLE: This needs to be modified. I will suggest to rephrase it as, “ Effects of Simvastatin on portal pressure in patients with cirrhosis”.\n2) ABSTRACT: Please avoid using abbreviations in the abstract. Moreover, abbreviations need to be written in full on their first appearance in a given article. In my opinion, the term PHT should therefore be expanded. The term “hypotensive agents” must be rephrased as “one of the portal hypotensive agents”.\n3) INTRODUCTION: In para 4, the authors write that simvastatin can be used safely in patients with chronic liver disease. I will need an explanation for this as at what stage can it be more effective and safer? This is because, advanced liver disease in itself leads to hypolipidemia which in itself is a worse prognostic indicator.\n4) METHODS: The sample size is small. I will recommend more participants. Regarding group 1, why were they segregated into two subgroups in terms of the dosage of simvastatin? If they were done so, why were the effects of the dosage on portal pressure not provided in the results/discussion sections? Moreover, was there any difference in the final outcomes because of the different dosages? The authors write that the patients were continued on their usual antihypertensive and diabetic medications. Were any potential drug-drug interactions excluded? Moreover, I will be interested in knowing the antihypertensive medications (esp ACEi/ARBs as they have antifibrotic and anti inflammatory effects).\n5) RESULTS: Th authors mention that all the patients included in the study had cirrhosis secondary to chronic viral hepatitis. Hence, the results can not be generalized to all forms of cirrhosis and either the title needs to be modified accordingly or more patients need to be recruited to better measure the etiology-outcome effect. In para 2, PVD in group 1 was 13.2 in contrast to group 2 where it was 14.8. Hence, there was a difference at baseline. This contradicts their statement that there was no difference in mean PVDs at baseline.\n6) DISCUSSION: The authors are requested to avoid repetition of the facts already mentioned in Introduction and Methods etc. The para 1 of discussion can therefore be either modified or removed. In para 3, the authors mention that PVD was higher in group 2 than in group 1. This is against their statement in Results as explained above. Moreover, as group 1 had a lower PVD at baseline, can the lowering effect of simvastatin be purely a chance finding? Therefore, it is suggested to include more patients in the study group.\n7) CONCLUSION: Please make the conclusion more succinct and comprehensive. Thank you.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-256
|
https://f1000research.com/articles/7-254/v1
|
01 Mar 18
|
{
"type": "Brief Report",
"title": "Dopamine synergizes with caffeine to increase the heart rate of Daphnia",
"authors": [
"Aman Kundu",
"Gyanesh Singh",
"Aman Kundu"
],
"abstract": "Dopamine is a key neurotransmitter, and is widely used as a central nervous system (CNS) agent. Dopamine plays an important role in humans, including a major role in reward and motivation behaviour. Several addictive drugs are well known to increase neuronal dopamine activity. We selected Daphnia, an important model organism, to investigate the effect(s) of selected CNS agents on heart rate. Dopamine’s effects on Daphnia’s heart has not been previously reported. Caffeine is a well-known and widely consumed stimulant. Ethanol is well known for its effects on both neurological and physiological processes in mammals. We tested the effect of dopamine on the heart rate of Daphnia, and compared its effect with caffeine and ethanol alone and in combination. Both caffeine and dopamine were found to instantly increase the heart rate of Daphnia in a dose-dependent manner. Interestingly, caffeine synergized with dopamine to increase Daphnia’s heart rate. As ethanol decreased the heart rate of Daphnia and dopamine increased the heart rate of Daphnia, we wanted to test the effect of these molecules in combination. Indeed, Dopamine was able to restore the ethanol-induced decrease in the heart rate of Daphnia. Effects of these CNS agents on Daphnia can possibly be correlated with similar effects in the case of mammals.",
"keywords": [
"Dopamine",
"heart",
"neurotransmitter",
"cardiac",
"central nervous system"
],
"content": "Introduction\n\nNeurotransmitters are the key mediators of communication between nerve cells. Because of their effect on brain and spinal cord, central nervous system (CNS) agents can be used to control or treat variety of medical conditions1. Stimulation of the hypothalamus can lead to cardiovascular disturbances, indicating a direct connection between the heart and the CNS2,3. Different types of rewards are known to increase the level of dopamine in the brain4. Daphnia are small crustaceans commonly known as “water fleas”, and are found in water bodies5. Daphnia is an ideal organism for research, as it has short life span, and can easily be cultured6. These organisms can feed on algae, yeast and bacteria5. More importantly, Daphnia are transparent, thus allowing clear visualization of different organs, including the heart7. The organs are protected by a thin membrane that allows the penetration of different compounds; therefore assisting with heart rate monitoring in real time5. Using a microscope that has computer-aided real-time imaging capabilities, the effect of various compounds can be observed on Daphnia‘s heart in real time. Daphnia’s life span is 40–50 days, which varies in different species and also changed with environmental conditions, especially temperature. Male and female Daphnia can easily be differentiated, as female Daphnia have brood pouch that holds eggs. These eggs develop into embryos, leading to the production of juveniles that attain sexual maturity within ten days.\n\nDopamine is important for normal cardiopulmonary response to exercise and is necessary for optimal high-intensity exercise performance. Blocking dopamine receptors appears to be detrimental to exercise performance8. Caffeine, by antagonizing adenosine A2A receptors, is known to augment dopamine signalling in the brain9,10. Even at routine doses, caffeine can enhance dopamine receptor accessibility in the mammalian CNS10. Caffeine has also been reported to normalize the heart rate of Daphnia, which is decreased by atropine and atenolol11. Ethanol is known to cause progressive weakness, difficulty in walking, and lowered heart rate12. Ethanol also inhibits calcium dependent neurotransmitter release, and, excitatory and inhibitory postsynaptic potentials in cultured spinal cord neurons13.\n\nThe aim of the present study was to investigate the effect of Dopamine on Daphnia’s heart rate, alone and in combination of caffeine and ethanol. The rationale behind this research was that both caffeine and ethanol are known to affect nervous system functions14, and dopamine is a major neurotransmitter.\n\n\nMethods\n\nDaphnia were isolated from Chitti Vai river of Punjab. For the isolation of Daphnia, 0.5–2.0 litres of river water was collected and transported to laboratory. Adult Daphnia were manually identified as per the standard identification features15, and filtered out using muslin cloth. These adults were cultured in 300 ml glass jars containing river water that was filtered with muslin cloth. Daphnia culture was supplemented with 0.5% yeast culture, added every third day. Yeast culture, in this case, was used as a food for Daphnia. Algae, yeast or bacteria are preferred food for Daphnia. Although, many workers use river water for Daphnia culture presuming that it would have better mineral composition, in our case, we were also able to culture Daphnia in aged tap water in the similar manner. Cultures were routinely monitored to ensure production of healthy Daphnia.\n\nTo investigate the effect of certain agents on the heart rate of Daphnia, real-time monitoring of changes in the heart rate of Daphnia is required. We used a microscope equipped with computer-aided real-time imaging capability (Magnus Live usb camera viewer, version 2.0, Magnus Analytics, New Delhi-110044, India), and for each reading heart rate was initially counted without any treatment (control). Subsequently, changes in the heart rate was monitored after the addition of selected agents. Each Daphnia was placed on the glass slide with 100 ul of water. The slide was observed in real time under the microscope at 40x or 100x magnification, and heart rate was counted. To avoid the effect of temperature or other environmental factors, counting was done after five seconds of starting the microscope. Subsequently, the microscope was switched off for five seconds, cardiovascular agents were added (see Table 1), and heart rate was counted again.\n\nA paired t test analysis was done to compare changes in heart rates upon treatment with different agents. Statistical analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego, CA, USA). P<0.05 was considered significant.\n\n\nResults and discussion\n\nDopamine’s effects on Daphnia’s heart has not been reported previously. We hereby report that dopamine instantly increases the heart rate of Daphnia in a dose-dependent manner, and a significant increase (25.7%) in the heart rate was observed, even at a low dose of 0.8 mg/ml (Figure 1). Caffeine showed a similar effect on Daphnia’s heart rate at a 10-times lower concentration than dopamine (28.5% increase at 0.08 mg/ml, Figure 2). Dopamine is the precursor of norepinephrine, and has been shown to augment heart activity by affecting beta-adrenergic receptors, in the case of a canine model16. Furthermore, dopamine can cause both relaxation and contraction of vascular smooth muscle. Dopamine is also known to augment heart activity, pulmonary pressure, and cardiac index in the case of normal and hypertensive individuals17.\n\nThis experiment was performed two times, and a paired t test analysis vs control indicated the following P values: 0.0070 (for 0.8 mg/ml), 0.0255 (1.6 mg/ml), 0.0424 (2.4 mg/ml), and 0.0344 (3.2 mg/ml). These values are statistically significant.\n\nThis experiment was done two times, and a paired t test analysis vs control revealed the following P values: 0.0406 (0.08 mg/ml), 0.0263 (0.16 mg/ml), 0.0367 (0.24 mg/ml), and 0.0189 (0.32 mg/ml). These values are statistically significant.\n\nCaffeine, in combination with dopamine, increased Daphnia’s heart rate more than when the agents were administered alone, which suggests a synergistic activity (Figure 3). Dopamine has also been previously reported to play a role in the responses of Drosophila to cocaine, nicotine or ethanol18.\n\nDaphnia’s heart rate was measured upon treatment with dopamine alone (red) or a combination of dopamine and caffeine (green). The concentration of caffeine (in combination with dopamine) was (A) 40 ug/ml and (B) 120 ug/ml. This experiment was performed two times, and a paired t test analysis vs control indicated the following P values: 0.0374 (0.8 mg/ml dopamine) and 0.0230 (1.6 mg/ml dopamine). These values are statistically significant.\n\nTo see the effect on the heart rate of Daphnia, ethanol was used at a concentration ranging from 2–8%, and was found to decrease the heart rate of Daphnia in a dose-dependent manner (Figure 4).\n\nThis experiment was done two times, and a paired t test analysis vs control indicate the following P values: 0.0152 (2% ethanol), 0.0059 (4% ethanol), 0.0130 (6% ethanol), and 0.0280 (8% ethanol). These values are statistically significant.\n\nWe observed that dopamine was able to rescue the ethanol-induced decrease in the heart rate of Daphnia, even at a concentration of 0.4 mg/ml (Figure 5).\n\nAt 2% ethanol, dopamine-induced increase in the heart rate was 62.5% compared to control, and 84.8% compared to ethanol-induced heart rate. At 4% ethanol, dopamine-induced increase in the heart rate was 4.3% compared to control, and 33.7% compared to ethanol-induced heart rate.\n\n\nConclusion\n\nThis fundamental investigation can be of enormous importance, as caffeine and ethanol are the most widely consumed psychoactive drugs, and dopamine is a master neurotransmitter that is known to be involved in variety of diseases19,20. It is possible that these psychoactive agents can have similar or more drastic effects in humans. It is, therefore, very important to urgently investigate the effect of these psychoactive agents, alone or in combination, in humans. Such studies can provide crucial information that can be used in a variety of clinical settings. For example, cases of alcohol or caffeine intoxication can be managed by dopamine therapy, treatment(s) of cardiac disorders may be different for alcoholics or coffeeholics, and patients undergoing dopamine therapy need to be regularly monitored for cardiothoracic status, and alcohol/caffeine consumption.\n\n\nData availability\n\nDataset 1: Effect of dopamine, caffeine and ethanol on the heart rate of Daphnia. Heart rates (beats per minute) was initially counted without any treatment (controls). Subsequently, changes in the heart rate was monitored after the addition of selected agents. DOI, 10.5256/f1000research.12180.d19418921",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe authors are thankful to Devika Chanu Khaidem of Zoological Survey of India (Kolkata, India) for her help while doing experiments.\n\n\nReferences\n\nSoddu E, Rassu G, Giunchedi P, et al.: From naturally-occurring neurotoxic agents to CNS shuttles for drug delivery. Eur J Pharm Sci. 2015; 74: 63–76. PubMed Abstract | Publisher Full Text\n\nArnsten AF, Wang M, Paspalas CD: Dopamine's Actions in Primate Prefrontal Cortex: Challenges for Treating Cognitive Disorders. Pharmacol Rev. 2015; 67(3): 681–96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOsteraas ND, Lee VH: Neurocardiology. Handb Clin Neurol. 2017; 140: 49–65. PubMed Abstract | Publisher Full Text\n\nPeinado AB, Rojo JJ, Calderón FJ, et al.: Responses to increasing exercise upon reaching the anaerobic threshold, and their control by the central nervous system. BMC Sports Sci Med Rehabil. 2014; 6: 17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStollewerk A: The water flea Daphnia -- a 'new' model system for ecology and evolution? J Biol. 2010; 9(2): 21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShaw JR, Pfrender ME, Ead BD, et al.: Daphnia as an emerging model for toxicological genomics. Adv Exp Biol. 2008; 2: 165–219. Publisher Full Text\n\nHarris KD, Bartlett NJ, Lloyd VK: Daphnia as an emerging epigenetic model organism. Genet Res Int. 2012; 2012: 147892. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTedjasaputra V, Bryan TL, van Diepen S, et al.: Dopamine receptor blockade improves pulmonary gas exchange but decreases exercise performance in healthy humans. J Physiol. 2015; 593(14): 3147–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZeitzer JM: Control of sleep and wakefulness in health and disease. Prog Mol Biol Transl Sci. 2013; 119: 137–54. PubMed Abstract | Publisher Full Text\n\nVolkow ND, Wang GJ, Logan J, et al.: Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain. Transl Psychiatry. 2015; 5(4): e549. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPodosinovikova NP, Beliaev VA, Dolgo-Saburov VB: [Studying intermediatory regulation of heart rhythm using Daphnia magna as the alternative test object]. Eksp Klin Farmakol. 2009; 72(6): 49–51. PubMed Abstract\n\nHorn-Hofmann C, Büscher P, Lautenbacher S, et al.: The effect of nonrecurring alcohol administration on pain perception in humans: a systematic review. J Pain Res. 2015; 8: 175–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNoori HR, Spanagel R, Hansson AC: Neurocircuitry for modeling drug effects. Addict Biol. 2012; 17(5): 827–64. PubMed Abstract | Publisher Full Text\n\nFerré S, O'Brien MC: Alcohol and Caffeine: The Perfect Storm. J Caffeine Res. 2011; 1(3): 153–162. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEbert D: Ecology, Epidemiology, and Evolution of Parasitism in Daphnia. Zoologisches Institut der Universität Basel, National Center for Biotechnology Information (US). 2005. ISBN-10: 1-932811-06-0. Reference Source\n\nGoldberg LI: Dopamine--clinical uses of an endogenous catecholamine. N Engl J Med. 1974; 291(14): 707–10. PubMed Abstract | Publisher Full Text\n\nHolloway EL, Polumbo RA, Harrison DC: Acute circulatory effects of dopamine in patients with pulmonary hypertension. Br Heart J. 1975; 37(5): 482–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBainton RJ, Tsai LT, Singh CM, et al.: Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila. Curr Biol. 2000; 10(4): 187–94. PubMed Abstract | Publisher Full Text\n\nBlum K, Thanos PK, Gold MS: Dopamine and glucose, obesity, and reward deficiency syndrome. Front Psychol. 2014; 5: 919. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRodan LH, Gibson KM, Pearl PL: Clinical Use of CSF Neurotransmitters. Pediatr Neurol. 2015; 53(4): 277–86. PubMed Abstract | Publisher Full Text\n\nKundu A, Singh G: Dataset 1 in: Dopamine synergizes with caffeine to increase the heart rate of Daphnia. F1000Research. 2018. Data Source"
}
|
[
{
"id": "31347",
"date": "09 Mar 2018",
"name": "Rafael Antonio Vargas",
"expertise": [
"Reviewer Expertise Cardiovascular physiology",
"neurophysiology",
"animal models"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe original article by Kundu and Singh focuses on the study the effects of cardiovascular drugs on the heart rate of Daphnia. The authors have used an interesting heart model: Daphnia. This model has some advantages in comparison with classical animal models such as rats, mice, dogs, cats, and others. This model could be interesting for researchers from undeveloped countries. The study shows the effect of dopamine, caffeine, and alcohol on Daphnia heart rate (HR), which has been studied in other animal models. In this case, it is shown that dopamine and caffeine increase HR, and alcohol reduces HR. Interestingly, dopamine restores the low HR ethanol-induced. The authors claim that it’s probably similar effects of this agents in humans, however, the Daphnia heart does not represent a direct analog of mammalian cardiac function, so the interpretation of the results of the present study in terms of mammals is problematic. I have two comments/questions:\nIt is mentioned in the introduction, that atropine and atenolol decrease HR and this is not true in humans: Atenolol, a beta blocker reduces HR, but atropine, a cholinolytic agent, increase HR, so this statement must be revised. It could be useful if the authors add the how HR was performed.\nIn general, after reading this submission, I consider that this is a short and interesting article about a simple model which will be useful for students, young researchers interested in alternatives to study cardiovascular function. After clarifying the comments the paper can be indexed.",
"responses": []
},
{
"id": "31349",
"date": "13 Apr 2018",
"name": "Mrinal K. Poddar",
"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 original article written by Kundu and Singh focuses on the study of the effects of cardiovascular drugs of the heart rate of Daphnia. In this study the authors have used an interesting heart model of Daphnia.\nThe article represents the synergistic effect of dopamine and caffeine to increase the heart rate in Daphnia, the tiny water fleas. Though, neither this model can be compare with mammals nor their organ function. The authors have tried to show some effects without mentioning the mechanism of effects. I think they should clarify the followings points:\n1. Authors have analyzed the data with a simple t test. Why they didn’t they approach with ANOVA test? Moreover, statistical analysis with only two observations is not sufficient. Observations should be made of at least four times for significance test. 2. In Figures, the vertical line given on top of each bar represents what? - SEM or SD? 3. Table 1 shows the lowest concentration of dopamine as 0.4mg/ml which is missing in the entire manuscript. Where is the result of 0.4 mg/ml? 4. Why did the authors choose 40µg or 120µg/ml instead of mg/ml that they have tested and represented in Figure 1. What is the reason of this sudden switch over? Nothing is clear to me! 5. In Figure 3 Why the authors have used the combination of lower concentration of caffeine (40µg/ml) with higher concentration of dopamine (1.6mg/ml) and higher concentration of caffeine (120 µg/ml) with lower concentration of dopamine (0.8mg/ml)? This needs clarification. 6. Figure 5 have the same queries like Figure 3 and it should be better in both the cases of combinations, keep one constant and vary the other one. 7. The Daphnia model does not directly resemble the mammalian system. The conclusion they have made in the manuscript requires rethinking and should be presented accordingly. 8. In the manuscript the authors didn’t mention about the treatment procedure. How have they administered the dopamine, caffeine or alcohol? What is the route of administration of these compounds? Are those administered or given in the culture medium? Depending on the route of administration for this particular model, the author should discuss about the mechanism of action of these agents on Daphnia heart rate or cardiovascular system.\n\nSuggestions:\nI think for indexing following works are needed to improve the manuscript: 1. Redesign the experiments as I suggested. 2. More observations (at least 4) for each experiment are needed. 3. Proper statistical analysis is essential. Simple t test is not enough as the experiment are in combination of different agents.",
"responses": []
},
{
"id": "35079",
"date": "27 Jun 2018",
"name": "Cecilia Scorza",
"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 “Dopamine synergizes with caffeine to increase the heart rate of Daphnia” done by Aman Kundu and Gyanesh Singh, investigates the effect of dopamine, caffeine and alcohol on Daphnia’s heart rate. Also the combination of caffeine and ethanol was tested. The rationale of the paper was to use the Daphnia’s heart rate to test treatment(s) of cardiac disorders in high consumers of coffee or alcohol. The importance and objectives of the study are well explained and methods are well described, although some aspects of this manuscript should be really improved and corrected.\n\nThe statistical Analysis applied is not correct; consequently I have some doubts if the results would be the same applying a correct statistical analysis. Authors used t-paired test to evaluate a dose-dependence curve in which more than two concentrations are compared. I suggest to authors apply One-Way ANOVA followed by a post hoc test (for example, Newman-Keuls). Additionally, authors indicated that the experiments were done two times. I understand that this means that a duplicate of each experiment were performed, is it correct?. If this is the case, a mean ± SD should be used to compare data. Please, correct.\nAnother important issue is about the synergism. Author concluded that dopamine synergizes with caffeine to increase the heart rate of Daphnia. But, synergism should be declared if a sub-threshold concentrations of DA and Caff is used. If not, only a potentiation is achieved. Please, correct the terminology of synergism or other experiments should be done using the combination of sub-threshold doses of DA and Caff.\n\nIt would be very important to add positive controls to test Daphnia’s heart rate, for example, noradrenaline or atropine, both are prototypical substances that increase heart rate in vertebrate animals.\n\nIn the conclusion: in addition to the purpose of this kind of assay, I wondering if Daphnia’s heart rate assay could be more suitable as a biomarker of toxicity instead of serve as a screening test of different drugs to alter Daphnia’s heart rate?",
"responses": []
}
] | 1
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https://f1000research.com/articles/7-254
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https://f1000research.com/articles/7-249/v1
|
28 Feb 18
|
{
"type": "Research Article",
"title": "Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells",
"authors": [
"Purnamawati Purnamawati",
"Jeanne Adiwinata Pawitan",
"Andhika Rachman",
"Septelia Inawati Wanandi",
"Purnamawati Purnamawati",
"Jeanne Adiwinata Pawitan",
"Andhika Rachman"
],
"abstract": "Background: Nowadays, umbilical cord- and adipose-derived stem cells (UCSCs and ASCs) are the most common sources of mesenchymal stem cells (MSCs). As part of the tumor microenvironment, MSCs are known to communicate with cancer cells via their secretomes. Increased activity of aldehyde dehydrogenase-1 (ALDH1) has been widely used as a common intrinsic stemness marker in normal and cancer stem cells. Our study aimed to elaborate on the effect of UCSC and ASC secretomes on the expression of ALDH1A3, as one of the important variants of ALDH1, TGF-β1 and TGF-β receptor type I (TβRI) in human breast cancer stem cells (BCSCs). Methods: UCSCs and ASCs were cultured in serum-free α-MEM media under standard conditions for 24 hours. The conditioned medium (CM) containing secretomes of UCSCs and ASCs were collected and added 50% (v/v) to the cultured of human BCSCs for 72 hours. The mRNA expressions of ALDH1A3, TGF-β1, and TβRI were determined using quantitative Reverse Transcriptase Polymerase Chain Reaction (q-RT-PCR). Results: We found that CM of UCSCs significantly increased the ALDH1A3 expression of BCSCs in parallel with the increase of TGF-β1 and TβRI expressions. Conversely, CM of ASCs had no significant effect on the ALDH1A3 expression, but significantly decreased TGF-β1 and TβRI expressions of BCSCs. These results contradict our published data on ALDH1A1, which is another important variant of ALDH1, as well as data of the pluripotency markers OCT4 and SOX2 expressions. Conclusions: UCSC and ASC secretomes have different regulation on ALDH1A3 expression in human BCSCs, which may be related to the autocrine TGF-β1 signaling in modulating cell proliferation and stemness of BCSCs. Further studies are required to evaluate factors involved in the differential effects of UCSC and ASC secretomes that regulate ALDH1A3 expression in relation to autocrine TGF-β1 signaling and aggressiveness of human BCSCs.",
"keywords": [
"UCSCs",
"ASCs",
"BCSCs",
"ALDH1A3",
"ALDH1A1",
"TGF-β1",
"TβRI",
"pluripotency"
],
"content": "Introduction\n\nMesenchymal stem cells (MSCs) are non-hematopoietic and adherent cells characterized by high CD90, CD105 and CD73, but lack of CD14, CD34 and CD45 expression. When treated with certain differentiation-stimulating factors, these cells will differentiate into adipocytes, chondrocytes, as well as osteocytes. MSCs show clinical feasibility, as studies on multiple animal models that yielded therapeutic efficacy gave rise to a series of clinical trials in a wide range of major diseases1. Umbilical cord and adipose tissue are two of the most common sources of MSCs, due to the fact that they can be non-invasively obtained and have minimal risk in terms of immunological and ethical issues. Apart from sharing the common properties of MSCs, umbilical cord-derived stem cells (UCSCs) are reported to have higher proliferation capability than adipose-derived stem cells (ASCs), whereas ASCs are able to differentiate to adipose tissue better than UCSCs2. Very recently, we have demonstrated that both ASCs and UCSCs expressed lower ALDH1A1 and OCT-4 than breast cancer stem cells (BCSCs), indicating that both types of MSCs have lower pluripotency compared to BCSCs (Wanandi SI, Purnamawati, Tamara A, Putri KT, Simadibrata D, in press).\n\nNowadays, MSCs are widely used for regenerative therapies due to their multipotent differentiation capacity. On top of that, MSCs are capable to secrete various paracrine signals for tissue regeneration and revascularization, such as chemoattractive, immunomodulatory, angiogenic, anti-apoptotic, and pro-survival factors. Nevertheless, MSC secretomes have also been reported to promote cancer progression and metastasis3. Accumulating evidence of cell-cell and paracrine interactions between MSC and cancer cells has indicated that MSCs can either induce or inhibit tumor progression3–9. Secretomes contained in conditioned medium (CM) of MSCs consist of various biologically active factors that have the same effectiveness as MSCs themselves10. In addition, secretomes of MSCs are considered safer to use since they do not contain cellular elements,11 making them free from possible mutations and transformation into cancer-associated fibroblast in a cancer microenvironment12.\n\nNormal and cancer stem cells share similar properties, such as self-renewal capacity and differentiation potential into multiple cell types. Activity of aldehyde dehydrogenase (ALDH) superfamily have been widely used as a marker of viable normal stem cells, as well as cancer stem cells13. In breast cancer, the existence of ALDH+ BCSCs often correlates with poor prognosis, progression, chemoradiation resistance, and metastasis. Chemotherapy resistance is due to the role of ALDH as a detoxifying enzyme, which mediates detoxification of toxic aldehyde intermediates that are produced in certain chemotherapeutic agents-treated cancer cells, while radioresistance occurs through direct removal of oxygen radicals and indirect production of antioxidant compound nicotinamide adenine dinucleotide (phosphate)14.\n\nALDH1A1 and ALDH1A3, two of ALDH1 superfamily isozymes, have been known to play important roles in the modulation of the retinoic acid signaling pathway, which can either support or suppress cancer growth14–16. In addition, they also serve as markers of stemness in CSCs13,17. The expression of ALDH1A1 was associated with advanced, triple-negative, and poor prognosis breast cancer after neoadjuvant chemotherapy18,19. ALDH1A1 expression was also found to be predictive of tumor responsiveness to cyclophosphamide treatment20,21. Meanwhile, the isoform of ALDH1A3 has been suggested to promote breast cancer progression via retinoic acid signaling22.\n\nTGF-β family members play a major regulatory role in various biological and physiological functions23. TGF-β physiologically exerts anticancer activities in normal and benign cells by prohibiting cell proliferation and by creating cell microenvironments that inhibit cell motility, invasion, and metastasis. However, along with the development and progression of tumors, various mutations or deletions occur in genes that encode various TGF-β signaling components. These lead to the loss of protective and cytostatic effects of TGF-β, which in turn alters TGF-β signaling to promote cancer progression, invasion, and tumor metastasis23,24. Recently, the mechanism of the TGF-β paradox may have been explained through Erk activation, which will auto-induce TGF-β in the microenvironment25. Auto-induction of TGF-β in benign or early stage cancer cells will create negative feedback of TGF-β signaling leading to growth arrest, whereas progression and metastasis in malignant cancer cells will be induced via a positive feedback loop25.\n\nUntil now, the interaction between MSC secretomes and BCSCs has not been fully understood. Our recent study has indicated that BCSCs treated with secretomes of ASCs expressed ALDH1A1 significantly higher compared to those treated with UCSCs, in accordance to OCT-4 and SOX2 expressions26. Those results showed that secretomes of ASCs contribute to pluripotency and viability of BCSCs more than those of UCSCs. To gain a better understanding of the effects of MSC secretomes on BCSCs, we conducted the present study on ALDH1A3 expression in association with TGF-β1 signaling pathways in BCSCs.\n\n\nMethods\n\nAccording to the Declaration of Helsinki 1964, this study has been approved by the Health Research Ethics Committee Faculty of Medicine Universitas Indonesia - Cipto Mangunkusumo Hospital (No. 205/UN2.F1/ETIK/2016).\n\nMSC specimens consisting of three ASCs and three UCSCs samples were obtained from HayandraLab and Stem Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital Faculty of Medicine Universitas Indonesia, Jakarta Indonesia, and have been characterized by the expression of stromal cell markers, i.e. CD73, CD90 positive and CD34 negative, as well as multidifferentiation capabilities to osteogenic, chondrogenic and adipocyte lineages as reported in our previous study26,27.\n\nBCSCs (ALDH+) were obtained from Cell Culture Laboratory for Cancer Stem Cells, Department Biochemistry and Molecular Biology Faculty of Medicine Universitas Indonesia, Jakarta Indonesia. The BCSC specimen has been isolated by ALDEFLUOR™ assay for subsequent downstream assessment (Fluorescence Activated Cell Sorting in Tsukuba University, 2015).\n\nMSCs were grown in Minimum Essential Medium Alpha (α-MEM) supplemented with 10% FBS, while BCSCs were grown in non-serum DMEM/F12 medium. Both cell cultures were supplemented with 1% penicillin-streptomycin and 1% amphotericin. Cells were incubated under standard conditions (5% CO2 and 37°C). The media were replaced every 3 days and cells were subcultured when confluence was obtained.\n\nEarly-passage human MSCs (P3-P5) were grown to 70–80% confluence in α-MEM medium with 10% FBS. Culture medium was removed and the cells were washed three times by PBS 1x to remove any residual serum. The cells were then grown in non-serum α-MEM medium under standard conditions for 24 hours. MSC-CM was centrifuged at 200xg for 10 min to remove cell debris and filtered using 0.22 μm filters. Subsequently, CM was stored at -20°C and used within 3 days. To prepare the CM for each experiment, CM was diluted with DMEM/F12 to obtain a 50% (v/v) concentration.\n\nBCSCs (ALDH+) were harvested and counted using an automatic cell counter (Luna®). About 5 x 105 cells with more than 90% viability were grown in non-serum DMEM/F12 medium. After 24 hours, the BCSC medium was replaced with 50% (v/v) MSCs-CM. After 72 hours of incubation, cells were harvested and counted using Tryphan blue exclusion dye assay. Cells that were grown in 50% (v/v) non-serum α-MEM medium were used as a control. The experiment was performed in triplicate.\n\nIsolation of total RNA was performed according to the manufacturer’s instruction (TriPure Isolation Reagent®, Roche). The RNA concentration was measured using a MicroDrop spectrophotometer (Thermo Scientific Skanlt Software for Varioskan™ Flash Multimode Reader). Quantitative reverse transcriptase PCR was performed using SYBR Green and reverse transcriptase enzyme (One-Step qRT-PCR Kit KAPA™SYBR®FAST). The cycling conditions were 5 minutes at 42°C for cDNA synthesis, 5 minutes at 95°C for inactivation of reverse transcriptase enzyme, then 40 cycles consisting of 30 seconds at 95°C for double stranded denaturation and 20 seconds at annealing gene temperature optimized for annealing stage, 20 seconds at 72°C for elongation stage. 18S rRNA was used as an internal control. The normalized fold expression was obtained using the 2-ΔΔCT (Livak) method. Primers used for qRT-PCR were obtained from IDT® (Table 1).\n\nAll relative gene expression data were analyzed using unpaired Student’s t-test, SPSS 20 and presented as mean ± standard error.\n\n\nResults\n\nBCSCs showed sphere formation within 2–3 days after plating (Figure 1a). UCSCs and ASCs used in this study adhered within hours after plating and displayed spindle-fibroblast-like morphology (Figure 1b–c). Both MSCs gradually fused into a single layer after cell confluence has been reached. Multidifferentiation capability of ASCs and UCSCs have been verified in our previous study26,27.\n\nMorphologies of BCSCs (a), UCSCs (b), and ASCs (c). About 1x105 cells were plated in each well of a 12-well plate and were grown under standard conditions as described under Materials and Methods. After 2–3 day incubation, cell morphology was observed under inverted microscope at 100x magnification. BCSCs, breast cancer stem cells; UCSCs, umbilical cord-derived stem cells; ASCs, adipose-derived stem cells; CM, conditioned medium.\n\nTreatment of UCSC-CM to BCSCs could significantly increase the relative expression of ALDH1A3 gene (1.79 times, p=0.001) compared with the control cells grown in 50% (v/v) non-serum α-MEM medium. In contrast, ASC-CM had no significant effect on ALDH1A3 gene expression in BCSCs (1.18 times, p=0.316) compared with the control cells. Nevertheless, the effect of ASC- was significantly lower than that of UCSC-CM (p=0.024) These results indicate that ALDH1A3 was distinctly expressed in BCSCs treated with UCSC- and ASC-CM.\n\nRelative expression of ALDH1A3 mRNA in BCSCs. Human BCSCs were treated with 50% (v/v) UCSC-CM and ASC-CM, respectively. As a control, BCSCs were treated in 50% (v/v) non-serum α-MEM medium. After 72-hour incubation, total RNA was isolated and quantitative reverse transcriptase PCR was performed to determine ALDH1A3 mRNA expression levels in human BCSCs.The Cq obtained was normalized to 18S rRNA and control cells. Data is presented as mean ± SE. Significance differences are considered at *p<0.05; **p<0.01 (Student’s t-test). BCSCs, breast cancer stem cells; UCSCs, umbilical cord-derived stem cells; ASCs, adipose-derived stem cells; CM, conditioned medium.\n\nIn attempt to analyze the effect of MSC-CM on TGF-β signaling, we determined the relative mRNA expressions of TGF-β1 and its receptor, TβRI. UCSC-CM significantly increases the relative expression of TGF-β1 (1.72 times, p=0.003) and TβRI (1.54 times, p=0.000). In contrast to this data, ASCs-CM significantly decreases the expression of TGF-β1 (0.64, p=0.003) and TβRI (0.76, p=0.014) in BCSCs compared with controls. Additionally, UCSC- and ASC-CM showed differential effects on either TGF-β1 (p=0.000) or TβRI (p=0.000) expression of BCSCs, suggesting that UCSC and ASC secretomes may be different in content and levels of growth factors, thereby influencing differential regulation of TGF-β1 and TβRI expressions.\n\nHuman BCSCs were treated with 50% (v/v) UCSC-CM and ASC-CM, respectively. As a control, BCSCs were treated in 50% (v/v) non-serum α-MEM medium. After 72-hour incubation, total RNA was isolated and quantitative reverse transcriptase PCR was performed to determine TGF-β1 and TβRI mRNA expression levels in human BCSCs.The Cq obtained was normalized to 18S rRNA and control cells. Data is presented as mean ± SE). Significance differences are considered at *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test). BCSCs, breast cancer stem cells; UCSCs, umbilical cord-derived stem cells; ASCs, adipose-derived stem cells; CM, conditioned medium.\n\n\nDiscussion\n\nRecently, the utilization of secretomes contained within MSC-CM has begun to be performed on various rejuvenation therapies. However, the possible consequences that may arise due to the interaction between biologically active factors (secretomes) and cancer cells has not yet been elucidated. As with MSCs, their secretomes have also been reported to either suppress or promote growth and development of cancer3. These contradictory effects may be due to different composition of secretomes, with one of the causes being the fact that they are obtained from different MSC sources4,28.\n\nIn this study, we found that secretomes within UCSC-CM have a higher ability to promote ALDH1A3 gene expression in BCSCs, indicating the higher paracrine signaling activity of UCSC secretomes when compared to those of ASCs (Figure 2). This result is in contrast with the effect of MSC secretomes on ALDH1A1 expression of BCSCs demonstrated in our previous study, which revealed that ALDH1A1 mRNA expression was significantly reduced by UCSC-CM and increased by ASC-CM26. In that study, we also found that ALDH1A1 expression is in line with the expression of OCT-4 and SOX2. Therefore, we suggest that unlike ALDH1A1, ALDH1A3 does not prominently contribute to the pluripotency of BCSCs. This has been verified by in silico analysis that showed that there is no direct interaction between ALDH1A3 and pluripotency markers, OCT4, SOX2, NANOG, and KLF4 (in press; Wanandi SI, Purnamawati, Tamara A, Putri KT, Simadibrata D). That study has also indicated that ALDH1A1 expression levels in MSCs were more similar to OCT4 rather than to ALDH1A3 levels, suggesting the role of ALDH1A1 on pluripotency.\n\nIn the present study, we highlighted that the effects of UCSC and ASC secretomes on ALDH1A3 were consistent with the expressions of TGF-β1 and its receptor, TβRI, in BCSCs (Figure 3). In spite of that, the effects of UCSC on ALDH1A3, TGF-β1, and TβRI expressions were opposite to those of ASC secretomes, in which UCSC up-regulated, while ASC secretomes down-regulated those gene expressions. Previous studies have reported that different MSC sources have different growth factor contents and levels4,28. Very recently, we have also demonstrated that UCSCs and ASCs expressed different levels of either ALDH1A1 or ALDH1A3 (in press; <Wanandi SI, Purnamawati, Tamara A, Putri KT, Simadibrata D>). These two isozymes of ALDH1 have newly been confirmed to have differential functional roles in facilitating aggressiveness of human breast cancer cells29. ALDH1A1 suppresses proliferation, metastatic properties and therapy resistance of breast cancer cells, whereas ALDH1A3 has predominant effect on ALDH activity. These results supported our previous study that presented the increase of viability and pluripotency in ASC secretomes-treated BCSCs in line with the increase of ALDH1A126.\n\nMoreover, the current study also underlines the effect of UCSC and ASC secretomes on TGF-β1 autocrine signaling in BCSCs, as revealed by TGF-β1 and its receptor, TβRI expressions (Figures 3A and B). We suggest that secretomes of UCSCs and ASCs presumably contained growth factors including TGF-β1 that could auto-induce TGF-β1 signaling in BCSCs. Due to TGF-β1 paradox, tumor proliferation could be stimulated or inhibited either via differential ERK depending on relative level of TGF-β1 present in tumor microenvironment25. A plausible explanation of TGF-β1 autocrine signaling induction in our BCSCs is as a cellular homeostasis against reduced cell viability and pluripotency due to enhanced ALDH1A3 and diminished ALDH1A1 expression after supplementation with UCSC secretomes26,28.\n\nIn conclusion, the differential effects of UCSC and ASC secretomes on ALDH1A3 expression in human BCSCs may be related to autocrine TGF-β1 signaling, as opposed to ALDH1A1 which regulates BCSC viability and pluripotency. In depth studies are further required to elaborate signaling factors within UCSC and ASC secretomes that specifically regulate ALDH1A1 and ALDH1A3 expressions in relation to TGF-β1 autocrine signaling and its impact on the aggressiveness of BCSCs.\n\n\nData availability\n\nDataset 1: Raw unedited images for Figure 1A, 1B, and 1C. DOI, 10.5256/f1000research.13609.d19456230\n\nDataset 2: Data for Figure 2 (ALDH1A3 Cq value). ALDH1A3 Cq was used to calculate ALDH1A3 mRNA expression levels using Livak formula as demonstrated in Figure 2 (Control: 50% (v/v) α-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells). DOI, 10.5256/f1000research.13609.d19456331\n\nDataset 3: Data for Figure 3A (TGF-β1 Cq value). TGF-β1 Cq was used to calculate TGF-β1 mRNA expression levels using Livak formula as demonstrated in Figure 3A (Control: 50% (v/v) α-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells). DOI, 10.5256/f1000research.13609.d19456432\n\nDataset 4: Data for Figure 3B (TβRI Cq value). TβRICq was used to calculate TβRI mRNA expression levels using Livak formula as demonstrated in Figure 3B (Control: 50% (v/v) α-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells). DOI, 10.5256/f1000research.13609.d19456533\n\nDataset 5: Data for Figure 2, Figure 3A, and Figure 3B (18S rRNA Cq value). 18S rRNA Cq was used to calculate ALDH1A3, TGF-β1, and TβRI mRNA expression levels using Livak formula (Control: 50% (v/v) α-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells). DOI, 10.5256/f1000research.13609.d19456634",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nPublication of this work was supported by the grant of International Indexed Publication for Final Assignment of the Postgraduate Student from Universitas Indonesia Year 2017 (557/UN2.R3.1/HKP.05.00/2017).\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\nWe thank Dr. dr. Novi Silvia Hardiany, M.Biomed (Dept of Biochemistry and Molecular Biology FKUI) dr.Isabella Kurnia Liem, M.Biomed., PA., Ph.D (Cell Medical Technology Integrated Service Unit, RSCM-FKUI), dr. Karina, SpBP-RE (HayandraLab) and Dr. dr. Reza Y. Purwoko, SpKK (Erpour Laboratory) for their generosity in providing BCSCs (ALDH+), USCSc, and ASCs, respectively.\n\n\nReferences\n\nMadrigal M, Rao KS, Riordan NH: A review of therapeutic effects of mesenchymal stem cell secretions and induction of secretory modification by different culture methods. J Transl Med. 2014; 12(1): 260.PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu L, Hu J, Zhao J, et al.: Side-by-side comparison of the biological characteristics of human umbilical cord and adipose tissue-derived mesenchymal stem cells. Biomed Res Int. 2013; 1–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZimmerlin L, Park TS, Zambidis ET, et al.: Mesenchymal stem cell secretome and regenerative therapy after cancer. Biochimie. 2013; 95(12): 2235–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhu W, Huang L, Li Y, et al.: Mesenchymal stem cell-secreted soluble signaling molecules potentiate tumor growth. Cell Cycle. 2011; 10(18): 3198–207. PubMed Abstract | Publisher Full Text\n\nOhta N, Ishiguro S, Kawabata A, et al.: Human umbilical cord matrix mesenchymal stem cells suppress the growth of breast cancer by expression of tumor suppressor genes. PLoS One. 10(5): e0123756. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDoi C, Maurya DK, Pyle MM, et al.: Cytotherapy with naive rat umbilical cord matrix stem cells significantly attenuates growth of murine pancreatic cancer cells and increases survival in syngeneic mice. Cytotherapy. 2010; 12(3): 408–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRhee KJ, Lee JI, Eom YW: Mesenchymal Stem Cell-Mediated Effects of Tumor Support or Suppression. Int J Mol Sci. 2015; 16(12): 30015–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKudo-Saito C: Cancer-associated mesenchymal stem cells aggravate tumor progression. Front Cell Dev Biol. 2015; 3: 23. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nShima H, Yamada A, Ishikawa T, et al.: Are breast cancer stem cells the key to resolving clinical issues in breast cancer therapy? Gland Surg. 2017; 6(1): 82–88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBednarz-Knoll N, Nastały P, Żaczek A, et al.: Stromal expression of ALDH1 in human breast carcinomas indicates reduced tumor progression. Oncotarget. 2015; 6(29): 26789–803. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTomita H, Tanaka K, Tanaka T, et al.: Aldehyde dehydrogenase 1A1 in stem cells and cancer. Oncotarget. 2016; 7(10): 11018–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAllahverdiyev AM, Bagirova M, Oztel ON, et al.: Aldehyde Dehydrogenase: Cancer and Stem Cells. InTech. [Internet] . 2012. [cited 2017 Jul 29]; Publisher Full Text\n\nKhoury T, Ademuyiwa FO, Chandraseekhar R, et al.: Aldehyde dehydrogenase 1A1 expression in breast cancer is associated with stage, triple negativity, and outcome to neoadjuvant chemotherapy. Mod Pathol. 2012; 25(3): 388–97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhao D, Mo Y, Li MT, et al.: NOTCH-induced aldehyde dehydrogenase 1A1 deacetylation promotes breast cancer stem cells. J Clin Invest. 2014; 124(12): 5453–65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarcato P, Dean CA, Pan D, et al.: Aldehyde dehydrogenase activity of breast cancer stem cells is primarily due to isoform ALDH1A3 and its expression is predictive of metastasis. Stem Cells. 2011; 29(1): 32–45. PubMed Abstract | Publisher Full Text\n\nRaha D, Wilson TR, Peng J, et al.: The cancer stem cell marker aldehyde dehydrogenase is required to maintain a drug-tolerant tumor cell subpopulation. Cancer Res. 2014; 74(13): 3579–90. PubMed Abstract | Publisher Full Text\n\nMarcato P, Dean CA, Liu RZ, et al.: Aldehyde dehydrogenase 1A3 influences breast cancer progression via differential retinoic acid signaling. Mol Oncol. 2015; 9(1): 17–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLebrun JJ: The Dual Role of TGFβ in Human Cancer: From Tumor Suppression to Cancer Metastasis. ISRN Mol Biol. 2012; 2012: 381428. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrincipe DR, Doll JA, Bauer J, et al.: TGF-β: Duality of function between tumor prevention and carcinogenesis. J Natl Cancer Inst. 2014; 106(2): djt369. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang Q, Yu N, Lee C: Mysteries of TGF-β Paradox in Benign and Malignant Cells. Front Oncol. 2014; 4: 94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPurnamawati P, Pawitan JA, Rachman A: Secretomes of Adipose and Umbilical Cord-Derived Stem Cells Affect ALDH1A1 Expression in Breast Cancer Stem Cells. Adv Sci Lett. 2017; 23(7): 6701–04. Publisher Full Text\n\nPawitan JA, Kispa T, Mediana D, et al.: Simple production method of umbilical cord derived mesenchymal stem cell using xeno-free materials for translational research. J Chem Pharm Res. 2015; 7(8): 652–6. Reference Source\n\nHeo JS, Choi Y, Kim HS, et al.: Comparison of molecular profiles of human mesenchymal stem cells derived from bone marrow, umbilical cord blood, placenta and adipose tissue. Int J Mol Med. 2016; 37(1): 115–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCroker AK, Rodriguez-Torres M, Xia Y, et al.: Differential Functional Roles of ALDH1A1 and ALDH1A3 in Mediating Metastatic Behavior and Therapy Resistance of Human Breast Cancer Cells. Int J Mol Sci. 2017; 18(10): pii: E2039. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPurnamawati P, Pawitan JA, Rachman A, et al.: Dataset 1 in: Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells. F1000Research. 2018. Data Source\n\nPurnamawati P, Pawitan JA, Rachman A, et al.: Dataset 2 in: Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells. F1000Research. 2018. Data Source\n\nPurnamawati P, Pawitan JA, Rachman A, et al.: Dataset 3 in: Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells. F1000Research. 2018. Data Source\n\nPurnamawati P, Pawitan JA, Rachman A, et al.: Dataset 4 in: Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells. F1000Research. 2018. Data Source\n\nPurnamawati P, Pawitan JA, Rachman A, et al.: Dataset 5 in: Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells. F1000Research. 2018. Data Source"
}
|
[
{
"id": "32467",
"date": "29 May 2018",
"name": "Albert D. Donnenberg",
"expertise": [
"Reviewer Expertise Cancer cell biology",
"regenerative medicine",
"flow cytometry",
"cGMP cell therapy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study investigated the effects of media conditioned by umbilical cord (UC)-derived and adipose-derived stromal cells on \"breast cancer stem cells.\" The measured outcomes were aldehyde dehydrogenase 1A3 (ALDH1A3), TGF-β1 and TβR1 messenger RNA. The methodology and data analysis are straight-forward and sound. The major finding was that culture in the presence of umbilical cord-derived stromal cell conditioned medium resulted in higher expression of ALDH1A3, TGF-β1, and TβR1 mRNA, compared to control medium. In contrast, adipose stromal cell conditioned medium resulted in slightly lower expression of TGF-β1, and TβR1 mRNA, but this was a modest effect. The authors conclude that different secretomes of UC- and adipose-derived stromal cells differentially regulate ALDH1A3 expression in breast cancer \"stem cells\", a point that is well-supported by their results. They further conclude that differential regulation of ALDH1A3 may be related to autocrine TGF-β1 signalling in modulating cell proliferation and stemness of breast cancer stem cells. This is speculative and based on a partial correlation of expression levels of the three measured mRNAs. If anything, the data speak against a causal relationship, as adipose-conditioned medium failed to down-regulate ALDH1A3 message, but modestly downregulated TGF-β1, and TβR1 mRNA. To their credit, the authors point out that the present results are inconsistent with their previous published data on ALDH1A1, OCT4 and SOX2 expression.\n\nThese results speak to the difficulty of modeling the interactions of stromal cells and cancer cells. Using freshly isolated human breast cancer cells, flow-sorted on the basis light scatter and expression of CD90, we found that co-injection of adipose stromal cells in a xenograft model increased tumorgenicity of active, but not resting tumor cells. In vitro, coculture of unpassaged breast cancer cells with adipose stromal cells greatly increased their proliferation1. The take-home is that the state of the cancer cells matters as much as the the signals provided by the stromal cells and microenvironment. To complicate matters further, stromal cells themselves can be polarized into pro- and anti-inflammatory states2 resulting in different secretomes. \nAt a minimum, the authors should provide detailed information about the preparation of their \"breast cancer stem cells\", as these are not standard reagents.\nAdditionally, UC- and adipose-conditioned media have not been characterized for cytokine/chemokine content. This is relatively simple to do, as multiplexed kits are commercially available and would significantly contribute to a more mechanistic understanding of the data.\nA minor consideration: wording such as \"nowadays\" and \"on top of that\" are rather colloquial for a scientific publication.\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? Partly",
"responses": []
},
{
"id": "35664",
"date": "17 Jul 2018",
"name": "Paola Marcato",
"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 results presented by Purnamawati et al., are potentially interesting; however, since the key findings of the paper hinges on the fact that breast cancer stem cells were used in the assays it is difficult to interpret the findings (i.e. no details of the source of cells or proof they are cancer stem cells was provided). Major concerns. 1. There is no details of the source of the cells, nor was the Aldefluor sorting of the cells shown. 2. Assay were not performed to confirm that cancer stem cells had been isolated (i.e. are these cells more tumorigenic than non-cancer stem cell counterparts? 3. Expression of ALDH1A3 should be compared in in the breast cancer stem cell versus non-cancer stem cell sorted populations to confirm that the Aldefluor+ cells have higher levels of ALHD1A3 than the Aldefluor- cells. 4. The culture conditions used to form mammospheres were not appropriate (should seed much lower number of cells in ultra-low adherent plates so that no cells are adherent.) Standard media for mammosphere assays in Mammocult media from Stemcell Technologies. Hence it is unclear if these are mammospheres.\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? 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/7-249
|
https://f1000research.com/articles/7-248/v1
|
28 Feb 18
|
{
"type": "Method Article",
"title": "A new environmental monitoring system for silkworm incubators",
"authors": [
"Alejandra Duque-Torres",
"Carlos Rodriguez-Pabon",
"Juan Ruiz-Rosero",
"Giselle Zambrano-Gonzalez",
"Martha Almanza-Pinzon",
"Oscar Mauricio Caicedo Rendon",
"Gustavo Ramirez-Gonzalez"
],
"abstract": "Silk is known as the queen of textiles due to its softness, durability, and luster. This textile is obtained from cocoons spun by larvae known as the silkworm. The combined effect of both temperature and humidity, determines the satisfactory growth of the silkworms and the production of good quality cocoons. For that rea- son, we propose a new prototype for silkworm incubators that monitors environmental conditions, created with Raspberry Pi due to its capabilities, features, and low cost. The prototype monitors the temperature, humidity, and luminosity in a silkworm incubator. The monitoring data are collected and saved on file hosting service, Google Drive, for subsequent analysis. Preliminary tests were gathered using the silkworm incubator of University of Cauca, Colombia.",
"keywords": [
"silkworm incubator",
"environmental conditions",
"monitoring",
"Raspberry-Pi",
"cloud"
],
"content": "Introduction\n\nSilk is known as the queen of textiles due to its softness, durability, and luster. Furthermore, the silk fibers provide characteristics that are superior to any other type of fiber (e.g., water absorbency, heat resistance, dyeing efficiency, and luster). This textile is obtained from cocoons spun by larvae known as silkworm (Bom- byx mori), which were discovered in China between 2600 and 2700 BC. The process of silk production is known as sericulture, beginning with the rearing of the silkworm1.\n\nThe silkworm is of great interest to the sericulture industry and the academic community. Because of this, there is an increased interest in knowing the combination of factors that affect growth and development of silkworms in different life stages, which in turn affects productivity, and quality of silk2.\n\nThe insects can only survive in specific conditions, defined by environmental factors such as temperature, relative humidity, or duration light. These environmental factors directly affect different activities such as feeding, dispersal, laying or development. Temperature is probably the environmental factor that exerts the most significant effect on the development of the insects3.\n\nIt is well known that the silkworm is highly sensitive to environmental variation1,2,4–6. Indeed, in an extreme natural fluctuation, it could be unable to survive because of its extended period of domestication, of about 5000 years5. The biological as well as physiology-related, characteristics (e.g., growth, development, productivity, and quality of silk), are influenced by the combination of temperature, air circulation, humidity, light duration, and gases.\n\nThe study of the effects of varying environmental conditions, will generate essential biological information that will allow production of cocoons in areas where the silkworm is of economic value.\n\nRecent progress in electronics, wireless communications, and production of small size sensors provides new opportunities to develop different monitoring systems (e.g., for homes, crops and the environment)7. A sensor is a device that can measure physical attributes and convert them into signals for the user. Sensors are the essential components of the environmental monitoring system of this proposal in addition to the embedded systems8.\n\nTo monitor the environmental conditions during the silkworm incubation and rearing process, we proposed an environmental conditions monitoring system that makes full use the concept of an open source and low-cost data acquisition and transmission system. We used an embedded platform, with cloud remote monitoring through the file hosting service Google drive, and the Internet of Things.\n\nWe describe the prototype for monitoring temperature, humidity relative, and light intensity in a silkworm incubator. Also, we provide the required tools for reproducibility of the system.\n\nIn the following sections, we describe a silkworm incubator, the materials used, prototype design, and results obtained. We also discuss the results and the possibility of future work around this topic.\n\n\nMethods\n\nFigure 1 shows the proposed conceptual model of the monitoring system. There are three main stages: the reading of sensors, the communication channel, and storage of data in the cloud. The system is scalable and it is possible to include other sensors and communication technologies. It consists of several sensors that record environmental parameters such as moisture, temperature, and luminosity in a silkworm incubator. The information is then uploaded to the cloud by Google Drive. We used two different sensors to collect data on temperature, humidity, and luminosity. Wi-Fi communication was used to upload the information to Google Drive. Users obtain information about the data recorded with a username and password through a smartphone or any device that supports the Google Drive application.\n\nThe silkworm incubator structure keeps the environment in uniform conditions for temperature and humidity (see Figure 2 and Figure 3). These are the most important variables to control. The water tank is located at the bottom, with a capacity for eight liters of water. The incubation chamber has nine metal mesh trays to allow air flow. The incubator brand is Incubapremium, manufactured in Colombia by Abraham Reyes Sanclemente incubation equipment specialist with 30 years' experience.\n\nSilkworms are distributed in trays that are about one meter wide and 60 cm long, which facilitates their handling. The materials used for their construction are aluminum and thermal insulation. The trays are placed to form shelves called swaths. Each tray, of the nine that make up the shelves, is separated by approximately 15 cm. The temperature inside the chamber is controlled by a heating system based on an electrical resistance. The humidity is controlled by a humidifier located at the bottom side of the chamber.\n\nTemperature and humidity plays a vital role in the rearing of the silkworms. As silkworms are cold-blooded animals, the temperature will have a direct effect on various physiological activities1. Humidity’s role in silkworm rearing is both direct and indirect5. The combined effect, of both temperature and humidity, largely determines the satisfactory growth of the silkworms and production of good-quality cocoons. It directly influences the silkworm’s physiological functions. The selected sensor was DHT22, which is an essential temperature and humidity digital low-cost sensing device. This sensor has the capability to obtain new data every 2 seconds.\n\nSilkworms, like other animals, are photosensitive and tend to crawl into dim light. They do not like strong light or complete darkness1,9–11. The breeding of silkworms in continuous light slows down growth and reduces both the weight of the larvae and the cocoon. We choose the luminosity sensor APDS-9301 due to the high precision in measurement and its low power consumption.\n\nRaspberry Pi version 3 (RPi3) was chosen for this project, due to its small size and low cost, compared to conventional PC and data loggers, plus zero cost of free Linux software11.\n\nRPi3, in comparison to most of the other Linux-based embedded systems, such as Arduino Due, Beagle Bone Black, and Intel Edison, has a better cost-benefit ratio, broader user community, and a more standard programming language and communication, many input-output pins and a graphics interface12. A picture of the RPi3 is shown in Figure 4.\n\nThe prototype hardware consisted of an RPi3 board inside a plastic box, nine sensors to measure temperature and humidity, and one luminosity sensor. The plastic box was used to protect the RPi3. All of the sensors were placed inside of the silkworm incubator. As aforementioned, the silkworm incubator has nine levels. Therefore, one temperature and humidity sensor was placed in each level and the luminosity sensor was placed in the incubator middle. In the initial design of the monitoring system, we considered necessary only a single light sensor due to the uniform light conditions for all levels.\n\nThe monitoring system was developed in three phases: the design of monitoring system architecture, hardware and software requirements, and the implementation.\n\nAll the hardware documentation such as schematics of circuits, types of sensor and material specifications used are available in GitHub, along with the documentation of the software developed, such as the communication algorithms between sensor and RP3, the storage algorithm, and the communication algorithm between RP3 and Google Drive service. Furthermore, instructions to install the different libraries necessary for the monitoring system are also contained here.\n\nArchitecture of the monitoring system. In the architectures of monitoring systems sensors, data gathering can vary, including data transmission (wired or wireless technologies), data storage and usage of intermediate devices. A particular monitoring system can also be supplemented with decision support system, which is responsible for data analysis, incubator environment condition determination and appropriate decision selection.\n\nIn our monitoring system, by the usage of RP3 the data from sensors using wired communication is transferred and uploaded to the cloud by Google Drive for further analysis (see Figure 5).\n\nHardware and software requirements. The hardware requirements are the materials aforementioned and the various sensors. The RP3 works with an SD card. It should work with any compatible SD card, although there are some guidelines that we followed from the official documentation for the Raspberry Pi, written by the Raspberry Pi Foundation with community.\n\nThe software requirements for the monitoring system are the installations of: the operative system for RP3, the library of the temperature and humidity sensors on RP3, the library of the light sensor, and the Google Drive on RP3. Furthermore, it is necessary to choose the programming language to use; we used Python language. However, other programming laugages can also be used, such as Ruby or C/C++. The instructions for installing all the software required are available in the GitHub repository as follows:\n\n1. Operative system for RP3: https://www.raspberrypi.org/documentation/installation/noobs.md\n\n2. Library for temperature and humidity sensor: https://github.com/Silkwormincubator/SilkWorm/wiki/Instructions-for-installing-the-DHT22-libraries-in-Raspberry-pi-3\n\n3. Library for light sensor: https://github.com/Silkwormincubator/SilkWorm/wiki/Installation--Light-sensor-apds9301\n\n4. Google Drive for RP3: https://github.com/Silkwormincubator/SilkWorm/wiki/Installation--Google-Drive\n\nThe main algorithm of the monitoring system works by initializing the sensors variables to zero; there is a query time where it collects sensor data. The program asks for the time elapsed and if the time elapsed is less to 5 minutes, the system accumulates the sensor values. When the time lapsed exceeds 5 minutes, the program calculates the average of collected sensors values (temperature, humidity, and light) and updates a txt file, which is synchronized with the file on Google Drive. The algorithm for this is available on our GitHub repository.\n\nFor a field test, worms were obtained through resources provided by the National Ph.D. Scholarship for Teachers granted by the Human Talent Training Network Project for Social and Productive Innovation in the Department of Cauca - InnovAcción Cauca. The monitoring system was used in one incubator. The condition environments of 200 worms was monitored for 25 days continuously.\n\n\nResults\n\nAs mentioned previously, the aim of this work was to implement a system that monitors the environmental conditions during the silkworm incubation process. The system was tested in real time.\n\nData was exported from the RPi3 to a plain .txt file in a Google Drive account and is available here. The data provides date, hour, temperatures measures from sensor one to sensor nine, humidity measures from sensor one to sensor nine, and the sensor light measure. Currently, our monitoring system is still working.\n\nEnvironmental condition uniformity inside the incubator is fundamental. However, through analysis of data, the generation of microclimates between incubator levels is evident, as shown in Figure 6 for temperature variance and Figure 7 for humidity variance. We provide more graphics in GitHub. This situation may be affect the biological as well as physiological characteristics (i.e., growth, development, productivity, and quality of silk) of the silkworm.\n\n\nConclusions\n\nIn this study, we describe a prototype for an environmental monitoring system created with Raspberry Pi. It allows recordings of temperature, humidity, and luminosity values in a silkworm incubator in natural conditions. In this work, we demonstrated the reliability offered by the Raspberry Pi in providing temperature, humidity and light readings in our field test, which was carried out for many days and in the University of Cauca silkworm incubators.\n\nThe present study only addresses one of the possible solutions for registering environmental parameters, which should minimize financial costs and human resources. We believe in the possibility of improving the general aspects of the incubator through the analysis of data provided by this monitoring system, as well as the quality of the breeding process of silkworms.\n\nOur proposed system has low cost and portability quality, which are essential for the recording of environmental conditions. Moreover, it supports useful features like remote data view and data analysis. In the next update of the system, we will implement an alarm system.\n\nThe proposed system offers new opportunities for researchers working in similar environments, i.e. those that need monitoring of environmental conditions accurately. The second important goal achieved is in this study is the automatic recording of the environmental conditions (temperature, humidity, and luminosity). This automatic recording is significant for researchers because it improves the analysis process to benefit its research results. Our device is directly programmable, and is based on open source codes, which increases the overall flexibility of the instrument. This feature is rarely present in commercial tools that provide only predetermined functions.\n\nWe believe that in the near future, researchers in other fields will be able to use an adapted version of this system.\n\n\nData and software availability\n\nAll data collected during this study and the all code for setting up the incubator are available on GitHub: https://github.com/Silkwormincubator/SilkWorm/\n\nArchived source code as at the time of publication: http://doi.org/10.5281/zenodo.116129413\n\nLicense: Creative Commons Attribution Share-Alike 4.0.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research is funded by Colciencias Doctoral scholarship 647-2014 for the PhD in Telematic Engineering at the Universidad del Cauca, Popayán, Colombia. The purchase of the materials was made with resources provided by the National Ph.D. Scholarship for Teachers granted by the Human Talent Training Network Project for Social and Productive Innovation in the Department of Cauca - InnovAcción Cauca.\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 the University of Cauca and the following research groups: the Geology, Ecology and Conservation research group (GECO), Telematic Engineering Group (GIT) and the Integrated Production Systems Research Group (SISINPRO). Also to the projects InnovAccion-Cauca and Technical Development for production of Innovative Organic Silk Products Project, from the General Royalties System, and the Government of Cauca (Colombia).\n\n\nReferences\n\nRahmathulla VK: Management of climatic factors for successful silkworm (Bombyx mori L.) crop and higher silk production: A review. Psyche (New York). 2012; 2012(2012): 12. Publisher Full Text\n\nRao CG, Seshagiri SV, Ramesh C, et al.: Evaluation of genetic potential of the polyvoltine silkworm (Bombyx mori L.) germplasm and identification of parents for breeding programme. J Zhejiang Univ Sci B. 2006; 7(3): 215–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu HI, Sharpe PJ, Wagner TL, et al.: Modeling insects development rates: a literature review and application of biophysical model. Entomol Soc Am. 1984; 77(2): 208–220. Publisher Full Text\n\nDe Postgraduados C: Effect of Temperature and Relative Humidity on. 2011; 1(311): 42–53.\n\nDezmirean D, Nagy A, Mărghitaş A: Diapause Storage Temperature Influence on Silkworm Larva Hatching Rate. Analele Universităţii din Oradea, Fascicula: Ecotoxicologie, Zootehnie şi Tehnologii de Industrie Alimntară. 2015; XIV B: 341–348. Reference Source\n\nHussain M, Khan SA, Naeem M, et al.: Effect of rearing temperature and humidity on fecundity and fertility of silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). Pak J Zool. 2011; 43(5): 979–985. Reference Source\n\nRuiz-Rosero J, Ramirez-Gonzalez G, Williams JM, et al.: Internet of things: A scientometric review. Symmetry. 2017; 9(12): 301. Publisher Full Text\n\nMesas-Carrascosa FJ, Verdú Santano D, Meroño JE, et al.: Open source hardware to monitor environmental parameters in precision agriculture. Biosyst Eng. 2015; 137: 73–83. Publisher Full Text\n\nFerdoush S, Li X: Wireless sensor network system design using Raspberry Pi and Arduino for environmental monitoring applications. Procedia Comput Sci. 2014; 34: 103–110. Publisher Full Text\n\nPasquali V, D’Alessandro G, Gualtieri R, et al.: A new data logger based on Raspberry-Pi for Arctic Notostraca locomotion investigations. Measurement (Lond). 2017; 110: 249–256. Publisher Full Text\n\nPereira RIS, Dupont IM, Carvalho PCM, et al.: IoT embedded linux system based on Raspberry Pi applied to real-time cloud monitoring of a decentralized photovoltaic plant. Measurement (Lond). 2018; 114: 286–297. Publisher Full Text\n\nVujović V, Maksimović M: Raspberry pi as a sensor web node for home automation. Comput Electr Eng. 2015; 44(Supplement C): 153–171. Publisher Full Text\n\nDuque-Torres A, Ruiz-Rosero J, Zambrano-Gonzalez G, et al.: A New Environmental Monitoring System For Silkworm Incubators (Version 2). zenodo. 2018. Data Source"
}
|
[
{
"id": "32803",
"date": "06 Apr 2018",
"name": "Enrico Petritoli",
"expertise": [
"Reviewer Expertise Raspberry-Pi",
"Public Lighting",
"Measurement",
"ADC Conversion",
"Energy Efficiency",
"Wireless Sensor Network"
],
"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'm happy to see a work like yours that realizes a \"climate chamber\" for silkworms. By climate chamber I mean a complex structured monitoring system for some entities useful for the life of the silkworms, and is able to suitably act to control the chamber and able to provide information of the entities on the internet.\n\nI have mixed feelings about the article: from one side the work is extremely clear and describes well all single steps made to realize the system, providing all the information to replicate the system, but from a scientific point of view, it does not seem innovative. The paper could be improved under many point of view as a study of reliability, consideration of the accuracy of the system and from a biological point of view explaining what could happen to animals if something is not fully managed, etc.\nAnyway, considering the effort made to do this system, considering that the application can be considered partially innovative and considering that you talk about preliminary test letting, you imagine that this is a first step of a longer research and I can consider it positive.\nThe current structure of the paper prevents me giving punctual improvements because, as I said, the paper is well written and important changes are no compatible with the time of the revision. The only thing that I ask is to improve your bibliography, is insufficient for an International scientific journal.\nI'll suggest many papers that you could insert in your, I admit, some of them are mine because I have a good experience in this field, but you are completely free to decide to insert them or not; anyway I wish that you expand your bibliography.\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": "93139",
"date": "16 Sep 2021",
"name": "LalBihari Barik",
"expertise": [
"Reviewer Expertise Lalbihari Barik",
"(Ph.D. Computer Science) has over 20 years of industrial & educationalexperience in the field of Java",
"network technologies",
"AI",
"IoT",
"ITS",
"Big Data",
"Cloud Computing",
"Self-Efficacy",
"and Stock Market analysis. He has worked on various web-driven projects whereexaMAIZE is one of his educational brand products. He honored the “IBM Drona Award 2008”",
"and “Developer Super Star 2011” in the national level software development program organizedby IBM. He serves as a resource person for many workshops in open-source software",
"processmining",
"etc. He received research granted projects and published and presented several researchpapers in National and International Journals and books."
],
"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 research is a new prototype for silkworm incubators that monitors environmental conditions storage of data in the cloud.\n\nThe method's description technically sounds as they use different sensors to collect data on temperature, humidity, and luminosity.\n\nThere are partial details provided to allow replication of the method development due to the clock speed processor compared to the latest version.\n\nAll the source data underlying the results are available to ensure partial reproducibility.\n\nThe conclusions are satisfactory as they implement the alarm system in the later phase.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-248
|
https://f1000research.com/articles/7-246/v1
|
28 Feb 18
|
{
"type": "Systematic Review",
"title": "Prostate specific antigen (PSA) kinetic as a prognostic factor in metastatic prostate cancer receiving androgen deprivation therapy: systematic review and meta-analysis",
"authors": [
"Andika Afriansyah",
"Agus Rizal Ardy Hariandy Hamid",
"Chaidir Arif Mochtar",
"Rainy Umbas",
"Andika Afriansyah",
"Agus Rizal Ardy Hariandy Hamid",
"Chaidir Arif Mochtar"
],
"abstract": "Aim: Metastatic prostate cancer (mPCa) has a poor outcome with median survival of two to five years. The use of androgen deprivation therapy (ADT) is a gold standard in management of this stage. Aim of this study is to analyze the prognostic value of PSA kinetics of patient treated with hormonal therapy related to survival from several published studies Method: Systematic review and meta-analysis was performed using literature searching in the electronic databases of MEDLINE, Science Direct, and Cochrane Library. Inclusion criteria were mPCa receiving ADT, a study analyzing Progression Free Survival (PFS), Overall Survival (OS), or Cancer Specific Survival (CSS) and prognostic factor of survival related to PSA kinetics (initial PSA, PSA nadir, and time to achieve nadir (TTN)). The exclusion criteria were metastatic castration resistant of prostate cancer (mCRPC) and non-metastatic disease. Generic inverse variance method was used to combine hazard ratio (HR) within the studies. Meta-analysis was performed using Review Manager 5.2 and a p-value <0.05 was considered statistically significant. Results: We found 873 citations throughout database searching with 17 studies were consistent with inclusion criteria. However, just 10 studies were analyzed in the quantitative analysis. Most of the studies had a good methodological quality based on Ottawa Scale. No significant association between initial PSA and PFS. In addition, there was no association between initial PSA and CSS/ OS. We found association of reduced PFS (HR 2.22; 95% CI 1.82 to 2.70) and OS/ CSS (HR 3.31; 95% CI 2.01-5.43) of patient with high PSA nadir. Shorter TTN was correlated with poor result of survival either PFS (HR 2.41; 95% CI 1.19 – 4.86) or CSS/ OS (HR 1.80; 95%CI 1.42 – 2.30) Conclusion: Initial PSA before starting ADT do not associated with survival in mPCa. There is association of PSA nadir and TTN with survival",
"keywords": [
"androgen deprivation therapy",
"metastasis",
"PSA kinetics",
"prostate cancer",
"survival",
"systematic review",
"meta analysis"
],
"content": "Introduction\n\nProstate cancer (PCa) is the second most common cancer in men, and the fourth most common cancer worldwide. More than one million men worldwide were diagnosed with PCa in 20121. The incidence of local-regional PCa has increased since the introduction of prostate specific antigen (PSA). This circumstance reduces the incidence of metastatic PCa2. PCa patient treated at early stages have a good prognosis with 5-year overall survival (OS) reaching 99%. In contrast, metastatic PCa patients generally experience a poor outcome. Several published studies showed a wide difference of survival, with median OS from two to five years3–5. Androgen deprivation therapy (ADT) becomes the standard treatment of patients with advanced PCa6,7, and with the first use reported by Huggins and Hodges in 19418.\n\nIn clinical practice, PSA is the most common diagnostic procedure to evaluate the disease and to predict the survival. PSA kinetics such as nadir PSA level, time to reach nadir (TTN), or specific PSA value after initiation of ADT might became a predictor of survival in several retrospective and clinical trial studies5,9–11. Some limitations were shown in the previous report of investigation for PSA kinetic to survival. They included patients with heterogeneous backgrounds (such as metastatic disease prior to surgical or radiation therapy), and the sample size was small. Therefore, we performed a systematic review and meta-analysis to evaluate the pooled effect of PSA kinetics of patient treated with hormonal therapy related to survival from several published studies.\n\n\nMethods\n\nThe systematic review was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines12. All studies in English were included. Retrospective cohorts, prospective cohort, randomized clinical trial (RCT), were eligible for inclusion for this review. The inclusion criteria were that (i) the participant of the study had metastatic PCa; (ii) patients were treated with ADT either using orchiectomy or luteinizing hormone-releasing hormone (LHRH) agonist with or without anti-androgen (AA); (iii) the studies outcome were either progresion free survival (PFS), overall survival (OS) or cancer specific survival (CSS); (iv) the studies had to analyze PSA kinetics (intial PSA prior to initiation of ADT, PSA nadir, and time to reach nadir (TTN) PSA). Studies analyzed in meta-analysis had to use adjusted analysis of prognostic factors, such as multivariate Cox regression, to overcome the confounding factors. Studies that analyzed patient with castration resistant PCa (CRPC) and non-metastatic disease were excluded.\n\nElectronic searched were performed in three databases: MEDLINE, Science Direct, and Cochrane Library from 1950 to 2016. This literature searching was conducted in March 2017. Gray literature and conference abstract, especially from urology oncology conference, were also searched. References list from included article were reviewed. We used the following search strategy: (prostate cancer OR adenocarcinoma prostate), (survival OR prognosis OR prognostic), (metastasis OR metastases OR metastatic), (PSA OR “Prostate Specific Antigen” OR nadir OR “initial PSA” OR kinetic). Two researchers (A.A and A.R.A.H) were indecently assessing the title and abstract of the paper. They agreed the studies included in the meta-analysis. Disagreement between the two review authors on the selection of studies was resolved by discussion with third authors (C.A.M) as a senior investigator. We used EndNote X6 for screening of duplicated studies.\n\nA data extraction table was created to extract data from each article. The data of study design, patient's characteristics, method of ADT, duration of follow up, outcomes of survival, and significant prognostic factors of PSA kinetics were collected from all included studies. For the observational studies, the quality of study was assessed using Newcastle-Ottawa Scale (NOS). There were three major components of this scale namely the selection of the group of the study, comparability, and assessment of the outcome. The quality of study assessed with number of stars based on NOS. A maximum 7 stars could be scored; 6 or 7 stars considered as high quality study, 4 – 5 stars corresponded with intermediate quality, and 0 – 3 stars showed low quality13.\n\nMeta-analysis was applied on studies with prognostic factor with similar outcome definition. I2 test was conducted in order to evaluate the heterogeneity, whilst for >30% a random effects model was applied, or otherwise, fixed effects model was done. Confounding in the individual studies was estimated using Hazard Ratio (HR) adjusted estimation, thus generic inverse variance method was used. We only combined data to estimate pooled effect of categorical parameters due to feasibility of statistical analysis. Studies that evaluated parameters but could not synthesize to meta-analysis were describe quantitatively. Meta-analysis was performed using Review Manager 5.2 from Cochrane Collaboration. A p-value < 0.05 was considered statistically significant.\n\n\nResults\n\nWe found 873 citations throughout database searching. No additional records identified through searching from reference list of included studies. Seventeen studies were found to be consistent to the inclusion criteria of the study, but seven studies could not be evaluated the in meta-analysis (Figure 1). Miyamoto et al.14 did not published the hazard ratio, and put the cumulative survival rate as the outcome. Six other studies used numerical parameters of PSA kinetics that cannot combine in the forest plot9,15–19. All of those studies were considered in qualitative synthesis. The characteristic of study is present in Table 1. Based on NOS, the quality of study included was good (Table 2).\n\nADT = androgen deprivation therapy; LHRH = luteinizing hormone releasing hormone; AA = anti androgen; CAB = combined anti androgen; PSA = prostate specific antigen; OS = overall survival; GS = Gleason score; TTN = time to nadir; NM = not mentioned; NSAID = non-steroidal anti inflammatory drug; ECOG PS = Eastern Cooperative Oncology Group Performance Status; PS = Performance Status; ALP = alkaline phosphatase; N1= regional nodal metastasis\n\nInitial PSA. Initial PSA before ADT treatment was evaluated in twelve studies4,9–11,16,17,19–25. However, we only put four studies in PFS outcome and four studies in CSS/OS outcome because the studies analyzed initial PSA as a categorical parameter. No significant association between initial PSA and PFS was found, and the studies were homogenous (I2=0%). In addition, there was no association between initial PSA and CSS/OS (Figure 2). In qualitative analysis, four studies analyzed the association between initial PSA and PFS. All of the studies did not find significant results for PSA and PFS16,17,19,25. The result was the same when we analyzed the studies for OS/CSS outcome9,11,16.\n\nForest plot of association between initial PSA and: A) Progression Free Survival Outcome; B) Cancer Specific Survival/Overall Survival.\n\nPSA Nadir. Six studies analyzed the effect of PSA nadir to influence survival using 0.2 ng/ml as a cut-off point5,10,11,20,22,23. Four studies analyzed the PSA nadir as a continuous variable15,17–19. Teoh et al. used cut-off point 2 ng/ml as a PSA nadir that influence the survival21. Bello et al. analysed nadir using 4 ng/ml as a threshold25. Meta-analysis of the studies found an association of reduced PFS of patient with high PSA nadir (HR 2.22; 95% CI 1.82 to 2.70). The studies appear homogenous in the forest plot. In addition, high PSA nadir had a negative impact on the OS/CSS outcome with HR 3.31 (95% CI 2.01–5.43) (Figure 3). In the studies using continuous measurement of PSA nadir, three studies found significant association of nadir PSA and survival15,18,19. However, studies by Koo et al. found no significant result17. Miyamoto et al. found the PSA nadir after first line hormonal therapy influenced survival14.\n\nForest plot of association between PSA nadir and: A) Progression Free Survival Outcome; B) Cancer Specific Survival/Overall Survival.\n\nTime to Nadir (TTN). A total of seven studies analyzed the relationship between TTN and survival5,10,16–18,20,21. Of the seven studies, two studies used 8 months5,10, one study used 9 months26, and one study used 12 months20 as a cut-off. Three studies analyzed TTN as a continuous variable16–18. Meta-analysis was performed with showing a shorter TTN correlated with poor survival for both PFS (HR 2.41; 95% CI 1.19 – 4.86) or CSS/OS (HR 1.80; 95%CI 1.42 – 2.30) (Figure 4). Studies using continuous variable of TTN showed a significant negative effect from shorter TTN on survival.\n\nForest plot of association between TTN and: A) Progression Free Survival Outcome; B) Cancer Specific Survival/Overall Survival.\n\n\nDiscussion\n\nNowadays, clinicians have use PSA not only for screening for PCa, but also for follow up of patients after the treatment. The PSA indicates PCa condition following radical treatment in localized disease, and hormonal treatment in metastatic condition. PSA has a prognostic value, and now has been widen to several parameters such as PSA nadir, TTN, PSA doubling time, and PSA response after the treatment. There is controversy among previous study about the utilization of the PSA kinetic after hormonal treatment for predicting the progression to CRPC and survival.\n\nThe meta-analysis performed in this study did not find an association between survival and high initial PSA. Significant heterogeneity was observed due to scattered cut off points of high initial PSA amongst the studies included. Several studies found significant association of initial PSA and survival in univariate analysis, but lost significant after multivariate analysis. This condition showed us the aggressiveness of the cancer has not reflected by PSA alone, and other measures such as Gleason score, PSA nadir, and PSA decline may need to be considered10,15. This finding was different in localized diseases. High initial PSA reflects disease burden and was found to be correlated with the pathological stage, Gleason score, and the risk of metastasis. The National Comprehensive Cancer Network (NCCN) guideline stratified the risk of localized disease based on PSA and that influences the treatment choice27.\n\nThe significant findings of this study showed that lower PSA nadir was associated with good prognosis after ADT treatment. However, due to the variety of PSA nadir threshold, we could not conclude the best optimal threshold of PSA nadir. Most of the papers in this meta-analysis were using below 0.2 ng/ml PSA nadir. Morote et al. analyzed 185 patients with metastatic prostate cancer and they found nadir PSA above 0.2 ng/ml was associated with 20 times likelihood progression to CRPC28. Moreover, Stewart et al. analyzed patient who received ADT due to biochemical recurrence after radical prostatectomy or radiation therapy, and they suggested PSA nadir above 0.2 ng/ml was associated with significant progression and mortality29. Keizman et al. used a different cut off for PSA nadir. They were using below 0.1 ng/ml because they found 4 times increased likelihood of biochemical or clinical progression in patients treated with intermittent ADT due to relapse after radical treatment30.\n\nOur findings found an association between longer time to get nadir PSA and survival. Longer time to nadir was associated with good prognosis. A study by Chung et al. found longer time to achieve nadir was a good prognosis for postoperative or post-radiation failure patients receiving ADT31. Possible mechanisms of longer time to nadir associated with a good prognosis was associated with differentiation of PCa cells. Rapid reduction of hormone sensitive cancer cells may induce an environment for the development of hormone resistant PCa cells. In addition, PCa cells that have potential to differentiate into castration resistant cell show a rapid reduction of PSA due to ablation of the androgen receptor. Thus, rapid reduction of PSA is associated to development of CRPC and has a poor prognosis32. This phenomenon is opposite to organ confined PCa receiving radical prostatectomy. In this setting, rapid decline of PSA result is associated with a better prognosis33.\n\nThis study has some methodological limitations. We did not analyze the method of administration of ADT due to heterogeneity of ADT administration and that might be influenced survival. Some of the PSA kinetics evaluated in this meta-analysis had significant high heterogeneity. The strengths of this study include (i) a high quality of study based on NOS scale; (ii) meta-analysis just included study with multivariate analysis (iii) several parameters that were associated with the survival were found in this study and might be evaluated in the future research.\n\n\nConclusion\n\nIn this study, the intial PSA before administering ADT did not influence the PFS or OS/CSS. Higher PSA nadir during ADT treatment was associated with shortened progression time and survival. A longer time to nadir is a good prognosis of progression and survival of mPCA treated with ADT.\n\n\nData availability\n\nDataset 1. Quality assessment (based on NOS), hazard ratio, and standard error of studies included in initial PSA parameter. For quality assessment a maximum 7 stars could be scored; 6 or 7 stars considered as high quality study, 4 – 5 stars corresponded with intermediate quality, and 0 – 3 stars showed low quality. 10.5256/f1000research.14026.d19555334\n\nDataset 2. Quality assessment (based on NOS), hazard ratio, and standard error of studies included in PSA nadir parameter. For quality assessment a maximum 7 stars could be scored; 6 or 7 stars considered as high quality study, 4 – 5 stars corresponded with intermediate quality, and 0 – 3 stars showed low quality. 10.5256/f1000research.14026.d19557335\n\nDataset 3. Quality assessment (based on NOS), hazard ratio, and standard error of studies included in time to nadir parameter. For quality assessment a maximum 7 stars could be scored; 6 or 7 stars considered as high quality study, 4 – 5 stars corresponded with intermediate quality, and 0 – 3 stars showed low quality. 10.5256/f1000research.14026.d19557736",
"appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nSupplementary File 1: Completed PRISMA checklist.\n\nClick here to access the data.\n\n\nReferences\n\nFerlay J, Soerjomataram I, Ervik M, et al.: GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 Lyon, France: International Agency for Research on Cancer, 2013; [cited 2016 11 January]. Reference Source\n\nBuzzoni C, Auvinen A, Roobol MJ, et al.: Metastatic Prostate Cancer Incidence and Prostate-specific Antigen Testing: New Insights from the European Randomized Study of Screening for Prostate Cancer. Eur Urol. 2015; 68(5): 885–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTangen CM, Faulkner JR, Crawford ED, et al.: Ten-year survival in patients with metastatic prostate cancer. Clin Prostate Cancer. 2003; 2(1): 41–5. PubMed Abstract | Publisher Full Text\n\nKadono Y, Nohara T, Ueno S, et al.: Validation of TNM classification for metastatic prostatic cancer treated using primary androgen deprivation therapy. World J Urol. 2016; 34(2): 261–7. PubMed Abstract | Publisher Full Text\n\nChoueiri TK, Xie W, D'Amico AV, et al.: Time to prostate-specific antigen nadir independently predicts overall survival in patients who have metastatic hormone-sensitive prostate cancer treated with androgen-deprivation therapy. Cancer. 2009; 115(5): 981–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeidenreich A, Bastian PJ, Bellmunt J, et al.: EAU guidelines on prostate cancer. Part II: Treatment of advanced, relapsing, and castration-resistant prostate cancer. Eur Urol. 2014; 65(2): 467–79. PubMed Abstract | Publisher Full Text\n\nMohler JL, Kantoff PW, Armstrong AJ, et al.: Prostate cancer, version 2.2014. J Natl Compr Canc Netw. 2014; 12(5): 686–718. PubMed Abstract | Publisher Full Text\n\nHuggins C, Hodges C: Studies on prostatic cancer II. The effects of castration on advanced carcinoma of the prostate gland. Arch surg. 1941; 43(2): 209–23. Publisher Full Text\n\nGlass TR, Tangen CM, Crawford ED, et al.: Metastatic carcinoma of the prostate: identifying prognostic groups using recursive partitioning. J Urol. 2003; 169(1): 164–9. PubMed Abstract | Publisher Full Text\n\nHong SY, Cho DS, Kim SI, et al.: Prostate-specific antigen nadir and time to prostate-specific antigen nadir following maximal androgen blockade independently predict prognosis in patients with metastatic prostate cancer. Korean J Urol. 2012; 53(9): 607–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHussain M, Tangen CM, Higano C, et al.: Absolute prostate-specific antigen value after androgen deprivation is a strong independent predictor of survival in new metastatic prostate cancer: data from Southwest Oncology Group Trial 9346 (INT-0162). J Clin Oncol. 2006; 24(24): 3984–90. PubMed Abstract | Publisher Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWells G, Shea B, O'Connell D, et al.: The Newcastle-Ottawa Scale (NOS) for assessing the quality if nonrandomized studies in meta-analyses. [cited 2016 May 2]. Reference Source\n\nMiyamoto S, Ito K, Miyakubo M, et al.: Impact of pretreatment factors, biopsy Gleason grade volume indices and post-treatment nadir PSA on overall survival in patients with metastatic prostate cancer treated with step-up hormonal therapy. Prostate Cancer Prostatic Dis. 2012; 15(1): 75–86. PubMed Abstract | Publisher Full Text\n\nPark YH, Hwang IS, Jeong CW, et al.: Prostate specific antigen half-time and prostate specific antigen doubling time as predictors of response to androgen deprivation therapy for metastatic prostate cancer. J Urol. 2009; 181(6): 2520–4; discussion 2525. PubMed Abstract | Publisher Full Text\n\nNayyar R, Sharma N, Gupta NP: Prognostic factors affecting progression and survival in metastatic prostate cancer. Urol Int. 2010; 84(2): 159–63. PubMed Abstract | Publisher Full Text\n\nKoo KC, Park SU, Kim KH, et al.: Predictors of survival in prostate cancer patients with bone metastasis and extremely high prostate-specific antigen levels. Prostate Int. 2015; 3(1): 10–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim KH, Han KS, Kim KH, et al.: The prognostic effect of prostate-specific antigen half-life at the first follow-up visit in newly diagnosed metastatic prostate cancer. Urol Oncol. 2015; 33(9): 383.e17–22. PubMed Abstract | Publisher Full Text\n\nMiller JI, Ahmann FR, Drach GW, et al.: The clinical usefulness of serum prostate specific antigen after hormonal therapy of metastatic prostate cancer. J Urol. 1992; 147 (3 Pt 2): 956–61. PubMed Abstract | Publisher Full Text\n\nTomioka A, Tanaka N, Yoshikawa M, et al.: Nadir PSA level and time to nadir PSA are prognostic factors in patients with metastatic prostate cancer. BMC Urol. 2014; 14: 33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTeoh JY, Tsu JH, Yuen SK, et al.: Survival outcomes of Chinese metastatic prostate cancer patients following primary androgen deprivation therapy in relation to prostate-specific antigen nadir level. Asia Pac J Clin Oncol. 2017; 13(2): e65–e71. PubMed Abstract | Publisher Full Text\n\nSasaki T, Onishi T, Hoshina A: Nadir PSA level and time to PSA nadir following primary androgen deprivation therapy are the early survival predictors for prostate cancer patients with bone metastasis. Prostate Cancer Prostatic Dis. 2011; 14(3): 248–52. PubMed Abstract | Publisher Full Text\n\nKwak C, Jeong SJ, Park MS, et al.: Prognostic significance of the nadir prostate specific antigen level after hormone therapy for prostate cancer. J Urol. 2002; 168(3): 995–1000. PubMed Abstract | Publisher Full Text\n\nKimura T, Onozawa M, Miyazaki J, et al.: Prognostic impact of young age on stage IV prostate cancer treated with primary androgen deprivation therapy. Int J Urol. 2014; 21(6): 578–83. PubMed Abstract | Publisher Full Text\n\nBello JO: Predictors of survival outcomes in native sub Saharan black men newly diagnosed with metastatic prostate cancer. BMC Urol. 2017; 17(1): 39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTeoh JY, Tsu JH, Yuen SK, et al.: Prognostic significance of time to prostate-specific antigen (PSA) nadir and its relationship to survival beyond time to PSA nadir for prostate cancer patients with bone metastases after primary androgen deprivation therapy. Ann Surg Oncol. 2015; 22(4): 1385–91. PubMed Abstract | Publisher Full Text\n\nMohler J, Antonorakis E, Armstrong A: NCCN Clinical Practice Guideline in Oncology. Prostate Cancer. Version 2.2017. 2017. 2017.\n\nMorote J, Esquena S, Abascal JM, et al.: Usefulness of prostate-specific antigen nadir as predictor of androgen-independent progression of metastatic prostate cancer. Int J Biol Markers. 2005; 20(4): 209–16. PubMed Abstract | Publisher Full Text\n\nStewart AJ, Scher HI, Chen MH, et al.: Prostate-specific antigen nadir and cancer-specific mortality following hormonal therapy for prostate-specific antigen failure. J Clin Oncol. 2005; 23(27): 6556–60. PubMed Abstract | Publisher Full Text\n\nKeizman D, Huang P, Antonarakis ES, et al.: The change of PSA doubling time and its association with disease progression in patients with biochemically relapsed prostate cancer treated with intermittent androgen deprivation. Prostate. 2011; 71(15): 1608–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChung CS, Chen MH, Cullen J, et al.: Time to prostate-specific antigen nadir after androgen suppression therapy for postoperative or postradiation PSA failure and risk of prostate cancer-specific mortality. Urology. 2008; 71(1): 136–40. PubMed Abstract | Publisher Full Text\n\nCrawford ED, Bennett CL, Andriole GL, et al.: The utility of prostate-specific antigen in the management of advanced prostate cancer. BJU Int. 2013; 112(5): 548–60. PubMed Abstract | Publisher Full Text\n\nVesely S, Jarolim L, Duskova K, et al.: The use of early postoperative prostate-specific antigen to stratify risk in patients with positive surgical margins after radical prostatectomy. BMC Urol. 2014; 14: 79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAfriansyah A, Hamid ARAH, Mochtar CA, et al.: Dataset 1 in: Prostate specific antigen (PSA) kinetic as a prognostic factor in metastatic prostate cancer receiving androgen deprivation therapy: systematic review and meta-analysis. F1000Research. 2018. Data Source\n\nAfriansyah A, Hamid ARAH, Mochtar CA, et al.: Dataset 2 in: Prostate specific antigen (PSA) kinetic as a prognostic factor in metastatic prostate cancer receiving androgen deprivation therapy: systematic review and meta-analysis. F1000Research. 2018. Data Source\n\nAfriansyah A, Hamid ARAH, Mochtar CA, et al.: Dataset 3 in: Prostate specific antigen (PSA) kinetic as a prognostic factor in metastatic prostate cancer receiving androgen deprivation therapy: systematic review and meta-analysis. F1000Research. 2018. Data Source"
}
|
[
{
"id": "34017",
"date": "15 May 2018",
"name": "Hong-Jun Li",
"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 meta-analysis, the authors analyzed the prognostic value of PSA kinetics in patients with mPCa. They concluded that higher PSA nadir during ADT treatment was associated with shortened progression time and survival. Although the topic was not new, the review has archival value. I have several concerns about the MS and I suggest major revision.\n\nIn the aim of the abstract, “hormonal therapy” should be replaced by “ADT” with regard to the homogeneity of the terms. In the introduction part, an explicit statement of the participants, interventions, comparisons, outcomes, and study design (PICOS) is essential. The ADT methods and cut-off point about the PSA indicators varied from different studies, which can result in potential bias. This limitation should be focused and comprehensively discussed. There were several grammar mistakes in the MS. For example, In the table 1. “Significance Prognostic Factor” should be “Significant Prognostic Factor”.\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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
},
{
"id": "33670",
"date": "21 May 2018",
"name": "Marniza Saad",
"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 seeks to show correlation between PSA kinetics (initial PSA, PSA nadir, time to nadir) and outcome in patients with metastatic prostate cancer (mPCa) receiving initial treatment with androgen-deprivation therapy (ADT) in terms of survival (PFS, CSS, OS). Seventeen studies were included based on the inclusion and exclusion criteria. The results showed that survival was correlated with PSA nadir and time to nadir (TTN) but not with initial PSA level. Low PSA nadir and long TTN are associated with improved survival. However, due to variation across the studies, no conclusion can be made on the cut-off level of PSA nadir and TTN.\nOverall it was well written. But some areas require review / modification:\nMethods\nThe inclusion criteria (ii) states that patients were treated with ADT (orchiectomy / LHRH agonists with or without anti-androgen). However, based on Table 1 there are two studies that used anti-androgen monotherapy1-2. Anti-androgen monotherapy is not included in the criteria. The studies seem to be rather heterogeneous in terms of the outcome measured and the definition of PSA kinetics. Table 1 summarized the studies nicely. But there are inconsistencies in the information given on individual studies in survival outcome column. I guess these are the outcome as reported by the studies. But for the purpose of this review, it would be useful to include on how the parameters studied in this review were defined / analysed in the individual studies i.e. the measurement of PSA kinetics and their definitions / cut-off.\n\nResults\nThere is variation in the survival outcome parameter reported by the studies - some reported median survival while others reported survival at various time-points e.g. 5-year OS. CSS has been combined with OS (Figures 2,3 & 4). I think this is not ideal since they do not refer to the same outcome but I assume this was done because the number of studies would be too small if they were to be assessed and reported separately. Need to check on the validity of the above with statistician.\n\nDiscussion\nIn para 4, it was stated that 'Our findings found an association between longer time to get nadir PSA and survival’. I suggest some comments are made on the reasonable time-points to categorise between short and long TTN to guide readers. Since there are quite a variation across the studies in the types of ADT used (ADT alone, ADT + anti-androgen, anti-androgen alone), good to add some comments on this and what data we may have on possible impact on PSA kinetics and survival. In the current era where chemohormonal and ADT in combination with abiraterone acetate have become the new standard of care for metastatic castrate sensitive prostate cancer, good to add some comments on this as we may not know how much the PSA kinetics may be prognostic of survival outcome in patients who are treated with these approaches instead of ADT alone and how the outcome of this review may or may not be applicable to these patients.\n\nThere are some errors in grammar / sentence structure / choice of words throughout the manuscript that should be reviewed and corrected. Some examples:\nPage 3 Introduction para 2: “In clinical practice……. PSA kinetics….might became a predictor of survival in several retrospective and clinical trial studies.”, should be written as “In clinical practice……. PSA kinetics….have been shown to be predictors of survival in several retrospective and prospective clinical studies.” Page 3 Methods under Search Strategy section: “Two researchers…were indecently assessing the title and abstract of the paper. They agreed the studies included in the meta-analysis.” This could perhaps be written as “The titles and abstracts of the papers were extensively assessed by two researchers. The studies for meta-analysis were selected based on the inclusion and exclusion criteria.”. Page 9 Discussion para 2: “Several studies found….., but lost significant after multivariate analysis.”, can be written as “Several studies found….., but the association was not significant after multivariate analysis.” or “Several studies found….., but lost its significance after multivariate analysis.”,\n\nSome figures were shown on page 7 - Datasets 1-3, but they are not explained / referred to in the text. These seem to be redundant. They are already documented on page 9 after the Conclusion.\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? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
},
{
"id": "33134",
"date": "21 May 2018",
"name": "Bannakij Lojanapiwat",
"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 demonstrated the importance of PSA kinetic in prognosis of prostate cancer that received ADT.\n\n1. One of important PSA kinetic is PSA doubling time; this should be in the analysis if possible.\n\n2. The sentence in discussion session” This condition showed us the aggressiveness of the cancer has not reflected by PSA alone, and other measures such as Gleason score, PSA nadir, and PSA decline may need to be considered.” As we know that Gleason score as the aggressiveness of cancer is one of very important factors in all CSS and OS, this should be more detail written in text.\n3. Volume of tumor is very important in combination upfront of chemotherapy /Noval hormonal therapy, this should be added in text that may be related with high initial PSA level.\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? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-246
|
https://f1000research.com/articles/7-244/v1
|
28 Feb 18
|
{
"type": "Research Article",
"title": "Alternative miRNAs? Human sequences misidentified as plant miRNAs in plant studies and in human plasma",
"authors": [
"Kenneth W. Witwer"
],
"abstract": "Background: A 2017 study reported that “Plant miRNAs found in human circulating system provide evidences of cross kingdom RNAi”. Analysis of two human blood plasma sequencing datasets was said to provide evidence for uptake of plant miRNAs into human plasma. The results were also purportedly inconsistent with contamination. Methods: Sequences from public datasets and miRNA databases were compared with results downloaded from the website of the reporting journal. Results: Only one putative plant miRNA (“peu-MIR2910) mapped consistently above background, and this sequence is found with 100% identity in a human rRNA. Several other rarer but consistently mapped putative plant miRNAs also have 100% or near 100% matches to human transcripts or genomic sequences, and some do not appear to map to plant genomes at all. Conclusions: Reanalysis of public data suggests that dietary plant xenomiR uptake is not supported, but instead confirms previous findings that detection of rare plant miRNAs in mammalian sequencing datasets is artifactual. Some putative plant miRNAs, including MIR2910 and MIR2911, may represent human sequence contamination or other artifacts in plant studies, emphasizing the need for rigorous controls and data filtering strategies when assessing possible xenomiRNAs.",
"keywords": [
"microRNA",
"xenomiR",
"diet",
"RNAi",
"biofluid",
"plasma"
],
"content": "Introduction\n\nReports of plant or other dietary miRNAs, or xenomiRs, entering mammalian circulation through the diet1–4 generated initial excitement for the xenomiR transfer hypothesis, yet negative results of replication and reproduction studies have cast doubt on xenomiR transfer as a general mechanism5–11. A prominent claim of xenomiR function1 has also failed rigorous reproduction7, unmasked as the result of an uncontrolled variable in the original experiment. Analyses of public datasets have revealed that studies of xenomiRs and other foreign-origin nucleic acids are fraught with artifacts: combinations of contamination, amplification or sequencing errors, permissive analysis pathways, and batch effects8,10,12–16. A particularly comprehensive study recently found that foreign miRNAs in human biofluids and tissues do not match human food consumption, are marked by batch effects, and are thus most parsimoniously explained as artifacts13. Studies of organisms with no exposure to plants have also found evidence of the same types of apparent plant contamination that plague some measurements of human samples8,17. Liu et al.18 mapped sequencing data from two studies of human plasma and other samples19,20 to various plant genomes using a 2010 plant miRNA database, PMRD21, concluding that previous reports of dietary xenomiR transfer are supported. In this brief report, these results are examined critically.\n\n\nMethods\n\nPlant mapping results from Liu et al.18 (total mapped counts) were downloaded from the BMC Genomics website. Accession numbers of sequencing datasets were checked against the publications of Ninomiya et al.19 and Yuan et al.20, as well as the Sequence Read Archive (SRA). Data were sorted and analyzed in Microsoft Excel for Mac 2011, Version 14.7.1. Plant miRNA sequences were obtained from miRBase22. Because certain plant sequences have been removed from miRBase because they have been identified as ncRNA degradation artifacts, the plant microRNA database (PMRD)21 was consulted; however, repeated attempts to access the site were unsuccessful, so information was retrieved instead from miRMaid23 or miRNEST 2.024. Supplementary File 1 contains the relevant count data from the Ninomiya et al.19 and Yuan et al.20 studies.\n\nAn earlier version of this article can be found on bioRxiv (https://doi.org/10.1101/120634).\n\n\nResults\n\nA cross-check of the source files and articles shows that the plasma data evaluated by Liu et al. were from 198 plasma samples, not 410 as reported. Ninomiya et al. sequenced six human plasma samples, six PBMC samples, and 11 cultured cell lines19. Yuan et al. sequenced 192 human plasma libraries (prepared from polymer-precipitated plasma particles)20. Each library was sequenced once, and then a second time to increase total reads. Counts were presented as reads per million mapped reads (rpm)20. In contrast, Liu et al. appear to have reported total mapped reads in their data table18. Yuan et al. also set an expression cutoff of 32 rpm (log2 rpm of 5 or above). With an average 12.5 million reads per sample (the sum of the two runs per library), and, on average, about half of the sequences mapped, the 32 rpm cutoff would translate to around 200 total reads in the average sample as mapped by Liu et al.18.\n\nConsulting the Liu et al. mapping table18 and the SRA, results from duplicate sequencing runs from the Yuan et al. dataset were combined, and two samples without reliable replicates were eliminated. A total of 1294 putative plant miRNAs had at least one mapped read in at least one of the remaining 190 samples. However, many of these miRNAs were identical orthologs or paralogs, and most were mapped at one or fewer rpm on average, and in only a small minority of samples. Across all samples, only one putative plant miRNA mapped above a median 200 read cutoff, roughly corresponding to the 32 rpm cutoff of Yuan et al. (Table 1). All other RNAs, including previously reported xenomiRs such as MIR159a, MIR168a, and the plant ribosomal degradation fragment MIR291125–27, were thus below the level of background noise established by the original investigators. Indeed, previously reported xenomiRs were mapped in few samples and below 1 rpm. The absence of these RNAs is confirmed by Liu et al.’s analysis of the Ninomiya, et al. study19, where MIR159a, MIR168a, and MIR2911 mapped in none of the plasma samples. The single putative plant miRNA that mapped above background levels in this study was, again, peu-MIR2910 (Table 2).\n\nHere, data from only 190 of 192 plasma samples were included, since all but the excluded 2 samples were successfully sequenced twice. miRNA inclusion criteria were: 1) Three total mapped reads according to Liu et al.’s data in at least 10% of the samples and 2) discoverable putative mature sequence through miRBase, miRMaid, or miRNEST 2.0. An “estimated median rpm” value was calculated based on median total counts, average reads, and the midoint of the reported mapping percentage range. miRNAs with perfect human matches are in red. Note that only MIR2910 consistently exceeds the rpm threshold set by Yuan et al..\n\nHere, all results are shown in the left half of the table, and plasma results on the right. miRNA inclusion criteria were: 1) Three total mapped reads according to Liu et al.’s data in at least 10% of the samples (cells and plasma together) and 2) discoverable putative mature sequence through miRBase, miRMaid, or miRNEST 2.0. “Avg rpm” is calculated from the total mapped reads and total reads per sample (not mapped reads). Putative miRNAs that met inclusion criteria in the Yuan et al. study are italicized, and sequences with perfect human matches are in red.\n\nSince only one plant miRNA appeared to map consistently above background, the inclusion threshold of Yuan et al. was relaxed to include all miRNAs with three or more mapped reads (Liu et al. data) in 10% or more of the samples from either study. These are rather permissive criteria but may at least screen out some false positives due to amplification and sequencing errors. All samples from the Ninomiya study were included, despite the fact that most were not plasma. 11 miRNAs satisfied these criteria for the Yuan et al. data (Table 1). (One low-mapping miRNA was excluded because its sequence could not be found in miRBase22,28, miRMaid23, miRNEST 2.024 or indeed through any searches attempted.) 10 satisfied the criteria from the Ninomiya study (Table 2), including one sequence that was part of another (compare ath-MIRf10046-akr and ath-MIRf10045-ak, Table 3). However, if only the plasma samples from the latter study are considered, three miRNAs remain (Table 2). In total, 15 putative miRNAs satisfied the permissive inclusion criteria, including five (Yuan only), four (Ninomiya only), and six (both) (Table 3).\n\nInclusion criteria were: 1) Three total mapped reads according to Liu et al.’s data in at least 10% of the samples in the respective studies and 2) discoverable putative mature sequence through miRBase, miRMaid, or miRNEST 2.0. miRNA status was considered unlikely if miRBase listed the miRNA as a non-miRNA or if the sequence mapped to non-miRNA regions. Human matches were exact (with an example given), “partial” (at least 15 nt stretches with 100% identity), or as otherwise described. Note that the ath-MIRf10046-akr is found within the ath-MIRf10045-akr sequence.\n\nAs miRNA discovery, validation, and annotation has advanced, numerous reported miRNAs have been reclassified as degradation fragments of other noncoding RNAs (ncRNAs). A classic example is MIR2911, a plant rRNA degradation fragment that has been misidentified as a microRNA. Interestingly, only 2 of the 15 miRNAs identified as plant miRNAs in this study are annotated in miRBase. Although some of these sequences may represent rare or unusually structured miRNAs, several are part of non-miRNA ncRNAs or other sequences that seem unlikely, at least at first glance, to give rise to microRNAs. Among the apparently misidentified miRNAs is MIR2910, the most abundant plant miRNA identified by Liu et al.. The MIR2910 sequence, UAGUUGGUGGAGCGAUUUGUC, is found in the highly conserved and expressed large subunit (LSU) rRNA of plants, and has been specifically removed from miRBase as a non-miRNA. Even the two identified miRNAs that remain in miRBase, MIR2916 and MIR894, are not above question. A 20 nucleotide stretch of MIR2916 map to rRNA, while the full MIR894 sequence appears to be found in a variety of plant transcripts.\n\nCuriously, several sequences did not map to the species to which they were ascribed by the PMRD21. Unfortunately, the PMRD could not be accessed directly during this study; however, other databases appear to provide access to its contents. Specifically, ptc-MIRf12412-akr and ptc-MIRf12524-akr did not map to Populus or to other plants. The poplar tree is also not a common dietary staple of human populations. In contrast, both sequences mapped with 100% identity and coverage to numerous human sequences (Table 3). ptc-MIRf10804-akr had numerous 100% identity human matches, plus a 1-mismatch alignment to the human miR-3929 precursor. Other miRNAs, including MIR2911, also displayed some lesser degree of matching to human transcripts or the genome. Strikingly, the putative MIR2910 sequence is not only a fragment of plant rRNA; it has a 100% coverage, 100% identity match in the human 18S rRNA (see NR_003286.2 in GenBank; Table 3). These matches of putative plant RNAs with human sequences are difficult to reconcile with the statement of Liu et al. that BLAST of putative plant miRNAs \"resulted in zero alignment hit\"18, suggesting that perhaps a mistake was made, and that the BLAST procedure was performed incorrectly.\n\n\nDiscussion\n\nIn mammalian studies, mapping of MIR2910 and other dubious plant miRNAs is best explained as mapping of human degradome fragments to plant RNAs that are in some cases genuine sequences but not miRNAs, and in other cases, human sequences that have contaminated plant RNA samples and databases. Re-analysis of the results of Liu et al.18 thus echoes the recent findings of Kang, Bang-Berthelsen, and colleagues13, as well as previous negative findings surrounding dietary xenomiRs, summarized above. A stringent data analysis procedure, such as filtering all reads against the ingesting organism genome/transcriptome with one or two mismatches, then requiring perfect matches of remaining reads against plant or other foreign organisms, would engender higher confidence that “foreign” RNAs are not simply amplification or sequencing artifacts. Indeed, pre-mapping to the ingesting organism’s genome may not be sufficient; as shown13, the largest number of xenomiRs in some human studies are from rodents, likely because of proximity in research laboratories. Therefore, it may be best to screen against mammalian sequences in general, and perhaps also against widespread microbe contaminants. Of course, even the most stringent analysis procedures cannot distinguish a physical contaminant from a “real” read; therefore strict process controls are also needed to assess possible contamination. In general, such controls have not been done in existing studies.\n\nThis report underlines the danger in assuming that xenomiRs in mammalian material originate from the diet. When the species and roles are reversed—for example, with the finding of human sequences in a list of poplar tree miRNAs—few analysts would conclude that poplar trees consume humans. The simplest explanation is that the sequenced plant material was contaminated with human nucleic acid. In the same way, the extremely low-level, variable, and batch-effect prone concentrations of several plant sequences in human plasma and tissue could be due to uptake from the diet, albeit at levels far too low to affect physiologic processes. However, artifact remains the simplest explanation.\n\n\nData availability\n\nData used in these analyses were downloaded from the supplementary materials of Liu et al.18 and are summarized in Supplementary File 1. Source data for the Ninomiya, et al. study19 are under DRA002550, DRX021324–DRX021346, and DRR023270–DRR023292 (DNA Data Bank of Japan), and, for the Yuan, et al. study20, available as GSE71008 (Gene Expression Omnibus).",
"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: Tabs for the miRNA counts from the Yuan et al. and Ninomiya et al. datasets.\n\nClick here to access the data.\n\n\nReferences\n\nZhang L, Hou D, Chen X, et al.: Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res. 2012; 22(1): 107–126. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChin AR, Fong MY, Somlo G, et al.: Cross-kingdom inhibition of breast cancer growth by plant miR159. Cell Res. 2016; 26(2): 217–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiang H, Zhang S, Fu Z, et al.: Effective detection and quantification of dietetically absorbed plant microRNAs in human plasma. J Nutr Biochem. 2015; 26(5): 505–512. PubMed Abstract | Publisher Full Text\n\nBaier SR, Nguyen C, Xie F, et al.: MicroRNAs Are Absorbed in Biologically Meaningful Amounts from Nutritionally Relevant Doses of Cow Milk and Affect Gene Expression in Peripheral Blood Mononuclear Cells, HEK-293 Kidney Cell Cultures, and Mouse Livers. J Nutr. 2014; 144(10): 1495–500. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSnow JW, Hale AE, Isaacs SK, et al.: Ineffective delivery of diet-derived microRNAs to recipient animal organisms. RNA Biol. 2013; 10(7): 1107–1116. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWitwer KW, McAlexander MA, Queen SE, et al.: Real-time quantitative PCR and droplet digital PCR for plant miRNAs in mammalian blood provide little evidence for general uptake of dietary miRNAs: Limited evidence for general uptake of dietary plant xenomiRs. RNA Biol. 2013; 10(7): 1080–1086. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDickinson B, Zhang Y, Petrick JS, et al.: Lack of detectable oral bioavailability of plant microRNAs after feeding in mice. Nat Biotechnol. 2013; 31(11): 965–967. PubMed Abstract | Publisher Full Text\n\nTosar JP, Rovira C, Naya H, et al.: Mining of public sequencing databases supports a non-dietary origin for putative foreign miRNAs: underestimated effects of contamination in NGS. RNA. 2014; 20(6): 754–757. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMicó V, Martín R, Lasunción MA, et al.: Unsuccessful Detection of Plant MicroRNAs in Beer, Extra Virgin Olive Oil and Human Plasma After an Acute Ingestion of Extra Virgin Olive Oil. Plant Foods Hum Nutr. 2016; 71(1): 102–8. PubMed Abstract | Publisher Full Text\n\nAuerbach A, Vyas G, Li A, et al.: Uptake of dietary milk miRNAs by adult humans: a validation study [version 1; referees: 3 approved]. F1000Res. 2016; 5: 721. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWitwer KW: Contamination or artifacts may explain reports of plant miRNAs in humans. J Nutr Biochem. 2015; 26(12): 1685. PubMed Abstract | Publisher Full Text\n\nLusk RW: Diverse and widespread contamination evident in the unmapped depths of high throughput sequencing data. PLoS One. 2014; 9(10): e110808. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKang W, Bang-Berthelsen CH, Holm A, et al.: Survey of 800+ data sets from human tissue and body fluid reveals xenomiRs are likely artifacts. RNA. 2017; 23(4): 433–445. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang Y, Wiggins BE, Lawrence C, et al.: Analysis of plant-derived miRNAs in animal small RNA datasets. BMC Genomics. 2012; 13: 381. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBağcı C, Allmer J: One Step Forward, Two Steps Back; Xeno-MicroRNAs Reported in Breast Milk Are Artifacts. PLoS One. 2016; 11(11): e0145065. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWitwer KW, Hirschi KD: Transfer and functional consequences of dietary microRNAs in vertebrates: concepts in search of corroboration: negative results challenge the hypothesis that dietary xenomiRs cross the gut and regulate genes in ingesting vertebrates, but important questions persist. Bioessays. 2014; 36(4): 394–406. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZheng LL, Deng KW, Deng AC, et al.: Exo-miRExplorer: A Comprehensive Resource for Exploring and Comparatively Analyzing Exogenous MicroRNAs. Front Microbiol. 2017; 8: 126. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu YC, Chen WL, Kung WH, et al.: Plant miRNAs found in human circulating system provide evidences of cross kingdom RNAi. BMC Genomics. 2017; 18(Suppl 2): 112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNinomiya S, Kawano M, Abe T, et al.: Potential small guide RNAs for tRNase ZL from human plasma, peripheral blood mononuclear cells, and cultured cell lines. PLoS One. 2015; 10(3): e0118631. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYuan T, Huang X, Woodcock M, et al.: Plasma extracellular RNA profiles in healthy and cancer patients. Sci Rep. 2016; 6: 19413. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang Z, Yu J, Li D, et al.: PMRD: plant microRNA database. Nucleic Acids Res. 2010; 38(Database issue): D806–D813. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKozomara A, Griffiths-Jones S: miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 2014; 42(Database issue): D68–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJacobsen A, Krogh A, Kauppinen S, et al.: miRMaid: a unified programming interface for microRNA data resources. BMC Bioinformatics. 2010; 11: 29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzczesniak MW, Makalowska I: miRNEST 2.0: a database of plant and animal microRNAs. Nucleic Acids Res. 2014; 42(Database issue): D74–D77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou Z, Li X, Liu J, et al.: Honeysuckle-encoded atypical microRNA2911 directly targets influenza A viruses. Cell Res. 2015; 25(1): 39– 49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang J, Hotz T, Broadnax L, et al.: Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911. Sci Rep. 2016; 6: 26834. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang J, Kongchan N, Primo Planta C, et al.: The atypical genesis and bioavailability of the plant-based small RNA MIR2911: bulking up while breaking down. Mol Nutr Food Res. 2017; 61(9): 1600974. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGriffiths-Jones S, Grocock RJ, van Dongen S, et al.: miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 2006; 34(Database issue): D140–4. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "31318",
"date": "08 Mar 2018",
"name": "Jens Allmer",
"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 ‘Alternative miRNAs? Human sequences misidentified as plant miRNAs in plant studies and in human plasma’ by Kenneth W. Witwer addresses the recurring claim of plant xeno-microRNAs found in human plasma. Such xenomiRs are allegedly ingested and enriched in human; and this time Liu et al. claim evidence for cross kingdom RNAi via this route. Thus far, similar claims could not stand up against thorough validation as, for example, we (Bagci and Allmer, 20161) and Kenneth W. Witwer (Auerbach et al. 20162) have shown before.\nHere, Witwer has a closer look at the article ‘Plant miRNAs found in human circulating system provide evidence of cross kingdom RNAi’ by Liu et al. 20173. Liu et al. 2017 claim that the plant miRNAs are not contamination, but only checked whether they contain sequencing adapters. Other sources of contamination were not considered. Additionally, Liu et al. 2017 reported to have used BLAST to check the plant miRNAs they found against the human genome without any similarities. Conversely, Witwer was able to find full-length identical alignments for these sequences in the human genome. For example, peu-MIR2910 is claimed by Liu et al to be conserved within comestible plants but a quick check on miRBase reveals that it is dead entry and not considered a miRNA anymore (http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0012633). Witwer correctly finds the mature sequence for this miRNA (UAGUUGGUGGAGCGAUUUGUC) in human while Liu et al. used the precursor sequence (UAGUUGGUGG AGCGAUUUGU CUGGUUAAUU CCGUUAACGA ACGAGACCUC AGCCUGCUA) for their search. I repeated the latter and found that the first 50 nucleotides are identical with several human RNAs (e.g.: NR_046235) which supports Witwer’s findings. It is of note, that PMRD is still not reachable and that the sequences I used were provided by Liu et al. in their commentary on Witwer’s article in biorxiv4.\nWitwer points out that mapping results were not normalized which is supported by the original manuscript by Liu et al. However, such normalization is of essence especially when the number of observations is very low (spurious even?).\nI fully support the conclusions Kenneth W. Witwer draws in this article.\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": "31319",
"date": "13 Apr 2018",
"name": "Claus H. Bang-Berthelsen",
"expertise": [
"Reviewer Expertise RNA 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\nK. W. Witwer presents some interesting findings in his brief report: Alternative miRNAs? Human sequences misidentified as plant miRNAs in plant studies and in human plasma. I consider the brief report scientifically good as well as well written.\nAdditionally, I would like to recognize the effort made by K. W. Witwer on examination of the methodology and critical reviewing a recent publication by Liu and colleagues in 20171. Here Liu et al. report plant cross kingdom RNAi in two human sRNA plasma sequencing studies. Here Witwer identify a number of methodology issues in the Liu et al. paper by reanalyzing the public available data that were used in their paper. Witwer concludes that the most likely explanation for the Liu et al. reported plant RNA results, detected in the human plasma datasets, are traces of human degradome fragments and not of plant origin.\nThe central conclusion in the manuscript are line with the findings that Kang et al. 20172 reported based on a massive analysis of public available sRNA datasets comprising 10 billion sequencing reads. All in all supporting the null hypothesis that presence of xenomiRs are technical/contamination artifacts.\nBased on the overall impression of the paper it is scientifically sound and I can fully support the conclusions.\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/7-244
|
https://f1000research.com/articles/6-1804/v1
|
05 Oct 17
|
{
"type": "Research Article",
"title": "Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection",
"authors": [
"Peter Wild",
"Andres Kaech",
"Elisabeth M. Schraner",
"Ladina Walser",
"Mathias Ackermann",
"Andres Kaech",
"Elisabeth M. Schraner",
"Ladina Walser",
"Mathias Ackermann"
],
"abstract": "Background: Herpesvirus capsids are assembled in the nucleus before they are translocated to the perinuclear space by budding, acquiring tegument and envelope, or releasing to the cytoplasm in a “naked” state via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” are essential steps for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of an alternative exit route. Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1) or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1) by transmission and scanning electron microscopy, employing freezing technique protocols that lead to improved spatial and temporal resolution. Results: Scanning electron microscopy showed the Golgi complex as a compact entity in a juxtanuclear position covered by a membrane on the cis face. Transmission electron microscopy revealed that Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced. Conclusions: The data strongly suggest that virions are intraluminally transported from the perinuclear space via Golgi complex-endoplasmic reticulum transitions into Golgi cisternae for packaging into transport vacuoles. Furthermore, virions derived by budding at nuclear membranes are infective as has been shown for HSV-1 Us3 deletion mutants, which almost entirely accumulate in the perinuclear space. Therefore, de-envelopment followed by re-envelopment is not essential for production of infective progeny virus.",
"keywords": [
"herpes virus",
"envelopment",
"egress pathway",
"endoplasmic reticulum",
"Golgi complex",
"intraluminal transport",
"brefeldin A"
],
"content": "Introduction\n\nThe Golgi complex plays a crucial role in the secretory pathway. Cargo is transported from the endoplasmic reticulum (ER) to the Golgi complex via vesicles that derive from ER exit sites (Bonifacino & Glick, 2004). Tubules are involved in anterograde as well as in retrograde transport (Lippincott-Schwartz et al., 1990; Lippincott-Schwartz et al., 1989). The sole paradigm of vesicular transport between ER and Golgi may evolve to account for the results of new technologies (Lippincott-Schwartz, 2011). Indeed, cargo may also be transported through an ER-Golgi intermediate compartment (ERGIC) (Hauri & Schweizer, 1992; Klumperman, 2000; Saraste et al., 2009). Whether the ERGIC is a stable structure, is under debate (Ben-Tekaya et al., 2005). There are also transitional elements connecting Golgi membranes to ER membranes (Pavelka & Roth, 2015; Polishchuk & Mironov, 2004; Vivero-Salmeron et al., 2008) possibly enabling direct transportation of cargo from ER cisternae into Golgi cisternae. Once in the Golgi cisternae, cargo is packaged into secretory granules for exocytotic release (Palade, 1975). Packaging of cargo is accompanied by loss of Golgi membranes. To maintain Golgi structure and function, multiple recycling processes take place (Orci et al., 1981). Although the structure and function of the Golgi complex has been investigated for decades, many uncertainties remain e.g. Golgi maturation and functionality of the trans Golgi network (TGN) (Emr et al., 2009).\n\nThe Golgi complex plays also a crucial role in herpes virus morphogenesis and intracellular transport (Roizman et al., 2014). Herpes viruses comprise the capsid, tegument and envelope, with embedded glycoproteins. Capsids are assembled in nuclei of host cells and transported to the Golgi complex concomitantly acquiring the envelope and tegument. Three diverse pathways have been proposed. In pathways 1 and 3 (summarized in Figure 11), capsids directed to the nuclear periphery are released into the perinuclear space (PNS) by budding at the inner nuclear membrane (INM) acquiring an electron dense envelope and tegument. In pathway 1, these perinuclear virions are intraluminally transported (Gilbert et al., 1994; Granzow et al., 1997; Radsak et al., 1996; Schwartz & Roizman, 1969; Stannard et al., 1996; Sutter et al., 2012; Whealy et al., 1991; Wild et al., 2002) into ER cisternae, whose membranes are connected to the outer nuclear membrane (ONM). These virions were suggested to be transported via ER-Golgi transitions into Golgi cisternae (Leuzinger et al., 2005; Wild et al., 2015; Wild et al., 2002). Importantly, intraluminal transportation requires mechanisms for preventing the viral envelope from fusion with the membrane the virions are transported along.\n\nIn pathway 2, capsids are released from the nuclear periphery into the cytoplasmic matrix via impaired nuclear pores (Leuzinger et al., 2005; Wild et al., 2005; Wild et al., 2009) or disrupted nuclear membranes (Borchers & Oezel, 1993; Klupp et al., 2011). Notably, pore impairment is the initial step in breakdown of the nuclear envelope (Terasaki et al., 2001) that takes place when HSV-1 infection proceeds (Maric et al., 2014). The capsids in the cytoplasmic matrix are transported to any site of the Golgi complex (Wild et al., 2002) and are enveloped either by wrapping (see below) or by budding into Golgi cisternae and/or vacuoles, which may enlarge to engulf multiple virions (Homman-Loudiyi et al., 2003; Leuzinger et al., 2005; Stannard et al., 1996; Sutter et al., 2012; Wild et al., 2015; Wild et al., 2002). Capsids bud, though less frequently, also at the ONM and RER membranes (Leuzinger et al., 2005; Wild et al., 2005), and are intraluminally transported as in pathway 1.\n\nAccording to pathway 3, virions in the PNS are de-enveloped by fusion of the viral envelope with the ONM or with adjacent ER membranes releasing capsid and tegument into the cytoplasmic matrix. These capsids then are re-enveloped by budding at membranes of the TGN (Mettenleiter et al., 2013) and endosomes (Albecka et al., 2016; Hollinshead et al., 2012) acquiring again an envelope and tegument. Concomitantly, a small concentric transport vacuole is formed enclosing the enveloped virion. This process is referred to as wrapping (Roizman et al., 2014).\n\nAlthough the phenotypes of the capsid transport across the ONM exhibit all characteristics of budding (Bonifacino & Glick, 2004; Harrison, 2015; Jahn et al., 2003; Kanaseki et al., 1997; Lee, 2010; Mayer, 2002) the de-envelopment theory is still favored. Fusion of the viral envelope with the ONM requires the glycoproteins gB and gH (Farnsworth et al., 2007). Nonetheless, various stages of capsid transportation across the ONM and ER membranes in the absence of the glycoproteins gB/gH have been shown. Furthermore, a substantial proportion of virions lacking gB/gH were reported to be in the cytoplasm and extracellular space. These two facts strongly suggest another pathway of virus particles out of the PNS. The lack of profound evidence for capsid transport across the ONM via fusion, and the growing evidence of Golgi to ER transitions, prompted us to investigate the ER and Golgi compartments by cryo-field emission scanning electron microscopy (Cryo-FESEM) and transmission electron microscopy (TEM), employing protocols for improved spatial and temporal resolution (Ellinger et al., 2010; Mueller, 1992). We show that the Golgi complex is a tightly packed organelle, that ER to Golgi transitions are formed, and that virions aggregate within the ER after exposure cells to brefeldin A (BFA), which disintegrates the Golgi complex within minutes (Hess et al., 2000), suggesting an intraluminal transportation route. Moreover, we observe that intraluminal virions are densely coated with a proteinaceous layer that arises during budding. Therefore, we propose that the significance of the dense coat is protecting the viral envelope from fusion with the membranes along which virions are transported, in a similar manner as clathrin protects coated vesicles from fusion.\n\n\nMaterials and methods\n\nVero cells and MDBK cells (European Collection of Cell Cultures) were grown in Dulbecco’s modified minimal essential medium (DMEM; Gibco, Bethesda, MD, USA) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) and 10% fetal bovine serum (FBS; Gibco). The Us3 deletion mutant R7041(ΔUs3) and the repair mutant R2641 (Longnecker & Roizman, 1987; Purves et al., 1987) were kindly provided by Bernard Roizman (The Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, Illinois, USA). Wild-type herpes simplex virus 1 (wt HSV-1) strain F (Ejercito et al., J. Gen. Virol. 2:357–364, 1968), R7041(ΔUs3) and R2641 were propagated in Vero cells, and bovine herpes virus 1 (BoHV-1: Metzler et al., Arch. Virol. 87: 205–217, 1986) in MDBK cells. Virus yields were determined by plaque titration.\n\n50 μm thick sapphire disks (Bruegger, Minusio, Switzerland) measuring 3 mm in diameter were coated with 8–10 nm carbon, obtained by evaporation under high vacuum conditions to enhance cell growth and to facilitate detachment of cells from the sapphire disks after embedding. Vero and MDBK cells were grown for 2 days on sapphire disks placed in 6 well plates. Cells were inoculated with R7041(ΔUs3), the repair mutant R2641, wt HSV-1 or BoHV-1 at a MOI of 5, incubated at 37°C, and fixed at 8 to 20 hpi by adding 0.25% glutaraldehyde to the medium prior to freezing in a high-pressure freezing unit (HPM010; Science Services, Munich, Germany) and processed as described in detail (Wild, 2008). In brief, the frozen water was substituted with acetone in a freeze-substitution unit (FS 7500; Boeckeler Instruments, Tucson, AZ, USA) at -88°C with acetone and subsequently fixed with 0.25% glutaraldehyde and 0.5% osmium tetroxide raising the temperature gradually to +2°C to achieve good contrast of membranes (Wild et al., 2001) , and embedded in epon at 4°C followed by polymerization at 60°C for 2.5 days. After removal of sapphire disks by immersion in liquid nitrogen, serial sections of 60 to 90 nm thickness were analyzed in a transmission electron microscope (CM12; FEI, Eindhoven, The Netherlands) equipped with a CCD camera (Ultrascan 1000; Gatan, Pleasanton, CA, USA) at an acceleration voltage of 100 kV.\n\nCells grown on sapphire disks were inoculated with wt HSV-1 at a MOI of 5 and incubated at 37°C. Stock solution (5 mg BFA solved in 0.5 ml methanol) was diluted with medium 1:10. One µl/ml medium (1µg/ml) of this solution was added to cell cultures at 5, 8, 12 and 16 hpi. Cells were high-pressure frozen at indicated times, and prepared for TEM. To quantify virus phenotypes and their location, 10 images of cellular profiles were taken at random from ultrathin sections of monolayers exposed to BFA from 5, 8, 12 or 16 hpi to 20 hpi of 5 independent experiments. Capsids budding at the INM, ONM, ER and Golgi membranes, capsids undergoing wrapping, as wells as virions in the PNS, ER, Golgi cisternae and vacuoles were counted. The means expressed per cellular profile were compared applying a Student’s t-test using GraphPad Prism 3 software.\n\nFor determination of infectious progeny virus produced after BFA exposure, cells were grown in 10 ml Falcon flasks, inoculated with wt HSV-1 at a MOI of 5, and exposed to BFA from 5, 8, 12 or 16 h to 20 hpi. Cells were harvested at 5, 8, 12, 16 and 20 hpi for determination of infectious progeny virus by plaque titration. Since the Golgi complex reacts immediately to BFA by disintegration, infectious viruses produced after BFA exposure were considered to have derived by budding at membranes other than those of the Golgi complex.\n\nVero cells were grown in 25 cm2 cell culture flasks for 2 days prior to inoculation with wt HSV-1, R7041(ΔUs3) or R2641 at MOI of 5. Cells were harvested at 9 to 12 hpi by trypsinization followed by centrifugation at 150 x g for 8 min. Pellet were re-suspended in 1 ml fresh medium, collected in Eppendorf tubes and fixed by adding 0.25% glutaraldehyde to the medium. The suspension was kept in the tubes at 4°C until cells were sedimented. After removal of the supernatant, cells were frozen in a high-pressure freezing machine EM HPM100 (Leica Microsystems, Vienna, Austria) as described in detail previously (Wild et al., 2012; Wild et al., 2009). Cells were fractured at -120°C in a freeze-fracturing device BAF 060 (Leica Microsystems) in a vacuum of 10-7 mbar. The fractured surfaces were partially freeze-dried (“etched”) at -105°C for 2 min, and coated with 2.5 nm platinum/carbon by electron beam evaporation at an angle of 45°. Some specimens were coated additionally with 4 nm of carbon to reduce electron beam damage during imaging at high magnifications. Specimens were imaged in an Auriga 40 Cross Beam system (Zeiss, Oberkochen, Germany) equipped with a cryo-stage (Leica Microsystems) at -115°C and an acceleration voltage of 5 kV using the inlens secondary electron detector.\n\nCells were grown for 2 days on 0.17 mm thick cover slips of 12 mm in diameter (Assistent, Sondheim, Germany) and inoculated with R7041(ΔUs3), wt HSV-1 or R2641 at a MOI of 5 and incubated at 37°C. After fixation with 2% formaldehyde for 25 min at room temperature, cells were briefly washed with PBS and stored in PBS at 4°C until further processing. Then, cells were permeabilized with 0.1% Triton-X-100 at room temperature for 7 min and blocked with 3% bovine serum albumin in PBS containing 0.05% Tween 20 (PBST). To ascertain infectivity, cells were labeled with antibodies against ICP4 (Life Technologies, Carlsbad, CA; USA), and Alexa 594 (Molecular Probes, Eugene, OR, USA) as secondary antibodies. To identify the Golgi complex, cells were incubated with recombinant monoclonal antibodies against the cis-Golgi protein GM130, abcam EP892Y (Abcam, Cambridge, UK) at a dilution of 1:1000 for 2 h at room temperature followed by incubation with Alexa 488 (Molecular Probes) as secondary antibodies, diluted 1:500, for 1 h at room temperature. After staining nuclei with 4',6-Diamidino-2-phenylindol (DAPI; Roche, Mannheim, Germany), cells were embedded in glycergel mounting media (Dako North America, Carpinteria, CA, USA) and 25 mg/ml 1,4-diazabicyclo [2.2.2] octane (DABCO; Fluka, Buchs, Switzerland). Specimens were analyzed using a confocal laser scanning microscope (SP2; Leica, Wetzlar, Germany). Images were deconvolved employing the deconvolution algorithm of the program suite Huygens Essential (SVI, Hilversum, The Netherlands).\n\n\nResults\n\nTo localize the Golgi complex, we first imaged the Golgi complex by confocal microscopy after labeling the cis-face with antibodies against the Golgi protein GM 130 in Vero cells. The Golgi complex was always found close to the nucleus in HSV-1, R7041(ΔUs3), or mock infected cell (Figure 1). Next, high resolution cryo-electron microscopy of freeze fractured cells demonstrated the bell-shaped form of the Golgi complex, and its localization close to the nucleus (Figure 2). This image also shows that the Golgi complex is a complex tightly packed entity with a diameter of approximately 6 µm. The Golgi complex is separated from the cytoplasmic matrix by an intact membrane covering the whole visible surface of the cis-face. The large dimension makes clear that the detected ultrastructural details depend on a large scale on how and where the Golgi complex is hit in a given section plane for studying by TEM. Both freeze-fracture planes and thin sections of central regions show that the membrane of the outermost cisterna covers the cis-side (Figure 3).\n\nConfocal microscopy of Vero cells, immunolabeled with antibodies against the Golgi protein GM130 (green) and ICP4 (red) at 12 hpi with HSV-1 (A) and after mock infection (B), showing the Golgi complex always in a juxtanuclear position. Bars: 1 µm.\n\nThe entire visible surface is covered by an intact membrane (m) except at the part it is broken away giving view to Golgi stacks (s). Bars: 200 nm.\n\nThe Golgi complex, as revealed in freeze-fracture planes (A) and in thin sections (B), is entirely covered at the cis-face by the membrane (m) of the outermost cisterna. Bars: 100 nm.\n\nThe Golgi complex undergoes dramatic changes during HSV-1 infection finally resulting in fragmentation and dispersion (Campadelli et al., 1993). However, the Golgi complex is not, or only minimal, involved in envelopment of R7041(ΔUs3) capsids (Wild et al., 2015). To identify ER-to-Golgi transitions, we thus imaged the Golgi complex in serial sections through wt HSV-1 or R7041(ΔUs3) infected cells by transmission electron microscopy after rapidly freezing and freeze-substitution applying a protocol especially suitable to visualize membranes (Wild et al., 2001). A series of images show that ER membranes continue into Golgi membranes. The ER runs from the perinuclear region into the membranes of the outermost Golgi stack (Figure 4A). The very same membranes continue again into ER membranes so that this Golgi cisterna is interconnected between ER lamellae. The membranes of the adjacent stack also turn into ER membranes. ER membranes also pass somewhere into central regions of Golgi fields where they are devoid of ribosomes (Figure 4BC). ER membranes may even connect two Golgi fields (Figure 4D). The ER forms, as its name implies, a network (Figure 4C) that connects to the PNS (Figure 5A).\n\n(A) The ER (er) runs from the nuclear (n) periphery towards a Golgi field (go), continuing into the membrane of the outermost stack and further into the cytoplasmic matrix. The membranes of the second stack continue also into ER membranes. (B) An ER cisterna runs through multiple small Golgi fields, whereby the ER membranes turn into Golgi membranes (thick arrows). (C) ER membranes forming a network continue into Golgi membranes. (D) ER membranes run through two Golgi fields, turning each time into Golgi membranes. Bars: 500 nm.\n\nVirions within the ER have been repeatedly shown, the first report dating back to the late 1960ties (Schwartz & Roizman, 1969). Virions were in the PNS or anywhere in ER cisternae after HSV-1 infection (Figure 5BC) as well as in Golgi cisternae of which membranes transit into ER membranes (Figure 5B). Virions accumulate in the PNS-ER compartment late in infection (Leuzinger et al., 2005; Wild et al., 2015), in the absence of Us3 (Reynolds et al., 2002; Wisner et al., 2009) or gB/gH (Farnsworth et al., 2007) or after disintegration of the Golgi complex by BFA (Chatterjee & Sarkar, 1992; Cheung et al., 1991; Jensen & Norrild, 2002; Whealy et al., 1991). To investigate the effect of BFA on virus release out of the PNS we exposed cells to BFA at 5, 8, 12 and 16 hpi with wt HSV-1, and harvested cells at 20 hpi for quantitative electron microscopic analysis and determination of infectious progeny virus. Electron microscopy revealed dilation of the PNS and ER containing many virions and amorphous material at 15 hpi (Figure 5D). At 17 hpi, the ER was congested with virions (Figure 5E). Quantitative analysis of phenotype distribution revealed that virions accumulate in the PNS-ER compartment after BFA administration in a time dependent manner. The number of intraluminal virions was 4 times higher when BFA was added at 8 hpi but 12 times higher than it was added at 16 hpi compared to that in untreated cells (Figure 6). The number of virus particles interacting with the ONM and ER membranes in BFA exposed cells was about twice as high as in controls whereas the number of capsids in the cytoplasmic matrix did not differ significantly. We thus conclude i) that the interactions at the ONM and ER membranes are budding capsids contributing to accumulation of virions in the PNS and ER, ii) that more capsids bud at the ONM and ER membranes because no Golgi membranes are available after Golgi disintegration induced by BFA, iii) that inhibition of virion release out of the PNS-ER compartment is due to a blockage of the intraluminal transportation pathway after Golgi disintegration, and iv) that the Golgi complex delivered components to budding sites prior to its disintegration by BFA.\n\nTEM of Vero cells at 9 hpi with R7041(ΔUs3) (A), at 16 hpi (B) and at 20 hpi (C) with wt HSV-1, and at 15 or 17 hpi with wt HSV-1 and BFA exposure (D and E). (A) The ER (er) runs from the nucleus (n) towards the cell periphery forming an entity with the PNS that contains a virion (v). (B) The ER contains virions. One capsid is in the stage of budding (b) into the ER. The ER continues into Golgi (go) membranes at two sites. One Golgi cisterna contains a virion (Vg), one virion has been derived by wrapping (Vw). Close to Golgi stacks, there is probably a virion (V?) of abnormal size. (C) One capsid buds (b) at the nuclear (n) periphery. The ER is dilated and filled with virions (Ve) and dense material: An ER membrane turns into a Golgi membrane (thick arrow). (D) After exposure to BFA from 8 to 15 hpi with wt HSV-1, the ER was dilated and contained some virions. (E) The ER was almost filled with virions after exposure to BFA from 8 to 17 hpi with wt HSV-1. Note that virions in the PNS and ER are covered by a dense coat hiding spikes whereas spikes are clearly apparent on virions in the extracellular space (C inset). Bars: 200 nm.\n\nBFA was added to monolayers at 5, 8, 12 or 16 hpi (MOI of 5) and incubated until 20 hpi. For control, inoculated cells were incubated for 20 h without addition of BFA. Cells were rapidly frozen at 20 hpi and processed for electron microscopy. The phenotypes of envelopment were counted in 10 cellular profiles of 5 independent experiments: Capsids budding at the INM (B-INM), at the ONM and ER membranes (B-ONM/ER) and at the Golgi complex (B-Golgi); virions in the PNS-ER compartment (PNS-ER); virions derived by wrapping (Wrapp); virions in Golgi cisternae or large vacuoles (Golgi); capsids in the cytoplasmic matrix (capsids).\n\nUs3 is not essential (Poon et al., 2006; Reynolds et al., 2002; Ryckman & Roller, 2004; Wisner et al., 2009). Us3 deletion mutants accumulating in the PNS are infective (Wild et al., 2015). Hence, it is reasonable to assume that wt HSV-1 virions in the PNS-ER compartment are also infective. To prove this idea, we determined infectious progeny virus by plaque titration at the time point of BFA administration and at 20 hpi. The Golgi complex completely disintegrates within less than 5 minutes after exposure of cells to BFA (Hess et al., 2000). Despite of Golgi disintegration infectious progeny virus was produced in a time depended manner. The later BFA was added the more infectious viruses were produced by 20 hpi (Figure 7). Since virions accumulated exceptionalness in the PNS-ER compartment in BFA exposed cells we conclude that virions derived by budding at nuclear membranes are infective.\n\nThe difference between virus yields (indicated with numbers) at the time of BFA addition and harvesting at 20 hpi is considered to be due to virus production after Golgi disintegration. These infectious virions correspond to the virions accumulating in the PNS-ER compartment. n = 4, p<0.01.\n\nThe nuclear envelope is part of the ER. The outer nuclear membrane (ONM) is stubbed with ribosomes. The ONM continues into ER cisternae, which, in turn, merge with Golgi cisternae. Golgi cisternae were also found to connect to the PNS via short ER-Golgi intermediates in R7041(ΔUs3) infected Vero cells (Figure 8ABC) and BoHV-1 infected MDBK cells (Figure 8D). ER-Golgi intermediates contained virus like particles (Figure 8B). The continuum between PNS and Golgi cisternae is considered likely to serve as a direct, short and efficient pathway to transport virions form the site of budding to Golgi cisternae for packaging.\n\n(A) Golgi (go) membranes continue (thick arrow) into the ONM (o) as well as towards the cytoplasm indicated by (?) because the destination is unknown. (B) Golgi membranes continue via ER membranes (er) into the ONM. The ER contains 4 virus-like particles. (C) Details of panel B. (D) PNS, ER and Golgi complex form an entity in a BoHV-1 infected MDBK cell (D: This figure has been reproduced with permission of P. Wild et al., Micron 33, 2002, Elsevier). Bars 200 nm.\n\nCapsids are postulated to be enveloped at the trans Golgi network (Mettenleiter et al., 2006) by a process designated wrapping. However, capsids can bud at any location of the Golgi complex (Figure 9B) and vacuoles as have been shown for HSV-1 (Leuzinger et al., 2005; Stannard et al., 1996), BoHV-1 (Wild et al., 2002) and pseudorabies virus (Klupp et al., 2008), and even at microsomes (Albecka et al., 2016; Hollinshead et al., 2012). The result of wrapping is a small concentric vacuole (Figure 9D) containing a single virion as shown elsewhere in detail (Leuzinger et al., 2005; Wild et al., 2005). Golgi cisternae and vacuoles can contain one to numerous virions in a given section plane (Figure 9A). The cavities at the trans face of the Golgi complex in Figure 9A are more likely to represent cisternae rather than vacuoles because of their shape and location. The virions had gained access either by budding or by intraluminal transportation via ER-Golgi intermediates, as might be the case also in Figure 9C. Note that the viral envelope including spikes are covered by a dense layer in narrow Golgi cisternae (Figure 9C) whereas spikes are visible on virions in wide Golgi cisternae or large vacuoles (Figure 5 inset) and in the extracellular space (Figure 9E) as shown previously (Leuzinger et al., 2005; Wild et al., 2005). From the facts that virions are within ER and Golgi cisternae, and that the ONM continues via ER membranes into Golgi membranes forming an entity, we postulate that virions can be intraluminally transported from the PNS via ER into Golgi cisternae.\n\nMany of them are tangentially (t) sectioned. (B) Budding BoHV-1 capsid at a Golgi membrane of the cis-face (arrow). (C) HSV-1 virion in a Golgi cisterna that connects to the ER (arrow). Note the dense content within the ER and Golgi cisterna indicating little loss of material during processing. (D) Concentric vacuole derived by wrapping containing a single BoHV-1 virion. The space between viral envelope and vacuolar membrane is always filled in well preserved cells. (E) Virions in a large vacuole or cisterna exhibiting clearly spikes even in tangentially (t) sectioned virions. Bars: 200 nm.\n\nER-to-Golgi transitions are not only established in infected cells, but also in cultured epithelial cells (Figure 10A) or in cells in organs, e.g. parathyroid gland, which was prepared by perfusion fixation (Wild et al., 1985) according to conventional protocols (Figure 10B). These observations suggest that ER to Golgi transitions may also serve as a direct transportation route e.g. for proteins to be finally released by exocytosis.\n\n(A) TEM image of a cultured epithelial cell in which the Golgi complex is embedded in the ER. The ER membranes (arrows) run into the Golgi complex, close to a structure that probably represents a tangential section of the Golgi organizing center. (B) TEM image of a parathyroid cell prepared according to conventional protocols showing Golgi membranes (go) continuing into ER membranes (er). Bars: 200 nm.\n\n\nDiscussion\n\nThe Golgi complex is among the first organelles that rapidly disintegrate during processing for electron microscopy after improper fixation and processing (Han et al., 2013; Wild et al., 2001). To minimize disintegration, we employed a technique that leads to improved retention of cellular material (Cope & Williams, 1969; Weibull et al., 1984), and to improved spatial and temporal resolution (Mueller, 1992). This is especially important for analytical studies of cells in which the Golgi complex is involved in highly dynamic processes such as packaging of proteins into granules (Ellinger et al., 2010; Orci et al., 1981; Wild et al., 1982) in the secretory pathway, or envelopment of capsids and vacuole formation (Figure 11) for delivery of hundreds of virions to the cell periphery (Leuzinger et al., 2005; Wild et al., 2015; Wild et al., 2002). Because of the difficulties in preservation of the Golgi ultrastructure (Ellinger et al., 2010; Han et al., 2013; Wild et al., 2001), its three-dimensional structure is poorly understood. Cryo-FESEM revealed the Golgi complex to be a complex tightly packed structure. The membrane of the outermost cisternae may completely cover the cis-face. TEM also revealed that the Golgi complex is embedded in the ER system, with multiple membrane connections forming a Golgi-ER entity.\n\n(1) Capsids bud at the INM into the PNS acquiring tegument and an envelope covered with a dense coat. These perinuclear virions are transported into the RER and further via Golgi transitions (1a) or the ERGIC (“hug-and-kiss”, 1b) into Golgi cisternae where they are packaged into transport vacuoles, which are detached from Golgi membranes by fission. The dense coat is shed off while vacuoles are transported to the cell periphery for exocytotic release of uncoated virions into the extracellular space. (2) Capsids gain direct access to the cytoplasmic matrix via dilated nuclear pores (dNP), and are transported to any site of the Golgi complex. They either bud into Golgi cisternae and vacuoles, respectively (2a) or are enveloped by a process designated wrapping (2b) that involves budding and concomitant formation of a small transport vacuole engulfing a single virion. Occasionally, capsids may bud at the OM or RER (2c), and the resulting virions are intraluminally transported as in pathway 1. (3) After budding at the INM, capsids and tegument of perinuclear virions are released into the cytoplasmic matrix via fusion of the viral envelopment (including dense coat) with the ONM (de-envelopment). Capsids are then re-enveloped at the trans Golgi network (TGN) by wrapping. Finally, vacuoles derived by fission from Golgi membranes transport virions to the cell periphery and release them into the extracellular space via exocytosis. The dense coat, which derived during the budding process at the INM and probably protects the viral envelope from fusion with membranes the virions are transported along, is shed of (de-coating) in transport vacuoles at latest when virions are released into the extracellular space. During budding at Golgi cisternae and vacuoles, a dense rim of tegument is closely attached to the inner layer of the viral envelope. No dense coat is formed so that spikes (glycoproteins) are readily seen in high resolution micrographs.\n\nThe Golgi complex fragments and disperses about 16 hpi with HSV-1 (Campadelli et al., 1993) adding additional difficulties for understanding Golgi function in herpes virus envelopment. To address the significance of the Golgi complex in virus envelopment and virus transportation, we thus investigated infected cells between 8 hpi (the approximate time of onset of envelopment) and 16 hpi. Our data clearly show that the Golgi complex is localized in a juxtanuclear position appearing as a compact entity by cryo-FESEM. Golgi membranes continue into ER membranes which in turn connect to the ONM forming a continuum between Golgi cisternae and PNS. Thus, the presence of virions within the PNS, ER cisternae and Golgi cisternae strongly suggests that the ER-to-Golgi transition is used as a direct intraluminal pathway to deliver virions from the PNS into Golgi cisternae (Figure 11, pathway 1). This idea is supported by the fact that about 80 HSV-1 virions per mean cell volume were within ER cisternae at 12 and 16 hpi but close to 300 by 24 hpi (Wild et al., 2015) suggesting that virus transportation out of the ER is inhibited after Golgi fragmentation (Campadelli et al., 1993). Virus transportation out of the ER is also drastically inhibited after BFA exposure. The ER delates and secretory protein transport from the ER to the Golgi complex is impeded after BFA treatment (Fujiwara et al., 1988; Misumi et al., 1986). Hence, the integrity of the Golgi complex is crucial for export of both secretory proteins and HSV-1 out of the ER suggesting that HSV-1 release from the ER to the Golgi complex follows a similar pathway as secretory proteins either by vesicle formation involving cop II (Klumperman, 2000) or equivalent, or via ER-Golgi transitions.\n\nAccording to the currently stressed herpes virus egress theory (Figure 11, pathway 3), formation of infectious herpes viruses follows a complicated uneconomic pathway involving primary envelopment by budding of capsids at the INM, de-envelopment of capsids by fusion of the viral envelope with the ONM releasing capsids and tegument into the cytoplasmic matrix, and re-envelopment by wrapping at the trans Golgi network. The interaction of the viral envelope with the ONM was described the first time in 1968 (Darlington & Moss, 1968) and identified as budding of capsids from the cytoplasmic matrix into the PNS. About 30 years later, it was tried to prove that this process is fusion (Kopp et al., 2002; Naldinho-Souto et al., 2006; Skepper et al., 2001). Fact is that the phenotypes of the process taking place at the ONM are identical with those at the INM (Darlington & Moss, 1968; Leuzinger et al., 2005; Wild et al., 2005; Wild et al., 2012) and that they show all characteristics of budding. Budding requires proteins that are able to induce positive and negative curvatures. Budding at the INM is driven by UL31/UL34 (Bigalke & Heldwein, 2016; Bigalke & Heldwein, 2017; Bigalke et al., 2014; Hagen et al., 2015). UL31 and UL34 are also present at the ONM even in cells infected with Us3 deletion mutants (Reynolds et al., 2002) those envelopes are unable to fuse with the ONM (Reynolds et al., 2002; Wisner et al., 2009). Therefore, the presence of UL31/UL34 at the ONM cannot be the result of membrane transportation from the INM to the ONM via budding and subsequent fusion as often used as arguments for the presence of viral proteins at the ONM. Furthermore, UL34 was shown to localize at the ER (Yamauchi et al., 2001) and that UL31 is required for its dislocation to the INM. Recently, it was accidentally proved that the virus interaction at the ONM and ER membranes is budding rather than fusion. Glycoproteins B and H (gB/gH), members of the quartet responsible for cell entry (Turner et al., 1998), have been claimed to be responsible for fusion of the viral envelope with the ONM (Farnsworth et al., 2007). Electron microscopy strikingly revealed virus interactions with the ONM and ER membranes showing all characteristics of budding in cells infected with a gB/gH deletion mutant. Since the viral envelope cannot fuse in the absence of gB/gH the phenotypes shown in Figure 2 of this report (Farnsworth et al., 2007) represent undoubtedly various stages of capsids budding from the cytoplasmic matrix into the PNS and ER cisternae. Furthermore, in Vero cells infected with the gB/gH deletion mutant, three times more virions were found in the cytoplasm compared to wild type infected Vero cells, and about a third in the extracellular space. Similar relations though less pronounced were found in other cell types absolutely implying another transportation route than that of de-envelopment by fusion of the viral envelope with the ONM.\n\nWrapping demands an enormous amount of membranes that would need to be provided to the TGN, which, to our knowledge, is not clear yet. However, capsids can be wrapped at diverse sides of the Golgi complex or can bud into Golgi cisternae and/or vacuoles via another pathway than wrapping. There are numerous reports showing HSV-1 virions in vacuoles and/or Golgi cisternae (Homman-Loudiyi et al., 2003; Leuzinger et al., 2005; Stannard et al., 1996; Sutter et al., 2012; Wild et al., 2015; Wild et al., 2002). Budding of capsids at various sites of Golgi membranes as well as virions in Golgi cisternae and vacuoles were also shown in pseudorabies virus infected cells (Klupp et al., 2008). These virions in all these vacuoles and/or Golgi cisternae did not arise by wrapping because wrapping results in a single virion in a concentric vacuole. They may have entered Golgi cisternae by budding into them or by intraluminal transportation via ER-to-Golgi transitions. Transient elements from cis and trans Golgi sides have been shown in various cells (Pavelka & Roth, 2015). It was also suggested that the cis-Golgi approaches the ER and contacts the ER exit sites in the yeast Saccharomyces cerevisiae to capture cargo for transportation to the Golgi complex (Kurokawa et al., 2014). This ‘hug-and-kiss’ behavior could be another route to transfer virions, which are intraluminally transported to ER exit sites, into the Golgi cisternae.\n\nIn cells prepared for improved resolution, we found no valid arguments for de-envelopment by fusion of the viral envelope with the ONM. There are a number of facts that argue clearly against the de-envelopment theory. First, the morphology of the process taking place at the INM and ONM are identical showing all characteristics for budding (Darlington & Moss, 1968; Leuzinger et al., 2005; Wild et al., 2005; Wild et al., 2012) but none for fusion (Haluska et al., 2006; Kanaseki et al., 1997) considering fundamentals of membrane bound transportation (Leabu, 2006; Peters et al., 2004; White, 1992). Second, virions have been repeatedly shown within ER cisternae (Gilbert et al., 1994; Granzow et al., 1997; Leuzinger et al., 2005; Radsak et al., 1996; Schwartz & Roizman, 1969; Stannard et al., 1996; Sutter et al., 2012; Wild et al., 2002). To reach this location, either virions need to be transported from the PNS into the ER, or capsids have to bud from the cytoplasmic matrix into the ER. If virions can be intraluminally transported out of the PNS the viral envelope must be protected from fusion with membranes the virions are transported along. If capsids have the ability to bud at ER membranes capsids are very likely to be able to bud at the ONM since the ONM is part of the ER. Third, virions can accumulate to large numbers in the PNS, e.g. in the absence of the protein kinase Us3 (Poon et al., 2006; Reynolds et al., 2002; Wild et al., 2015). Strangely enough, these virions are infective (Reynolds et al., 2002; Ryckman & Roller, 2004; Wild et al., 2015) despite the inability of the envelope to fuse with the ONM (Wisner et al., 2009) clearly contradicting the theory that de- and re-envelopment is essential to become infective (Klupp et al., 2011). Fourth, the equivalent to budding is the formation of coated pits resulting in coated vesicles (Owen & Luzio, 2000; Pearse et al., 2000). Coated vesicles derive e.g. from the plasma membrane and are transported towards the Golgi complex where they fuse with Golgi membranes (Orci et al., 1981). However, coated vesicles must be uncoated to gain the ability for fusion. The coat consists of clathrin that drives formation of coated pits and finally coated vesicles and protects them from fusion. Clathrin was claimed to be involved in envelopment of HSV-6 capsids at Golgi membranes together with viral proteins (Mori et al., 2008). Budding of HSV-1 capsids at the INM is driven by the nuclear envelopment complex, UL31/UL34, (Bigalke & Heldwein, 2015; Bigalke et al., 2014; Hagen et al., 2015) located at the nuclear rim (Mou et al., 2009).\n\nBudding at the INM, ONM and ER membranes starts with deposition of dense substances that finally result in a dense coat at the viral envelop. The dense coat is readily seen on virions in the PNS, ER and, inconsistently, in Golgi cisternae and large vacuoles containing many virions. The dense coat suggests that it protects virions from fusion with membranes the virions is transported along but allows virus transportation from the PNS into ER and Golgi cisternae. However, in large Golgi cisternae and/or vacuoles, many virions are without dense coat. Instead, spikes are visible like at virions in the extracellular space. Budding at Golgi membranes takes place without dense coat formation.\n\n\nConclusions\n\nGolgi membranes interconnect with ER membranes in cells infected with HSV-1, a Us3 deletion mutant thereof or with BoHV-1 as well as in uninfected cells. The ER continues into the ONM, that turns into the INM at sites of nuclear pores. Consequently, the PNS, ER and Golgi complex forms an entity that can be only visualized by TEM, either when the membranes of all three compartments are luckily hit in the same plane of a given section, or by 3D-reconstruction after imaging of serial sections, focused ion beam (FIB) microscopy or electron tomography. Fact is that virions are intraluminally transported out of the PNS into the ER. This implies that the viral envelope needs to be protected from fusion with the membranes the virions are transported along. Thus, the significance of the dense coat, which derives during budding of capsids at nuclear membranes, is protecting the viral envelope from fusion in a similar manner as clathrin protects coated vesicles from fusion. The ER-to-Golgi transitions, virions in Golgi cisternae with a similar dense coat as virions in the PNS and ER, and inhibition of virion transportation out of the PNS and ER after disruption of the Golgi complex by BFA, strongly suggest that virions are intraluminally transported from the PNS through the ER into Golgi cisternae. We propose that this is the only pathway for virions out of the PNS, and that capsids gain direct access to the cytoplasm from the nucleus via dilated nuclear pores and impaired nuclear envelope.\n\n\nData availability\n\nDataset 1: Raw images for Figure 1–Figure 5, Figure 8–Figure 10. DOI, 10.5256/f1000research.12252.d179644 (Wild et al., 2017a)\n\nDataset 2: Raw values for Figure 6 and Figure 7. DOI, 10.5256/f1000research.12252.d179645 (Wild et al., 2017b)",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by the Foundation for Scientific Research at the University of Zürich, Switzerland.\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 Bernard Roizman, University of Chicago, for providing the Us3 deletion mutant.\n\n\nReferences\n\nAlbecka A, Laine RF, Janssen AF, et al.: HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis. Traffic. 2016; 17(1): 21–39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBen-Tekaya H, Miura K, Pepperkok R, et al.: Live imaging of bidirectional traffic from the ERGIC. J Cell Sci. 2005; 118(Pt 2): 357–367. PubMed Abstract | Publisher Full Text\n\nBigalke JM, Heldwein EE: Structural basis of membrane budding by the nuclear egress complex of herpesviruses. 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}
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[
{
"id": "26712",
"date": "10 Oct 2017",
"name": "Charles Grose",
"expertise": [
"Reviewer Expertise Varicella zoster virus"
],
"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\nProfessor Wild and his collaborators are well known for their expertise in electron microscopy. I also note that three other scientists who developed cryo-TEM won the Nobel Prize this year. The SEM and TEM images in this manuscript are excellent. I enjoyed reviewing their data. I have heard Professor Wild present his results at conferences. However, I can say upfront that most recent herpesvirus data appear to support pathway #3 in Figure 11, which is not the pathway preferred by the Wild lab. For example, the authors do not consider that both the Elliott HSV lab and the C. Grose VZV lab have found evidence for Rab11 on the vesicles that transport enveloped virus. If the above two labs are correct, the pathway #1 in Figure 11 cannot be completely correct because the drawings do not include the endocytic pathway. Thus, the experiment requested in Comment 6 needs to be performed to resolve this question. Overall, the suggestion is made that the Wild lab take a look at some of the VZV articles and include them in their discussion, even if the Wild lab disagree with the conclusions of the VZV lab.\n\nFigure 1. Confocal microscopy of Vero cells. The text states that both WT and mutant virus were tested but only one virus is shown in Figure 1. Which virus is shown? If only WT virus is shown, may wish to add confocal with mutant virus.\n\nFigures 4 and 5. Virions in ER cisternae. The C. Grose lab has published several articles about VZV encapsidation, VZV capsid transport from nucleus to cytoplasm and subsequent envelopment. These articles should be reviewed and cited by Wild et al. First of all, Wild should show us whether he finds viral particles between the INM and the ONM. For VZV, Harson and Grose (1995) have clearly shown that capsids with a thin envelope can be found between the INM and the ONM. See Harson and Grose, Figure 5, panels C, D and E. Note increase in diameter of particle between panels C and E. Panel F is important because panel F shows a thin-enveloped particle still within the ER cisternae and clearly beyond the ONM. Thus, the VZV micrograph agrees with the data from Wild about “Virions are within ER cisternae.”\n\nData not shown by Wild lab. Harson and Grose have shown that VZV capsids without a thin envelope can be found in the cytoplasm. The capsids do not appear to be within any Golgi derived compartment. See Figure 6 panel A, (Harson & Grose, 1995). This micrograph provides evidence against the Wild hypothesis that herpesviruses are always retained within compartments derived from the ER or Golgi. There is one micrograph in the Wild manuscript that appears to resemble the VZV micrograph. See Wild manuscript Figure 9B, which shows a BoHV capsid (without a thin envelope) in the cytoplasm adjacent to a Golgi (or ER/Golgi) membrane. This single BoHV micrograph would seem to invalidate the Wild hypothesis that BoHV always is transported within ER or Golgi compartments?\n\nDifferences between viral particles in ER cisternae and viral particles in vesicles near the outer cell membrane. As noted above, data from the Grose lab agree with data from the Wild lab that viral particles are found in ER cisternae. But the Grose lab finds differences between particles in ER cisternae and particles in vesicles near the outer cell membrane. ER cistern are irregularly shaped. Particles within ER cisternae have a thin envelope without an obvious electron dense outer coat (presumably the viral glycoproteins). Vesicles containing viral particles near the outer cell membrane often are circular with a single outer membrane. See Figure 8 panel A in Harson & Grose (1995), for a clear VZV example. The vesicle contains 4 particles, 2 of which are perfectly enveloped, with clearly defined capsid, tegument and envelope with electron-dense outer coat. These particles are slightly larger in diameter than the thin-enveloped particles seen in the irregular ER cisternae. The VZ virions in the circular vesicles are similar to the BoHV virions shown by Wild in his Figure 9 panels D and E. In other words, there clearly are two types of enveloped particles: particles with thin-envelope and particles with a thicker electron-dense envelope.\n\nVirions are within Golgi cistern? Wild discusses viral particles with spikes and viral particles without obvious spikes. Spikes presumably represent mature glycoproteins. One possibility that Wild has not considered is that viral particles may be routed in different pathways. Some viral particles may enter the large Golgi-like cisternae. Other viral particles may be wrapped in individual vesicles. Why does there need to be a single pathway for all viral particles?\n\nLack of data about endocytic or autophagy pathways. There are now HSV and VZV papers that provide evidence for involvement of the endocytic and/or autophagy pathways in viral egress. The Elliott lab has shown that both Rab5 and Rab11 are involved in the final envelopment process for HSV. Further, they did not find co-localization of capsids with the TGN marker TGN46. See Hollinshead et al. 2012. The C. Grose lab confirmed that a fraction of purified VZ virions were positive for the Rab11 protein, presumably because the purified virions retained remnants of a Rab11-positive vesicle. See VZV article by Buckingham et al, 2016. The C. Grose lab also found the LC3-II protein in the same purified virion fraction and hypothesized that the virions were housed in amphisome-like vesicles, which contain both Rab11 and LC3-II proteins in their membrane. The fact that a HSV lab and a VZV lab both found Rab11 in the virion greatly strengthens the observation. These data are presented because they suggested that the Wild lab cannot determine the origin of the vesicles containing either HSV or BoHV virions by using only TEM and SEM. The Wild lab will need to use either immuno-TEM or alternatively isolate the HSV and BoHV particles, then examine them for proteins such as Rab11 and LC3-II by immunoblotting.\n\nFigure 11. Schematic drawings. If the Elliott lab and the Grose lab are correct that purified HSV or VZV contain Rab11 protein, then schematic pathway #1 in Figure 11 cannot be correct. Also, if the Elliott lab and the Grose lab are correct, then secondary envelopment must involve an intersection of the virus with a pathway outside of the ER/Golgi pathway. Strongly suggest that the Wild lab investigate whether any of the vacuoles that contain either HSV or BoHV are positive for the Rab11 protein. Electron microscopy alone is not sufficient.\n\nFigure 7. Virus titers. cell by endocytosis. The titers at 8hpi probably represent viral particles formed after some actual HSV glycoprotThe data about the different titers with and without BFA may need re-interpretation. The titer is much lower when BFA was added at 5hpi. Most of these particles would be capsids and capsids with thin-envelopes. A titer of 1000 PFU/ml seems reasonable for capsids, which could enter a ein biosynthesis. Some VZV glycoproteins undergo their biosynthesis within 2 hours. See article from Grose lab by Yao et al, 1993. Presumably HSV glycoproteins are synthesized at the same rate. Therefore, addition of BFA at 8hpi would not prevent formation of substantial amounts of HSV glycoproteins, which would allow envelopment of viral particles, even by 8 hpi. In other words, I do not think that the BFA experiments add much weight to the arguments about which pathway HSV takes\n\nBovine herpesvirus data. I do find inclusion of the BoHV micrographs to be informative. The BoHV data appear to resemble the VZV data more closely than the HSV data. Remember that BoHV is a member of the varicellovirus subfamily.\n\nSchematic pathway for VZV trafficking. Please take the time to look over the VZV pathway illustrated in Figure 8, in article by Buckingham et al, 2016. It resembles pathway #3 by the Wild group.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "3508",
"date": "15 Mar 2018",
"name": "Peter Wild",
"role": "Author Response",
"response": "Professor Wild and his collaborators are well known for their expertise in electron microscopy. I also note that three other scientists who developed cryo-TEM won the Nobel Prize this year. The SEM and TEM images in this manuscript are excellent. I enjoyed reviewing their data. I have heard Professor Wild present his results at conferences. However, I can say upfront that most recent herpesvirus data appear to support pathway #3 in Figure 11, which is not the pathway preferred by the Wild lab. For example, the authors do not consider that both the Elliott HSV lab and the C. Grose VZV lab have found evidence for Rab11 on the vesicles that transport enveloped virus. If the above two labs are correct, the pathway #1 in Figure 11 cannot be completely correct because the drawings do not include the endocytic pathway. Thus, the experiment requested in Comment 6 needs to be performed to resolve this question. Overall, the suggestion is made that the Wild lab take a look at some of the VZV articles and include them in their discussion, even if the Wild lab disagree with the conclusions of the VZV lab. Response: Thank you for the compliment. It is great honour to have become influenced by Jacques Dubochet, one of the Nobel prize winners on cryo-TEM. I am also very grateful for the impact of another Nobel prize winner, George Palade, whose laureate speech entitled “Variation on a common theme in the secretory pathway” published in Science,1975, is still a must to be read by people working in this field, including herpes virologists. The intention of this work is to demonstrate connectivity between the PNS and the Golgi complex that may be used as a direct transport pathway for virions derived by budding at nuclear membranes. Budding at nuclear membranes followed by intraluminal transport is referred to as pathway 1. That does not mean that our lab prefers pathway 1. We also showed that capsids gain access to the cytoplasmic matrix via impaired nuclear pores and impaired nuclear envelope, respectively. These capsids follow envelopment pathway 2. In this pathway, the endocytic pathway will be included (we did not because, we shall discuss it in detail in another manuscript). Pathway 3 is obsolete, because the process at the outer nuclear membrane is budding not fusion as accidentally proved by Farnsworth et all, 2007, overlooking the process taking place at the ONM (see Fig.12). Figure 1. Confocal microscopy of Vero cells. The text states that both WT and mutant virus were tested but only one virus is shown in Figure 1. Which virus is shown? If only WT virus is shown, may wish to add confocal with mutant virus.Response: The location of the Golgi complex depends on the cell, not on the virus. Nevertheless, we included micrographs of Golgi labelled with antibodies against the cis and the trans face in cells infected with HSV-1 and the Us3 deletion mutant thereof. Fragmentation of the Golgi complex depends on the virus. E.g. in Us3 deletion mutant infected cells, the Golgi complex enlarges, A manuscript addressing the significance of Us3 on the Golgi complex is in progress. Figures 4 and 5. Virions in ER cisternae. The C. Grose lab has published several articles about VZV encapsidation, VZV capsid transport from nucleus to cytoplasm and subsequent envelopment. These articles should be reviewed and cited by Wild et al. First of all, Wild should show us whether he finds viral particles between the INM and the ONM. Response: We have shown many times virions between the INM an ONM as well as in adjacent cisternae. However, we never found virions with a thin envelope. This might be related to the different fixation and preparation procedure.For VZV, Harson and Grose (1995) have clearly shown that capsids with a thin envelope can be found between the INM and the ONM. See Harson and Grose, Figure 5, panels C, D and E. Note increase in diameter of particle between panels C and E. Panel F is important because panel F shows a thin-enveloped particle still within the ER cisternae and clearly beyond the ONM. Thus, the VZV micrograph agrees with the data from Wild about “Virions are within ER cisternae.” Data not shown by Wild lab. Harson and Grose have shown that VZV capsids without a thin envelope can be found in the cytoplasm.Response: We have shown capsids in the cytoplasm in this manuscript (Figs.4B and 9B) and plotted the total number of capsids in the cytoplasm in Fig. 6. We also showed capsids in many articles published earlier, e.g. Wild et al. 2005 (impairment of nuclear pores) or Leuzinger et al.2005 (The dual pathway). In these articles the difference between budding at Golgi membranes and wrapping by Golgi membranes is clearly shown. The capsids do not appear to be within any Golgi derived compartment. See Figure 6 panel A, (Harson & Grose, 1995). This micrograph provides evidence against the Wild hypothesis that herpesviruses are always retained within compartments derived from the ER or Golgi. There is one micrograph in the Wild manuscript that appears to resemble the VZV micrograph. See Wild manuscript Figure 9B, which shows a BoHV capsid (without a thin envelope) in the cytoplasm adjacent to a Golgi (or ER/Golgi) membrane. This single BoHV micrograph would seem to invalidate the Wild hypothesis that BoHV always is transported within ER or Golgi compartments?Response: As discussed above, we postulate 2 pathways: 1) Budding at nuclear membranes followed by intraluminal transportation, 2) Release via impairs nuclear envelope followed by budding into Golgi (and other) membranes, or by wrapping (see Leuzinger et al, 2005, Wild et.al. 2005 and 2009). In introduction, we inserted the pathway via vesicle formation. We have also observed some formation of vesicles late in infection. Differences between viral particles in ER cisternae and viral particles in vesicles near the outer cell membrane. As noted above, data from the Grose lab agree with data from the Wild lab that viral particles are found in ER cisternae. But the Grose lab finds differences between particles in ER cisternae and particles in vesicles near the outer cell membrane. ER cistern are irregularly shaped. Particles within ER cisternae have a thin envelope without an obvious electron dense outer coat (presumably the viral glycoproteins). Vesicles containing viral particles near the outer cell membrane often are circular with a single outer membrane. See Figure 8 panel A in Harson & Grose (1995), for a clear VZV example. The vesicle contains 4 particles, 2 of which are perfectly enveloped, with clearly defined capsid, tegument and envelope with electron-dense outer coat. These particles are slightly larger in diameter than the thin-enveloped particles seen in the irregular ER cisternae. The VZ virions in the circular vesicles are similar to the BoHV virions shown by Wild in his Figure 9 panels D and E. In other words, there clearly are two types of enveloped particles: particles with thin-envelope and particles with a thicker electron-dense envelope. Response: Using freezing and substitution techniques we did not find differences in the viral envelope of virions in the PNS and ER. The virus particles in Harson at al. differ most probably because of preservation quality (in panel 8A much of the substances are lost) and of the section plane.Virions are within Golgi cistern? Wild discusses viral particles with spikes and viral particles without obvious spikes. Spikes presumably represent mature glycoproteins. One possibility that Wild has not considered is that viral particles may be routed in different pathways. Some viral particles may enter the large Golgi-like cisternae. Other viral particles may be wrapped in individual vesicles. Why does there need to be a single pathway for all viral particles?Response: As stated above, we propose two diverse pathways indicated in Fig, xx by 1 and 2. However, we do not agree with the postulation, that de-envelopment followed by re-envelopment is essential for production of infectious progeny virus because Us3 deletion mutants are fully effective although 98% of these virions accumulate in the PNS and adjacent ER. Lack of data about endocytic or autophagy pathways. There are now HSV and VZV papers that provide evidence for involvement of the endocytic and/or autophagy pathways in viral egress. The Elliott lab has shown that both Rab5 and Rab11 are involved in the final envelopment process for HSV. Further, they did not find co-localization of capsids with the TGN marker TGN46. See Hollinshead et al. 2012. The C. Grose lab confirmed that a fraction of purified VZ virions were positive for the Rab11 protein, presumably because the purified virions retained remnants of a Rab11-positive vesicle. See VZV article by Buckingham et al, 2016. The C. Grose lab also found the LC3-II protein in the same purified virion fraction and hypothesized that the virions were housed in amphisome-like vesicles, which contain both Rab11 and LC3-II proteins in their membrane. The fact that a HSV lab and a VZV lab both found Rab11 in the virion greatly strengthens the observation. These data are presented because they suggested that the Wild lab cannot determine the origin of the vesicles containing either HSV or BoHV virions by using only TEM and SEM. The Wild lab will need to use either immuno-TEM or alternatively isolate the HSV and BoHV particles, then examine them for proteins such as Rab11 and LC3-II by immunoblotting. Response: The intention of this manuscript is to discover a possible intraluminal transportation route from the PNS into Golgi cisternae. No more no less. By no means does it question the existence of an “endocytic” pathway. Therefore, there is no need to repeat experiments other groups have done as cited for HSV-1 in introduction (Albecka et al. 2016, Holinstead et al., 2012). We will describe in detail this pathway in another manuscript. Considering membrane recycling, it would be a great surprise if an “endocytic” pathway would not exist. However, I do not think that capsids bud at endosomes per se. Possibly, they rather bud at membrane compartments that contain recycled (endosomal) membrane components. Figure 11. Schematic drawings. If the Elliott lab and the Grose lab are correct that purified HSV or VZV contain Rab11 protein, then schematic pathway #1 in Figure 11 cannot be correct. Also, if the Elliott lab and the Grose lab are correct, then secondary envelopment must involve an intersection of the virus with a pathway outside of the ER/Golgi pathway. Strongly suggest that the Wild lab investigate whether any of the vacuoles that contain either HSV or BoHV are positive for the Rab11 protein. Electron microscopy alone is not sufficient.Response: The endocytic pathway will be included in Fig. 11. For simplicity, we removed pathway 3 because we did not find correct proves it. Instead, we introduced Figure 2 of Farnsworth et al., 2007 showing transport of capsids across the ONM and ER membranes in the absence of the fusion proteins gB/gH and accumulation of virions in the PNS-ER compartment.Figure 7. Virus titers. cell by endocytosis. The titers at 8hpi probably represent viral particles formed after some actual HSV glycoprotThe data about the different titers with and without BFA may need re-interpretation. The titer is much lower when BFA was added at 5hpi. Most of these particles would be capsids and capsids with thin-envelopes. A titer of 1000 PFU/ml seems reasonable for capsids, which could enter a ein biosynthesis. Some VZV glycoproteins undergo their biosynthesis within 2 hours. See article from Grose lab by Yao et al, 1993. Presumably HSV glycoproteins are synthesized at the same rate. Therefore, addition of BFA at 8hpi would not prevent formation of substantial amounts of HSV glycoproteins, which would allow envelopment of viral particles, even by 8 hpi. In other words, I do not think that the BFA experiments add much weight to the arguments about which pathway HSV takesResponse: Glycoprotein of HSV-1start to be synthesised prior to 4 hpi and are localized at the Golgi complex (unpublished data) which is in agreement with the biosynthesis of glycoproteins in VZV. In the work of de Oliviera et al., 2008, gH was found in the cytoplasm and at the nuclear periphery at 4hpi. The proteins at nuclear membranes are used for envelopment after disintegration of the Golgi complex. The only way capsids can be enveloped after disintegration of the Golgi complex is by budding at nuclear and ER membranes. An important question is: where the capsids get their envelope after fragmentation of the Golgi complex in HSV-1 infection (Campadelli-Fiume et al., 1993)? Bovine herpesvirus data. I do find inclusion of the BoHV micrographs to be informative. The BoHV data appear to resemble the VZV data more closely than the HSV data. Remember that BoHV is a member of the varicellovirus subfamily. Response: The images of BoHV nicely show connectivity between PNS and Golgi via RER as well as the juxtanuclear position of the Golgi complex. Therefore, it is an important image to understand the relation between Golgi, ER, and PNS. ER-to Golgi transitions have been shown in many cells and are not induced by herpes viruses (see also comments of Gareth Griffiths). Schematic pathway for VZV trafficking. Please take the time to look over the VZV pathway illustrated in Figure 8, in article by Buckingham et al, 2016. It resembles pathway #3 by the Wild group.Response: Pathway 3 was omitted in Fig. 11 because it is not our idea, and because the process at the ONM is not fusion (de-envelopment) as shown using a mutant lacking the fusion proteins gB/gH that results in accumulation of virions in the PNS (see Fig.12). By the way, to distinguish between budding and fusion is as easy as to distinguish between the right and left shoe."
}
]
},
{
"id": "26714",
"date": "20 Oct 2017",
"name": "Gareth Griffiths",
"expertise": [
"Reviewer Expertise EM",
"cell biology",
"virology",
"cellular 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\nThe paper by Wild and colleagues investigates the question of how Herpes virus assembles, a story that has been under debate for decades. This family of DNA viruses assemble in the nucleus and thereafter opinions differ as to how the virus gets out of the cell. The bulk of the data are based on high pressure freezing and freeze substitution and are of exceptionally high quality.\nHowever, I had a hard time trying to figure out what the point of the paper was. The title suggests a virus-induced ‘transition’ between ER and Golgi. I don't understand what they conclude about the ER-Golgi nor what it means for the virus. Virus-induced implies a difference between infected and uninfected cells. The only image of an uninfected cell is the IF image in Fig 1. I don't see much difference in the pattern of GM-130 labeling, a marker of early Golgi compartments. They show no EM images of uninfected cell Golgi.\nI had a serious problem also trying to understand how they could distinguish between the three models shown in Fig 11. I could not follow their logic. In my opinion to do this in a satisfactory was they would need to use host cell markers for the different Golgi compartments with use of some viral membrane markers. To be able to give directionality to their static images they would also need e.g an inducible system whereby they could block some stages of assembly and then release the block and follow kinetically where the accumulated viral proteins move to-with respect to host compartment markers. No compartment of the Golgi can be unequivocally identified without a marker.\nThe paper is not well described. For example the two virus mutants R7041 and R2641 are not well described. Why are they interesting? At the end of the discussion we learn that Us3 is a kinase. I could not find the interpretation for the acronym PNS, I eventually figured out it means peri nuclear space.\nIn the background part they describe how gB/gH are reported as being essential for de-envelopment –the fusion step after budding into the nuclear envelope. When these proteins are missing one would expect enveloped virus to accumulate in the NE/ER. Yet they write- under this condition lots of capsids (without membrane) accumulate- and even infectious virus- so there must be an alternative exit route. As a question of logic the reader may ask: maybe the idea that these proteins are essential for de-envelopment is wrong?\n\nTheir description of the data on Us3 deletion mutants is not well explained.\nThe close connection between the ER and the Golgi in uninfected cells has been seen by many EM studies, going back to Albert Claude 1970, Rambourg and many others. It is also not true as stated in the 1st paragraph of their Discussion that the Golgi ‘disintegrates rapidly after improper fixation’-unless one forgets to add the aldehyde. They show a nice example of normal fixation in Fig 10B. I take issue with their claim that the 3D structure of the Golgi is poorly understood because of poor preservation. It is poorly understood because the organelle is complex and because one lacks systems to identify the different compartments in the same sections. I of course accept their claim that freeze substitution is the preferred option.\nThe images from BFA treated cells are impressive but I’m not sure that I get a real take home message from these experiments. The Golgi fuses back with the ER; there is accumulation of budded virions in the ER. I cannot see a clear interpretation.\nThe long paragraph in the beginning of page 10, dealing with the mutants is especially difficult to follow- e.g what does ‘accidentally proved’ mean?\nAmong the many issues I have with their three models (Fig 11) I ask: how do they distinguish between budding into a Golgi compartment and wrapping?\n\nModel 1 is the only one devoid of A. cytoplasmic capsids and B. ‘wrapping’ profiles. Doesn't this rule out this model?\nThey poorly describe how they quantify virus particles in the different locations by EM. They must surely be referring the budding profiles to membrane length or compartment volume? (Page 4).\nThe Conclusion part is also very difficult to relate to.\nBottom of page 8 Dilates mis-spelt as ‘Delates’.\nBeginning of page 7 there is no word I know of in English – Exceptionalness’.\nIt is a pity that such technically nice work is not supported by clarity of writing.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "3434",
"date": "28 Feb 2018",
"name": "Peter Wild",
"role": "Author Response",
"response": "Thank you for carefully reading the manuscript. You are right that the title may imply virus-induced formation of ER-to-Golgi transitions. However, it also may imply that viruses utilize existing transitions to transport virions from the PNS (abbreviation explained in introduction) into Golgi cisternae. Golgi structure does not differ in uninfected and infected cells except that in infected cells Golgi cisternae contain virions, which is clearly shown in Figs. 3 and 8D. Cells harvested or fixed e.g. 6 hpi (hours post inoculation) does not necessarily mean, that the cell under study is infected or that the infection occurred at the very moment of inoculation. Therefore, the Golgi complexes shown in Figs 3 and 8D are not (or not yet) involved in envelopment or transportation although capsids have been produced yet not obviously released from the nucleus. Immunolabelling does also not differ between uninfected and infected cells early in infection. However, the Golgi complex fragments about 16 hpi with wt HSV-1 as shown by Campadelli-Fiume et al., 1993. The reaction of the Golgi complex to infection with Us3 deletion mutant differs, which will be addressed in another article. The significance of Us3 will be included in introduction. To use markers for the different Golgi compartments is an interesting issue. As shown in Fig. 1, the cis-Golgi disappears by 16 hpi, whereas the trans-Golgi became fragmented. In contrast, both cis- and trans-Golgi enlarge after infection with the Us3 deletion mutant. The significance of Us3 on the Golgi complex will be addressed elsewhere. Markers of the viral envelope do not really help to locate the virions because they are present in the cytoplasm without virions, and even before the egress of virions starts (see e.g. de Oliveira et al., 2008). For simplicity we will show in Fig. 11 only the 2 pathways we propose: 1) the intraluminal pathway via ER-to-Golgi transitions, and 2) the pathway involving capsid release via impaired envelope (starting by pore dilation) followed by envelopment at Golgi membranes either by wrapping or budding. We have published enough data to support the direct nucleus to cytoplasm capsid translocation as well as to differentiate between wrapping and budding at Golgi membranes and vacuolar membranes. Numerous articles have been published using EM techniques and immunolabelling techniques on the light microscopic and EM level to try to support the idea of the envelopment – de-envelopment – re-envelopment pathway. Therefore, we do not repeat these experiments. The idea of de-envelopment (fusion of the viral envelope of virions in the PNS with the ONM releasing capsids and tegument into the cytoplasmic matrix) came from Stackpole (1968) to explain the presence of naked capsids in the cytoplasm. However, there is no correct prove that the process taking place at the ONM is fusion. Fact is that the processes taking place at the ONM is identical to that taking place at the INM showing all characteristics of budding as shown in detail earlier (Leuzinger et a., 2005). Farnsworth et al., 2007 wanted to prove that the fusion proteins gB/gH are responsible for the fusion of the viral envelope with the ONM leading to accumulations of virions in the PNS. They show in their Fig. 2. - now included in this paper as Fig. 12 - accumulation of virions in the PNS. “Accidentally” - or by mistake -, they overlooked about a dozen of virus interactions at the ONM. If these interactions were fusion virions would not have accumulated in the PNS. Consequently, 1) the process taking place at the ONM is budding not fusion, 2) gB/gH play other roles in the egress pathway than mediating fusion of the viral envelope with the ONM, and 3) the envelopment – de-envelopment – re-envelopment theory is incorrect, as shown earlier using Us3 deletion mutants. Us3 deletion mutants are infective although they almost completely accumulate in the PNS implying that Us3 null virions are not de-enveloped and re-enveloped. The same is true for the virions originating by budding at the INM after disintegration of the Golgi complex by BFA. We have revised the manuscript implementing your suggestion, and simplified the complicated paragraph dealing with the false interpretation that gB/gH promote fusion of the viral envelop with the ONM The “simple” take home massages are: Virions derive by budding at the INM membrane are infective. The process taking place at the ONM is budding, not fusion. The de-envelopment – re-envelopment theory is not valid."
},
{
"c_id": "3506",
"date": "15 Mar 2018",
"name": "Peter Wild",
"role": "Author Response",
"response": "Comment: The paper by Wild and colleagues investigates the question of how Herpes virus assembles, a story that has been under debate for decades. This family of DNA viruses assemble in the nucleus and thereafter opinions differ as to how the virus gets out of the cell. The bulk of the data are based on high pressure freezing and freeze substitution and are of exceptionally high quality.However, I had a hard time trying to figure out what the point of the paper was. The title suggests a virus-induced ‘transition’ between ER and Golgi. I don't understand what they conclude about the ER-Golgi nor what it means for the virus. Virus-induced implies a difference between infected and uninfected cells. Response: Thank you for carefully reading the manuscript. You are right that the title may imply virus-induced formation of ER-to-Golgi transitions. However, it also may imply that viruses utilize existing transitions to transport virions from the PNS into Golgi cisternae. Virions are transported out of the PNS into ER cisternae. ER-Golgi transitions may thus be used to transport virions directly into Golgi cisternae. There are evidences that this idea is likely to be correct. The only image of an uninfected cell is the IF image in Fig 1. I don't see much difference in the pattern of GM-130 labeling, a marker of early Golgi compartments. They show no EM images of uninfected cell Golgi.Response: Golgi structure does not differ in uninfected and infected cells except that in infected cells Golgi cisternae contain virions, which is clearly shown in Figs. 3 and 8D. Cells harvested or fixed e.g. 6 hpi (hours post inoculation) does not necessarily mean, that the cell under study is infected or that the infection occurred at the very moment of inoculation. Therefore, the Golgi complexes shown in Figs 3 and 8D are not (or not yet) involved in envelopment or transportation although capsids might have been produced yet not obviously released from the nucleus and transported into the Golgi complex. Immunolabelling does also not differ between uninfected and infected cells early in infection. However, the Golgi complex fragments about 16 hpi with wt HSV-1 as shown by Campadelli-Fiume et al., 1993. The Golgi complex enlarges after infection with Us3 deletion mutant, which will be addressed in another article. The revised Fig. shows the cis and trans Golgi after infection with HSV-1 and the Us3 deletion mutant. I had a serious problem also trying to understand how they could distinguish between the three models shown in Fig 11. I could not follow their logic. Response: We can only distinguish between two pathways: 1) Via budding at nuclear membranes followed by intraluminal transportation, and 2) capsid release via impaired nuclear envelope (that starts by pore dilation) followed by envelopment at Golgi membranes or membranes of the ER. This can be easily done based on the diverse phenotypes. For details see Leuzinger 2005, Wild 2002, 2005, 2009Pathway 3 (which is omitted in the revised manuscript) is not our idea. The idea of de-envelopment (fusion of the viral envelope of virions in the PNS with the ONM releasing capsids and tegument into the cytoplasmic matrix) came from Stackpole (1968) to explain the presence of naked capsids in the cytoplasm. However, there is no correct prove that the process taking place at the ONM is fusion.. In my opinion to do this in a satisfactory was they would need to use host cell markers for the different Golgi compartments with use of some viral membrane markers. Response: Where is no need to distinguish between Golgi compartments in this context. The question is: can virions be transported from the ER into the Golgi to what compartment ever. However, to use markers for the different Golgi compartments is an interesting issue considering Golgi fragmentation as shown in the revised Fig 1. (work is in progress). Markers of the viral envelope do not really help to locate the virions because they are present in the cytoplasm without virions, and even before the egress of virions starts (see e.g. de Oliveira et al., 2008). To be able to give directionality to their static images they would also need e.g an inducible system whereby they could block some stages of assembly and then release the block and follow kinetically where the accumulated viral proteins move to-with respect to host compartment markers. Response: As stated above, viral proteins are not useful to track virus particles. This is shown in the revised Fig. 1 showing the viral protein gB throughout the cytoplasm. To localize the 3 viral compartments (capsid, tegument, envelope) we constructed a triple coloured virus (Oliveira et al.2008). No compartment of the Golgi can be unequivocally identified without a marker. Response: In an image as shown in Fig. 9A one can easily identify the cis-and trans-face. The paper is not well described. For example the two virus mutants R7041 and R2641 are not well described. Why are they interesting? At the end of the discussion we learn that Us3 is a kinase. Response: The knowledge of the significance of Us3 is not important considering ER-to-Golgi transitions. One important aspect is that Us3 downregulates phospholipid biosynthesis leading to enlargement of the Golgi complex if Us3 is absent as shown in the revised Fig. 1. The significance of Us3 on Golgi assembly early in infection is under study. R2641 is the repair mutant and does not need any explanation. The significance of Us3 is described in introduction of the revised manuscript. I could not find the interpretation for the acronym PNS, I eventually figured out it means peri nuclear space. Response: It was and still is in “introduction”In the background part they describe how gB/gH are reported as being essential for de-envelopment –the fusion step after budding into the nuclear envelope. When these proteins are missing one would expect enveloped virus to accumulate in the NE/ER. Yet they write- under this condition lots of capsids (without membrane) accumulate- and even infectious virus- so there must be an alternative exit route. As a question of logic the reader may ask: maybe the idea that these proteins are essential for de-envelopment is wrong? Response: Yes, this idea is wrong. The process taking place at the outer nuclear membrane shows all characteristics of budding, none of fusion. It takes place in the absence of the fusion proteins gB and gH leading to accumulation of virions in the PNS. This is a clear indication for budding even for those who are not familiar with the fundamentals of membrane bound transportation. By the way, to distinguish between budding and fusion is as easy as to distinguish between the right and the left shoe. Their description of the data on Us3 deletion mutants is not well explained. Response: The only things we will show is ER-to-Golgi transitions, which we found often in cells infected with the Us3 deletion mutant. This has possibly something to do with the enlargement of the Golgi in the absence of Us3.The close connection between the ER and the Golgi in uninfected cells has been seen by many EM studies, going back to Albert Claude 1970, Rambourg and many others. Response: In my opinion, the question is not whether connections between ER and Golgi exist. The question is whether these connections are used for transport of cargo from the ER into Golgi cisternae. Considering the enormous amount of proteins produced and secreted in the exocrine pancreas (1.2 litter per day in humans) it seems to me likely that such a route exists. It is also not true as stated in the 1st paragraph of their Discussion that the Golgi ‘disintegrates rapidly after improper fixation’-unless one forgets to add the aldehyde. Response: This is certainly true. One can destroy the Golgi complex e.g. by using an inappropriate buffer during fixation. I know that you did excellent studies on Golgi structure and function. Unfortunately, there are too many papers published showing inadequate preservation of Golgi structure, including papers dealing with the egress of herpes viruses. They show a nice example of normal fixation in Fig 10B. I take issue with their claim that the 3D structure of the Golgi is poorly understood because of poor preservation. It is poorly understood because the organelle is complex and because one lacks systems to identify the different compartments in the same sections. I of course accept their claim that freeze substitution is the preferred option. Response: The complicated 3D structure plays certainly an important role that the Golgi is poorly understood.The images from BFA treated cells are impressive but I’m not sure that I get a real take home message from these experiments. The Golgi fuses back with the ER; there is accumulation of budded virions in the ER. I cannot see a clear interpretation. Response: The accumulation of virions is the result of impaired transportation out of the PNS-ER compartment. Since the process taking place at the ONM is budding not fusion it is reasonable to assume that the intraluminal transport to the Golgi complex is impaired as was shown also for proteins. Furthermore, it is also an additional indication that the process at the ONM is not fusion because it is unlikely that disintegration of the Golgi would have an impact on the fusion process.Us3 deletion mutants are infective although they almost completely accumulate in the PNS implying that Us3 null virions are not de-enveloped and re-enveloped. The same is true for the virions originating by budding at the INM, ONM and ER membranes after disintegration of the Golgi complex by BFA.The real take home message is that the theory of de-re-envelopment is essential for production of infectious progeny virus is not valid. The long paragraph in the beginning of page 10, dealing with the mutants is especially difficult to follow- e.g what does ‘accidentally proved’ mean? Response: Accidentally means the following: Farnsworth et al., 2007 wanted to prove that the fusion proteins gB/gH are responsible for the fusion of the viral envelope with the ONM leading to accumulations of virions in the PNS. They show in their Fig. 2. - now included in this paper as Fig. 12 - accumulation of virions in the PNS. “Accidentally” - or by mistake -, they overlooked about a dozen of virus interactions at the ONM and ER. If these interactions were fusion virions would not have accumulated in the PNS. Consequently, 1) the process taking place at the ONM is budding not fusion, 2) gB/gH play other roles (if any?) in the egress pathway than mediating fusion of the viral envelope with the ONM, and 3) the envelopment – de-envelopment – re-envelopment theory is incorrect, as shown earlier using Us3 deletion mutants (see above).Among the many issues I have with their three models (Fig 11) I ask: how do they distinguish between budding into a Golgi compartment and wrapping? Response: Capsids can bud into vacuoles and cisternae of any size even if they contain already virions. During wrapping, the viral envelope and the vacuole are concomitantly formed. The result is a concentric vacuole containing a single virion. The space between virion and vacuolar membrane is filled by dense material that probably had driven the process. For details see Leuzinger et al. 2005 (JV), and Wild et al. 2002 (Micron).Model 1 is the only one devoid of A. cytoplasmic capsids and B. ‘wrapping’ profiles. Doesn't this rule out this model?Response: Model 1 is the intraluminal pathway: Capsids arise by budding at the INM, ONM or ER membranes and are intraluminally transported. The destination is considered likely to be Golgi cisternae via ER-Golgi transitions.In model 2, capsids gain access to the cytoplasmic matrix via impaired nuclear envelope that starts by dilation of nuclear pores. See Leuzinger 2005, Wild 2005, 2009.They poorly describe how they quantify virus particles in the different locations by EM. They must surely be referring the budding profiles to membrane length or compartment volume? (Page 4).Response: We expressed the number of virus particles per cellular profile. I know that this is not very appropriate. However, this was done by many other authors addressing herpes virus envelopment. Since it is obvious that virions accumulate in the PNS we consider the data to be sufficiently accurate. In the study about the significance of Us3 on the biosynthesis of phospholipids, we expressed the morphometric data per mean nuclear and cell volume, respectively (Wild et al., 2012). The Conclusion part is also very difficult to relate to.Bottom of page 8 Dilates mis-spelt as ‘Delates’.Beginning of page 7 there is no word I know of in English – Exceptionalness’.It is a pity that such technically nice work is not supported by clarity of writing. Response: It is difficult to write an article about a process that was misinterpreted for decades ignoring the fundamentals of membrane bound transportation, and that was pushed to an untouchable dogma.We have revised the manuscript implementing your suggestion, and simplified the complicated paragraph dealing with the false interpretation that gB/gH promote fusion of the viral envelop with the ONM. We also made clear in the conclusions that the significance of many viral proteins must be re-investigated in the light of correct interpretation of viral-membrane interactions."
}
]
}
] | 1
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https://f1000research.com/articles/6-1804
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https://f1000research.com/articles/6-1972/v1
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08 Nov 17
|
{
"type": "Research Note",
"title": "Draft genomes of two Australian strains of the plant pathogen, Phytophthora cinnamomi",
"authors": [
"Amy L. Longmuir",
"Peter L. Beech",
"Mark F. Richardson",
"Amy L. Longmuir",
"Mark F. Richardson"
],
"abstract": "Background: The oomycete plant pathogen, Phytophthora cinnamomi, is responsible for the destruction of thousands of species of native Australian plants, as well as several crops, such as avocado and macadamia, and has one of the widest host-plant ranges of the Phytophthora genus. The currently available genome of P. cinnamomi is based on an atypical strain and has large gaps in its assembly. To further studies of the pathogenicity of this species, especially in Australia, more robust assemblies of the genomes of more typical strains are required. Here we report the genome sequencing, draft assembly, and preliminary annotation of two geographically separated Australian strains of P. cinnamomi. Findings: Some 308 million raw reads were generated for the two strains. Independent genome assembly produced final genomes of 62.8 Mb (in 14,268 scaffolds) and 68.1 Mb (in 10,084 scaffolds), which are comparable in size and contiguity to other Phytophthora genomes. Gene prediction yielded > 22,000 predicted protein-encoding genes within each genome, while BUSCO assessment showed 82.5% and 81.8% of the eukaryote universal single-copy orthologs to be present in the assembled genomes, respectively. Conclusions: The assembled genomes of two geographically distant isolates of Phytophthora cinnamomi will provide a valuable resource for further comparative analysis and evolutionary studies of this destructive pathogen, and further annotation of the presented genomes may yield possible targets for novel pathogen control methods.",
"keywords": [
"Phytophthora genome",
"plant pathogen",
"Phytophthora cinnamomi"
],
"content": "Introduction\n\nPhytophthora cinnamomi is a highly virulent plant pathogen that has a devastating impact of the Australian ecosystem, namely in the south-western areas of Western Australia and much of the south and east coasts of Victoria and New South Wales1. In the south west Botanical Province of Western Australia, alone, over 40% of the 5710 plant species present have been shown to be susceptible to P. cinnamomi2. Significant genetic and phenotypic variation can occur within a signal clonal linage of P. cinnamomi3 and susceptibility of a given host plant species has been shown to vary from site to site4. Furthermore, despite the general lack of crossing during sexual reproduction, P. cinnamomi excels at adapting to new environments and developing virulence to new host species through asexual growth, making it a deadly and difficult-to-control pathogen. Unravelling how P. cinnamomi is able to adapt so quickly, and remain virulent to a wide range of hosts in Australia, is an important research goal.\n\nThe currently available genome of P. cinnamomi var. cinnamomi (Joint Genome Institute (JGI); NCBI Accession no. PRJNA68241) is based on the Rans isolate from Sumatra in 1922, which has been in culture for many decades and may not be representative of the current pathogenic strains present in Australia. Here we report and make available two Australian P. cinnamomi genomes, isolated from geographically very separate areas with different available host species. After analyses of genetic differences between these two P. cinnamomi genomes, it may be that key genes or gene families under high evolutionary pressure can be identified; this may aid further studies on more effective control of this pathogen.\n\n\nSample collection and sequencing\n\nTwo isolates of P. cinnamomi were selected from areas of infection on either side of the Australian continent: one from the Brisbane Ranges in southeastern Australia (DU054, A2 mating type)5 and the other from southwestern Western Australia (WA94.26, A2 mating type), both Deakin University culture collection. These isolates were selected to represent possible genetic diversity of P. cinnamomi in Australia arising from geographic isolation, and possible variation of selective pressures due to different host species. Isolates were maintained on V8 agar (V8A) [50 ml unclarified V8 ‘Original’ Juice (Campbells, Australia), 0.5 g CaCO3 and 7.5 g biological agar per 500 mL of distilled water] at 25°C in darkness, as per 5. Genomic DNA was isolated from hyphae using a DNeasy Plant Mini Kit (Qiagen), following the manufacturers protocol. Illumina TruSeq Nano library preparation and sequencing on an Illumina HiSeq 2500 platform were performed by the Australian Genome Resources Facility (Walter and Eliza Hall Institute, Parkville, Australia) generating ~154 million (2x 150bp) raw reads per isolate. Raw reads are available in the NCBI Short Read Archive (SRA) under the Bioproject Accession: PRJNA413098\n\n\nGenome assembly\n\nRaw sequencing data for each isolate was first pre-processed using Trimmomatic v0.336 with the following parameters: ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:4 AVGQUAL:30 MINLEN:36, to remove Illumina adapters and filter reads based on quality scores (Phred score). Only reads with average Phred > 30 were retained. To ensure only the desired P. cinnamomi genomes were assembled, a second round of pre-processing was conducted to remove potential contaminants. MetaPhlAn v27, was run with default settings and identified the Paenibacillus genus as a likely contaminate. Using BBMap v0.35 (BBMap - Bushnell. B), we mapped the Trimmomatic-filtered reads to the closest species match (Paenibacillus sp., JDR-2, GenBank accession: GCA_000023585.1, with 2.7% and 2.0% of DU054 and WA94.26 reads mapping, respectively; these Paenibacillus reads were subsequently removed. The remaining reads were then mapped using BBMap to the human genome (GRCh38; NCBI accession: GCA_000001405.15), with < 0.5% (~ 430,000 reads from DU054 and ~ 630,000 from WA94.26) being mapped and subsequently removed from the data set. Thus, the final set of reads (DU054, 149 million reads; WA94.26, 151 million reads) used for the assembly contained highly quality paired-end reads not belonging to either human or bacterial contaminants.\n\nDe novo contig assembly of the two genomes was conducted independently, using IDBA-UD v1.1.08. IDBA-UD was run using the following parameters: --mink 20 --maxk 100 --step 20 --min_contig 500 --min_support 2 --min_count 3. Briefly, these conducted a multiple K-mer assembly from k = 20 up to k = 100; only assembled contigs above 500 bp and those with a minimum depth coverage ≥ 3 were kept. As heterogeneous data can increase redundancy in genome assemblies, the IDBA-UD assembled contigs were run through the Redundans pipeline v0.12c9 with the following parameters: -threads 4 -min_length 500. Redundans uses paired-end mapping data to reduce assembled sequence redundancy and scaffold contigs into longer less fragmented sequences. The final assembled genome of DU054 was 62.80 Mb in 14,269 scaffolds with an N50 of 9,951; the longest scaffold was 1.54 Mb in length (Table 1). For WA94.26, the final genome was 68.07 Mb in length, in 10,085 scaffolds with the largest being 1.54 Mb and an N50 of 20,813. GC content remained consistent, at ~ 53%, between both isolate genomes across both assemblies and before and after processing with Redundans. The quality, as measured by the above metrics, of the presented genomes is comparable to the previously available P. cinnamomi var. cinnamomi Rans isolate genome (JGI).. The final genome assemblies are available under the NCBI Bioproject Accession: PRJNA413098.\n\nWe used the BUSCO (benchmarking universal single-copy orthologs) pipeline v1.2210 with the default e-value cutoff of 0.01, to assess the completeness of the assembled genomes and compared the results to the previously available Rans isolate. Utilizing the set of 429 conserved eukaryotic single-copy orthologs (hereafter BUSCOs), the analysis indicated 82.5% and 81.8% BUSCO completeness for DU054 and WA94.26 genomes, respectively. For DU054, 335 complete BUSCOs (including single-copy and duplicated BUSCOs) and 19 fragmented BUSCOs were identified, and 333 complete and 19 fragmented BUSCOs in WA94.26 (Table 2). Overall, we find comparable levels of BUSCO completeness with the Rans isolate, suggesting our two Australian isolate assemblies are as complete references as that currently available.\n\n\nPreliminary genome annotation\n\nIn the absence of any available high quality ESTs (expressed sequence tags) or transcriptome (gene expression) data for P. cinnamomi, we conducted a preliminary protein-coding sequence prediction using GeneMark-ES v4.3211, which utilises a self-training algorithm to identify exon, intron and intergenic regions as well as initiation and termination sites. GeneMark-ES was run using the default settings and a database of predicted gene models (i.e., predicted polypeptides) was constructed for DU054 and WA94.26 genomes. An initial 23,414 gene models were identified in DU054 and 22,573 in WA94.26. Of these, 14,735 pairs of predicted gene models appear to be orthologous between the two genomes (reciprocal best-hit Blastp10, e value ≤ 1e-5). As a preliminary verification of these gene model builds, we identified orthologous counterparts to eight available Phytophthora genomes with more complete annotations (P. infestans12, P. kernoviae13, P. lateralis14, P. nicotianae15, P. parasitica (P1569_v1; Broad Institute), P. ramorum16, P. sojae16 and P. cinnamomi var. cinnamomi). Accordingly, we used OrthoFinder v1.1.1017 with default parameters, except we used DIAMOND18 as the alignment program with the diamond_more_sensitive flag. OrthoFinder first identifies ‘orthogroups’ (an extension of orthologues to include groups of genes descended from a single gene in the last common ancestor of a group of species17) and then orthologues between each pair of species in the comparison. OrthoFinder assigned 88.5% (170,769) of the genes found in all the species to 19,089 orthogroups, and of these 50% of all the genes were contained in orthogroups, which had 10 or more genes within them. We found 2,931 orthogroups that contained genes for each of the species, and of these 1,309 orthogroups consisted entirely of single copy genes, see associated data repository19. Using these single copy orthogroups gene trees were first constructed then the species tree was inferred using the distance-based implemented by fastme20. The resultant species tree (see associated data repository19) exhibits strong congruence to the Phytophthora phylogeny recently published by 21, providing more evidence that the genome assembly and preliminary annotation conducted here is valuable.\n\n\nConclusions\n\nIn summary, we present the genome assembly of two geographically separated isolates of Phytophthora cinnamomi from Australia, representing the first genome assembly of an Australian-isolated strain. These high-quality genomes will act as a valuable resource, particularly for the further identification and analysis of protein-encoding genes, which are expressed during plant infection, such as members of the avirulence gene families22. These gene families are of specific interest in the development of novel and effective pathogen control mechanisms.\n\n\nData availability\n\nRaw reads are available in the NCBI SRA under the Bioproject Accession: PRJNA413098\n\nThe final assemblies are available at DDBJ/EMBL/GenBank under the accessions, PDCY00000000 and PDCZ00000000 and under the Bioproject Accession: PRJNA413098.\n\nSupporting data, including OrthoFinder analysis and BUSCO assessment results can be found in the associated data repository: doi, 10.4225/16/59d15a6917a5e19. Data are available under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0).",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCahill DM, Rookes JE, Wilson BA, et al.: Phytophthora cinnamomi and Australia’s biodiversity: impacts, predictions and progress towards control. Aust J Bot. 2008; 56(4): 279–310. Publisher Full Text\n\nShearer BL, Crane CE, Barrett S, et al.: Phytophthora cinnamomi invasion, a major threatening process to conservation of flora diversity in the South-west Botanical Province of Western Australia. Aust J Bot. 2007; 55(3): 225–238. Publisher Full Text\n\nHüberli D, Tommerup IC, Dobrowolski MP, et al.: Phenotypic variation in a clonal lineage of two Phytophthora cinnamomi populations from Western Australia. Mycol Res. 2001; 105(9): 1053–1064. Publisher Full Text\n\nShearer B, Dillon M: Susceptibility of plant species in Banksia woodlands on the Swan Coastal Plain, Western Australia, to infection by Phytophthora cinnamomi. Aust J Bot. 1996; 44(4): 433–445. Publisher Full Text\n\nRookes JE, Wright ML, Cahill DM: Elucidation of defence responses and signalling pathways induced in Arabidopsis thaliana following challenge with Phytophthora cinnamomi. Physiological and Molecular Plant Physiology. 2008; 72(4–6): 151–161. Publisher Full Text\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSegata N, Waldron L, Ballarini A, et al.: Metagenomic microbial community profiling using unique clade-specific marker genes. Nat Meth. 2012; 9(8): 811–814. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeng Y, Leung HC, Yiu SM, et al.: IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth. Bioinformatics. 2012; 28(11): 1420–1428. PubMed Abstract | Publisher Full Text\n\nPryszcz LP, Gabaldón T: Redundans: an assembly pipeline for highly heterozygous genomes. Nucleic Acids Res. 2016; 44(12): e113. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimao FA, Waterhouse RM, Ioannidis P, et al.: BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31(19): 3210–3212. PubMed Abstract | Publisher Full Text\n\nLomsadze A, Ter-Hovhannisyan V, Chernoff YO, et al.: Gene identification in novel eukaryotic genomes by self-training algorithm. Nucleic Acids Res. 2005; 33(20): 6494–6506. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaas BJ, Kamoun S, Zody MC, et al.: Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans. Nature. 2009; 461(7262): 393–398. PubMed Abstract | Publisher Full Text\n\nSambles C, Schlenzig A, O’Neill P, et al.: Draft genome sequences of Phytophthora kernoviae and Phytophthora ramorum lineage EU2 from Scotland. Genom Data. 2015; 6: 193–194. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuinn L, O’Neill PA, Harrison J, et al.: Genome-wide sequencing of Phytophthora lateralis reveals genetic variation among isolates from Lawson cypress (Chamaecyparis lawsoniana) in Northern Ireland. FEMS Microbiol Lett. 2013; 344(2): 179–185. PubMed Abstract | Publisher Full Text\n\nLiu H, Ma X, Yu H, et al.: Genomes and virulence difference between two physiological races of Phytophthora nicotianae. Gigascience. 2016; 5: 3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTyler BM, Tripathy S, Zhang X, et al.: Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis. Science. 2006; 313(5791): 1261–1266. PubMed Abstract | Publisher Full Text\n\nEmms DM, Kelly S: OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol. 2015; 16(1): 157. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuchfink B, Xie C, Huson DH: Fast and sensitive protein alignment using DIAMOND. Nat Methods. 2015; 12(1): 59–60. PubMed Abstract | Publisher Full Text\n\nLongmuir A, Beech P, Richardson M: Supporting data for \"Draft Genomes of two Australian strains of the plant pathogen, Phytophthora cinnamomi\". [data collection], 2017. Publisher Full Text\n\nLefort V, Desper R, Gascuel O: FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program. Mol Biol Evol. 2015; 32(10): 2798–800. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcCarthy CGP, Fitzpatrick DA: Phylogenomic Reconstruction of the Oomycete Phylogeny Derived from 37 Genomes. mSphere. 2017; 2(2): pii: e00095-17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBos JI, Kanneganti TD, Young C: The C‐terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana. Plant J. 2006; 48(2): 165–176. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "27739",
"date": "17 Nov 2017",
"name": "Nicolás Daniel Ayub",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work was carried out professionally and resulted in good draft genomes of two pathogen strains belonging to Phytophthora genus. In my opinion, this article is an important contribution to future studies about the molecular mechanism involved in Phytophthora-plant interaction. Particularly, in the first steps of pathogen adhesion, where the virulence factors related to this are little known.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3471",
"date": "01 Mar 2018",
"name": "Mark Richardson",
"role": "Author Response",
"response": "Thank you for the positive review."
}
]
},
{
"id": "27740",
"date": "20 Nov 2017",
"name": "Erik Andreasson",
"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 data adds information about this important organism in the standard format to report a draft genome these days so it looks fine. They used hiseq and sequence coverage (BUSCO) looks appropriate and expected although there are relatively large differences between the two isolates (i.e .different final genome sizes and busco completeness scores). One suggestion is to add information on how many libraries they sequenced, and if it was only paired end and not also mate paired.\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? 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": "3470",
"date": "01 Mar 2018",
"name": "Mark Richardson",
"role": "Author Response",
"response": "Thank you for the positive review, we have added the additional information you have requested."
}
]
},
{
"id": "28065",
"date": "28 Nov 2017",
"name": "David J. Studholme",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript announces the availability of genomic sequence data from Phytophthora cinnammomi strains DU054 and WA94.26. This is a useful resource for researchers interested in this important pathogen. The authors have deposited and made available the raw sequence data in SRA and their assemblies in GenBank, which is commendable. The genome annotation and protein sequences do not appear to be deposited in GenBank, however. This does not preclude publication, but when discussing predicted genes in the manuscript, the authors should be up-front about this or provide full details of the annotations in supplementary data or deposit them in some public repository.\nOne oversight that the authors should be aware of is the previous publication of two genome sequences of this species, one of which (MP94-48) is from Australia. See Studholme et al. (2015)1. So, the authors' assertion (in their Conclusions section) that this is the first genome assembly from an Australian strain should be revised. The authors should also include those two assemblies in their comparisons of assembly quality metrics. And also please revise the several other mentions of previously sequenced genome throughout the text in the light of the additional two previously sequenced genomes. Also, it would be interesting to assess how similar or different all these four available P.c. genome sequences are to each other, e.g. by calculating pairwise ANIs.\nSome specific points that should be addressed around the methodology:\nWhy were reads mapped against the human genome? Why should contamination from human DNA be more prevalent or likely than from other organisms?\n\nThe authors make good efforts to remove contaminating Paenibacillus sequence reads. Interestingly, we also observed contamination of Phytophthora genomic DNA with this bacterial genus. However, the authors go on to claim that the data contained \"highly quality reads not belonging to ... bacterial contaminants\". Their approach does not remove non-Paenibacillus bacterial contaminants.\n\nPlease cite a reference to support the claim that \"heterogeneous data can increase redundancy in genome assemblies\". It is not entirely clear what this statement means, precisely, and in any case it is not self-evident and needs to be supported by peer-reviewed publication.\n\nThe use of BUSCO version 1.22 is questionable, given that versions 2 and 3 are now available. Furthermore, rather than using the general Eukaryote set of BUSCOs, the authors should use the Stramenopile set.\n\nThe completeness of the genome assemblies is rather poor (only < 65% of expected genes are present intact in a single copy). It would be useful to compare/benchmark this against other available Phytophthora genome sequences. For example, our recent sequencing of P. ramorum genomes, we found around 81- 85% of Stramenopile BUSCOs were intact and single-copy in each genome (See PubMed ID 28243575).\n\nTowards the end of page 4, the authors claim that the \"preliminary annotation ... is valuable\". I agree and would go further to say that not just the annotation but the genome sequencing per se is valuable. I would also suggest including a brief explanation of how/why the presented data is valuable.\n\nThe authors say that their annotation is valuable, but the annotation has not apparently been deposited in a public repository. Therefore, please either make this valuable resource available, or remove the claim that it is valuable.\n\nSome very minor points:\nIn the Introduction, it was not obvious to me what is meant by a \"Botanical Province\". Please consider explaining this term.\n\nPlease add an apostrophe after \"manufacturers\".\n\nAt several places in the text, the authors write \"parameters\" when they really mean \"parameter values\" or \"options\" or \"switches\". Please check and revise.\n\nPlease write \"high-quality\" not \"highly quality\".\n\nOn page 3, the authors say that no gene expression data are available for this species. This is untrue, since EST data (i.e. expressed sequence tags) are available. Furthermore, in the SRA, there are several RNAseq datasets available:\n\nIllumina HiSeq 2000 sequencing; RNAseq analysis of germinating cysts of Phytophthora cinnamomi 1 ILLUMINA (Illumina HiSeq 2000) run: 46,420 spots, 4.2M bases, 3.5Mb downloads Accession: ERX709652 Select item 14623972.\nIllumina HiSeq 2000 paired end sequencing 1 ILLUMINA (Illumina HiSeq 2000) run: 9.9M spots, 1.8G bases, 1.1Gb downloads Accession: ERX943317 Select item 1426113.\nPhytophthora cinnamomi library 1 ILLUMINA (Illumina HiSeq 2000) run: 88.1M spots, 17.6G bases, 10.3Gb downloads Accession: SRX124562 Select item 1426104.\nPhytophthora cinnamomi library 1 ILLUMINA (Illumina HiSeq 2000) run: 30,453 spots, 6.1M bases, 2.6Mb downloads Accession: SRX124561 Select item 1426095.\nPhytophthora cinnamomi library1 ILLUMINA (Illumina HiSeq 2000) run: 50.6M spots, 10.1G bases, 5.9Gb downloads Accession: SRX124560 Select item 1426086.\nPhytophthora cinnamomi library 2 ILLUMINA (Illumina HiSeq 2000) runs: 38.5M spots, 7.7G bases, 4.5Gb downloads Accession: SRX124559\n\nWhen quoting N50 values, please include the units. For example, the N50 for DU054 was 9,951 bp or nt.\n\nThe authors refer to (on page 4) \"more complete annotations\" of several species. Among these examples is P. lateralis and a citation of our paper (PubMed 23678994) about the sequencing of this species' genome; however, I would not agree that its annotation is \"more complete\".\n\nOn page 3, second paragraph, the authors write \"the available genome\". It is not the \"genome\" that is available; rather it is the \"genome sequence\".\nOnce the authors have addressed all these issues, I would be very pleased to see this indexed.\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? 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": "3469",
"date": "01 Mar 2018",
"name": "Mark Richardson",
"role": "Author Response",
"response": "Thank you very much for providing a thorough review and pointing out several oversights we made. We have endeavoured to rectify these as you will see from our responses below. Importantly, we have included the preliminary gene prediction results in the associated public repository with the current supplementary materials. We have also revised the manuscript to include the additional genomes from Studholme et al. 2015 and included them in a comparative BUSCO completeness assessment. We feel a more comprehensive comparative analysis (including ANIs) is beyond the scope of this research note, but this will be part of a future paper. For clarity, the below responses are separated into Major and Minor subheadings and numbered as per points in the review in order to avoid duplication of text. Major:1. Contamination for human DNA should not be more prevalent than any other. As this was one of the first times we cultured this species we carried out this pre-filtering to ascertain whether or not we had any inadvertent contamination. The results show this was not the case.2. We find it interesting that the reviewer has also detected Paenibacillus contamination during their work.2. While removing contamination through mapping to the Paenibacillus genome alone would not warrant our statement, this is not what we did. We used MetaPHlAn to first screen our raw reads to identify which, if any, bacterial species might be present. Only Paenibacillus could be detected. Thereby, once removed, we are confident that no other bacterial contamination exists. If others had been identified with MetaPHlA then they could be removed in the same way. 3. We have added a citation to this extent and clarified what we mean in the text. 4. We have repeated this analysis with version 3.02 and used the suggested ortholog set. 5. With respect, we feel that the reviewer’s statement that the completeness was poor is unfounded, especially if we consider that they are not making a ‘like for like’ comparison by comparing results from the eukaryotic set to those from the stramenopile set. Nevertheless, the updated BUSCO analysis using the stramenopile set reveals the genome assemblies presented here have BUSCO completeness of ~91 to 94 %, which falls within the range for the previous P. cinnamomi assembles (86 -97% completeness, see Table 2 for full comparison). 6. Thank you for this suggestion, we have done so. 7. This is a very valid point. We have now included the preliminary gene predictions with the supplementary data. Minor: We have changed this to the more commonly understood ‘ecoregion’ Done Done Done We have removed this statement. Done We have removed this statement. Addressed"
}
]
}
] | 1
|
https://f1000research.com/articles/6-1972
|
https://f1000research.com/articles/6-2079/v1
|
01 Dec 17
|
{
"type": "Case Report",
"title": "Case Report: Dual nebulised antibiotics among adults with cystic fibrosis and chronic Pseudomonas infection",
"authors": [
"Nina Mann",
"Shirley Murray",
"Zhe Hui Hoo",
"Rachael Curley",
"Martin J. Wildman",
"Nina Mann",
"Shirley Murray",
"Zhe Hui Hoo",
"Rachael Curley"
],
"abstract": "Pulmonary exacerbations in adults with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa (Psae) infection are usually treated with dual intravenous antibiotics for 14 days, despite the lack of evidence for best practice. Intravenous antibiotics are commonly associated with various systemic adverse effects, including renal failure and ototoxicity. Inhaled antibiotics are less likely to cause systematic adverse effects, yet can achieve airway concentrations well above conventional minimum inhibitory concentrations. Typically one inhaled antibiotic is used at a time, but dual inhaled antibiotics (i.e. concomitant use of two different inhaled antibiotics) may have synergistic effect and achieve better results in the treatment of exacerbations. We presented anecdotal evidence for the use of dual inhaled antibiotics as an acute treatment for exacerbations, in the form of a case report. A female in her early thirties with CF and chronic Psae infection improved her FEV1 by 5% and 2% with two courses of dual inhaled antibiotics to treat exacerbations in 2016. In contrast, her FEV1 changed by 2%, –2%, 0% and 2%, respectively, with four courses of dual intravenous antibiotics in 2016. Baseline FEV1 was similar prior to all six courses of treatments. The greater FEV1 improvements with dual inhaled antibiotics compared to dual intravenous antibiotics suggest the potential role of using dual inhaled antibiotics to treat exacerbations among adults with CF and chronic Psae infection, especially since a greater choice of inhaled anti-pseudomonal antibiotics is now available. A previous study in 1985 has looked at the concomitant administration of inhaled tobramycin and carbenicillin, by reconstituting antibiotics designed for parenteral administration. To our knowledge, this is the first literature to describe the concomitant use of two different antibiotics specifically developed for delivery via the inhaled route.",
"keywords": [
"Cystic fibrosis",
"nebuliser therapy",
"pulmonary exacerbations",
"Pseudomonas aeruginosa"
],
"content": "Introduction\n\nCystic fibrosis (CF) is a genetic condition whereby ~80% of mortalities are primarily due to lung disease1. People with CF are prone to recurrent respiratory infections (termed ‘pulmonary exacerbations’), which leads to progressive lung damage and respiratory failure2. This is especially so after Pseudomonas aeruginosa (Psae) is acquired3.\n\nAlthough there is a lack of evidence for best practice in treating exacerbations among adults with CF and chronic Psae infection4, two weeks of dual intravenous antibiotics are generally used for synergistic effect4,5. The European CF Society recommend against using inhaled antibiotics to treat exacerbations, due to concerns that increased mucus plugs during exacerbations may prevent antibiotics from reaching smaller airways5.\n\nThere is scant research on using inhaled antibiotics to treat exacerbations. A Cochrane review found only four relevant studies, with inadequate sample sizes to demonstrate efficacy6. Nonetheless, a large observational study in North America found that ~24% of exacerbations are treated with inhaled antibiotics7. Inhaled antibiotics have several advantages. Systemic adverse effects e.g. allergic reactions, gastrointestinal manifestations, ototoxicity and renal failure are common with intravenous antibiotics8,9 but rare with inhaled antibiotics10. Higher antibiotic concentrations within the airways are achieved via inhaled route, which may be beneficial in overcoming resistance11. Inhaled route also overcomes difficulties associated with venous access.\n\nTypically one inhaled antibiotic is used at a time, and someone on multiple inhaled antibiotics would alternate between those antibiotics10. Of the four studies identified in the Cochrane review, only one study with 18 participants looked at concomitant administration of two inhaled antibiotics (tobramycin and carbenicillin)6. Yet dual inhaled antibiotics (i.e. concomitant use of two different inhaled antibiotics) may have synergistic effect, thus achieve better results. We report on an adult with CF and chronic Psae infection who achieved good results when treating exacerbations using dual inhaled antibiotics.\n\n\nCase report\n\nA Caucasian female in her early thirties with F508del/Class I mutation, pancreatic insufficiency and CF related diabetes also fulfilled the Leeds criteria12 for chronic Psae infection. Despite high objective adherence to nebulised dornase alfa 2.5mg once daily and alternating Promixin® 1megaunit twice daily/TOBI® 300mg twice daily (82.9% two years ago, 96.3% in the previous year; measured with an I-neb®), stable BMI around 23.1 and reasonable glycaemic control (HbA1c 47 in month 1 of the follow-up period), there was a trend of declining %FEV1. Her annual best FEV1 was 47%, 44% and 42% over the previous three years. There was no evidence of allergic bronchopulmonary aspergillosis (ABPA) or other CF complications compromising her %FEV1.\n\nIn month one, Promixin® was switched to nebulised AZLI® 75mg thrice daily. She had two courses (28 days) of intravenous antibiotics throughout the previous year. She agreed to three-monthly intravenous antibiotics in the follow-up year to try to arrest the FEV1 decline. She had 14 days of intravenous Tazocin 4.5g thrice daily and colomycin 2megaunit thrice daily in month one (%FEV1 improved from 38% to 40%), and again in month 4 (%FEV1 declined from 40% to 38%).\n\nShe felt less well with increased sputum volume and dyspnoea at the start of month six. Her %FEV1 was 39%. She agreed to try 14 days of concomitant nebulised AZLI® 75mg thrice daily and TOBI® 300mg twice daily. Her %FEV1 improved to 45% at day 7 and 44% at day 14. Her symptoms resolved by day 14.\n\nShe felt well but her %FEV1 declined to 39% during her next clinic review at the end of month seven. She went on another 14-day course of intravenous Tazocin 4.5g thrice daily and colomycin 2megaunit thrice daily. At day 14, her %FEV1 remained 39%. Another 14-day course of concomitant nebulised AZLI® and TOBI® was started in month 8. Her %FEV1 improved to 41% at day 7 and day 14, despite developing viral coryzal symptoms at day 12. With 14 days of IV Tazocin 4.5g thrice daily and tobramycin 480mg once daily in month 11, her %FEV1 improved from 39% at day 1 to 41% at day 14.\n\n\nDiscussion\n\nIn this case, %FEV1 improvement following acute treatment of exacerbations with dual inhaled antibiotics (mean 3.5% over two courses) was somewhat higher than with dual intravenous antibiotics (mean 0.5% over four courses), despite similar baseline %FEV1. The %FEV1 improvement also occurred despite severe background lung disease (high resolution CT in month eight showed extensive bronchiectasis and baseline %FEV1 was ~40%).\n\nAlthough she reported symptomatic improvement during her first dual inhaled antibiotics course, we did not formally measure symptomatic responses to treatments with a validated tool. The sample size is too small for null hypothesis significance testing, and regression to the mean is potentially a threat to our results. Our results are nonetheless intriguing and suggest that dual inhaled antibiotics could potentially have a role in treating exacerbations among adults with CF and chronic Psae infection. With the increasing number of inhaled anti-pseudomonal antibiotics available, e.g. nebulised levofloxacin13, different combinations of concomitant inhaled antibiotics can be used in the future for synergistic effect.\n\nLike all medications, there are adverse events associated with inhaled antibiotics. There is a case report of acute respiratory distress syndrome potentially due to inhaled colistin14. However, localised adverse events with inhaled antibiotics are usually mild, e.g. bronchoconstriction which tends to resolve spontaneously within hours or can be controlled by pre-dosing with nebulised bronchodilator10.\n\nHigh adherence to inhaled therapies probably contributed to the good clinical response from dual inhaled antibiotics observed in this case. Real-world adherence with long-term inhaled antibiotics among adults with CF is only 35–50%15,16. Someone who is already struggling with a single inhaled antibiotic is unlikely to cope with dual inhaled antibiotics, thus may derive less benefit. However, adherence to short-term drug regimen tends to be higher17. Adults with CF might be able to summon adequate self-regulation during a 14-day dual antibiotics course to really focus on their nebuliser use18.\n\nIn conclusion, the %FEV1 improvements observed in this case report provide anecdotal evidence that dual inhaled antibiotics could potentially be a treatment option for exacerbations among adults with CF and chronic Psae infection. Given the lack of good quality evidence regarding optimum exacerbations treatments and the theoretical advantages of using inhaled antibiotics, this warrants further investigations.\n\n\nConsent\n\nWritten informed consent for publication of her clinical details 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\nElborn JS: Cystic fibrosis. Lancet. 2016; 388(10059): 2519–2531. PubMed Abstract | Publisher Full Text\n\nBhatt JM: Treatment of pulmonary exacerbations in cystic fibrosis. Eur Respir Rev. 2013; 22(129): 205–216. PubMed Abstract | Publisher Full Text\n\nMayer-Hamblett N, Kronmal RA, Gibson RL, et al.: Initial Pseudomonas aeruginosa treatment failure is associated with exacerbations in cystic fibrosis. Pediatr Pulmonol. 2012; 47(2): 125–134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlume PA, Mogayzel PJ Jr, Robinson KA, et al.: Cystic fibrosis pulmonary guidelines: treatment of pulmonary exacerbations. Am J Respir Crit Care Med. 2009; 180(9): 802–808. PubMed Abstract | Publisher Full Text\n\nDöring G, Flume P, Heijerman H, et al.: Treatment of lung infection in patients with cystic fibrosis: current and future strategies. J Cyst Fibros. 2012; 11(6): 461–479. PubMed Abstract | Publisher Full Text\n\nRyan G, Jahnke N, Remmington T: Inhaled antibiotics for pulmonary exacerbations in cystic fibrosis. Cochrane Database Syst Rev. 2012; 12: CD008319. PubMed Abstract | Publisher Full Text\n\nWagener JS, Rasouliyan L, VanDevanter DR, et al.: Oral, inhaled, and intravenous antibiotic choice for treating pulmonary exacerbations in cystic fibrosis. Pediatr Pulmonol. 2013; 48(7): 666–673. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeckham D, Whitaker P: Drug induced complications; can we do more? J Cyst Fibros. 2013; 12(6): 547–558. PubMed Abstract | Publisher Full Text\n\nRoehmel JF, Schwarz C, Mehl A, et al.: Hypersensitivity to antibiotics in patients with cystic fibrosis. J Cyst Fibros. 2014; 13(2): 205–211. PubMed Abstract | Publisher Full Text\n\nQuon BS, Goss CH, Ramsey BW: Inhaled Antibiotics for Lower Airway Infections. Ann Am Thorac Soc. 2014; 11(3): 425–434. PubMed Abstract | Publisher Full Text | Free Full Text\n\nConway SP: Evidence for using nebulised antibiotics in cystic fibrosis. Arch Dis Child. 1999; 80(4): 307–309. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee TW, Brownlee KG, Conway SP, et al.: Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. J Cyst Fibros. 2003; 2(1): 29–34. PubMed Abstract | Publisher Full Text\n\nElborn JS, Flume PA, Cohen F, et al.: Safety and efficacy of prolonged levofloxacin inhalation solution (APT-1026) treatment for cystic fibrosis and chronic Pseudomonas aeruginosa airway infection. J Cyst Fibros. 2016; 15(5): 634–640. PubMed Abstract | Publisher Full Text\n\nMcCoy KS: Compounded colistimethate as possible cause of fatal acute respiratory distress syndrome. N Engl J Med. 2007; 357(22): 2310–2311. PubMed Abstract | Publisher Full Text\n\nDaniels T, Goodacre L, Sutton C, et al.: Accurate assessment of adherence: self-report and clinician report vs electronic monitoring of nebulizers. Chest. 2011; 140(2): 425–432. PubMed Abstract | Publisher Full Text\n\nQuittner AL, Zhang J, Marynchenko M, et al.: Pulmonary medication adherence and health-care use in cystic fibrosis. Chest. 2014; 146(1): 142–151. PubMed Abstract | Publisher Full Text\n\nHaynes RB, McDonald HP, Garg AX: Helping patients follow prescribed treatment: clinical applications. JAMA. 2002; 288(22): 2880–2883. PubMed Abstract | Publisher Full Text\n\nBaumeister RF, Bratslavsky E, Muraven M, et al.: Ego depletion: is the active self a limited resource? J Pers Soc Psychol. 1998; 74(5): 1252–1265. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "29445",
"date": "04 Jan 2018",
"name": "Tim Lee",
"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 useful case report highlighting the potential for treating certain pulmonary exacerbations in people with cystic fibrosis with inhaled antibiotics. The background is well written. References are appropriate and balanced.\nThe case is well described and the adherence data prior to the exacerbations is helpful. The clinical response in terms of FEV1 is well reported and favourable by comparison to response to iv therapy.\nParagraph 2 of the \"Case Report\" section should in my view more clearly specify that the colomycin 2MU three times a day was given intravenously rather than nebulised alongside the intravenous Tazocin for 14 days in month 1 and month 4, given the overall title of the case report and the similar iv/nebulised dosages for colomycin. It would also be instructive to know whether the intravenous antibiotics were administered at home or in hospital.\nIn paragraphs 3 and 4 of the \"Case Report\" section, it would be useful to know if the subject was also taking nebulised dornase alpha during this period, i.e. was she achieving six nebulised therapies per day? Also, if available, adherence measures during these 2 week pulses of inhaled dual antibiotic therapies would be useful, as many clinicians do have concerns that this intensity of nebulised medications may be difficult to achieve for many people with CF, despite the good clinical response seen here.\nThe conclusions, acknowledgements of limitations, are all well written and appropriate. In summary this case report is valuable and will be of interest to the CF community. I support publication but would recommend the clarifications I have suggested are considered.\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": [
{
"c_id": "3315",
"date": "04 Jan 2018",
"name": "Zhe Hui Hoo",
"role": "Author Response",
"response": "We thank Dr Lee for the review and very useful comments which will guide our revision of the case report.We will clarify in Paragraph 2 (and 4) of the \"Case Report\" section that colomycin 2MU thrice daily was given intravenously, and likely for tobramycin 480mg once daily (in paragraph 4).We will also clarify the Paragraphs 2 and 3 of the \"Case Report\" section that once daily nebulised dornase alfa was continued throughout the dual nebulised antibiotics; and report the total number nebuliser doses taken during the 2 week pulses of dual inhaled dual antibiotic therapies."
}
]
},
{
"id": "30297",
"date": "30 Jan 2018",
"name": "Freddy Frost",
"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\nMann et al report an interesting case demonstrating that inhaled antibiotics may be useful for the treatment of acute pulmonary exacerbations in CF. The authors accurately describe the background and much of the evidence relating to the use of inhaled antibiotics in acute exacerbations including the Cochrane review of 2012. However, the Cochrane review preceded the most recent clinical trial in this field. The study demonstrated and discussed both pulmonary and renal benefits for nebulized tobramycin in the treatment of exacerbations and the authors should consider including it in the introduction. 1\n\nIn the fourth paragraph it should be clarified whether “typically one inhaled antibiotic is used at a time” refers to the routine practice in the chronic setting or previous studies in the acute setting.\nThe case itself is well described however the authors may wish to consider including some relevant extra information. Firstly, had the patient cultured any other organisms aside from P. aeruginosa in their sputum in the preceding 12 months? Secondly, were all the treatments completed at home or in the inpatient setting? Differing settings with increased physiotherapy input as an inpatient could theoretically account for some of the differences. Finally, were other adjunctive therapies such as oral corticosteroids used in the management of each exacerbation.\nThe discussion is balanced and recognises the limitations of the case as well as potential adverse events related to nebulised therapy. Overall, this is an interesting case worthy of publication and we wholeheartedly agree with the authors that this is a management strategy that warrants further investigation.\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? Partly",
"responses": [
{
"c_id": "3388",
"date": "30 Jan 2018",
"name": "Zhe Hui Hoo",
"role": "Author Response",
"response": "We thank Drs Frost and Nazareth for the review and very useful comments which will guide our revision of the case report.We will include 2014 pilot study of nebulised vs IV tobramycin in the 'Introduction'. We will also alter the 4th paragraph of the 'Introduction' to clarify that the first sentence was referring to inhaled antibiotics in chronic settings whilst the second sentence referred to inhaled antibiotics in acute settings.We will include the extra information relating to microbiology results, clarify the treatment setting (all treatments were at home except for the last course of IV antibiotics in month 11) and clarify the use of any adjunctive therapies."
}
]
},
{
"id": "29543",
"date": "08 Feb 2018",
"name": "Theodore G. Liou",
"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 case report of a patient with CF during a 6 month period during which she was treated with multiple antibiotics in several regimens that seemed to differ with each treatment episode.\n\nThe introduction is well written and informative. The case history itself raises many questions. There are fewer details of the patient’s baseline history than desired. The lack of a specific age is not typical for case reports. It would be nice to know whether her genotype was F508del homozygous or heterozygous with a second, preferably identified, mutation. The report of high adherence of the patient is open to doubt because of the poor track record of clinicians detecting non-adherence in an accurate way, and confirmation of the degree of adherence, perhaps with a medication possession report, would be helpful.\n\nThe key portion of the history detailing treatments with various antibiotics, regimens and dosing routes is interesting but lacks some key details. It is not entirely clear what triggered the more intensive 6 months. There is reference to symptoms and signs often associated with acute exacerbations, the introduction seems to focus at least in part on exacerbations, and the discussion refers to “acute treatment of exacerbations,” but the patient’s course is not described in a way as to identify when pulmonary exacerbations were specifically diagnosed or treated. This detracts from helping readers understand the circumstances for which different treatments were initiated.\n\nIn the discussion, the authors discuss the possible effects of dual inhaled antibiotics, but I find it difficult to associate such use with a superior outcome even on an anecdotal basis because many drugs and regimens were employed in response to what seems to have been multiple clinical situations during the 6-month case history period.\n\nI think the report could be improved by providing more patient history prior to the 6-month period of focus, better assessment of her level of adherence, clear definition of pulmonary exacerbation as used in this patient’s case and clearer description of triggers for different treatments as well as more precise descriptions of outcomes. An additional outcome to consider including would be the results of subsequent bacterial cultures and any changes in resistance patterns.\n\nA study of dual inhaled antibiotics compared to intravenous would be of interest. However, there are likely to be many barriers and difficulties developing a generally usable protocol. Some mention of these barriers might be helpful to explain why such a study has not been done.\n\nFinally, to help readers in areas of the world where trade names are not the same or the medications are not available, Promixin, Azli, Tobi, Tazocin and Coly-Mycin should be identified by their generic names in addition to the trademarked names. It would be better, I think, if they were referred to by their generic names in preference to the trade names.\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? 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? Partly",
"responses": [
{
"c_id": "3407",
"date": "09 Feb 2018",
"name": "Zhe Hui Hoo",
"role": "Author Response",
"response": "We thank Dr Liou for the review and we will iterate the case report taking into account the comments.In Sheffield whereby we only provide care for around 200 adults with CF, we only have a female adult with her exact age (in years) and her F508del/Class I mutation. Thus, to maintain the anonymity of the patient (which was the basis on which she provided consent), we could only provide an approximate age and specify that she was heterozygous for F508 mutation in the first sentence of the 'case report' section. In any case, whether the patient is 30 years of age or 33 years of age (which is the age range we consider to sit within 'early thirties') is irrelevant to the conclusion of the case report or to the interpretation of her FEV1. By stating that she has a Class II mutation (i.e. F508del) and Class I mutation in the first sentence of the 'case report' section, we have also specified that she has 'severe genotype' according to the definition of Castellani et al. J Cyst Fibros 2008, 7:179-196.In Sheffield, we measure nebuliser adherence using I-neb (as specified in the second sentence of the 'case report' section). Electronic data capture is generally considered the 'gold standard' measure for adherence and MPR is not a \"better assessment of adherence\" compared to electronic data capture. The study by Siracusa et al. J Cyst Fibros 2015;14:621-6 have demonstrated that MPR data may be unreliable. In that study, ivacaftor adherence according to electronic data capture was only around 60% but MPR suggested adherence of 84%. There was also no correlation between adherence data captured using electronic data capture and MPR. Since we have provided EDC adherence data, we do not think MPR data will add further information regarding her adherence. We will clarify the trigger for the more intensive treatment period and clarify which of the treatment was used to treat an acute exacerbation. We will also provide results for her subsequent bacterial cultures and any changes in resistance patterns.A small study using dual nebulised antibiotics has been done in 1985, as identified by the 2012 Cochrane review. We agree that any study for the treatment of pulmonary exacerbation is challenging. The STOP program in the US appears to be making breakthroughs in starting to define best practices in the treatment of pulmonary exacerbations, suggesting that such studies are difficult but not impossible to execute."
}
]
}
] | 1
|
https://f1000research.com/articles/6-2079
|
https://f1000research.com/articles/6-1940/v1
|
02 Nov 17
|
{
"type": "Research Note",
"title": "Rheumatoid arthritis in an adult patient with mosaic distal 18q-, 18p- and dicentric ring chromosome 18",
"authors": [
"Alanna Chau",
"KH Ramesh",
"Anand D Jagannath",
"Shitij Arora",
"Alanna Chau",
"KH Ramesh",
"Anand D Jagannath"
],
"abstract": "Ring chromosome 18 has a highly variable phenotype, depending on the extent of distal arm deletions. It is most commonly presented as a combination of 18p- and distal 18q- syndrome. IgA deficiency and autoimmune diseases have been previously described in these patients. Seven cases of juvenile rheumatoid arthritis (JRA) have been reported. Here we report the first case of late onset rheumatoid arthritis (RA) in a 32 year old Dominican woman with hypothyroidism, vitiligo, IgA deficiency, interstitial lung disease (ILD), cystic bronchiectasis, and features consistent with 18p- and distal 18q syndrome. Comparative genome hybridization analysis showed a del(18p11.21p11.32), dup(18q11.21-q22.1), and del(18q22.1-q23). Chromosomal analysis and fluorescence in situ hybridization showed three cell lines. One cell line was detected with a dicentric ring chromosome, another with duplication of the long arm and no short arm, and lastly a long arm terminal deletion of 18. The multiple autoimmune findings in our patient lends further support to the idea of loci on chromosome 18 playing a role in autoimmune disease expression. Late onset RA and ILD in a patient with chromosome 18 abnormalities are novel findings and are additional conditions to be aware of in this population.",
"keywords": [
"ring chromosome 18",
"rheumatoid arthritis",
"interstitial lung disease"
],
"content": "Introduction\n\nChanges in the structure of chromosome 18 are implicated in a number of conditions affecting health and development. 18p- and distal 18q- syndrome has been estimated to occur in 1/50,000 and 1/40,000 live births, respectively. The characteristics of 18p- syndrome are wide ranging and include speech delay, holoprosencephaly spectrum, micrognathia, ptosis, flat nasal bridge, wide mouth with short upper lip, excessive dental caries, large protruding ears, and skeletal abnormalities. 18q- syndrome also presents with a wide variety of clinical features that commonly includes foot anomalies, carp like mouth, midface hypoplasia, cleft palate, cleft lip, inner epicanthal folds, slanted palpebral fissures, narrow or atretic external auditory canals and low set ears. Features common to both syndromes include intellectual disability, short stature, microcephaly, tone abnormalities, seizures, hearing loss and cardiac defects. The phenotypic severity of either condition appears to be correlated with the amount of genetic material affected1.\n\nRing chromosome 18, or r(18), is a rarer condition that most commonly forms when there is breakage in both chromosome arms, fusion of those breakpoints and the subsequent loss of the distal fragments2. Ring chromosomes can also result from terminal deletions as well as contiguous duplication, with some of these cases demonstrating inversion of these duplications and thus an inv dup del rearrangement mechanism. As a result of the inconsistent amount of duplication and hemizygosity of the distal ends, the r(18) phenotype is extremely variable. Clinical characteristics are typically a combination of 18p- and distal 18q- syndrome3.\n\nIgA deficiency and immunological diseases such as type 1 diabetes mellitus (T1DM), juvenile rheumatoid arthritis (JRA), Grave’s disease, hypothyroidism, and vitiligo have been reported in a number of individuals with 18p-, 18q-, and to a lesser extent r(18)3–8. These numerous reports may represent chance relationships or suggest true genetic linkages. To the best of our knowledge, there have been seven reported cases of chromosome 18 abnormalities and JRA (see Table 1). Here we report the first case of late onset rheumatoid arthritis (RA) associated with mosaic 18p-, distal 18q-, and r(18) in a 32 year old Dominican woman with intellectual disability, hypothyroidism, vitiligo, IgA deficiency, interstitial lung disease (ILD), cystic bronchiectasis, and features consistent with both 18p- and distal 18q- syndrome.\n\n+ = present, - = absent, ? = not reported; F = female, M = male, R = right, L = left; ANA = antinuclear antibodies, RF = rheumatoid factor, CCP = cyclic citrullinate peptide; ASD = atrial septal defect, IQ = intelligence quotient, MR = mental retardation ** Possible drug related etiology, occurred after trimethoprim sulfamethoxazole use with intermittent reoccurrences\n\n\nCase report\n\nThe patient was a 32-year-old Dominican woman who presented to the emergency room with fever, hypoxia, wheezing, shortness of breath, cough productive of yellow sputum, and coarse breath sounds. She was hospitalized three months prior for community acquired pneumonia. She was subsequently admitted to the general medicine service for management of presumed healthcare associated pneumonia.\n\nShe immigrated with her family to the US from the Dominican Republic 2 years ago. Unfortunately, we have no access to previous medical records and past medical history was obtained from her mother. The patient is the second child of a healthy non-consanguineous couple. At the time of birth, mother and father were 25 and 30 years old, respectively. Prenatal ultrasound was not done. She was born full term with birth weight of 2948 g. At birth, the patient was found to have an abnormal head shape, enlarged heart and heart murmur that resolved by 4 years after taking an unspecified oral medication. Cleft palate and left clubfoot were surgically repaired at age 3 and 4 years, respectively. Dentition was initially normal, however teeth fell early or had extensive caries. At age 9 years skin and hair depigmentation began and she was diagnosed with vitiligo. She is hypothyroid and maintained on levothyroxine. At age 18 years monthly menses began. Over the years hearing has deteriorated, necessitating louder cues to respond. At age 19 years she began to develop morning pain and swelling in her knees. Symptoms progressed to left shoulder, bilateral wrists, proximal interphalangeal and metacarpophalangeal joints. At age 31 years she was diagnosed with rheumatoid arthritis (RA) by the rheumatology service. At presentation she was on prednisone 5mg daily, methotrexate 17.5mg weekly, status post 2 doses of adalimumab 40mg every 2 weeks. Acetaminophen and diclofenac used as needed. She was previously also on sulfasalazine 1000mg twice daily but was discontinued due to aggressiveness. She was diagnosed with mild intermittent asthma in the past year.\n\nShe began to walk at age 4 years. She never attended school, has a vocabulary of 10–12 words, follows basic commands, independently feeds, dresses, and bathes herself. She has a 37 year old brother with mild learning disability; he completed school, works and lives independently. There was no family history of similar congenital defects or autoimmune disorders.\n\nOn physical examination, her height was 135 cm and weight was 53 kg. Head was microcephalic with circumference of 51 cm. She was nonverbal and appears to fall under severe-profound intellectual disability. Skin, head and body hair was hypopigmented with a few patches of pigmentation and a large 2x1 cm left neck nevus. Midface is hypoplastic. Eyes were symmetrical with a left limbal dermoid cyst. Mouth was carp like with downturning corners. Residual posterior cleft palate and split uvula were present. Dentition was poor with several teeth broken, missing, or carious. No murmurs were appreciated. On lung exam, bilateral basilar crackles and scattered wheezes were appreciated. There was full range of motion in limbs and normal muscle tone. There was mild tenderness in left shoulder. Surgically corrected left foot noted.\n\nLaboratory results showed positive antinuclear antibody (ANA) at a titer of <1:40, positive rheumatoid factor (RF) at 81.1 IU/mL, negative anti-citrullinated cyclic protein (anti-CCP) at 16AU, elevated erythrocyte sedimentation rate at 42 mm/hr and C-reactive protein at 1.3 mg/dL. Quantitative immunoarray revealed IgA deficiency at 88.5mg/dL, normal IgM and IgG.\n\nRadiographic evaluation revealed osteopenia of left foot, ankle, hands and wrists. Images of knees revealed small left and trace right joint effusion. Transthoracic echocardiogram showed moderate tricuspid valve regurgitation and moderate pulmonary hypertension.\n\nDuring this admission, high resolution CT chest showed scattered areas of cystic bronchiectasis and bilateral right upper lobe predominant ground glass opacities. She was subsequently treated for bronchiectasis exacerbation with zosyn for a total of 7 days. She improved clinically and was discharged with extensive follow up appointments.\n\nHer primary care physician referred her to the genetics department in our hospital for suspected chromosomal abnormalities. Comparative genome hybridization (aCGH) was done using the custom designed Agilent 44,000 oligonucleotide probes microarray. Probes were placed approximately every 50–100 kb across the entire euchromatic genome with a resolution of 500 kb. The probe density at clinically relevant regions was about 5–10 kb, thus increasing the resolution to 50 kb in targeted regions based on hg19. The aCGH revealed a 14 Mb deletion at 18p11.21-p11.32 (148963-14188180)x1, a 47.8Mb duplication at 18q11.21-q22.1 (18542074-66367715)x3, and a 11.6 Mb deletion at 18q22.1-q23 (66377285-78010032)x1 (Figure 1a). Standard chromosomal analysis and fluorescence in situ hybridization (FISH) revealed a mosaic condition. One cell line contained a dicentric ring chromosome. Another cell line showed duplication of the long arm and no short arm, 46XX, r(18)(q11.3q23) (Figure 1b–c). A third cell line was detected with terminal long arm deletion, 46XX del(18)(q22). Her mother had a normal analysis performed with her consent, alongside the patients’ analysis. The paternal sample was not available.\n\nA. CGH panel illustrates deletions on distal arms (red) and duplication of proximal long arm (blue). B. Karyotype in metaphase showing a ringed chromosome 18. C. FISH analysis showing a ringed chromosome 18.\n\n\nDiscussion\n\nThe described chromosomal abnormalities are most likely de novo, since maternal analysis was normal and her father was normal in appearance and health. Furthermore, ring chromosomes usually arise de novo, with only 1% inherited9. Of the inherited cases, 90% are maternal since the presence of a ring blocks spermatogenesis and induces infertility in males.\n\nConsistent with previously reported cases of r(18) (see Table 1), our patient had many of the characteristics associated with both 18p- and distal 18q- syndrome. The features shared between the two conditions include intellectual disability, short stature, microcephaly, IgA deficiency, autoimmune disorders (RA, vitiligo, hypothyroidism), and likely conductive hearing loss. Features specific to 18p- include excessive dental caries, while those specific to distal 18q- include cleft palate, carp shaped mouth, and clubfoot. Duplication did not appear to cause any distinguishing Edwards syndrome manifestations such as clenched fist, rocker bottom feet, severe organ involvement, and failure to thrive.\n\nThe unique feature of our patient was her late onset of RA compared to the early JRA previously reported4–8,10. Our patient was RF positive, anti-CCP negative and met 9/10 of the 2010 American College of Rhematology (ACR) Clinical Classification Criteria for RA6. To the best of our knowledge there have been seven reported, and six published, cases of RA associated with chromosome 18 abnormalities (Table 1). Daentl first mentioned an unpublished case of JRA in a patient with 18p- and IgA deficiency7. In the first published case, Finley et al. reported a 9-month-old female with 18p-, swelling and contractures in many joints, fever, rash, and hepatosplenomegaly, consistent with JRA6. JRA has subsequently been associated with cases of 18q- as well5,7,9.\n\nThe exact genes involved with RA and autoimmune disease development in chromosome 18 abnormalities are still not well defined. There is a suggested link between PTPN2 (protein tyrosine phosphatase non-receptor type 2), located at 18p11.2-11.3, and RA and T1DM11,12. Genome wide studies have shown evidence for the association of the PTPN2 locus with RA susceptibility in both Japanese and European populations11,13. A case similar to ours was reported by Jain et al. with de novo r(18), del(18q23-18qter) and del(18p11.3-18pter) associated with hyperthyroidism, T1DM, vitiligo, and IgA deficiency, but not RA3. Their case had a more distal deletion that likely spared PTPN2. Another autoimmune critical region was proposed on 18p, with molecular breakpoints at 12,316,423-1,231,7830; interestingly, PTPN2 is not in this region12. The genetic basis of autoimmune disease is not as well established in 18q- compared to 18p-. On the long arm, Merriman et al. proposed a locus at 18q12-21 that influences development of autoimmune diseases14. Another gene of interest is NFATc1 (nuclear factor of activated T cells) at 18q23, implicated in maintaining the programmed death receptor (PD-1) and ligand (PD-L) pathway that is essential for regulatory T cells to terminate immune responses and protect against autoimmunity15,16. It is difficult to establish a definitive genotype-phenotype association, but it appears plausible that this proposed autoimmune critical region and PTPN2 on the short arm, as well as NFATc1 on the long arm may play a part in the autoimmune diseases seen in our patient.\n\nOverall, adult RA has a poorer outcome compared to JRA17. Mortality rates in RA patients are increased due to medication related infections, gastrointestinal bleeding as well as extra-articular pulmonary, renal disease, and cardiovascular manifestations17. Our patient was also found to have ILD, bronchiectasis, and pulmonary hypertension. To the best of our knowledge, there is no known connection between interstitial lung pathologies and chromosome 18 abnormalities. However, ILD and bronchiectasis are known extra-articular manifestations of RA. In a population study, the lifetime risk of developing ILD was 7.7% for RA patients and 0.9% for non-RA subjects18. The classic presentation is a reticular, reticulonodular or honeycomb pattern in the lung bases.\n\nThe patient was also on several medications known to cause drug-induced interstitial lung disease (DI-ILD) including: methotrexate, adalimumab, sulfasalazine, and diclofenac19. The predominantly bilateral patchy ground-glass opacities with upper lobe predominance in our patient do suggest a hypersensitivity pneumonitis picture. Unfortunately, ground-glass opacities are non-specific and pattern of involvement on HRCT does not always correspond to histological findings. Medications known to cause DI-ILD were discontinued in our patient, with further management focusing on better characterizing the extent and etiology of ILD and continuation of steroid therapy.\n\nOur report describes the first case of late onset RA associated with mosaic 18p-, 18q- and dicentric r(18). The complex rearrangements were detected by aCGH, karyotype and FISH. Her syndrome has features of both 18q- and 18p-, including multiple autoimmune disorders that support the idea of genetic loci on chromosome 18 playing a role in disease expression. Additionally, the finding of ILD - whether caused by RA, drug exposure, or an unexplored linkage - is an important condition to be aware of in patients with chromosome 18 abnormalities and autoimmune diseases. Ultimately, further studies are required to better define the genotype-phenotype associations that will further the clinical management of these patients.\n\n\nConsent\n\nWritten and informed consent was obtained from the mother, who was the designated health care proxy prior to publishing this case report, for publication of any potentially identifiable clinical data that may be associated.",
"appendix": "Author contributions\n\n\n\nAC wrote the manuscript. KR contributed with the cytogenetics analysis. AJ and SA were involved in the care of this patient. AC and SA conceptualized the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to thank the patient and her family for their participation.\n\n\nReferences\n\nKline AD, White ME, Wapner R, et al.: Molecular analysis of the 18q- syndrome--and correlation with phenotype. Am J Hum Genet. 1993; 52(5): 895–906. PubMed Abstract | Free Full Text\n\nGuilherme RS, Meloni VF, Kim CA, et al.: Mechanisms of ring chromosome formation, ring instability and clinical consequences. BMC Med Genet. 2011; 12: 171. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJain N, Reitnauer PJ, Rao KW, et al.: Autoimmune polyendocrinopathy associated with ring chromosome 18. J Pediatr Endocrinol Metab. 2011; 24(9–10): 847–50. PubMed Abstract | Publisher Full Text\n\nRecalcati MP, Valtorta E, Romitti L, et al.: Characterisation of complex chromosome 18p rearrangements in two syndromic patients with immunological deficits. Eur J Med Genet. 2010; 53(4): 186–91. PubMed Abstract | Publisher Full Text\n\nRosen P, Hopkin RJ, Glass DN, et al.: Another patient with chromosome 18 deletion syndrome and juvenile rheumatoid arthritis. J Rheumatol. 2004; 31(5): 998–1000. PubMed Abstract\n\nFinley SC, Finley WH, Johnson JC, et al.: Rheumatoid arthritis in the 46, XX, 18p- syndrome. Clin Genet. 1972; 3(6): 465–9. PubMed Abstract | Publisher Full Text\n\nPetty RE, Malleson P, Kalousek DK: Chronic arthritis in two children with partial deletion of chromosome 18. J Rheumatol. 1987; 14(3): 586–7. PubMed Abstract\n\nHansen US, Herlin T: Chronic Arthritis in a Boy with 18q- Syndrome. J Rheumatol. 1994; 21(10): 1958–9. PubMed Abstract\n\nCaba L, Rusu C, Plăiaşu V 5th, et al.: Ring autosomes: some unexpected findings. Balkan J Med Genet. 2012; 15(2): 35–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCzakó M, Riegel M, Morava É, et al.: Patient with rheumatoid arthritis and MCA/MR syndrome due to unbalanced der(18) transmission of a paternal translocation t(18;20)(p11.1;p11.1). Am J Med Genet. 2002; 108(3): 226–8. PubMed Abstract | Publisher Full Text\n\nCobb JE, Plant D, Flynn E, et al.: Identification of the Tyrosine-Protein Phosphatase Non-Receptor Type 2 as a Rheumatoid Arthritis Susceptibility Locus in Europeans. PLoS One. 2013; 8(6): e66456. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHasi-Zogaj M, Sebold C, Heard P, et al.: A review of 18p deletions. Am J Med Genet C Semin Med Genet. 2015; 169(3): 251–64. PubMed Abstract | Publisher Full Text\n\nOkada Y, Terao C, Ikari K, et al.: Meta-analysis identifies nine new loci associated with rheumatoid arthritis in the Japanese population. Nat Genet. 2012; 44(5): 511–6. PubMed Abstract | Publisher Full Text\n\nMerriman TR, Cordell HJ, Eaves IA, et al.: Suggestive Evidence for Association of Human Chromosome 18q12-q21 and Its Orthologue on Rat and Mouse Chromosome 18 With Several Autoimmune Diseases. Diabetes. 2001; 50(1): 184–94. PubMed Abstract | Publisher Full Text\n\nCody JD, Sebold C, Heard P, et al.: Consequences of chromsome18q deletions. Am J Med Genet C Semin Med Genet. 2015; 169(3): 265–80. PubMed Abstract | Publisher Full Text\n\nFrancisco LM, Sage PT, Sharpe AH: The PD-1 pathway in tolerance and autoimmunity. Immunol Rev. 2010; 236(1): 219–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrahalad S, Glass DN: Is juvenile rheumatoid arthritis/juvenile idiopathic arthritis different from rheumatoid arthritis? Arthritis Res. 2002; 4(Suppl 3): 303–10. Publisher Full Text | Free Full Text\n\nBongartz T, Nannini C, Medina-Velasquez YF, et al.: Incidence and mortality of interstitial lung disease in rheumatoid arthritis: A population-based study. Arthritis Rheum. 2010; 62(6): 1583–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchwaiblmair M, Behr W, Haeckel T, et al.: Drug Induced Interstitial Lung Disease. Open Respir Med J. 2012; 6: 63–74. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "27904",
"date": "23 Nov 2017",
"name": "György Kosztolányi",
"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 well documented case report which may contribute to our knowledge on the clinical consequences of chromosome 18 abnormalities. I suggest to accept the MS for publication on condition that the authors consider a major and some minor remarks for modification.\nMajor remark\nThe routine cytogenetics description is too short. Nothing is written about the percentages of the \"mosaic cell lines” (as the authors write), although one of the unique characteristics of ring chromosomes is their dynamic nature. As a result of mitotic difficulties, a ring chromosome is subject of additional cytogenetic mutations, resulting in continuous generation of secondary aneuploidy cells. Accordingly, this dynamic mutations series may manifest themselves as \"mosaic cell lines”, however, the survival of such cells as cell line, as well as the explanation of their presence being \"cell lines” is questionable. I would recommend to refer to this wildely accepted explanation for the presence of differentially shaped chromosomes in patients with ring chromosome, at least as an alternativ possibility. (e.g. Kosztolányi G: Does \"ring syndrome\" exist? Hum.Genet. 1987; 75:174-179)\n\nMinor remark\nSome peculiarity of the case should be highlighted with more emphasis. E.g., Detecting a chromosome abnormality in a patient at the age of 32 is rare – it should be highlighted in the paper. Also the extreme severity of the somatic and mental underdevelopment should be pointed on abstract.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "27903",
"date": "27 Nov 2017",
"name": "Jannine D. Cody",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes a single 32 year old woman with Ring 18 and rheumatoid arthritis. Overall the manuscript is well written, however there are several items that need further work before publicaiton.\nMost of the literature on Ring 18 has been omitted. The paper would benefit from a better review of Ring 18 as opposed to mostly 18p- and 18q-. Has the lung disease eve been reported?\n\nThe methods and data that determined the actual chromosome content are missing. The percent that each cell type is present in the blood as well as the FISH studies definitively demonstrating each should be included. From the text it is not clear if the “duplication of the long arm” is present as a ring or an isochromosome. The aCGH showing net copy number suggests that not all of the long arm is duplicated since a larger proportion of cells have an 18q terminal deletion than have an 18p terminal deletion. What percent of the cells actually have a ring chromosome?\n\nThe interpretation of the FISH data needs to be described more fully. Why are there two BCL2 probes? I don’t see a D18Z1 signal?\n\nThe table needs to be made more clear in its title that it only includes RA cases and not all Ring 18 or 18q-, 18p- cases (at least I think that is the intent).\n\nIn the first paragraph of the discussion, the last sentence needs a reference.\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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1940
|
https://f1000research.com/articles/7-232/v1
|
27 Feb 18
|
{
"type": "Method Article",
"title": "Identification of microsatellite loci in sea anemones Aulactinia stella and Cribrinopsis albopunctata (family Actiniidae)",
"authors": [
"Ekaterina S. Bocharova",
"Alexey A. Sergeev",
"Aleksandr A. Volkov",
"Alexey A. Sergeev",
"Aleksandr A. Volkov"
],
"abstract": "From the DNA libraries enriched by the repeat motifs (AAAC)6, (AATC)6, (ACAG)6, (ACCT)6, (ACTC)6, ACTG)6, (AAAT)8, (AACT)8, (AAGT)8, (AGAT)8, for two viviparous sea anemones Aulactinia stella and Cribrinopsis albopunctata, 41 primer pairs were developed. These primer pairs resulted in the identification of 41 candidate microsatellite loci in either A. stella or C. albopunctata. Polymorphic loci were identified in both sea anemone species for 13 of the primer pairs and can be applicable for population genetics researches.",
"keywords": [
"cnidaria",
"sea anemones",
"microsatellites",
"primers",
"Aulactinia",
"Cribrinopsis"
],
"content": "Introduction\n\nSea anemones are known to live in clonal or partially clonal populations (Bocharova, 2015; Bocharova 2016; Bocharova & Mugue, 2012). Data based on sequences of mitochondrial (12S rRNA, 16S rRNA and cytochrome oxidase III) and nuclear (18S rRNA and 28S rRNA) genes, which are successfully used in phylogenetic research, are not applicable to population genetics studies because of the high amount of monomorphic samples. Sometimes it is not evident that a population is clonal, for instance, in the parthenogenetic populations of Aulactinia stella (Verrill, 1864) in the White and the Barents Seas (Bocharova & Mugue, 2012; Bocharova, 2015). Representatives of other species can combine sexual and asexual (clonal) reproduction in response to environmental changes (Bocharova & Kozevich, 2011). For Cribrinopsis albopunctata Sanamyan et Sanamyan, 2006 there is no data about its asexual or parthenogenetic reproduction and populations of this species usually consist of males and females. Thus, these two species are characterized by different reproductive modes. The development of polymorphic microsatellite markers resulted in the design of 41 primer pairs, which were subsequently screened using DNA from both A. stella and C. albopunctata to assess primer utility in different species and populations of the same species.\n\n\nMethods\n\nFor this research, sea anemone specimens were collected in Avachinsky Bay of Kamchatka Peninsula at the depths of 11–18 meters and identified in vivo. The total DNA was extracted from the samples, which were preserved in 96% ethanol, using the Wizard SV Genomic DNA Purification System (Promega) with previous desiccation and grinding. Extracted genomic DNA was exposed to fragmentation by Covaris S-Series (Covaris, USA) resulting in average distribution of fragment lengths of about 150–200 bp, which were additionally estimated by capillary electrophoresis (Nanofor-05, Syntol, Russia) with non-denaturing polymer and intercalating dye. DNA libraries were prepared by using TruSeq DNA LT Sample Prep Kit (Illumina, USA). Then the libraries were enriched for the repeat motifs (AAAC)6, (AATC)6, (ACAG)6, (ACCT)6, (ACTC)6, ACTG)6, (AAAT)8, (AACT)8, (AAGT)8, (AGAT)8 and screened according to the protocol described in Glenn & Schable (2005). The fragments containing hybridization of biotinylated oligonucleotides with tandem microsatellite repeats was separated by magnetic Streptavidin M-280 Dynabeads (Dynal, Oslo, Norway). The enriched genomic DNA libraries were treated using Miseq Kit v2 for 300 cycles in the paired-end read mode (250 + 250 bp) for MiSeq next-generation sequencing (Illumina, USA).\n\nSequences obtained were analyzed for the repeat regions by NGS analysis tool Geneious 10.2.3, which also compared sequences to determine the existence of duplicates. This software was also used to create 41 primer pairs flanking the repeat regions of interest. Primers were named Act ## and numbered sequentially.\n\nThe total DNA from pedal disc tissues of five A. stella specimens and three C. albopunctata specimens was extracted by Wizard SV Genomic DNA Purification System (Promega, USA) following the manufacturer’s protocol. Extracted DNA was amplified using the newly created primers. An amount of 50 ng of the extracted DNA was amplified in 20 µL reactions with 1x SmarNGTaq Buffer (Dialat Ltd., Russia), 25 µM of each of four deoxyribonucleoside triphosphates, 2 mM MgCl2, 0.1 µM of each fluorescent labeled forward and unlabeled reverse primers, and 1 unit SmarNGTaq polymerase (Dialat Ltd., Russia). Amplification of all the microsatellite loci was performed by Touchdown PCR with the following conditions: 96°C for 3 minutes for initial denaturation, followed by 30 cycles at 96°C for 10s, 62°C for 30s (with a 0.2°C decrease in the second step of each cycle), 72°C for 10s; 10 cycles at 96°C for 10s, 56°C (with a 0.2°C increase in the second step of each cycle) for 30s, 72°C for 10s; 20 cycles at 96°C for 10s, 56°C for 30s, 72°C for 10s; 72°C for 10 minutes; ending with a 4°C soak.\n\nOne µL of PCR product was added to 24 µL of deionized formamide Hi-Di (Applied Biosystems, USA) and 1 µL of Liz-labeled ladder SD-450 (Syntol, Russia) and denatured at 95°C for 3 minutes. Products were visualized in 3500 Genetic Analyzer (Applied Biosystems, USA) using POP7 gel polymer.\n\n\nValidation\n\nAnalysis of the obtained chromatograms was performed by GenMapper Software (ThermoFisher Scientific, USA). Of the 41 primer pairs developed, 5 (12.2%) resulted in poor or no amplification in both A. stella and C. albopunctata. Almost half (56.1%) of the remaining loci successfully amplified was monomorphic in the two species. Finally, 13 primer pairs appeared to amplify polymorphic microsatellite loci at combined panels for the two species (Table 1).\n\n\nData availability\n\nThe raw data is available:\n\n- https://doi.org/10.5281/zenodo.1171106 (Bocharova et al., 2018a). The dataset contains three files in different formats (*.scv, *.geneious,*.fasta) with primer sequences of all 41 STR loci for Aulactinia stella and Cribrinopsis albopunctata.\n\n- http://doi.org/10.5281/zenodo.1144120 (Bocharova et al., 2018b). The dataset includes *.fsa files (the Prism Genetic Analyzer 3500, Applied Biosystems) of pooled DNA PCR product of 13 STR polymorphic loci for Aulactinia stella and Cribrinopsis albopunctata.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work is supported by Russian Fund for Basic Research 16-04-01685.\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\nAdditional support came from the Syntol Company (www.syntol.ru) due to help of Vera Ustinova and Julia Monakhova with DNA libraries preparation and MiSeq sequencing. Special thanks are given to Nadya and Karen Sanamyan (Kamchatka Branch of Pacific Geographical Institute, Far-Eastern Branch of the Russian Academy of Sciences) for collecting and identifying of these anemone samples.\n\n\nReferences\n\nBocharova ES: Reproductive Biology and Genetic Diversity of the Sea Anemone Aulactinia stella (Verrill, 1864). Hydrobiologia. 2015; 759(1): 27–38. Publisher Full Text\n\nBocharova ES: Reproduction of sea anemones and other hexacorals. In book: The Cnidaria, Past, Present and Future. 2016; 239–248. Publisher Full Text\n\nBocharova ES, Kozevich IA: Modes of reproduction in sea anemones (Cnidaria, Anthozoa). Biology Bulletin. 2011; 38(3): 849–860. Publisher Full Text\n\nBocharova ES, Mugue NS: Sea anemones Aulactinia stella (Verrill, 1864) (Hexacorallia, Actiniidae) can brood offspring from other individuals of the same species. Dokl Biol Sci. 2012; 444(1): 173–175. PubMed Abstract | Publisher Full Text\n\nBocharova ES, Sergeev AA, Volkov AA: Primer sequences of 41 STR loci of two viviparous sea anemones Aulactinia stella and Cribrinopsis albopunctata. 2018a. Data Source\n\nBocharova ES, Sergeev AA, Volkov AA: 13 STR polymorphic loci for Aulactinia stella and Cribrinopsis albopunctata [Data set]. Zenodo. 2018b. Data Source\n\nGlenn TC, Schable NA: Isolating microsatellite DNA loci. In: Methods Enzymol, Molecular Evolution: Producing the Biochemical Data, Part B. (Zimmer EA, Roalson EH, eds). Academic Press, San Diego, CA. 2005; 395: 202–222. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "31280",
"date": "23 Mar 2018",
"name": "Boris Levin",
"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\nMS ‘Identification of microsatellite loci in sea anemones Aulactinia stella and Cribrinopsis albopunctata (family Actiniidae)’ by E.S. Bocharova et al. is a methodical study related to the search and testing of polymorphic microsatellite loci in the sea anemones of above mentioned species of Actiniidae. Microsatellites are informative genetic markers for population genetic studies. Such approach is useful for obtaining information on many fundamental issues related to the reproductive strategy and spread of clonal organisms. Unlike well-studied commercial species, the biology of sea anemones is still remained unexplored in many aspects. Theory explaining the evolution of a larval stage and their long-distance dispersal ability requires genetic examination. The clonal nature of these organisms impedes application of mtDNA polymorphism and requires the search for other informative genetic markers such as microsatellites. The methods used in this study are modern and reproducible. Manuscript is well written and concise. Apparently, developed STR markers will be suitable in studies of other sea anemone species. I recommend the MS to be indexed.\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": "33174",
"date": "18 Apr 2018",
"name": "Fabián H. Acuña",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is a concise study concerning with the identification of microsatellite loci in sea anemones Aulactinia stella and Cribrinopsis albopunctata, both belonging to family Actiniidae, from Avachinsky Bay of Kamchatka Peninsula. In the introduction the authors said \"Sea anemones are known to live in clonal or partially clonal populations\" but this is valid only for some (not all) sea anemone species and it is not possible to generalize. The metodology is detailed and all the source data are available to ensure full reproducibility. The authors mention that total DNA from pedal disc tissues of studied species was extracted, but they must justify why they choose this part and not other (column, oral disc or tentacles) of examined sea anemones for DNA extraction. The text is correctly written and obtained results are well exposed in the text and table 1. This article is a valuable contribution for sea anemone genetic population investigations and merit approval for indexing.\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": "33175",
"date": "24 Apr 2018",
"name": "Svetlana V. Malysheva",
"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 the title implies the article describes the method of microsatellite loci isolation from the DNA of two sea anemone species, which are not the model objects. The goal of the study is to bring some information about 41 microsatellite loci and corresponding primers for PCR than can be used for different species of sea anemones, which genomes have not been sequenced yet. It is reported that 13 of 41 microsatellite loci were polymorphic for the two species but possibly will be suitable for population genetic studies of other sea anemones. Much attention is given to the otimization of PCR conditions and this procedure is described in detail. In my opinion, this article is interesting and can be useful for scientists who deal with sea anemones or other representatives of Anthozoa because it includes elaborate technique for microsatellite loci isolation as well as prepared loci for microsatellite analysis. The manuscript is certainly worthy for publication.\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/7-232
|
https://f1000research.com/articles/7-231/v1
|
27 Feb 18
|
{
"type": "Clinical Practice Article",
"title": "Intraoperative frozen section as a reliable ancillary technique in salivary gland surgery: A cross sectional study",
"authors": [
"Andrea Marzullo",
"Gabriella Serio",
"Luigi Madami",
"Federica Pezzuto",
"Francesco Fortarezza",
"Nicola Quaranta",
"Maria Luisa Fiorella",
"Teresa Lettini",
"Leonardo Resta",
"Massimo Marrelli",
"Marco Tatullo",
"Luigi Santacroce",
"Andrea Marzullo",
"Gabriella Serio",
"Luigi Madami",
"Federica Pezzuto",
"Francesco Fortarezza",
"Nicola Quaranta",
"Maria Luisa Fiorella",
"Teresa Lettini",
"Leonardo Resta",
"Massimo Marrelli",
"Marco Tatullo"
],
"abstract": "Background. Salivary glands tumours are uncommon, frequently benign lesions, prevalently located in the parotid gland (80%). Surgical decision making is based on the patient’s history, examination findings, imaging and fine needle aspiration (FNA). FNA is a pre-operative method with good ability in detecting malignancy. During surgery, therefore, Frozen section (FS) can differentiate benign lesions from malignant tumours, to reduce incorrect treatments, to increase the chances of conservative surgery and to better evaluate surgical margins. The aim of our study is to demonstrate the accuracy of the FS procedure in surgery of the salivary glands and to stress the need for dedicated pathology units specialized in lesions of the oral cavity. Methods. The study included 499 patients who underwent surgery from May 2005 and October 2014. An intra-operative frozen section procedure was done for 288 of them. All frozen sections were compared with the final results. The cases were classified by site, nature of the lesion and histotype, according to the WHO classification. Comparison was made between the intra-operative and the definitive diagnosis. Results. Of the 288 FS procedures, 259 were for neoplastic lesions, 199 of which benign and 60 malignant, and 29 for non-neoplastic lesions. Of the 259 neoplastic FS results, 2 were shown to be false positives and 2 were diagnosed as different malignant types. Of the 29 non-neoplastic FS results, 4 were false negatives. Conclusions. Our results showed that the accuracy of frozen section procedure is 98% for salivary glands tumors. The highest concordance between frozen section and the definitive diagnosis was for inflammatory processes (99%), pleomorphic adenoma (98%), Warthin’s tumor (97%) and malignant neoplasms (96%). In conclusion, based on these findings, frozen section of the salivary glands may be proposed as a routine procedure and should be used in decision-making.",
"keywords": [
"Salivary gland tumors",
"frozen section",
"differential diagnosis",
"Warthin’s tumor",
"pleomorphic adenoma"
],
"content": "Introduction\n\nSalivary gland tumors are rare lesions that occur mainly in the major salivary glands; 80% of tumors occur in the parotid gland, and these are prevalently benign.\n\nThe parotid gland histology is complex: there are i) abundant intralobular and extralobular adipose tissues, which increase in relative volume with age, ii) randomly distributed lymphoid aggregates, and iii) lymph nodes that occasionally contain ducts or salivary acini. Therefore, it is often difficult to distinguish a neoplastic lesion from a non-neoplastic lesion, as well as a benign lesion from a malignant lesion, especially in view of the morphological variability of salivary tumors. However, a correct differential diagnosis, safely and promptly executed, is crucial for the entire patient’s management, both clinical and surgical, including possible tissue regeneration1–5.\n\nThe clinical approach to salivary lesions is supported by imaging techniques, such as ultrasound, computed tomography or magnetic resonance imaging; these can provide greater definition of the lesion, but are not always sufficient to formulate a definitive diagnosis. Therefore, it is necessary to resort to pre-surgical techniques to better define a salivary lesion.\n\nFine needle aspiration (FNA), which can be performed at the time of the initial clinical consultation, can be used both as a diagnostic test and as a guideline in selecting the patient’s management: surgical vs follow-up without surgery. The FNA technique demonstrates high sensitivity and specificity (80% and 97%) for benign tumors, but is not very sensitive for malignant neoplasms (sensitivity ranges from 54% to 92%; specificity 87% to 98%). False-negative rates range from 2% to 31% and false positive rates from 0% to 7%6–11.\n\nFNA is a simple, safe procedure that does not require the use of local anesthesia, and can be performed either blinded or under ultrasound guidance. It is important to remember that the methodological approach, clinical needle aspiration skills and experience of the pathologist are the elements that affect the definitive diagnosis. Frozen section (FS) is a less rapid, more invasive intraoperative diagnostic procedure, but precisely because of this, it is a guarantee of better histological results. It allows surgery to be performed in a targeted treatment continuum1,9–10.\n\nOnce again, it should be emphasized that salivary tumors are a heterogeneous group of lesions that necessitate the examination of many sections. Early diagnosis is essential, to establish the correct histological type of salivary glands lesion, in order to achieve proper planning of the surgical treatment, which may also involve the regional lymph nodes, and adjacent tissues11. Variable percentages of effectiveness of the FS method applied to the diagnosis of salivary glands lesions are reported in the literature. The reliability rate ranges from 40% to 100%; this variability is often attributed to the pathologist's experience or to the technician responsible for slide preparation12–16.\n\nIn this study we assessed the diagnostic accuracy of FS; specifically, the concordance between FS and the definitive histological diagnosis, as well as verifying the importance of being able to rely on a team of experts, to reduce false positive or negative cases.\n\n\nMethods\n\nBetween May 2005 to October 2014, 499 patients (275 males and 224 females, mean age 54±17.2 years) suffering from localized masses in the salivary glands were recruited at the Complex Unit of Otolaryngology at the University of Bari (Italy).\n\nInclusion criteria were related to the clinical aspects of the first access diagnosis: only patients with localized masses developed in the major salivary glands regions were included. Exclusion criteria were a previous history of cancer or any suspicion of infectious disease as main noxa of the mass. Also, patients reporting smoking habits were excluded as well (Figure 1).\n\nWe selected 288 salivary lesions (out of the total 499) operated on by the same team of surgeons and pre-analyzed with FS.\n\nTo reduce bias, in 90% of cases, the intraoperative examination was performed by the same team of pathologists. In all cases, the radiological examination posed an indication for surgical treatment and suggested a provisional diagnosis (i.e. benign vs malignant). FNA was not considered because if done at all, it was at non-dedicated centers and there was a high number of \"non-diagnostic\" results.\n\nOur study was aimed to assess if and how many were the neoplastic lesions, how many were benign and/or malignant, and if some non-neoplastic lesions were also diagnosed. Our attention was also directed towards the identification of some false positive/negative results. The accuracy of FS procedure was compared, in all the reported cases, with the traditional histological assay. Our null-hypothesis was aimed to assess that FS of the salivary glands may be proposed as a routine procedure and should be used in the decision-making process.\n\nThe FS procedure, also called cryosection, is a commonly used procedure to perform rapid microscopic analysis of a specimen: it is mostly used as a first-look diagnostic tool in intraoperative oncological surgery. The protocol adopted for performing intraoperative FS was a standardized method: i) the biopsy was fixed and cut in a cryostat at -25°-28°C, ii) the samples were cut into 5 micrometers thick slices, iii) the samples were subjected to haematoxylin-eosin staining. (Figure 2).\n\n(A–C) Left parotidectomy surface that includes a well-demarcated nodule measuring 3cm in diameter and showing translucent appearance of the cut surface; (D) The intraoperative frozen section (FS) technique and (E) corresponding hematoxylin-eosin section; (F–G) Pathology preliminary FS report compatible with pleomorphic adenoma.\n\nThe medical report was obtained, in all patient cases, within 10–15 minutes. Each sample was stored with labels detailing the biodata and the macroscopic characteristics of the excised lesion.\n\n\nResults\n\nWe reported 288 patients with salivary lesions operated on by the same team of surgeons and pre-analyzed with FS, in order to make a comparative analysis between the two different techniques.\n\nFS was useful to indicate the correct surgical treatment of 269 nodules (93.4%) of the parotid gland, 14 nodules (4.9%) of the submandibular gland and 5 lesions (1.7%) that involved the minor salivary glands located in the palatal mucosa. Using the FS method, a correct diagnosis was obtained in 280 cases (97.2%) (Table 1).\n\nThe highest concordance between the FS and the definitive diagnosis was for pleomorphic adenoma (98%), Warthin’s tumor (97%), inflammatory processes (99%) and malignant neoplasms (96%). In 8 cases (2.8%), the \"provisional diagnosis\" with intraoperative FS and the definitive diagnosis were discordant. The false negative results for FS consisted of the following: 1 inflammatory process, actually diagnosed as Warthin’s tumor; 1 normal tissue, actually diagnosed as an arteriovenous malformation; 1 reactive lymphoid tissue, actually diagnosed as a non-Hodgkin’s lymphoma; and 1 sialometaplasia, actually diagnosed as a squamous cell carcinoma. Two false positive results were obtained: in both these cases squamous metaplasia in pleomorphic adenoma was interpreted as malignant (Table 2). Thus, the sensitivity and specificity for malignancy were assessed to 97%. Patients with a positive FS diagnosis underwent lymphadenectomy. The FS diagnosis of malignancy was not confirmed at histology in two cases: 1 carcinoma, actually a NH-Lymphoma; 1 NH-Lymphoma, actually a small cell carcinoma. In these cases, only one patient received overtreatment.\n\nAnalyzing the definitive diagnosis vs FS, the false positive cases were found to have been diagnosed by a non-dedicated pathologist, as also one case of Warthin’s cancer, defined as an inflammatory process.\n\n\nDiscussion\n\nThis study was conducted in order to assess the diagnostic accuracy, sensitivity and specificity of FS examination, used as a preoperative method to guide the surgeon in the choice of treatment of salivary lesions. A further aim of the study was to analyse the diagnostic accuracy of FS examination to pose the diagnosis of malignancy. Previous studies have compared the accuracy of FS examination with FNA cytology, observing a greater reliability of the FS technique13. In fact, FNA shows too high a number of false negatives due to factors related to: i) triage errors, ii) hypocellularity of the material, iii) interpretation errors10,11. Some studies have demonstrated an excellent effectiveness of a FS examination, obtaining a specificity of 99% and a sensitivity of 90%, other authors have even reported maximum specificity and sensitivity, equal to 100%16,17.\n\nIn our study, we excluded FNA as a perioperative examination because the number of \"inadequate\" tissues was too high. In our experience, FS could replace FNA, reducing the risk of inappropriate surgery, of unnecessary adjuvant radiotherapy, as well as reducing National Health System costs18.\n\nBased on the results described in this study, the FS examination shows a high reliability in the diagnosis of salivary gland tumors. FS is particularly useful in cases of differential diagnosis between neoplastic and non-neoplastic lesions; it also shows a good reliability in the differential diagnosis between benign and malignant neoplasms.\n\nDespite our study limitations, related to the small sample size and related to the single unique center participating to this study, our results are in agreement to those reported by various authors, and they also highlight the need to be able to rely on a dedicated team of clinicians and pathologists15–20.\n\n\nConclusions\n\n“Misinterpretation” was observed in those cases diagnosed by pathologists with no specific experience of head and neck tumors. Our work group experience emphasizes the importance of intraoperative examination in order to define the histotype and the cancer margins, permitting the performance of effective, predictable surgery of the salivary glands.\n\n\nEthical statement\n\nIn Italy, directors of clinical research units and those responsible for clinical services may access data records for research purposes if patients have previously signed a consent form that confirming that they allow this use of their data. Therefore, no specific ethical approval is required for this study, and all patients included in the study signed written informed consent allowing their data to be used in future research.\n\n\nData availability\n\nDataset 1: Raw data underlying the study, including final diagnosis and FS diagnosis. DOI, 10.5256/f1000research.13043.d19293521",
"appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors are grateful to Mary V. Pragnell, BA, for language assistance.\n\n\nReferences\n\nBarnes L, Evenson JW, Reichart P, et al.: Pathology and Genetics. Head and Neck Tumours. WHO Classification of Tumours. Lyon, 2005. Reference Source\n\nPinkston JA, Cole P: Incidence rates of salivary gland tumors: results from a population-based study. Otolaryngol Head Neck Surg. 1999; 120(6): 834–403. PubMed Abstract | Publisher Full Text\n\nSpiro RH: Salivary neoplasms: overview of a 35-year experience with 2,807 patients. Head Neck Surg. 1986; 8(3): 177–844. PubMed Abstract | Publisher Full Text\n\nLee RJ, Tan AP, Tong EL, et al.: Epidemiology, Prognostic Factors, and Treatment of Malignant Submandibular Gland Tumors: A Population-Based Cohort Analysis. JAMA Otolaryngol Head Neck Surg. 2015; 141(10): 905–12. PubMed Abstract | Publisher Full Text\n\nPerniconi B, Coletti D, Aulino P, et al.: Muscle acellular scaffold as a biomaterial: effects on C2C12 cell differentiation and interaction with the murine host environment. Front Physiol. 2014; 5: 354. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBall AB, Fish S, Thomas JM: Malignant epithelial parotid tumours: a rational treatment policy. Br J Surg. 1995; 82(5): 621–23. PubMed Abstract | Publisher Full Text\n\nTan LG, Khoo ML: Accuracy of fine needle aspiration cytology and frozen section histopathology for lesions of the major salivary glands. Ann Acad Med Singapore. 2006; 35(4): 242–48. PubMed Abstract\n\nMegerian CA, Maniglia AJ: Parotidectomy: a ten year experience with fine needle aspiration and frozen section biopsy correlation. Ear Nose Throat J. 1994; 73(6): 377–380. PubMed Abstract\n\nHowlett DC, Trantafyllou A: Evaluation: Fine Needle Aspiration Cytology, Ultrasound-Guided Core Biopsy and Open Biopsy Techniques. Adv Otorhinolaryngol. 2016; 78: 39–45. PubMed Abstract | Publisher Full Text\n\nFoundakowski C, Castaño J, Abouyared M, et al.: The role of indeterminate fine-needle biopsy in the diagnosis of parotid malignancy. Laryngoscope. 2014; 124(3): 678–81. PubMed Abstract | Publisher Full Text\n\nFakhry N, Antonini F, Michel J, et al.: Fine-needle aspiration cytology in the management of parotid masses: evaluation of 249 patients. Eur Ann Othorynolaryngol Head Neck Dis. 2012; 129(3): 131–35. PubMed Abstract | Publisher Full Text\n\nSantacroce L, Luperto P, Fiorella ML, et al.: [Carcinoma of unknown origin++ with latero-cervical metastasis. Diagnostic problems. Retrospective analysis of 110 cases of latero-cervical tumefaction]. Clin Ter. 2000; 151(3): 199–201. PubMed Abstract\n\nFakhry N, Santini L, Lagier A, et al.: Fine needle aspiration cytology and frozen section in the diagnosis of malignant parotid tumours. Int J Oral Maxillofac Surg. 2014; 43(7): 802–5. PubMed Abstract | Publisher Full Text\n\nHillel AD, Fee WE Jr: Evaluation of frozen section in parotid gland surgery. Arch Otolaryngol. 1983; 109(4): 230–232. PubMed Abstract | Publisher Full Text\n\nOlsen KD, Moore EJ, Lewis JE: Frozen section pathology for decision making in parotid surgery. JAMA Otolaryngol Head Neck Surg. 2013; 139(12): 1275–78. PubMed Abstract | Publisher Full Text\n\nMostaan LV, Yazdani N, Madani SZ, et al.: Frozen section as a diagnostic test for major salivary gland tumors. Acta Med Iran. 2012; 50(7): 459–62. PubMed Abstract\n\nCarvalho MB, Soares JM, Rapoport A, et al.: Perioperative frozen section examination in parotid gland tumors. Sao Paulo Med J. 1999; 117(6): 233–37. PubMed Abstract | Publisher Full Text\n\nArabi Mianroodi AA, Sigston EA, Vallance NA: Frozen section for parotid surgery: should it become routine? ANZ J Surg. 2006; 76(8): 736–39. PubMed Abstract | Publisher Full Text\n\nSchmidt RL, Hunt JP, Hall BJ, et al.: A systematic review and meta-analysis of the diagnostic accuracy of frozen section for parotid gland lesions. Am J Clin Pathol. 2011; 136(5): 729–738. PubMed Abstract | Publisher Full Text\n\nTatullo M, Marrelli M, Amantea M, et al.: Bioimpedance Detection of Oral Lichen Planus Used as Preneoplastic Model. J Cancer. 2015; 6(10): 976–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarzullo A, Serio G, Madami L, et al.: Dataset 1 in: Intraoperative frozen section as a reliable ancillary technique in salivary gland surgery: A cross sectional study. F1000Research. 2018. Data Source"
}
|
[
{
"id": "31226",
"date": "26 Mar 2018",
"name": "Michele Cassano",
"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\nInteresting paper about the importance of frozen section in the diagnosis of Salivary glands lesions. The casistic is very wide and the results are innovative also if many guidelines report the need of performing Fine Needle Aspiration in the preoperative setting of a salivary gland lesion and I do not think that we can actually derogate from this guideline. Anyway this paper is worthy of indexing but some points need to be reviewed:\nThe first sentence of the \"FS process\" in method section should be moved to the introduction to complete the paragraph about the frozen section.\n\nIn the inclusion criteria the authors report that just major salivary glands lesions were analyzed but in the outcomes data they state that 5 cases were minor salivary glands glands in the palatal mucosa. Please clarify this point.\n\nIn the discussion the authors state that the FS is used as \"pre-operative method\". I think that is more correct to define it an intraoperative method.\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": "32697",
"date": "24 Apr 2018",
"name": "Kyung-Ja Cho",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is recommending frozen section as a primary evaluation of salivary gland tumors, and it is considered out of date and unethical. Many salivary gland tumors share histologic features and have dintinct expression or molecular characteristics. Ancillary tests are occasionally necessary. Accurate diagnosis before operation, if possible, is needed for proper patient management.\n\nCore needle biopsy of the salivary gland tumors is recently substituting aspiration cytology. Authors need to comment the usefulness of such preoperative diagnostic procedures.\n\nThree exclusion criteria from selection seem irrelevant with diagnosis of salivary gland tumors.\n\nThe distribution of tumors appear very biased even after exclusion. Mucoepidermoid carcinoma is the most common malignancy worldwide, while squamous cell carcinoma of salivary gland is rare.\n\nIt is difficult to be convinced that most of lymphomas, and all small cell carcinomas and carcinoma ex pleomorphic adenomas were accurately diagnosed by frozen section. Illustrations or other back-up data are needed.\n\nIs the background of the cases’ 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 conclusion balanced and justified on the basis of the findings? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-231
|
https://f1000research.com/articles/7-228/v1
|
26 Feb 18
|
{
"type": "Case Report",
"title": "Case Report: “Incognito” proteus syndrome",
"authors": [
"Michelangelo Vestita",
"Angela Filoni",
"Nicola Arpaia",
"Grazia Ettorre",
"Domenico Bonamonte",
"Angela Filoni",
"Nicola Arpaia",
"Grazia Ettorre",
"Domenico Bonamonte"
],
"abstract": "Proteus syndrome (PS) is a postnatal mosaic overgrowth disorder, progressive and disfiguring. It is clinically diagnosed according to the criteria reported by Biesecker et al. We describe the case of a 49-year-old woman who presented with a 10-year history of pauci-symptomatic infiltrating plaque lesions on the sole and lateral margin of the left foot, which had been diagnosed as a keloid. The patient had a positive history for advanced melanoma and a series of subtle clinical signs, such as asymmetric face, scoliosis, multiple lipomas on the trunk, linear verrucous epidermal nevi, and hyperpigmented macules with a mosaic distribution. Even if the clinical presentation was elusive, she had enough criteria to be diagnosed with PS. This case describes the first evidence, to the best of our knowledge, of pauci-symptomatic PS in adulthood, reports its rare association with advanced melanoma, and illustrates the importance of even minor cutaneous clinical signs, especially when atypical, in formulating the diagnosis of a complex cutaneous condition such as this.",
"keywords": [
"proteus syndrome",
"cerebriform",
"keloid",
"diagnosis",
"elusive",
"hidden"
],
"content": "Introduction\n\nProteus syndrome (PS) is a postnatal mosaic overgrowth disorder, which was originally described by Cohen and Hayden in 19791. In 1983, the syndrome was named after a minor Greek deity who had the power to change his appearance2. The occurrence of this disorder is sporadic, with a prevalence of less than one per million3. PS is a progressive, disfiguring disorder caused by a somatic point mutation in AKT1 leading to gene activation. The product of this gene is involved in cell proliferation and apoptosis suppression, acting through the mammalian target of rapamycin signaling pathway, which may explain the overgrowths in this syndrome4. Clinically, this disorder is characterized by typically asymmetric, disproportionate, postnatal overgrowth of tissues derived from any of the three germ layers. While skin, bone, connective, and adipose tissues are most commonly involved, some patients present with overgrowths of the central nervous system, spleen, thymus, or colon. In addition, patients may also present with a range of tumors, pulmonary complications, and a striking predisposition to deep vein thrombosis and pulmonary embolism5. PS is clinically diagnosed according to the criteria described by Biesecker et al.6 (Table 1).\n\n\nCase\n\nA 49-year-old woman presented with a 10-year history of pauci-symptomatic infiltrating plaque lesions on the sole and lateral margin of the left foot (Figure 1 and Figure 2). The lesions simulated and had been misdiagnosed as keloids, but there was no history of trauma to the area. The patient reported that similar lesions had affected her great-grandmother. The patient had a positive history for stage IV melanoma, and she had finished chemotherapy7 just 3 months before our observation. Physical examination also revealed multiple lipomas on the trunk, linear verrucous epidermal nevi, and hyperpigmented macules with a mosaic distribution. Additionally, she presented with an asymmetric face, dysmorphic skull with frontal-parietal hyperostosis, dropped shoulders, scoliosis, and a stiff spine. Her legs were asymmetric with disproportionate overgrowth, the left leg being longer than the right one and having ectatic veins. In addition, computed tomography documented uterine fibromas, and abdominal magnetic resonance imaging demonstrated hepatic angiomatosis. A skin biopsy specimen from the left foot stained with hematoxylin and eosin revealed remarkable hyperkeratosis, epidermal hyperplasia, dermoepidermal fibrosis with extensive sclerosis of the reticular dermis, thickened collagen bundles, and fat-cell entrapment (Figure 3). We made the diagnosis of Proteus syndrome. No therapeutic intervention was carried out.\n\nHematoxylin and eosin (magnification x100).\n\n\nDiscussion\n\nTo meet the diagnostic criteria for PS, a patient must fulfill all three general criteria, plus a single criterion from category A or two criteria from category B or all three criteria from category C (Table 1). Even though our patient had not previously sought medical care, when she presented to us her condition fulfilled the criteria, and the diagnosis of PS was confirmed.\n\nThis case illustrates the importance of even minor cutaneous clinical signs, especially when atypical. They should not be overlooked because, together with other clinical and diagnostic findings, they may lead to the diagnosis of a specific condition. This is especially true in mosaic diseases, such as PS, in which the wide variety of tissue types and cells that are involved may not be apparent at the first examination. Subtle cutaneous forms of PS have been described in infants4, but to the best of our knowledge, this is the first case in which the cutaneous signs remained elusive in adulthood. We do not know whether the chemotherapy she had been administered had somehow altered the lesion morphology7, but that seems unlikely, as that occurred in adulthood and the patient referred no significant changes in shape and volume.\n\nCorrect recognition of pauci-symptomatic “incognito” PS is essential, as PS patients are known to be exposed to a higher risk to develop tumors1–4, such as meningiomas, breast and ovarian cancer, parotid adenoma, and others. We do not know whether melanoma occurrence is facilitated by PS, and the literature provides scarce data on this. The association between PS and melanoma in this case is either a novel finding or an incidental coexistence. At present our patient reached the 24 months follow up with no clinical or instrumental signs of recurrence.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCohen MM Jr, Hayden PW: A newly recognized hamartomatous syndrome. Birth Defects. 1979; 15(5B): 291–296. PubMed Abstract\n\nNguyen D, Turner JT, Olsen C, et al.: Cutaneous manifestations of Proteus syndrome. Correlations with general clinical severity. Arch Dermatol. 2004; 140(8): 947–953. PubMed Abstract | Publisher Full Text\n\nDoucet ME, Bloomhardt HM, Moroz K, et al.: Lack of mutation-histopathology correlation in a patient with Proteus syndrome. Am J Med Genet A. 2016; 170(6): 1422–1432. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRodenbeck DL, Greyling LA, Anderson JH, et al.: Early Recognition of Proteus Syndrome. Pediatr Dermatol. 2016; 33(5): e306–310. PubMed Abstract | Publisher Full Text\n\nBiesecker LG, Sapp JC: Proteus Syndrome. 2012.\n\nBiesecker LG, Happle R, Mulliken JB, et al.: Proteus Syndrome: diagnostic criteria, differential diagnosis and patient evaluation. Am J Med Genet. 1999; 84(5): 389–395. PubMed Abstract\n\nGuida M, Cramarossa A, Fistola E, et al.: High activity of sequential low dose chemo-modulating Temozolomide in combination with Fotemustine in metastatic melanoma. A feasibility study. J Transl Med. 2010; 8: 115. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "31217",
"date": "28 Feb 2018",
"name": "Marco Marcasciano",
"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\nProteus syndome is a complex multi-organ mosaic disorder, characterized by progressive overgrowth and disfiguring. The authors did a nice job in describing the occurrence of such a multi-faceted condition in its (rare) minimal symptomatic presentation. To suspect and correctly diagnose a Proteus in view of (apparently) so few clues is indeed challenging. Since the repercussions are relevant (increased incidence of malignancies) the educational message conveyed here is an important one: never underestimate the importance and significance of a given set of signs and symptoms, as subtle as they may be.\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": "4356",
"date": "14 Jan 2019",
"name": "Leslie Biesecker",
"role": "Reader Comment",
"response": "It is always helpful to see interesting clinical reports. A few things that are puzzling about this report is that the patient does not appear to meet the clinical criteria for this condition. It should be noted that the authors cited our 1999 criteria from AJMG, although those were superseded by the criteria published in 2006 (PMID 16883308). Irrespective of that suboptimal citation, the patient probably meets the general criteria but does not meet specific criterion A because while she probably has a connective tissue nevus, it is not a cerebriform connective tissue nevus (see photos in PMID16883308 for examples that do have this attribute), she does not have two criteria from specific category B nor does she have three criteria from category C. Therefore, I would conclude that clinically, this patient has a segmental (or mosaic) overgrowth disorder and not necessarily Proteus syndrome. The second puzzling aspect of this report is that the molecular etiology of Proteus syndrome was elucidated eight years ago (PMID 21793738), yet this test was not performed on this patient. While there can be valid reasons for not performing such testing in a clinical context, to claim a patient has a diagnosis in a scientific publication without confirming that diagnosis molecularly is problematic. Should the authors be unable to access clinical testing for this patient, we would be willing to provide that free of charge should this lady be willing to enroll in our research study. As we have documented, diagnostic confusion between Proteus syndrome and related overgrowth disorders is a common problem (PMID 15372514). PIK3CA-related overgrowth spectrum (PMID 25557259) is probably 100X more common than is Proteus syndrome, and inexperienced clinicians frequently confuse these entities. Finally, these authors claim that \"This case describes the first evidence, to the best of our knowledge, of pauci-symptomatic PS in adulthood...\" I would suggest that the authors review PMID 24850616 for a prior example of just that. Again, the authors are to be congratulated for identifying this interesting patient, though the lessons learned may be quite distinct from that which they assert."
}
]
},
{
"id": "31219",
"date": "08 Mar 2018",
"name": "Giuseppe Micali",
"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\nIt is an interesting and well-presented case report of pauci-symptomatic, previously overlooked Proteus syndrome in a 49-year-old woman. Proteus syndrome is a rare, complex disorder with multisystem involvement and remarkable clinical variability. The authors highlight the relevance of the recognition of minor cutaneous clinical signs for the diagnosis of minimal forms of the disease, as several complications, some of which life-threatening, may potentially occur in these patients.\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/7-228
|
https://f1000research.com/articles/6-1584/v1
|
29 Aug 17
|
{
"type": "Opinion Article",
"title": "Developing WHO guidelines: Time to formally include evidence from mathematical modelling studies",
"authors": [
"Matthias Egger",
"Leigh Johnson",
"Christian Althaus",
"Anna Schöni",
"Georgia Salanti",
"Nicola Low",
"Susan L. Norris",
"Matthias Egger",
"Leigh Johnson",
"Christian Althaus",
"Anna Schöni",
"Georgia Salanti",
"Nicola Low"
],
"abstract": "In recent years, the number of mathematical modelling studies has increased steeply. Many of the questions addressed in these studies are relevant to the development of World Health Organization (WHO) guidelines, but modelling studies are rarely formally included as part of the body of evidence. An expert consultation hosted by WHO, a survey of modellers and users of modelling studies, and literature reviews informed the development of recommendations on when and how to incorporate the results of modelling studies into WHO guidelines. In this article, we argue that modelling studies should routinely be considered in the process of developing WHO guidelines, but particularly in the evaluation of public health programmes, long-term effectiveness or comparative effectiveness. There should be a systematic and transparent approach to identifying relevant published models, and to commissioning new models. We believe that the inclusion of evidence from modelling studies into the Grading of Recommendations Assessment, Development and Evaluation (GRADE) process is possible and desirable, with relatively few adaptations. No single “one-size-fits-all” approach is appropriate to assess the quality of modelling studies. The concept of the ‘credibility’ of the model, which takes the conceptualization of the problem, model structure, input data, different dimensions of uncertainty, as well as transparency and validation into account, is more appropriate than ‘risk of bias’.",
"keywords": [
"World Health Organization",
"guidelines",
"mathematical modelling",
"study quality",
"GRADE"
],
"content": "Introduction\n\nMathematical models have a long history in public health1. In 1760, Daniel Bernoulli developed a model of smallpox transmission and control. William Hamer published a measles transmission model in 1906 and Ronald Ross a model of malaria transmission in 1908. In recent years, the number of publications related to mathematical modelling has increased steeply. Today, mathematical modelling studies are not restricted to infectious diseases but address a wide range of questions.\n\nThe World Health Organization (WHO) provides recommendations on many public health, health system and clinical topics. WHO guidelines are developed using processes and methods that ensure the publication of high-quality recommendations, as outlined in the WHO Handbook for Guideline Development2. WHO uses the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to rate the certainty of a body of evidence and to produce information that is used by guideline panels to formulate recommendations, based on the balance of benefits and harms and other considerations3.\n\nMany of the questions addressed in mathematical modelling studies are relevant to the development of guidelines. Increasingly, WHO and other guideline developers need to decide whether and how the results of mathematical modelling studies should be included in the evidence base used to develop recommendations. We reviewed the 185 WHO guidelines that were approved by the Guidelines Review Committee from 2007 to 2015: 42 (23%) referred to mathematical modelling studies. However, these studies were rarely formally assessed as part of the body of evidence, and quality criteria for modelling studies were often lacking. A major barrier to the incorporation of evidence from mathematical modelling studies into guidelines is the perceived complexity of the methods used to construct and analyse these studies. At present, there are no widely agreed methods for, or approaches to, the evaluation of the results of mathematical modelling studies, and to their integration with primary data to inform guidelines and recommendations. In April 2016 WHO organized a workshop in Geneva, Switzerland to discuss when and how to incorporate the results of modelling studies into WHO guidelines (see Acknowledgements for names of participants; see the Meeting Report). Specifically, the following three questions were discussed at the workshop:\n\n(1) When is it appropriate to consider modelling studies as part of the evidence that supports a guideline?\n\n(2) How should the quality and risk of bias in mathematical modelling studies be assessed?\n\n(3) How can the GRADE approach be adapted to assess the certainty of a body of evidence that includes the results of modelling and to formulate recommendations?\n\nThe role of modelling in economic evaluation is well recognised in guideline development and at WHO, and was therefore excluded from discussions. At the workshop, we considered the results of a survey of experts (see Box 1) and a rapid literature review (see below). In this paper, which reflects the opinions of the authors but not necessarily that of all workshop participants, we first define models and modelling studies. We then address the three questions outlined above and conclude with some recommendations on the use of evidence from modelling studies in guidelines development.\n\nThe survey was conducted between March 17 and April 4, 2016. It consisted of 10 questions: four on the characteristics of the respondents, three on the role of mathematical models in guideline development, two questions on quality criteria for mathematical models and one on the challenges in using mathematical modelling in guideline development (see Figure S1). Using snowball sampling, mathematical modellers, epidemiologists, guideline developers and other experts were invited to participate in the survey. A total of 151 individuals from 28 countries and 87 different institutions responded. About half of respondents were modellers, and the other half users of the results from modelling studies. The majority of respondents (58%) had been part of a guideline development group in the past.\n\nNinety-five percent of respondents answered yes to the question “Should mathematical modelling inform guidance for public health interventions?” and 60% indicated that findings of mathematical modelling studies can sometimes provide the same level of evidence as those of empirical research studies. When asked to list situations in which mathematical modelling could be particularly useful for the development of guidelines, the absence of empirical data on the effectiveness, cost-effectiveness and impact of an intervention, and on the comparative effectiveness of different interventions was most frequently mentioned. We also asked about situations where mathematical modelling studies should not be used or have been inappropriately used in the development of guidelines. Respondents reported that modelling should not be used “to cover up” for the lack of evidence from empirical research, and due emphasis should be given to the uncertainty of model predictions. When asked about the five most important criteria for the quality of reporting of modelling studies, respondents mentioned that the model structure should be clearly described and justified, the important sources of uncertainty reported, and model validity addressed. Assumptions should be clearly stated, justified and discussed and the sources of parameter estimates described. Finally, respondents identified the interpretation of results from modelling studies, the evaluation of their quality and the communication of uncertainty as major challenges in using mathematical modelling in guideline development. These challenges would be best addressed by including at least one modelling expert in guideline development groups.\n\n\nWhat is a mathematical modelling study?\n\nUsing a common terminology across different disciplines, for example infectious disease modelling and modelling in chronic disease, will facilitate the assessment, evaluation and comparison of mathematical modelling studies. We define a mathematical model as a “mathematical framework representing variables and their interrelationships to describe observed phenomena or predict future events”4. Mathematical modelling studies are studies that address defined research questions using mathematical modelling, for example the potential of HIV testing with immediate antiretroviral therapy to reduce HIV transmission5, or the likely impact of different screening practices on the incidence of cervical cancer6. In contrast, statistical modelling is typically concerned with associations between variables assessed in empirical studies, rather than an understanding of wider phenomena or systems. The results from statistical analyses of empirical data often inform mathematical models. Mathematical modelling studies also increasingly integrate statistical models into complex models to relate the model output to data.\n\n\nRole of mathematical modelling studies in guideline development\n\nMathematical models typically address questions that cannot easily be answered with randomized controlled trials (RCTs) or observational studies. Table 1 lists specific situations and examples where the results of mathematical modelling are particularly relevant to guideline development, based on the survey, published examples and the Geneva workshop. Mathematical modelling can overcome some of the limitations of results obtained from the carefully controlled settings in which RCTs are typically conducted. First, the main trial results provide an average effect estimate that applies to a specific intervention and study population. Mathematical modelling studies can be used to extrapolate from the results of RCTs to different target groups and settings, to long term outcomes, and to bridge the gap between efficacy and (long-term) effectiveness7. Second, interventions to prevent and control infectious diseases have non-linear effects. RCTs that address short term effects at the individual level might not be suitable for estimating the longer term effects of introducing an intervention, say a vaccine, in a whole population if indirect herd effects influence the incidence of infection and hence the impact of the intervention8,9. Third, rapid guidance is often needed early in outbreaks or public health emergencies when relevant interventions for prevention or management might simply not have been evaluated. The results of mathematical modelling studies can be used to draft emergency guidelines or to assess the epidemic potential of new outbreaks10.\n\nSource: WHO expert survey and consultation.\n\nThe findings of mathematical modelling studies are only as good as the data and assumptions that inform them. Guideline recommendations should therefore not be based on the outputs of models when uncertainty in the empirical data has not been appropriately quantified, when the model makes implausible assumptions or has not been validated adequately, or when the model predictions vary widely over a plausible range of parameter estimates.\n\n\nAssessing the quality of a mathematical modelling study: Rapid review\n\nWe performed a rapid review of the methodological literature to identify criteria that are proposed to assess the “quality” of mathematical modelling studies (see Table S1 for the detailed search strategy). Specifically, we aimed to identify criteria proposed to assess the quality of single mathematical modelling studies, including best practice standards or criteria for assessing risk of bias or reporting quality and criteria proposed to assess the quality of a body of evidence from mathematical modelling studies. We were also interested in identifying checklists or other instruments developed to assess the quality of mathematical modelling studies.\n\nWe identified 20 relevant articles (see Figure 1 for a flow chart of the identification of eligible articles)9,11–28. Most gave recommendations for good modelling practice and were compiled by a task force in a consensus process or based on a systematic or narrative review of the literature. The widely cited 2003 paper by Weinstein and colleagues organized 28 recommendations under the headings “structure”, “data”, and “validation”15. A questionnaire or checklist was not included. A subsequent series of seven articles9,22–26,28 by the joint International Society for Pharmacoeconomics and Outcomes Research (ISPOR) and Society for Medical Decision Making (SMDM) task force elaborated upon these recommendations, providing detailed advice on conceptualizing the model, state transition models, discrete event simulations, dynamic transmission models, parameter estimation and uncertainty, and transparency and validation. The 79 recommendations are summarized in the first article of the series28.\n\nWe identified four articles16,18,21,27 that present comprehensive frameworks of good modelling practice, with detailed justifications of the items covered and attributes of good practice. They include signalling or helper questions to facilitate the critical appraisal of published modelling studies: the number of questions ranges from 38 in Caro et al.16 to 66 questions in Bennett and Manuel21. The four frameworks cover similar territory, including items related to the problem concept, model structure, data sources and synthesis of the evidence, model uncertainty, consistency, transparency and validation (Table 2). Two of the frameworks include sponsorship and conflicts of interest16,21.\n\nIn a qualitative study Chilcot et al.11 performed in-depth interviews with 12 modellers from academic and commercial sectors, and model credibility emerged as the central concern of decision-makers using models. Respondents agreed that developing an understanding of the clinical situation or disease process being investigated is paramount in ensuring model credibility, highlighting the importance of clinical input during the model development process11.\n\n\nModel comparisons and modelling consortia\n\nPublished mathematical models addressing the same issue may reach contrasting conclusions. In this situation, careful comparison of the models may lead to a deeper understanding of the factors that drive outputs and conclusions. Ideally, the different modelling groups come together to explore the importance of differences in the type and structure of their models, and of the data used to parameterize them29–31. For example, several groups of modellers have investigated the impact of expanding access to antiretroviral therapy (ART) on new HIV infections. The HIV Modelling Consortium compared the predictions of several mathematical models simulating the same ART intervention programs to determine the extent to which models agree on the epidemiological impact of expanded ART30. The consortium concluded that although models vary substantially in structure, complexity, and parameter choices, all suggested that ART, at high levels of access and with high adherence, has the potential to substantially reduce new HIV infections in the population30. There was broad agreement regarding the short-term epidemiologic impact of ART scale-up, but more variation in longer-term projections and in the efficiency with which treatment can reduce new infections. The impact of ART on HIV incidence long-term is expected to be lower if models: (i) allow for heterogeneity in sexual risk behaviour; (ii) are age-structured; (iii) estimate a low proportion of HIV transmission from individuals not on ART with advanced disease (at low CD4 counts); (iv) are compared to what would be expected in the presence of HIV counselling and testing (compared to no counselling and testing); (v) assume relatively high infectiousness on ART; and (vi) consider drug resistance30,32,33.\n\n\nAssessing mathematical modelling studies using the GRADE approach\n\nGRADE was conceived with the intention of creating a uniform system to assess a body of evidence to support guideline development in response to a confusing array of different systems in use at that time34. It has since been adopted by over 90 organisations, including WHO. GRADE addresses clinical management questions, including the impact of therapies and diagnostic strategies, diagnostic accuracy questions (i.e., the accuracy of a single diagnostic or screening test), the (cost-) effectiveness and safety of public health interventions, and questions about prognosis.\n\nThe GRADE approach encompasses two main considerations: the degree of certainty in the evidence used to support a decision and the strength of the recommendation. The degree of certainty, i.e., the confidence in or quality of a body of evidence, is rated as “high”, “moderate”, “low”, or “very low” based on an assessment of five dimensions: study limitations (risk of bias), imprecision, inconsistency, indirectness, and publication bias. The initial assessment is based on the study design: RCTs start as high certainty and observational studies as low certainty. Based on the assessments of the five dimensions, RCTs may be down-rated and observational studies up- or down-rated. Judgment is required when assessing the certainty of the evidence, taking into account the number of studies of higher and lower quality and the relative importance of the different dimensions in a given context. The second consideration is the strength of the recommendation, which can be “strong” or “conditional”, for or against an intervention or test, based on the balance of benefits and harms, certainty of the evidence, the relative values of persons affected by the intervention, resource considerations, acceptability and feasibility, among others35.\n\nWe believe that evidence from mathematical modelling studies could be assessed within the GRADE framework and included in the guideline development process. Specifically, guideline groups might include mathematical modelling studies as an additional study category, in addition to the categories of RCTs and observational studies currently defined in GRADE. The dimensions of indirectness, inconsistency, imprecision and publication bias are applicable to mathematical modelling studies, but criteria may need to be adapted. The concept of bias relates to results or inferences from empirical studies36. “Lack of credibility” may therefore be a more appropriate term for modelling studies than “risk of bias”. The assessment of the credibility of a model is informed by a comprehensive quality framework and should cover the conceptualization of the problem, model structure, input data, different dimensions of uncertainty, as well as transparency and validation (Table 2). The framework should be tailored to each set of modelling studies by adding or omitting questions and developing review-specific guidance on how to assess each criterion. The certainty of the body of evidence from modelling studies can then be classified as high, moderate, low, or very low. In the evidence-to-decision framework a distinction should be made between observed outcomes from empirical studies and modelled outcomes from modelling studies (see an example).\n\n\nConclusions and recommendations\n\nBased on the discussions and presentations at the workshop in Geneva, the survey and rapid systematic review, we believe a number of conclusions can be formulated.\n\n1. The use of modelling studies should routinely be considered in the process of developing WHO guidelines. Findings of mathematical modelling studies can provide important evidence that may be highly relevant. Evidence from modelling studies should be considered specifically in the absence of empirical data directly addressing the question of interest, when modelling based on appropriate indirect evidence may be indicated. Examples for such situations include the evaluation of long-term effectiveness, and the impact of one or several interventions (comparative effectiveness), for example in the context of public health programmes where RCTs are rarely available.\n\n2. Modelling may be more acceptable and more influential in situations where immediate action is called for, but little direct empirical evidence is available, and may arguably be more acceptable in public health than in clinical decision making. In these situations (for example, the HIV, Ebola, or Zika epidemics) funding is also likely to become available to support dedicated modelling studies.\n\n3. The use of evidence from mathematical models should be carefully considered and there should be a systematic and transparent approach to identifying existing models that may be relevant, and to commissioning new models.\n\n4. No single “one-size-fits-all” approach is appropriate to assess the quality of modelling studies. Existing frameworks and checklists may be adapted to a set of modelling studies by adding or omitting questions. In some situations, the approach will need to be developed de novo.\n\n5. Additional expertise will typically be required in the systematic review groups or guideline development groups to appropriately assess the credibility of modelling studies and interpret their results.\n\n6. The credibility of the models should not be evaluated only by modellers, and not only by modellers involved in the development of these models.\n\n7. The inclusion of evidence from modelling studies into the GRADE process is possible and desirable, with relatively few adaptations. GRADE is simply rating the certainty of evidence to support a decision and any type of evidence can in principle be included.\n\n8. The certainty of the evidence for modelling studies should be assessed and presented separately in summaries of the evidence (GRADE evidence profiles), and classified as high, moderate, low, or very low certainty.\n\n9. The GRADE dimensions of certainty (imprecision, indirectness, inconsistency and publication bias) and the criteria defined for their assessment are also relevant to modelling studies.\n\n10. For modelling studies, the concept of the ‘credibility’ of the model, which takes the structure of the model, input data, dimensions of uncertainty, as well as transparency and validation into account, is more appropriate than ‘study limitations’ or ‘risk of bias’.\n\n11. When summarizing the evidence, a distinction should be made between observed and modelled outcomes.\n\nWe look forward to discussing these recommendations with experts and stakeholders and to developing exact procedures and criteria for the assessment of modelling studies and their inclusion in the GRADE process.",
"appendix": "Competing interests\n\n\n\nSusan L. Norris is a member of the GRADE working group. No other competing interests were disclosed.\n\n\nGrant information\n\nThe work reported in this article and the expert consultation meeting in Geneva, Switzerland, were funded by the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (WHO/TDR).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are grateful to Helen Ward and Tom Trikalinos who chaired the April 2016 expert meeting in Geneva and to all who participated in the meeting: Patrick Bossuyt, David Fisman, Gordon Guyatt, Tim Hallett, Mark Helfand, Rod Jackson, Veena Manja, Holger Schünemann, Julie Ann Simpson, Christopher Dye, Philippa Easterbrook, Nathan Ford, Daniel Hogan, and Gretchen Stevens. All authors of this article also participated.\n\n\nSupplementary material\n\nTable S1. Search strategy in MEDLINE from inception to January 2016 without language restrictions, combining terms for mathematical models with terms for quality assessment and health care decision-making.\n\nClick here to access the data.\n\nFigure S1. Questionnaire of the online survey on the use of mathematical modelling in guidelines for public health decision making.\n\nClick here to access the data.\n\n\nReferences\n\nSmith DL, Battle KE, Hay SI, et al.: Ross, macdonald, and a theory for the dynamics and control of mosquito-transmitted pathogens. PLoS Pathog. 2012; 8(4): e1002588. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: WHO Handbook for Guideline Development. Geneva, 2014. Reference Source\n\nGuyatt GH, Oxman AD, Vist GE, et al.: GRADE: An emerging consensus on rating quality of evidence and strength of recommendations. BMJ. 2008; 336(7650): 924–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEykhoff P: System identification; parameter and state estimation. Chester: John Wiley & Sons Ltd., 1974. Reference Source\n\nGranich RM, Gilks CF, Dye C, et al.: Universal voluntary HIV testing with immediate antiretroviral therapy as a strategy for elimination of HIV transmission: a mathematical model. Lancet. 2009; 373(9657): 48–57. PubMed Abstract | Publisher Full Text\n\nCanfell K, Barnabas R, Patnick J, et al.: The predicted effect of changes in cervical screening practice in the UK: results from a modelling study. Br J Cancer. 2004; 91(3): 530–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEgger M, Moons KG, Fletcher C, et al.: GetReal: from efficacy in clinical trials to relative effectiveness in the real world. Res Synth Methods. 2016; 7(3): 278–81. PubMed Abstract | Publisher Full Text\n\nWeinstein MC: Recent developments in decision-analytic modelling for economic evaluation. Pharmacoeconomics. 2006; 24(11): 1043–53. PubMed Abstract | Publisher Full Text\n\nPitman R, Fisman D, Zaric GS, et al.: Dynamic transmission modeling: a report of the ISPOR-SMDM Modeling Good Research Practices Task Force--5. Value Health. 2012; 15(6): 828–34. PubMed Abstract | Publisher Full Text\n\nCamacho A, Kucharski AJ, Funk S, et al.: Potential for large outbreaks of Ebola virus disease. Epidemics. 2014; 9: 70–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChilcott J, Tappenden P, Rawdin A, et al.: Avoiding and identifying errors in health technology assessment models: qualitative study and methodological review. Health Technol Assess. 2010; 14(25): iii–iv, ix–xii, 1–107. PubMed Abstract | Publisher Full Text\n\nWeinstein MC, Toy EL, Sandberg EA, et al.: Modeling for health care and other policy decisions: uses, roles, and validity. Value Health. 2001; 4(5): 348–61. PubMed Abstract | Publisher Full Text\n\nPhilips Z, Ginnelly L, Sculpher M, et al.: Review of guidelines for good practice in decision-analytic modelling in health technology assessment. Health Technol Assess. 2004; 8(36): iii–iv, ix–xi, 1–158. PubMed Abstract | Publisher Full Text\n\nGoldhaber-Fiebert JD, Stout NK, Goldie SJ: Empirically evaluating decision-analytic models. Value Health. 2010; 13(5): 667–74. PubMed Abstract | Publisher Full Text\n\nWeinstein MC, O’Brien B, Hornberger J, et al.: Principles of good practice for decision analytic modeling in health-care evaluation: report of the ISPOR Task Force on Good Research Practices--Modeling Studies. Value Health. 2003; 6(1): 9–17. PubMed Abstract | Publisher Full Text\n\nJaime Caro J, Eddy DM, Kan H, et al.: Questionnaire to assess relevance and credibility of modeling studies for informing health care decision making: An ISPOR-AMCP-NPC good practice task force report. Value Health. 2014; 17(2): 174–82. PubMed Abstract | Publisher Full Text\n\nBilcke J, Beutels P, Brisson M, et al.: Accounting for Methodological, Structural, and Parameter Uncertainty in Decision-Analytic Models: A Practical Guide. Med Decis Mak. 2011; 31(4): 675–92. PubMed Abstract | Publisher Full Text\n\nPhilips Z, Bojke L, Sculpher M, et al.: Good practice guidelines for decision-analytic modelling in health technology assessment: a review and consolidation of quality assessment. Pharmacoeconomics. 2006; 24(4): 355–71. PubMed Abstract | Publisher Full Text\n\nChilcott J, Brennan A, Booth A, et al.: The role of modelling in prioritising and planning clinical trials. Health Technol Assess. 2003; 7(23): iii, 1–125. PubMed Abstract | Publisher Full Text\n\nForsberg HH, Aronsson H, Keller C, et al.: Managing health care decisions and improvement through simulation modeling. Qual Manag Health Care. 2011; 20(1): 15–29. PubMed Abstract | Publisher Full Text\n\nBennett C, Manuel DG: Reporting guidelines for modelling studies. BMC Med Res Methodol. 2012; 12: 168. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSiebert U, Alagoz O, Bayoumi AM, et al.: State-transition modeling: a report of the ISPOR-SMDM Modeling Good Research Practices Task Force--3. Value Health. 2012; 15(6): 812–20. PubMed Abstract | Publisher Full Text\n\nBriggs AH, Weinstein MC, Fenwick EA, et al.: Model parameter estimation and uncertainty: a report of the ISPOR-SMDM Modeling Good Research Practices Task Force--6. Value Health. 2012; 15(6): 835–42. PubMed Abstract | Publisher Full Text\n\nKarnon J, Stahl J, Brennan A, et al.: Modeling using Discrete Event Simulation: A Report of the ISPOR-SMDM Modeling Good Research Practices Task Force--4. Value Health. 2012; 15(6): 821–7. PubMed Abstract | Publisher Full Text\n\nRoberts M, Russell LB, Paltiel AD, et al.: Conceptualizing a model: a report of the ISPOR-SMDM Modeling Good Research Practices Task Force--2. Value Health. 2012; 15(6): 804–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEddy DM, Hollingworth W, Caro JJ, et al.: Model transparency and validation: a report of the ISPOR-SMDM Modeling Good Research Practices Task Force--7. Value Health. 2012; 15(6): 843–50. PubMed Abstract | Publisher Full Text\n\nRamos MC, Barton P, Jowett S, et al.: A Systematic Review of Research Guidelines in Decision-Analytic Modeling. Value Health. 2015; 18(4): 512–29. PubMed Abstract | Publisher Full Text\n\nCaro JJ, Briggs AH, Siebert U, et al.: Modeling good research practices--overview: a report of the ISPOR-SMDM Modeling Good Research Practices Task Force--1. Value Health. 2012; 15(6): 796–803. PubMed Abstract | Publisher Full Text\n\nMandelblatt JS, Cronin KA, Bailey S, et al.: Effects of mammography screening under different screening schedules: model estimates of potential benefits and harms. Ann Intern Med. 2009; 151(10): 738–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEaton JW, Johnson LF, Salomon JA, et al.: HIV treatment as prevention: systematic comparison of mathematical models of the potential impact of antiretroviral therapy on HIV incidence in South Africa. PLoS Med. 2012; 9(7): e1001245. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlthaus CL, Turner KM, Schmid BV, et al.: Transmission of Chlamydia trachomatis through sexual partnerships: a comparison between three individual-based models and empirical data. J R Soc Interface. 2012; 9(66): 136–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHontelez JA, Lurie MN, Bärnighausen T, et al.: Elimination of HIV in South Africa through expanded access to antiretroviral therapy: a model comparison study. PLoS Med. 2013; 10(10): e1001534. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaw MG, Prestage G, Grulich A, et al.: Modelling the effect of combination antiretroviral treatments on HIV incidence. AIDS. 2001; 15(10): 1287–94. PubMed Abstract | Publisher Full Text\n\nAtkins D, Best D, Briss PA, et al.: Grading quality of evidence and strength of recommendations. BMJ. 2004; 328(7454): 1490. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndrews JC, Schünemann HJ, Oxman AD, et al.: GRADE guidelines: 15. Going from evidence to recommendation-determinants of a recommendation’s direction and strength. J Clin Epidemiol. 2013; 66(7): 726–35. PubMed Abstract | Publisher Full Text\n\nGuyatt GH, Oxman AD, Vist G, et al.: GRADE guidelines: 4. Rating the quality of evidence--study limitations (risk of bias). J Clin Epidemiol. 2011; 64(4): 407–15. PubMed Abstract | Publisher Full Text\n\nSamur S, Klebanoff M, Banken R, et al.: Long-term clinical impact and cost-effectiveness of obeticholic acid for the treatment of primary biliary cholangitis. Hepatology. 2017; 65(3): 920–928. PubMed Abstract | Publisher Full Text\n\nGetsios D, Blume S, Ishak KJ, et al.: Cost effectiveness of donepezil in the treatment of mild to moderate Alzheimer's disease: a UK evaluation using discrete-event simulation. Pharmacoeconomics. 2010; 28(5): 411–27. PubMed Abstract | Publisher Full Text\n\nPalmer AJ, Roze S, Valentine WJ, et al.: The CORE Diabetes Model: Projecting long-term clinical outcomes, costs and cost-effectiveness of interventions in diabetes mellitus (types 1 and 2) to support clinical and reimbursement decision-making. Curr Med Res Opin. 2004; 20 Suppl 1: S5–26. PubMed Abstract | Publisher Full Text\n\nSmolen HJ, Cohen DJ, Samsa GP, et al.: Development, validation, and application of a microsimulation model to predict stroke and mortality in medically managed asymptomatic patients with significant carotid artery stenosis. Value Health. 2007; 10(6): 489–97. PubMed Abstract | Publisher Full Text\n\nLowy A, Munk VC, Ong SH, et al.: Effects on blood pressure and cardiovascular risk of variations in patients’ adherence to prescribed antihypertensive drugs: role of duration of drug action. Int J Clin Pract. 2011; 65(1): 41–53. PubMed Abstract | Publisher Full Text\n\nSchuetz CA, van Herick A, Alperin P, et al.: Comparing the effectiveness of rosuvastatin and atorvastatin in preventing cardiovascular outcomes: estimates using the Archimedes model. J Med Econ. 2012; 15(6): 1118–29. PubMed Abstract | Publisher Full Text\n\nBarnett JC, Alvarez Secord A, Cohn DE, et al.: Cost effectiveness of alternative strategies for incorporating bevacizumab into the primary treatment of ovarian cancer. Cancer. 2013; 119(20): 3653–61. PubMed Abstract | Publisher Full Text\n\nDidden E, Ruffieux Y, Hummel N, et al.: Prediction of Real-World Drug Effectiveness Pre-Launch: Case study in Rheumatoid Arthritis. Value Health. 2017; submitted.\n\nTrotter CL, Gay NJ, Edmunds WJ: Dynamic models of meningococcal carriage, disease, and the impact of serogroup C conjugate vaccination. Am J Epidemiol. 2005; 162(1): 89–100. PubMed Abstract | Publisher Full Text\n\nElbasha EH, Dasbach EJ: Impact of vaccinating boys and men against HPV in the United States. Vaccine. 2010; 28(42): 6858–67. PubMed Abstract | Publisher Full Text\n\nHouben RM, Dodd PJ: The Global Burden of Latent Tuberculosis Infection: A Re-estimation Using Mathematical Modelling. PLOS Med. 2016; 13(10): e1002152. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCassini A, Plachouras D, Eckmanns T, et al.: Burden of Six Healthcare-Associated Infections on European Population Health: Estimating Incidence-Based Disability-Adjusted Life Years through a Population Prevalence-Based Modelling Study. PLoS Med. 2016; 13(10): e1002150. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnston SC, Mendis S, Mathers CD: Global variation in stroke burden and mortality: estimates from monitoring, surveillance, and modelling. Lancet Neurol. 2009; 8(4): 345–54. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "25463",
"date": "20 Sep 2017",
"name": "Wilma A. Stolk",
"expertise": [
"Reviewer Expertise Epidemiology / mathematical modelling",
"with focus on neglected tropical 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\nIn this opinion article, the authors discuss when and how to incorporate the results of modelling studies into WHO guidelines, by addressing three questions: (1) When is it appropriate to consider modelling studies as part of the evidence that supports a guideline? (2) How should the quality and risk of bias in mathematical modelling studies be assessed? (3) How can the GRADE approach be adapted to assess the certainty of a body of evidence that includes the results of modelling and to formulate recommendations? Based on findings from a web-based expert survey, a rapid literature review to identify criteria for assessing the “quality” of mathematical modelling studies, and on discussions and presentations at a workshop on the topic that was held April 2016 in Geneva, the authors conclude that modelling studies should indeed routinely be considered in the process of developing WHO guidelines, particularly in the evaluation of public health programmes, long-term effectiveness or comparative effectiveness. As for other types of evidence taken into consideration, there should be a systematic and transparent approach to identifying existing models that may be relevant and the quality and credibility of models should be systematically assessed. Relatively few adaptations are needed in the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to rate the certainty of a body of evidence and to produce information that is used by guideline panels to formulate recommendations, based on the balance of benefits and harms and other considerations.\nMINOR COMMENTS:\nRecommendation 4 is “No single ‘one-size-fits-all’ approach is appropriate to assess the quality of modelling studies. Existing frameworks and checklists may be adapted to a set of modelling studies by adding or omitting questions. In some situations, the approach will need to be developed de novo.” I’d prefer to turn it around: based on existing frameworks and checklists, generic criteria can be developed to assess the quality of modelling studies, although – depending on the situation –questions may have to be added or omitted. I am not convinced that in some situations a completely new approach is needed, and this would also not be advisable. The authors should either delete the last statement, or explain under which circumstances such a new approach is needed, ideally illustrated with an example.\n\nRecommendation 8 is “The certainty of the evidence for modelling studies should be assessed and presented separately in summaries of the evidence (GRADE evidence profiles), and classified as high, moderate, low, or very low certainty.” In the text, the authors state that RCTs start as high certainty and observational studies as low certainty, although this certainty score may be up- or down-rated based on detailed assessment of five dimensions. Is it possible to give an indication of where modelling studies would start, with a justification? If not, can the authors describe factors to be considered when determining the start class?\n\nThe questionnaire of the online survey on the use of mathematical modelling in guidelines for public health decision making is included as Figure S1, which combines a series of screen shots. The quality of this figure is poor and I recommend to include the questionnaire as a text document.\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": "3421",
"date": "26 Feb 2018",
"name": "Susan Norris",
"role": "Author Response",
"response": "Reviewer 1: Wilma A. Stolk, Erasmus MC, Department of Public Health, University Medical Center Rotterdam, Rotterdam, Netherlands Approved Authors’ response: Thank you for reviewing and approving our paper. See below our responses to your comments. We have made changes in the text via tracked changes. In this opinion article, the authors discuss when and how to incorporate the results of modelling studies into WHO guidelines, by addressing three questions: (1) When is it appropriate to consider modelling studies as part of the evidence that supports a guideline? (2) How should the quality and risk of bias in mathematical modelling studies be assessed? (3) How can the GRADE approach be adapted to assess the certainty of a body of evidence that includes the results of modelling and to formulate recommendations? Based on findings from a web-based expert survey, a rapid literature review to identify criteria for assessing the “quality” of mathematical modelling studies, and on discussions and presentations at a workshop on the topic that was held April 2016 in Geneva, the authors conclude that modelling studies should indeed routinely be considered in the process of developing WHO guidelines, particularly in the evaluation of public health programmes, long-term effectiveness or comparative effectiveness. As for other types of evidence taken into consideration, there should be a systematic and transparent approach to identifying existing models that may be relevant and the quality and credibility of models should be systematically assessed. Relatively few adaptations are needed in the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to rate the certainty of a body of evidence and to produce information that is used by guideline panels to formulate recommendations, based on the balance of benefits and harms and other considerations. Authors’ response: Thank you. This is a nice summary of our paper.MINOR COMMENTS: Recommendation 4 is “No single ‘one-size-fits-all’ approach is appropriate to assess the quality of modelling studies. Existing frameworks and checklists may be adapted to a set of modelling studies by adding or omitting questions. In some situations, the approach will need to be developed de novo.” I’d prefer to turn it around: based on existing frameworks and checklists, generic criteria can be developed to assess the quality of modelling studies, although – depending on the situation –questions may have to be added or omitted. I am not convinced that in some situations a completely new approach is needed, and this would also not be advisable. The authors should either delete the last statement, or explain under which circumstances such a new approach is needed, ideally illustrated with an example. Authors’ response: Thank you. We agree and have deleted the last statement on page 9. Recommendation 8 is “The certainty of the evidence for modelling studies should be assessed and presented separately in summaries of the evidence (GRADE evidence profiles), and classified as high, moderate, low, or very low certainty.” In the text, the authors state that RCTs start as high certainty and observational studies as low certainty, although this certainty score may be up- or down-rated based on detailed assessment of five dimensions. Is it possible to give an indication of where modelling studies would start, with a justification? If not, can the authors describe factors to be considered when determining the start class? Authors’ response: Thank you, we have addressed this issue as follows on page 9:“We propose that within the GRADE system, modelling studies start at low certainty, and it is then possible to increase or decrease the certainty of modelling studies based on a set of criteria. The development of these criteria was beyond the scope of this article; a GRADE working group is addressing this issue (http://www.gradeworkinggroup.org/).“ The questionnaire of the online survey on the use of mathematical modelling in guidelines for public health decision making is included as Figure S1, which combines a series of screen shots. The quality of this figure is poor and I recommend to include the questionnaire as a text document. Authors’ response: Thank you. There is no text document for the survey but we have enlarged the screen shots to increase their readability (pages 22-27)."
}
]
},
{
"id": "25673",
"date": "15 Nov 2017",
"name": "Joseph W Hogan",
"expertise": [
"Reviewer Expertise Biostatistics"
],
"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 to incorporate findings from mathematical modeling studies into the development of WHO guidelines and other processes related to evaluating and developing public health policies. They argue that evidence from modeling studies should be included in the Grading of Recommendations Assessment, Development and Evaluation (GRADE) process, and make specific recommendations to that effect. The authors argue that model credibility is more appropriate than risk of bias for evaluating strength of evidence generated by modeling studies. The paper is based on discussions and findings from a meeting of modeling experts in Geneva in 2016; the authors were also participants in the meeting.\n\nThe paper lays out a structured argument for incorporating modeling studies into the evidence base, particularly for formulating WHO recommendations related to treatment of HIV. The authors start by providing a review of how models are used in various fields, with suggestions about how they can inform guideline development. They address the question of what constitutes a modeling study. A comprehensive accounting of published literature on assessment of models is provided. Finally, they give recommendations for how models can be evaluated using the GRADE approach, with specific conclusions about important issues such as when modeling studies should be used as part of the evidence base and how their credibility should be assessed.\n\nGiven the sweeping variety of models used in published studies about HIV treatments, policies and interventions, the authors are to be applauded for putting forward a framework for having this conversation. It will promote broader understanding of how models work and how they can be most optimally used for informing treatment guidelines.\n\nAt the crux of their argument is the claim that evidence generated from models should be judged in terms of model credibility rather than on risk of bias. This argument raises several important issues. First, what constitutes a mathematical model? If a model is to be evaluated on its credibility, we need a definition to work from. Second, what kinds of output from models should be considered evidence, and how should the quality of that evidence be judged and ultimately weighed against or combined with evidence generated from randomized trials and observational cohort data?\n\nWhat is a mathematical model?\n\nAccording to the authors, mathematical modeling is “a mathematical framework representing variables and their interrelationships to describe observed phenomena or predict future events.”\n\nOn its face this is surely true, but for the purpose of understanding whether and how models should add to the evidence base, it’s too broad. This definition covers a vast assortment of mathematical models, ranging from validated descriptions of natural phenomena (where the mathematical relationships are known and directly observable) to representations of progression from HIV infection to death (comprising known and unknown mathematical components, many of which cannot be directly observed).\n\nConsider three examples for illustration:\n\nThe mathematical representation of radioactive decay is known and can be written down explicitly. The model enables accurate and replicable predictions of future observations. The mathematical model for absorption of a specific drug is typically not known, but empirical studies have shown that it is possible to approximate the systematic variation using nonlinear equations. These models incorporate known information about physiology and properties of a specific drug, but are necessarily oversimplified representations of drug absorption because there are unobservable characteristics of individuals that affect absorption. The models can be used to make reliable predictions on average, but require unexplained variation to be reflected in terms of prediction intervals. Now consider a model of the population dynamics of HIV infection and disease progression. This process also follows a mathematical model, but the model itself is highly complex. Unlike radioactive decay or rate of drug absorption, the mathematical representations of several components of the underlying processes are essentially unknown.\n\nMoreover, much of the data needed to inform the models are either unobserved (e.g. timing of HIV infection) or only sparsely observed (e.g. individual-level viral load).\n\nAll of these are mathematical models, but definitions must distinguish between them. Otherwise there is an implied equivalence that lends more credibility than is deserved to models that are heavily reliant on unverified assumptions about the mathematical structure underlying the dynamic system being modeled. A more systematic classification of model types would therefore be helpful.\n\nWhile the authors' definition of mathematical model is overly broad, the definition of statistical model, used to contrast with mathematical models, is too narrow. A statistical model is used to characterize sources of variation in observed data. It is based on a probabilistic representation of the data generating mechanism, which is itself a mathematical model. Theory and methods of statistical inference provides a rigorous and transparent set of techniques for parameter estimation, prediction of future outcomes, extrapolation (e.g. for causal inference), and uncertainty quantification. The last of these, uncertainty quantification, is a critical and frequently missing component of predictions based on mathematical models.\n\nFor the purposes of generating evidence for WHO recommendations, the main difference between a mathematical model and a statistical model is that mathematical models tend to have broader scope and incorporate higher dimensions of complexity, but rely more heavily on assumptions about underlying mathematical structure than on individual-level data. Statistical models tend to have less mathematical complexity and more narrow scope, and are typically fitted to a single (possibly large) set of observed individual-level data drawn from the target population(s) of interest. A mathematical structure underlies both statistical and mathematical models, and both can be used for prediction of future outcomes and for causal policy comparisons.\n\nShould models be judged on 'risk of bias'?\n\nThe authors propose that evidence generated from mathematical models should be weighted more heavily toward model credibility than risk of bias.\n\nMany mathematical models are over-parameterized relative to the amount of data used to fit them; hence multiple configurations of parameter values can be lead to very similar predictions. Mathematical models are typically calibrated to observed population-level data (e.g. annual HIV incidence rate for the target population), but the formal rules for doing this seem to vary across application.\n\nFor many consumers of model-based outputs, this is a significant methodologic concern that goes directly to the question of credibility. If multiple model configurations can generate similar predictions, which configuration is the most credible one? It seems reasonable that model-based outcomes such as 10-year predictions of HIV incidence need to be evaluated on their own terms. If coupled with a formal process for back-checking or recalibrating existing models this would surely add value, and would possibly strengthen model identifiability (i.e., provide evidence in favor of one set of model parameters over another).\n\nA more general justification for incorporating risk of bias into model evaluation can be found in Coveney et al 1 (page 4), who provide a general rubric for assessing quality of scientific evidence in the age of big data, emphasizing ‘acceptance of the theory based on concordance between the predictions and the measurements.’\n\nModel calibrations at the time of model fitting partially fulfill this objective, but post-hoc evaluation of model predictions must play an important role in establishing credibility.\n\nThe process of combining and comparing models is highly innovative and likely to have a positive impact on whether the results will be well received. This kind of cooperation and collaboration, exemplified recently by the Modelling Consortium, is perhaps unique to the mathematical modeling community. Evidence generated by these kinds of activities can form an important part of the evidence base.\n\nSummary\nThe authors have provided a thorough case for including results from mathematical modeling into the formal evidence base used for making health recommendations, especially as they relate to HIV. The paper is based on findings from a recent conference and a comprehensive survey of extant literature.\n\nThe main critiques are that the definition of mathematical model is far too broad, and that bias (or risk of bias) needs to be incorporated into the evaluation criteria. Formal methods for uncertainty quantification are critical as well.\n\nMathematical models are prevalent and influential in the HIV literature; hence a discussion about whether and how to place their findings in the broader evidence base is needed and welcome. This paper provides a necessary starting point.\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": "3422",
"date": "26 Feb 2018",
"name": "Susan Norris",
"role": "Author Response",
"response": "Joseph W Hogan, Department of Biostatistics and Center for Statistical Sciences, Brown University School of Public Health, Providence, RI, USA Approved with Reservations Authors’ response: Thank you very much for reviewing our paper and making such thoughtful comments. We address your reservations one by one below. We have made changes in the text via tracked changes. The authors propose to incorporate findings from mathematical modeling studies into the development of WHO guidelines and other processes related to evaluating and developing public health policies. They argue that evidence from modeling studies should be included in the Grading of Recommendations Assessment, Development and Evaluation (GRADE) process, and make specific recommendations to that effect. The authors argue that model credibility is more appropriate than risk of bias for evaluating strength of evidence generated by modeling studies. The paper is based on discussions and findings from a meeting of modeling experts in Geneva in 2016; the authors were also participants in the meeting. Authors’ response: First, we would like to stress that our article is an opinion piece, and does not reflect an official position of the World Health Organization or any other body. In the introduction we write: “In this paper, which reflects the opinions of the authors… “. Also, we tried to keep the recommendations fairly general, rather than specific and prescriptive. For example, we refrained from recommending a specific instrument to assess the quality of modeling studies. We conclude by saying that “We look forward to discussing these recommendations with experts and stakeholders and to developing exact procedures and criteria for the assessment of modelling studies and their inclusion in the GRADE process.” The paper lays out a structured argument for incorporating modeling studies into the evidence base, particularly for formulating WHO recommendations related to treatment of HIV. The authors start by providing a review of how models are used in various fields, with suggestions about how they can inform guideline development. They address the question of what constitutes a modeling study. A comprehensive accounting of published literature on assessment of models is provided. Finally, they give recommendations for how models can be evaluated using the GRADE approach, with specific conclusions about important issues such as when modeling studies should be used as part of the evidence base and how their credibility should be assessed.Authors’ response: Thank you, this is a nice outline of the paper.Given the sweeping variety of models used in published studies about HIV treatments, policies and interventions, the authors are to be applauded for putting forward a framework for having this conversation. It will promote broader understanding of how models work and how they can be most optimally used for informing treatment guidelines. Authors’ response: Thank you very much. At the crux of their argument is the claim that evidence generated from models should be judged in terms of model credibility rather than on risk of bias. This argument raises several important issues. First, what constitutes a mathematical model? If a model is to be evaluated on its credibility, we need a definition to work from. Second, what kinds of output from models should be considered evidence, and how should the quality of that evidence be judged and ultimately weighed against or combined with evidence generated from randomized trials and observational cohort data? Authors’ response: We agree that these are central issues. Regarding the outputs from models that should be considered evidence, please note that we distinguish between mathematical models and modelling studies. The latter address well defined questions and outcomes, such as the impact of HIV testing and immediate antiretroviral therapy on HIV incidence or the impact of different screening strategies on the incidence of cervical cancer. In other words, the modeling outputs that constitute relevant evidence will depend on the question addressed in the modeling studies. In the revised version we write.GRADE provides a well-defined framework for weighing evidence from randomized trials and observational studies, as discussed in the section on “Assessing mathematical modelling studies using the GRADE approach”. Of note, randomized trials and observational studies are assessed separately. In general, guideline development groups will focus on randomized evidence if such evidence is available from several trial, and only consider observational studies in the absence of substantial randomized evidence. Similarly, evidence from mathematical modelling studies will be considered primarily if other studies cannot answer the question. Statistically combining evidence from different study types is not foreseen in GRADE, and beyond the scope of our article.What is a mathematical model? According to the authors, mathematical modeling is “a mathematical framework representing variables and their interrelationships to describe observed phenomena or predict future events.” On its face this is surely true, but for the purpose of understanding whether and how models should add to the evidence base, it’s too broad. This definition covers a vast assortment of mathematical models, ranging from validated descriptions of natural phenomena (where the mathematical relationships are known and directly observable) to representations of progression from HIV infection to death (comprising known and unknown mathematical components, many of which cannot be directly observed). Consider three examples for illustration: The mathematical representation of radioactive decay is known and can be written down explicitly. The model enables accurate and replicable predictions of future observations. The mathematical model for absorption of a specific drug is typically not known, but empirical studies have shown that it is possible to approximate the systematic variation using nonlinear equations. These models incorporate known information about physiology and properties of a specific drug, but are necessarily oversimplified representations of drug absorption because there are unobservable characteristics of individuals that affect absorption. The models can be used to make reliable predictions on average, but require unexplained variation to be reflected in terms of prediction intervals. Now consider a model of the population dynamics of HIV infection and disease progression. This process also follows a mathematical model, but the model itself is highly complex. Unlike radioactive decay or rate of drug absorption, the mathematical representations of several components of the underlying processes are essentially unknown. Moreover, much of the data needed to inform the models are either unobserved (e.g. timing of HIV infection) or only sparsely observed (e.g. individual-level viral load). All of these are mathematical models, but definitions must distinguish between them. Otherwise there is an implied equivalence that lends more credibility than is deserved to models that are heavily reliant on unverified assumptions about the mathematical structure underlying the dynamic system being modeled. A more systematic classification of model types would therefore be helpful.Authors’ response: Thank you for these three examples. We agree that the definition by Pieter Eykhoff is fairly broad and covers all three categories. However, we feel it is clear from the title and text of our paper that we are primarily concerned with models of the second and third category, i.e. more complex mathematical models that are relevant to WHO guidelines. Within these categories, the level of abstraction and complexity of models and their credibility will of course vary (see also examples in Table 1). We have now made explicit the distinction that we make between mathematical models and mathematical modelling studies at the beginning of the section, “What is a mathematical modelling study?” (page 4): A broad definition of a mathematical model is a “mathematical framework representing variables and their interrelationships to describe observed phenomena or predict future events”.5 We make a distinction between a mathematical model and mathematical modelling studies, which we define as studies that address defined research questions using mathematical models with a considerable degree of complexity and abstraction. At the Geneva workshop participants discussed different types of mathematical models in detail, based on a presentation by one of the authors (CA) on “The anatomy of mathematical modelling studies”. The workshop report and slides and be found at http://apps.who.int/iris/bitstream/10665/258987/1/WHO-HIS-IER-REK-2017.2-eng.pdf. Unfortunately, the link to the report and slides was incorrect in the F1000research paper. CA discussed model dichotomies, based on the book by Ben Bolker (Ecological Models and Data in R, 2008, Princeton University Press), and illustrated these using case studies from the Ebola crisis – see copy of one of his slides at the end of this response. In the discussion, workshop participants argued that guideline groups will often not be able to differentiate between different model dichotomies, and that this is not essential: guideline groups “just have to know what information models can provide and what value can be placed in that information.” However, we recommend that experts in mathematical modelling should support guideline groups (see recommendation 5 on page 9). We agree with the referee that guideline developers should carefully assess the credibility of models, and that models that “are heavily reliant on unverified assumptions about the mathematical structure underlying the dynamic system” are not credible. Our review of the methodological literature (see Table 2 in the paper) showed that the published frameworks of good modelling practice consistently emphasize the importance of the rationale for the model structure, the structural assumptions and uncertainty, the model transparency and validation etc. See also our recommendations 4, 5 and 6 on p 9. We added a more explicit reference and the correct link to the Workshop report, (page 3, last line): A detailed workshop report is available from WHO4. We also expanded the section, “What is a mathematical modelling study” to clarify our view on the need for classifying mathematical modelling studies (page 4, last paragraph): Workshop participants discussed whether it might be helpful for guideline groups to classify mathematical models in terms of their scope (for example descriptive versus predictive) or technical approach (for example static versus dynamic)8. Discussants argued that a good understanding of what information models can provide and what level of confidence can be placed in that information was more important than a taxonomy of models4. While the authors' definition of mathematical model is overly broad, the definition of statistical model, used to contrast with mathematical models, is too narrow. A statistical model is used to characterize sources of variation in observed data. It is based on a probabilistic representation of the data generating mechanism, which is itself a mathematical model. Theory and methods of statistical inference provides a rigorous and transparent set of techniques for parameter estimation, prediction of future outcomes, extrapolation (e.g. for causal inference), and uncertainty quantification. The last of these, uncertainty quantification, is a critical and frequently missing component of predictions based on mathematical models. Authors’ response: We agree with the reviewer’s comment about assessing uncertainty in mathematical models and state this explicitly (page 5, paragraph 2). For the purposes of generating evidence for WHO recommendations, the main difference between a mathematical model and a statistical model is that mathematical models tend to have broader scope and incorporate higher dimensions of complexity, but rely more heavily on assumptions about underlying mathematical structure than on individual-level data. Statistical models tend to have less mathematical complexity and more narrow scope, and are typically fitted to a single (possibly large) set of observed individual-level data drawn from the target population(s) of interest. A mathematical structure underlies both statistical and mathematical models, and both can be used for prediction of future outcomes and for causal policy comparisons. Authors’ response: We are grateful to the referee for this insightful and well-phrased comment about the relevance of the terms ‘statistical modelling’ and ‘mathematical modelling’ to WHO guidelines. We have taken the liberty of paraphrasing the comment to revise this section (page 4) as follows: Mathematical modelling studies may address complex situations and tend to rely more heavily on assumptions about underlying mathematical structure than on individual-level data. Examples include investigating the potential of HIV testing with immediate antiretroviral therapy to reduce HIV transmission6, or the likely impact of different screening practices on the incidence of cervical cancer7. Statistical modelling is typically concerned with characterizing sources of variation and associations between variables in observed individual-level data drawn from a target population of interest. Statistical models tend to be narrower in scope than mathematical models. Both statistical and mathematical models can be used to predict future outcomes and to compare different policies. The results from statistical analyses of empirical data often inform mathematical models. Mathematical modelling studies also increasingly integrate statistical models to relate the model output to data. Should models be judged on 'risk of bias'? The authors propose that evidence generated from mathematical models should be weighted more heavily toward model credibility than risk of bias. Authors’ response: Yes, we believe that the concept of model credibility is more useful than the more narrow concept of risk of bias (RoB). However, we think there is a mis-understanding here: the assessment RoB of specific studies also has a role. The RoB concept is widely used in the context of randomized controlled trials and observational studies that aim to make causal inference, and dedicated “RoB tools” have been developed to assess the risk of bias of studies included in systematic reviews (see references 1,2 below and www.riskofbias.info). These tools are based on relatively few well-defined biases. In the case of randomized trials they include selection bias, performance bias, detection bias, attrition bias and reporting bias (1). In the context of mathematical modelling studies, the risk of bias of empirical studies contributing parameter estimates is important and should be considered, for example in sensitivity analyses. On the other hand, many other and additional aspects are important when assessing the trustworthiness or credibility of mathematical models. These aspects are listed in Table 2, based on a review of published frameworks developed to assess good modelling practice. Please note that we use the term credibility as applied by the International Society for Pharmacoeconomics and Outcomes Research (ISPOR) to assessment of studies for decision making (3). These frameworks include assessments of the quality of the data used to parameterize a model. For example, Bennett and Manuel (4) and Philips et al (5) include several questions to that effect: Where choices have been made between data sources, are these justified appropriately? Where data from different sources are pooled, is this done in a way that the uncertainty relating to their precision and possible heterogeneity is adequately reflected? Has the quality of the data been assessed appropriately? The questionnaire proposed by Caro et al (6) asks Are the data used in populating the model suitable for your decision problem? All things considered, do you agree with the values used for the inputs? Similarly, the framework of Ramos and colleagues (7) includes the following questions: Have transition probabilities and intervention effects been derived from representative data sources for the decision problem? Have parameters relating to the effectiveness of interventions derived from observational studies been controlled for confounding? We have clarified our position and the role of RoB assessments as follows on page 8: The concept of bias relates to results or inferences from empirical studies, including randomized controlled trials and observational studies38,39 and is too narrow in the context of assessing mathematical modelling studies.40 “Credibility”, a term used by ISPOR,41 may therefore be more appropriate for modelling studies than “risk of bias”. The assessment of the credibility of a model is informed by a comprehensive quality framework and should cover the conceptualization of the problem, model structure, the input data and their risk of bias, different dimensions of uncertainty, as well as transparency and validation (Table 2). 1. Higgins JP, Altman DG, Gotzsche PC, et al. The Cochrane Collaboration’s tool for assessing risk of bias in randomised trials. BMJ 2011; 343:d5928.2. Sterne JA, Hernán MA, Reeves BC, et al. ROBINS-I: A tool for assessing risk of bias in non-randomised studies of interventions. BMJ 2016; 355:i4919.3. Aronson N, Grant MD. Tools for health care decision making: observational studies, modeling studies, and network meta-analyses. Value Health 2014; 17:141–142.4. Bennett C, Manuel DG: Reporting guidelines for modelling studies. BMC Med Res Methodol. 2012; 12: 168.5. Philips Z, Bojke L, Sculpher M, et al.: Good practice guidelines for decision- analytic modelling in health technology assessment: a review and consolidation of quality assessment. Pharmacoeconomics. 2006; 24(4): 355–71.6. Jaime Caro J, Eddy DM, Kan H, et al.: Questionnaire to assess relevance and credibility of modeling studies for informing health care decision making: An ISPOR-AMCP-NPC good practice task force report. Value Health. 2014; 17(2): 174–82.7. Ramos MC, Barton P, Jowett S, et al.: A Systematic Review of Research Guidelines in Decision-Analytic Modeling. Value Health. 2015; 18(4): 512–29.Many mathematical models are over-parameterized relative to the amount of data used to fit them; hence multiple configurations of parameter values can be lead to very similar predictions. Mathematical models are typically calibrated to observed population-level data (e.g. annual HIV incidence rate for the target population), but the formal rules for doing this seem to vary across application. Authors’ response: We agree – model concept, structure and parsimony are important elements when evaluating the credibility of mathematical models. Validation and predictive validity are also very important – again see Table 2.For many consumers of model-based outputs, this is a significant methodologic concern that goes directly to the question of credibility. If multiple model configurations can generate similar predictions, which configuration is the most credible one? It seems reasonable that model-based outcomes such as 10-year predictions of HIV incidence need to be evaluated on their own terms. If coupled with a formal process for back-checking or recalibrating existing models this would surely add value, and would possibly strengthen model identifiability (i.e., provide evidence in favor of one set of model parameters over another). Authors’ response: We agree and, again, believe that these issues are covered by the frameworks we present in Table 2. A more general justification for incorporating risk of bias into model evaluation can be found in Coveney et al 1 (page 4), who provide a general rubric for assessing quality of scientific evidence in the age of big data, emphasizing ‘acceptance of the theory based on concordance between the predictions and the measurements.’ Model calibrations at the time of model fitting partially fulfill this objective, but post-hoc evaluation of model predictions must play an important role in establishing credibility. Authors’ response: The timely piece by Coveney and Dougherty is really a critique of “’blind’ big data projects” and a plea for “the elucidation of the multiscale and stochastic processes controlling the behaviour of complex systems, including those of life, medicine and healthcare.” We could not agree more and argue that insights from the latter (mathematical models) should inform the development of WHO guidelines. The process of combining and comparing models is highly innovative and likely to have a positive impact on whether the results will be well received. This kind of cooperation and collaboration, exemplified recently by the Modelling Consortium, is perhaps unique to the mathematical modeling community. Evidence generated by these kinds of activities can form an important part of the evidence base. Authors’ response: We completely agree and have stressed this point in our paper. SummaryThe authors have provided a thorough case for including results from mathematical modeling into the formal evidence base used for making health recommendations, especially as they relate to HIV. The paper is based on findings from a recent conference and a comprehensive survey of extant literature. The main critiques are that the definition of mathematical model is far too broad, and that bias (or risk of bias) needs to be incorporated into the evaluation criteria. Formal methods for uncertainty quantification are critical as well. Authors’ response: Thank you again for reviewing our paper. See our responses above. We hope that based on our responses and the changes made in the manuscript you will be able to approve our contribution.Mathematical models are prevalent and influential in the HIV literature; hence a discussion about whether and how to place their findings in the broader evidence base is needed and welcome. This paper provides a necessary starting point."
}
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}
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https://f1000research.com/articles/6-1584
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https://f1000research.com/articles/7-224/v1
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23 Feb 18
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{
"type": "Opinion Article",
"title": "Social determinants and BCG efficacy: a call for a socio-biological approach to TB prevention",
"authors": [
"Jennifer B. Dowd",
"Helen A Fletcher",
"Delia Boccia",
"Helen A Fletcher",
"Delia Boccia"
],
"abstract": "A high burden of TB mortality persists despite the long-term availability of the bacillus Calmette-Guérin (BCG) vaccine, whose efficacy has been highly variable across populations. Innovative and alternative approaches to TB prevention are urgently needed while optimal biomedical tools continue to be developed. We call for new interdisciplinary collaborations to expand and integrate our understanding of how social determinants influence the biological processes that lead to TB disease, how this translates into differential BCG efficacy and, ultimately, how social protection interventions can play a role in reducing the global burden of TB. After providing an overview of the immune pathways important for the establishment of a response to the BCG vaccine, we outline how social determinants and psychosocial stressors can contribute to the observed variation in BCG efficacy above and beyond these biological factors. We conclude by proposing a new interdisciplinary research model based on the integration of social epidemiology theories with biomedical knowledge.",
"keywords": [
"tuberculosis",
"BCG",
"social determinants",
"social protection",
"psychosocial",
"immunity",
"vaccine",
"psychosocial stress"
],
"content": "Introduction\n\nImprovements in the prevention of Tuberculosis (TB) remain an urgent global public health priority, with the disease killing an estimated 1.5 million people annually1. This high burden of mortality persists despite the long-term availability of the bacillus Calmette-Guérin (BCG) vaccine, whose efficacy has been highly variable across populations. Recent trials of the vaccine candidate MVA85A, designed to boost BCG efficacy, showed high levels of immunogenicity in UK adults but poor levels in South African infants, highlighting a frustrating but common inconsistency of responses across populations2,3. This inconsistency of response is not limited to TB vaccines but is also observed in malaria and HIV vaccine development, where vaccine candidates often appear highly effective in early phase trials and yet perform below expectation in efficacy trials4. Calls to solve the puzzle of heterogeneous response to TB vaccination have traditionally focused on the technology of vaccine design and the interaction of Mycobacterium tuberculosis (M.tb) with the immune system at the molecular level5,6. These are crucial areas of investigation, but we argue that such large gaps in translation from the laboratory to human populations require new approaches to understand how social and biological variables interact to shape immune response and vaccine efficacy.\n\nWhile research on both the social and biological determinants of TB risk is well developed, thus far the two lines of enquiry have proceeded largely independently. The social epidemiology of TB typically examines social determinants of risk for exposure, diagnosis, and treatment, such as malnutrition, poor ventilation and overcrowding, and barriers in access to health care7. The more limited literature on social factors and TB vaccination focuses on factors such as vaccine distribution and uptake, but not immune response or vaccine efficacy8, and does not attempt to understand how social determinants may influence the expression of biological markers relevant to BCG efficacy and risk of TB disease. To date, variability in BCG efficacy remains explained in terms of interference from previous exposure to tuberculous or non-tuberculous mycobacteria6. Meanwhile outside of the context of TB, psychosocial and social influences on host immunity have been well described9–11, but research on socio-environmental factors and the biology of TB vaccine response is lacking.\n\nIn this paper, after providing an overview of the immune pathways important for the establishment of a response to the BCG vaccine, we propose a new focus on social determinants and psychosocial stressors that can contribute to the observed variation in BCG efficacy above and beyond these biological factors. We conclude by proposing a new interdisciplinary research model based on the integration of social epidemiology theories with biomedical knowledge.\n\n\nBCG immune response and vaccine efficacy\n\nWhile TB disease is a classic example of a disease of poverty, it is ultimately the failure of the immune system to contain infection which leads to active disease. Although we do not have a validated immune correlate of BCG vaccine efficacy, we know from studies of host-genetic susceptibility that cellular immunity plays a critical role in protection from TB disease12. Defects in the IFN-γ and IL-12 pathways, T cell and NK cell defects (GATA2 deficiency) and defects in monocytes and dendritic cells (IRF8 deficiency and CGD) are associated with susceptibility to mycobacterial disease12. Vaccination with BCG leads to the expansion of both classical antigen specific CD4+ and CD8+ T cells and non-classical cells such as CD1 and HLA-E restricted T cells13. There is also an NK response following immunization with BCG, likely driven by IL-2 secretion from mycobacterial antigen specific CD4+ T cells14. BCG can modify the innate immune response through induction of epigenetic changes in monocytes and dendritic cells15. A role for BCG vaccine induced IFN-γ in protection from TB disease has been suggested in an infant study where both BCG-specific IFN-γ secretion and Ag85A specific IgG were associated with lower risk of developing TB disease14. The BCG vaccine can therefore interact and modify all the key immune cells and pathways identified in human genetic studies to be critical for protection from TB disease.\n\nRecently, specific correlates of immune risk for TB disease have been identified in BCG vaccinated infants aged 10 weeks16 and 6 months14 enrolled in TB vaccine efficacy trials in the Western Cape of South Africa. Immune markers associated with TB risk include CD4+ T cell activation14, increased monocyte to lymphocyte ratio16,17 CMV positivity on ELISPOT18, and Type I Interferon19. These immune correlates highlight a role for the host immune environment in TB disease risk. Pre-vaccination inflammation has been associated with lower efficacy of hepatitis B, yellow fever and HIV vaccines20–22. Inflammation has also been associated with altered CD4 T cell response following immunization with BCG23. Known environmental drivers of T cell activation include viral infection24,25, previous exposure to mycobacteria, age and proximity to the equator. However, there are also well-documented social and psychosocial determinants of these immune biomarkers in other contexts, which we describe below.\n\n\nThe role of socioeconomic and psychosocial factors on immunity\n\nSocioeconomically disadvantaged populations face both material and psychosocial threats to adequate host immunity9–11. Stress is defined as an event or environmental demand that exceeds an individual’s perceived ability to cope, eliciting physiological stress responses from the hypothalamic-pituitary-adrenal (HPA) axis and the autonomic nervous system to deal with the threat26. There is abundant evidence from developed countries that chronic stressors and negative emotions such as anxiety and depression directly influence the immune system, including greater susceptibility to viral and bacterial infection and inhibited response to vaccines9,27,28. These stressors have also been found to be associated with other biomarkers of immunity and inflammation including TNFA, IL6 and IL1B, decreased NK cell function, as well as impaired control over latent viral infections9,11,29–31. Increased monocyte counts and monocyte to lymphocyte ratios have also been found in individuals with depressive symptoms, a common consequence of chronic stress and social deprivation32,33. Importantly for vaccination of infants, prenatal maternal stress has been shown to influence postnatal immunity in offspring, though this literature is currently more developed in animals than in humans34,35. Despite the high burden of deprivation and psychosocial stressors in areas where TB is endemic, the links between these exposures and biological immune response to BCG vaccine has not been explored.\n\nThese important but seldom overlapping literatures present a unique opportunity to elucidate how the social environment contributes biologically to variability in BCG vaccine efficacy. As an example of potential insights from such intersection, infection and immune response to one latent herpesvirus, cytomegalovirus (CMV), has been associated with both socioeconomic status and stress in developed countries11,36. Recent work suggests that CD4+ T cell activation is associated with risk of TB in BCG vaccinated infants in South Africa, with CMV infection emerging as one important driver of this T-cell activation14. These associations suggest a testable pathway from socioeconomic status and maternal stress, CMV reactivation, and impaired immune response to BCG vaccine in infants37,38.\n\n\nNew opportunities for TB prevention: a “social technology” approach\n\nFrom the pathway above, it could be argued that poverty reduction strategies, such as social protection, directly impacting socioeconomic status and maternal mental health could potentially play a role in increasing BCG efficacy. Social protection has been defined as a range of policies that enable people to cope with and recover from shocks, with the objective of moving people out of extreme poverty and interrupting the transgenerational transmission of inequalities39. Social protection includes widely used poverty-reduction strategies in both low and middle-income countries and encompasses both social security and social assistance interventions. In the latter group, cash transfer interventions are currently the most popular form of social protection40. These typically consist of the provision of regular, non-contributory, monetary benefits to households living in poverty and extreme poverty. They can be given unconditionally or conditionally on a number of educational (i.e. children school enrollment and attendance) and health behaviour requirements (i.e. access to maternal and child care services) that beneficiaries have to meet in order to remain enrolled. Today it is well acknowledged that cash transfers can improve beneficiaries’ socioeconomic status41. More recent literature suggests that cash transfers can significantly improve maternal mental health42,43 as well as self-perceived happiness among beneficiaries44,45, pointing to potential benefits on biological markers of stress and immunity.\n\nBased on these promising links, we call for the establishment of an interdisciplinary partnership, preliminarily named the “Social Technology Lab”, aiming to address the knowledge gaps in the pathways linking social protection, BCG immunological response, and TB disease. The ultimate goal of this research effort is the development and testing of a new vaccine R&D model based on the combination of innovative biotechnology tools with poverty reduction strategies, using BCG as proof of concept. We argue that such an innovative paradigm can provide crucial insights into how social factors manifest biologically via immune response to TB vaccine and potentially help explain the puzzle of wide variation in responses. Ultimately, this interdisciplinary approach will increase our understanding of how social determinants influence host immunity and TB risk over the life course, informing the integration of upstream structural and downstream biomedical intervention strategies.\n\n\nConclusions\n\nA more effective TB vaccine is considered the real game changer in the fight against the disease, however the development and delivery of new products may take decades even with considerable scientific and financial investment. Recent simulations suggest that if existing biomedical tools, including current BCG, were combined synergistically with social protection interventions, this could result in a significant acceleration in the decline of TB incidence globally46. Innovative and alternative approaches to TB prevention are urgently needed while optimal biomedical tools continue to be developed. An interdisciplinary model that expands and integrates our understanding of how social determinants influence the biological processes that lead to TB disease, how this translates into differential BCG efficacy and, ultimately, how this influence can be affected by social protection interventions holds promise for reducing the global burden of TB disease.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis paper is supported by pump-priming funding from the VALIDATE network (to P.I. Delia Boccia).\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\nGlobal tuberculosis report 2015. (World Health Organization). Reference Source\n\nTameris MD, Hatherill M, Landry BS, et al.: Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet. 2013; 381(9871): 1021–1028. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlack GF, Weir RE, Floyd S, et al.: BCG-induced increase in interferon-gamma response to mycobacterial antigens and efficacy of BCG vaccination in Malawi and the UK: two randomised controlled studies. Lancet. 2002; 359(9315): 1393–1401. PubMed Abstract | Publisher Full Text\n\nLi S, Plebanski M, Smooker P, et al.: Editorial: Why Vaccines to HIV, HCV, and Malaria Have So Far Failed-Challenges to Developing Vaccines Against Immunoregulating Pathogens. Front Microbiol. 2015; 6: 1318. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavenne T, McShane H: Why don’t we have an effective tuberculosis vaccine yet? Expert Rev Vaccines. 2016; 15(8): 1009–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaufmann SH: Future Vaccination Strategies against Tuberculosis: Thinking outside the Box. Immunity. 2010; 33(4): 567–577. PubMed Abstract | Publisher Full Text\n\nHargreaves JR, Boccia D, Evans CA, et al.: The social determinants of tuberculosis: from evidence to action. Am J Public Health. 2011; 101(4): 654–662. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAntai D: Inequitable childhood immunization uptake in Nigeria: a multilevel analysis of individual and contextual determinants. 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}
|
[
{
"id": "31152",
"date": "21 Mar 2018",
"name": "Aric A. Prather",
"expertise": [
"Reviewer Expertise Psychoneuroimmunology"
],
"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\nThere is no question that tuberculosis prevention serves as a pressing global health priority, and while the BCG vaccine is moderately effective, the variability in response among those inoculated raises important questions about what accounts for such variation. Dowd and colleagues make a strong case for the need to investigate more systematically the role of social determinants and psychosocial stressors in predicting BCG response. The call for a “Social Technology Lab” would fill a knowledge gap around how and when social determinants affect vaccination efficacy; however, it raises questions about at what level of measurement one would need to employ to get meaningful answers. Population scientists are often hampered by space constraints around questionnaires to obtain the necessary contextual information to understand psychological constructs, including psychological stress, and there is a need for a common vernacular when discussing stress (see Epel et al., in press1). Similar harmonization around other relevant social factors likely tied to vaccination response is needed. Nevertheless, an interdisciplinary approach to understanding BCG efficacy is well warranted.\n\nThere are a couple of minor concerns that, if addressed, could improve the paper. In paragraph 5, it would be helpful to specify the direction in which immune markers are associated with TB risk. In the following paragraph, the definition of stress could be more specific. In the context of psychosocial factors, stress occurs when one’s perceived demands exceed one’s ability to cope (Lazarus & Folkman, 1984), which often results in a coordinated physiologic and behavioral response to adaptively cope with the perceived demands. Stress as an event, i.e., stressor, is relevant but presumably the stress-immune link of interest in psychological in nature.\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": "33031",
"date": "29 May 2018",
"name": "Marie-Claude Rousseau",
"expertise": [
"Reviewer Expertise Epidemiology",
"BCG vaccination"
],
"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 opinion article, Dowd et al. propose a new approach for tuberculosis (TB) prevention, based on BCG vaccination and poverty reduction strategies. The article is well written, logically organized, and scientifically sound. After reviewing the evidence on immune pathways involved in protection from TB, they describe host factors associated with vaccine efficacy, and address socioeconomic and psychosocial factors affecting immunity. They argue convincingly that there exist several research gaps about the pathways connecting social factors, the immune response elicited by the BCG vaccine, and TB prevention. They recommend the establishment of a new “Social Technology Lab”, an interdisciplinary partnership aiming to address these knowledge gaps. This is a promising and innovative proposal in the context of the worldwide burden of TB and obstacles faced toward designing a more efficacious TB vaccine.\nThe authors’ rationale rests on the highly variable efficacy of BCG, as observed across several populations. They provide credible evidence that differences in vaccine efficacy may very well be related to varying socioeconomic and psychosocial factors across study populations. They also document that the host immune environment can modulate TB risk and thus modify the effect of the BCG vaccine. One aspect that seems to be missing from this conceptual framework is that vaccine characteristics are likely related to vaccine efficacy. Indeed, some authors have reported that BCG substrain and route of vaccination influenced BCG virulence and efficacy1,2,3,4. The article would be strengthened by including a few sentences describing the state of knowledge on vaccine characteristics and how they relate to efficacy. This could be integrated in the section “BCG immune response and vaccine efficacy”. Although it is a minor issue for the current article, this aspect is relevant for the future activities of the Social Technology Lab and should be integrated in its investigations.\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/7-224
|
https://f1000research.com/articles/7-223/v1
|
23 Feb 18
|
{
"type": "Case Report",
"title": "Case Report: Enlarging symmetrical masses of the palate of idiopathic etiology",
"authors": [
"Michelangelo Vestita",
"Eleonora Nacchiero",
"Michele Maruccia",
"Giuseppe Giudice",
"Eleonora Nacchiero",
"Michele Maruccia",
"Giuseppe Giudice"
],
"abstract": "We report the case of a 33 year-old woman who came to our attention with slowly enlarging exophytic masses of the palate, histologically characterized by sub-epithelial fibrous proliferation with packed collagen bundles and increased fibroblasts number. We describe the condition of idiopathic fibrous hyperplasia, its diagnosis and its surgical treatment, which in our case was carried out with the aid of a custom made thermal printed plaque used as a scaffold.",
"keywords": [
"fibrous hyperplasia",
"palate",
"idiopathic",
"gengival hyperplasia"
],
"content": "Introduction\n\nIdiopathic fibrous hyperplasia is a rare benign condition, characterized by a slow and progressive increase in gingival volume1,2. It manifests as a rosy swelling of hard consistency while, at histological examination, it is characterized by a proliferation of fibroblasts in a myxomatous stroma. We describe and discuss a case of idiopathic fibrous hyperplasia of the palate.\n\n\nCase\n\nA 33 year-old woman came to our attention with slowly enlarging exophytic masses of the palate, which had begun to grow 2 years before and caused her disturbances of phonation as well as in swallowing solids and liquids. The patient did not take any drugs; however she had been a frequent user of nonsteroidal anti-inflammatory drugs for the last 3 years because of chronic back pain. Remote personal and family histories were negative, except for recurrent gastric nuisance and back pain.\n\nClinical and rhinoscopy examination demonstrated bilateral and symmetrical exuberant hypertrophic tissue, of hard consistency and rosy color, at the posterior-later area of the palate, with a tendency to coalesce medially. This tissue was contiguous to the adjacent gingiva (Figure 1).\n\nComputerized tomography scan showed such lesions to be limited to the mucosal palate, with no underlying bone involvement (Figure 2).\n\nAn incisional biopsy demonstrated a sub-epithelial fibrous proliferation with packed collagen bundles and increased fibroblasts number (Figure 3). We concluded for a diagnosis of localized idiopathic fibrous hyperplasia. We treated the patient with a personalized approach using surgical resection and insetting of a thermal-printed palate plaque (Figure 4). We obtained good functional results at 20 days post-op (Figure 5), and no sign of recurrence at the 12 months follow up (Figure 6).\n\nConsistent with idiopathic fibrous hyperplasia.\n\n\nDiscussion\n\nTwo main subtypes of fibrous hyperplasia are known: generalized and localized. Generalized form has a genetic predisposition, appears at decimal or definitive dental eruption, and usually demonstrates a tendency to recur after surgery1–4. Secondary forms associated to pregnancy, scurvy, leukemia and drugs are also known1–3. Among the latter, various chemotherapy agents can elicit secondary forms5, including ipilimumab and vemurafenib6. However, no reports link gingival fibrous hyperplasia to the drugs administered in our patient. The localized form has its onset from the second decade, does not generally recur after surgery, and normally is not associated to genetic predisposition,7,8 although investigations to exclude syndromes commonly associated to gingival fibromatosis should always be carried out in our experience; these include Laband, Rutherfurd, Cross and Ramon syndromes5. In both the localized and the generalized forms, local factors such as dental plaques, caries, and the action of chemical substances and their metabolites might contribute to the onset in susceptible patients6. The precise pathogenic mechanism of idiopathic forms is unknown, but it appears to confine to the gingival and mucosal fibroblasts with no involvement of the periodontal ligament or the palate underlying bones1,2.\n\nRegardless of the etiology, excess tissue removal is the treatment of choice in localized fibrous hyperplasia, either by scalpel or by CO2 laser7,8. Our personal approach includes the use of a thermal-printed palate plaque (Figure 4), to be left in place for 20 days after surgery (Figure 5), which in our experience yields excellent hemostasis by exerting compression and, at the same time, functions as a scaffold that promotes and guides second intention healing, preventing recurrence of exuberant tissue growth. The long term (12 months) results reported in our case testify to the efficacy of such an approach.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCekmez F, Pirgon O, Tanju IA: Idiopathic gingival hyperplasia. Int J Biomed Sci. 2009; 5(2): 198–200. PubMed Abstract | Free Full Text\n\nSapp JP, Eversole LR, Wysocki GP: Connectivetissue lesions. Contemporary Oral and Maxillofacial Pathology. 2004; 294–297, Mosby, London, UK 2nd edition. Reference Source\n\nPappachan B, Narayan JV, Nayak A: Idiopathic gingival fibromatosis: A neglected case. Indian J Radiol Imaging. 2002; 12: 335–338.\n\nHart TC, Pallos D, Bozzo L, et al.: Evidence of genetic heterogeneity for hereditary gingival fibromatosis. J Dent Res. 2000; 79(10): 1758–1764. PubMed Abstract | Publisher Full Text\n\nGuida M, Cramarossa A, Fistola E, et al.: High activity of sequential low dose chemo-modulating Temozolomide in combination with Fotemustine in metastatic melanoma. A feasibility study. J Transl Med. 2010; 8: 115. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMangold AR, Bryce A, Sekulic A: Vemurafenib-associated gingival hyperplasia in patient with metastatic melanoma. J Am Acad Dermatol. 2014; 71(5): e205–206. PubMed Abstract | Publisher Full Text\n\nShetty AK, Shah HJ, Patil MA, et al.: Idiopathic gingival enlargement and its management. J Indian Soc Periodontol. 2010; 14(4): 263–265. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPalaia G: Idiopathic fibrous hyperplasia of the palate. Ann Stomatol (Roma). 2013; 4(Suppl 2): 35. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "31157",
"date": "26 Feb 2018",
"name": "Ilaria Mataro",
"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 believe the article is well structured and describes an interesting presentation of a rare benign condition.\nI must congratulate the authors on the quality and quantity of the photographic material. The case is really well documented with clinical pictures, histologic images and radiologic findings.\nI have personally treated a couple of these cases but often experienced short to medium term recurrence. The alternative treatment described by the authors (the use of a guiding plaque after removal) is interesting and seems to warrant better long term outcome.\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": "31155",
"date": "08 Mar 2018",
"name": "André Salval",
"expertise": [
"Reviewer Expertise Plastic surgery",
"burns",
"aesthetic surgery"
],
"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 Report is of scientific interest and well written. A more detailed explanation of therapy would be appreciated. About the thermal printed palate plaque for instance: what material is it made of? Is it a mold or 3D printed? Is it placed immediately after surgery and left in place 24 hours a day? Is thermal plaque therapy a novel approach or are there any references about this kind of therapy?\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/7-223
|
https://f1000research.com/articles/7-221/v1
|
23 Feb 18
|
{
"type": "Case Report",
"title": "Case Report: Evaluation strategies and cognitive intervention: the case of a monovular twin child affected by selective mutism",
"authors": [
"Micaela Capobianco",
"Luca Cerniglia",
"Micaela Capobianco"
],
"abstract": "The present work describes the assessment process, evaluation strategies, and cognitive intervention on a 9 years old child with selective mutism (SM), a monovular twin of a child also affected by mutism. Currently, the cognitive behavioral multimodal treatment seems the most effective therapeutic approach for children diagnosed with selective mutism (Capobianco & Cerniglia, 2018). The illustrated case confirms the role of biological factors involved in mutacic disorder but also highlights the importance of environmental influences in the maintenance of the disorder with respect to relational and contextual dynamics (e.g. complicity between sisters, family relationships). The article discusses furthermore the importance of an early diagnosis as a predictor of positive treatment outcomes.",
"keywords": [
"Selective mutism",
"twins",
"assessment",
"intervention."
],
"content": "Introduction\n\nSelective mutism (SM) is a developmental disorder of biological and environmental etiopathogenesis. It is characterized by the child’s significant inability to speak in specific social situations (for instance at school) or in non-familiar circumstances. The clinical and research data confirm that SM is a complex etiological multifactorial disorder whose maintenance is connected with the interaction of biological (familiarity with shyness, preterm birth) (Capobianco & Cerniglia, 2017) and environmental early factors (anxiety and overprotectiveness of the mother) (Capobianco et al., 2010; Capobianco et al., 2017; Capozzi et al., 2017; Pizzuto & Capobianco, 2008) Analysis of the underlying thoughts and emotions in mutacic behavior reveals fear of judgment, fear of failure and being mocked, shame, depression, feelings of worthlessness or inadequacy,catastrophizing, and confirms the comorbidity between selective mutism and internalizing symptoms. The typical age of onset for selective mutism is between 3–6 years, even though the disorder gets usually diagnosed belatedly during the school age (around 7–8 years). It seems that the twins condition in presence of frequent associated additional biological and environmental conditions (prematurity, maternal stress, isolation) plays a crucial role in aggravating mutacic difficulties (Capobianco & Cerniglia, 2017; Capobianco & Cerniglia, 2018). The possibility to improve and change the symptomatology of SM-diagnosed children is strongly related to the severity of the disorder and the biological environmental factors implicated in each single case. Cognitive-behavioral intervention on the child has as its purpose to change the resulting cognitive biases and emotional states, but parallel efforts on the environmental dynamics that determine the disorder maintenance are needed (family and school).\n\n\nCase report\n\nF is a child with a belated diagnosis of selective mutism (diagnosed after the third grade of elementary school). The mutacic symptomatology appears particularly in the school environment, in interaction with teachers and peers during teaching activities. She has a monovular twin sister (L) with the same diagnosis. She is born at 36 weeks without any particular pre- or perinatal problems. The mother describes F’s development as regular through the “normal” stages and does not recall any developmental problems in the motor and/or linguistic realms during F’s first years of life (Capobianco et al., 2017; Pizzuto & Capobianco, 2008).\n\nIn short, F has always been a very shy girl, showing difficulties and fear in relating to non- familiar people . F has always perceived similar situations with an intense discomfort, anxiety and concern and has learned to react through inhibition, behavioral and verbal withdrawal. As a result of functional evaluation (cognitive and projective tests), spontaneous observation and exploration of the ABC (through various simulation techniques with characters, drawings, written questions and cartoons) emerges fear of judgment, fear of failure and being mocked, being made fun of, (“everyone will laugh at me”), shame, and the fact that others might notice this feeling. Depressiveness and catastrophizing prevail over the possible consequences on others of her speaking, her perception of inadequacy and her feelings of worthlessness (“I am afraid to make a mistake”, “I didn’t study”, “I am not capable”). F shows her discomfort via “critical-provocative” behavior especially when her sister L is present, although the twins seem to have a strong complicity: “a mute complicity” in keeping the mutism part.\n\nMaternal relationship. The mother’s behavior represents an important maintenance factor of F’s mutism. The girl shows indeed an anxious-dependent attachment style (Pattern C) towards the mother. The mother actually appears to be anxious and worried showing a pressure attitude to make the girl speak (sometimes even punitive), and she often replaces F in her responsibilities. Another important aspect is the confrontation with F’s twin sister. The mother often tends to compare the two sisters. This creates conflict situations and a quest for attention by F, who uses various maternal affection manipulation strategies.\n\nMaternal thoughts and emotional states. During the interviews, the mother shows extreme attention to maintaining a positive image in front others and a propensity to avoid feelings of shame to which she gives a meaning of failure, devaluation and humiliation. These aspects are strongly connected to her life story and particularly to experiences of abandonment and maltreatment. Her necessity not to feel ashamed and her focus on avoiding situations, in which she could be exposed to that risk, have an extreme importance to the mother of F and L.\n\nThe belated diagnosis has led F to reinforce her behavioral style such that, by now, has became habitual and an integral part of her way of being, and the basis of an equilibrium reached in the dependent relationship with her mother and her twin sister. Behaviors of passivity, lack of interest, creativity and pro-activeness, are products of the extended time in missing use of verbal language as a necessary means to develop the formation of concepts, thoughts, complex thoughts, and metacognition. Despite F’s functional evaluation, she has a normal intelligence level but her neuropsychological profile indicates poor cognitive functioning.\n\n\nTherapy\n\n1. To change dysfunctional thoughts of:\n\na) “Catastrophizing” and “Overgeneralization” compared to the consequences of speaking and the judgment of others: to speak does not necessarily provoke “negative judgments” by others. At this point it is necessary to reflect on different hypotheses of other one’s judgments and on the fact that talking can bring about different consequences that can often be more “useful” than not talking at all. Additionally the consideration of the fact that it is “normal” that we might not be liked by others, but that this does not and should not damage our personal value and capacities is considered. Moreover the reflection on shame as being a natural emotion, that can appear in particular moments, is considered. (emotion acceptance process).\n\nb) “Feelings of worthlessness” and sense of “incapacity” to speak: the promotion of autonomy and self-esteem. Not always do others laugh at what we say and if it happens that someone doesn’t share the same opinion this doesn’t mean that the content of speaking doesn’t have any value, independent from ones self-esteem and a negative or positive evaluation of contingent situations (for example interrogation, speaking in front of strangers, etc.)\n\n2. To understand and label emotions:\n\nDuring the event simulation (to play and draw), F was asked at various times to indicate the emotion the character feels in that particular moment (by indicating the faces for each emotion or writing) with the scope to reflect carefully on the connection between events-emotions and their alternative consequences.\n\n- Psycho-educational meetings with the parents of F and L on selective mutism disorder: F does not speak because she refuses to or throws tantrums as she feels a “discomfort” that makes her unable to speak. These meetings are mainly focused on changing the parents’ interpretation and thoughts on F’s mutacic behaviors.\n\n- Indications on the parents’ behavior at home:\n\n1. To adopt a neutral attitude towards the non-speaking, to neither underline it frequently as a problem nor show a punitive approach;\n\n2. No replacement of F in her daily activities and relationships. Whenever any person asks her a question it is important to leave space for the girl and never insist that she answers verbally, nor reply on her behalf. To involve her in the conversation whilst accepting other ways of communication (gestures, drawings, written text);\n\n3. Home working and autonomy promotion: to let F gradually initiate small daily activities: for example pay at the newsstand, make a phone call, ask for information, etc.;\n\n4. To increase social interactions with F’s peers, if possible, separately from her sister and to create various individual spaces and locations;\n\n5. To avoid comparisons between the two girls. To dedicate a single and separate location for and with F.\n\nIt is very important to have a common approach both at home as at school to enforce the effects of intervention:\n\nTeachers behavior in the classroom: a) neutral attitude towards the non-speaking enforcing the non-verbal communication with them and between F’s peers; b) use of various disciplines (drawing, writing, open questions, multiple choice); c) promote and create activities in small groups with at least one peer with whom the girl talks to or gets along with.\n\nSelective mutism in the developmental age is a complex problem that depends on the interaction of multiple individual and environmental issues that are interweaved in various ways in the life of the girl. Therefore there is a strong necessity to explore all life situations, to attempt to involve the school, parents and teachers and to reconcile between therapeutic objectives and demands of the various life context of the child. The main scope of intervention in the various social areas of F has collided against specific difficulties in the family-(e.g. the experiences of suffering of the mother) and school environment. It is often difficult to help parents to implement social relationships with the class or with other children. In F’s case the twin condition, the diagnosis and the belated intervention, have been important factors in the maintenance of the disorder. The cognitive intervention has a strong connection with the evaluation of thoughts and the emotions underlying the mutacic disorder. Neuropsychological and emotional evaluation through structured tests that demand verbal methods and sufficient collaboration is not easy in a condition where there is no verbal communication (Pizzuto & Capobianco, 2008). It is necessary to use various answering procedures (e.g. written, non-verbal, drawings) whilst keeping the maximum of validity during the administration of the tests. The evaluation of beliefs, cognitive biases and the behaviors of the child are fundamental to be able to structure the treatment program and it is necessary to “create” additional functional strategies and procedures to explore these aspects in each individual child.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the parent 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\nAcknowledgments\n\nThe authors acknowledge the work of Dr. Liesbeth Elsink for the translation of this case report into English language.\n\n\nReferences\n\nCapobianco M, Cerniglia L: Early language development in preterm children without neurological damage: a longitudinal study [version 1; referees: 2 approved]. F1000Res. 2017; 6: 2169. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCapobianco M, Cerniglia L: Cognitive,emotional and behavioral iussues in selective mutism: a narrative review on elements of a multimodal intervention. Interacti Stud. (in press, next iussue). 2018. Reference Source\n\nCapobianco M, Pizzuto EA, Devescovi A: Gesture–speech combinations and early verbal abilities. Interact Stud. 2017; 18(1): 55–76. Publisher Full Text\n\nCapobianco M, Riccio G, Devescovi A: Early communicative and language development in preterm infants without neurological damage. J Appl Res Intellect Disabil. 2010; 23(5): 513.\n\nCapozzi F, Manti F, Di Trani M, et al.: Children's and parent's psychological profiles in selective mutism and generalized anxiety disorder: a clinical study. Eur Child Adolesc Psychiatry. 2017; 1–9. PubMed Abstract | Publisher Full Text\n\nPizzuto EA, Capobianco M: Is pointing “just” pointing? Unraveling the complexity of indexes in spoken and signed discourse. Gesture. 2008; 8(1): 82–103. Publisher Full Text"
}
|
[
{
"id": "31146",
"date": "26 Feb 2018",
"name": "Mirco Fasolo",
"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\n26/02: This peer review report was mistakenly published with an Approved with Reservations status. It has now been corrected to an Approved.\nI have reviewed the Case Report “Evaluation strategies and cognitive intervention: the case of a monovular twin child affected by selective mutism”.\n\nI think that the paper is well written and offers a relevant clinical and research contribution to studies on selective mutism, a little know disorder that consists in an “inability to talk in unfamiliar contexts”.\nThe case of a child monovular twin is very interesting because it allows to evidence the link between biological and environmental factors.\nI accept the work but I think that:\nIt is necessary to insert more details on the clinical case.\n\nThe child is a twin and interaction with her sister is a though factor for maintaining the mutacic behaviour, so, the relationships between the two sisters must be better presented.\n\nThe cognitive-behavioural is the best approach for children with mutism. The authors can mention also others approaches for comparison (e.g. psychodynamic approach).\n\nEarly diagnosis and surgery of selective mutism must be briefly presented.\n\nSome misprints are present in the text.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
},
{
"id": "31484",
"date": "06 Mar 2018",
"name": "Marco Bozzali",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Capobianco & Cerniglia investigates the selective mutism phenomenon in twins. The study is interesting and well written and it improves the knowledge about mutacic difficulties in children. I accept the paper with minor revision:\n\nThe Authors in the Conclusion section of the manuscript should better clarify the modalities of intervention on the two twins interaction.\n\nA table with neuropsychological scores should be added to the manuscript to better clarify the patient’s cognitive profile.\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": "31340",
"date": "12 Mar 2018",
"name": "Deny Menghini",
"expertise": [
"Reviewer Expertise Neurodevelopmental 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\nAbstract:\nPlease avoid References in the abstract.\n\nIntroduction:\nPlease avoid self-quotations in the Introduction. For example, cite other studies on etiological factors as Krysanski VL A brief review of selective mutism literature. J Psychol. 2003 Jan; 137(1):29-40.1\n\nDiagnostic criteria for “selective mutism” definition are needed: include Diagnostic and Statistical Manual of Mental Disorders, DSM-5; APA, 2013, p. 195.\n\nCite other studies on single-cases: e.g. see Joseph PR. Selective mutism: the child who doesn’t speak at school. Pediatrics 1999; 104:308–309 2\n\nPlease check the year in Capobianco & Cernaglia (2017). In the Reference list is dated 2018.\n\nAdd references for the comorbidity of internalizing symptoms and selective mutism: see Diliberto RA, Kearney CA. Anxiety and oppositional behavior profiles among youth with selective mutism. J Commun Disord. 2016 Jan-Feb;59:16-23. 3\n\nPlease, include other studies on the relationship between twins and mutism: e.g. Albrigtsen V, Eskeland B, Mæhle M. Ties of silence--Family lived experience of selective mutism in identical twins. Clin Child Psychol Psychiatry. 2016 Apr;21(2):308-234; Gensthaler A, Maichrowitz V, Kaess M, Ligges M, Freitag CM, Schwenck C. Selective Mutism: The Fraternal Twin of Childhood Social Phobia. Psychopathology. 2016;49(2):95-107.5\n\nLast paragraph of the introduction: Please include references on the beneficial effect of CBT: see Walkup JT, Albano AM, Piacentini J, et al. Cognitive behavioral therapy, sertraline, or a combination in childhood anxiety. N Engl J Med 2008; 359:2753–2766.6\n\nDetails on the cognitive behavioral multimodal therapy are needed.\n\nCase report:\n\nPlease describe in details neuropsychological measures (e.g. IQ) and instruments adopted for the assessment (anxiety scales as MASC, semi-structured interview as K-sads).\n\nAvoid to mention “projective tests”. Projective measures are not believed scientifically valid measures and they are not adopted as standard procedures for the assessment or diagnosis in literature and guidelines.\n\nAvoid self-quotation in the “case report” session.\n\nA brief description of familial history of psychopathological disorder (especially anxiety) shoul be included.\n\nResults:\nPlease include a session of the main results of the present study obtained after the intervention and the measures used to verify the efficacy of the treatment.\n\nDiscussion:\nIn the Discussion, describe the significance of present findings in light of what was already known about the topic (previous literature), and explain new understanding about the problem.\n\nPlease, include limitations of the study.\n\nFor example, the lack of cognitive measures and psychopathological instruments (especially anxiety) for the assessment and the post-treatment evaluation.\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? 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? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/7-221
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https://f1000research.com/articles/7-121/v1
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29 Jan 18
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{
"type": "Method Article",
"title": "Using regulatory genomics data to interpret the function of disease variants and prioritise genes from expression studies",
"authors": [
"Enrico Ferrero"
],
"abstract": "The identification of therapeutic targets is a critical step in the research and developement of new drugs, with several drug discovery programmes failing because of a weak linkage between target and disease. Genome-wide association studies and large-scale gene expression experiments are providing insights into the biology of several common and complex diseases, but the complexity of transcriptional regulation mechanisms often limit our understanding of how genetic variation can influence changes in gene expression. Several initiatives in the field of regulatory genomics are aiming to close this gap by systematically identifying and cataloguing regulatory elements such as promoters and enhacers across different tissues and cell types. In this Bioconductor workflow, we will explore how different types of regulatory genomic data can be used for the functional interpretation of disease-associated variants and for the prioritisation of gene lists from gene expression experiments.",
"keywords": [
"bioconductor",
"r",
"rstats",
"regulatory genomics",
"functional genomics",
"genetics",
"gwas",
"transcriptomics",
"integration",
"multiomics"
],
"content": "Abbreviations\n\nCAGE: cap analysis of gene expression; DHS: DNase I hypersensitive site; eQTL: expression quantitative trait locus; GWAS: genome-wide association study; PheWAS: phenome-wide association study; SLE: systemic lupus erythematosus; SNP: single nucleotide polymorphism; TSS: transcription start site\n\n\nIntroduction\n\nDiscovering and bringing new drugs to the market is a long, expensive and inefficient process1,2. Increasing the success rates of drug discovery programmes would be transformative to the pharmaceutical industry and significantly improve patients’ access to medicines. Of note, the majority of drug discovery programmes fail for efficacy reasons3, with up to 40% of these failures due to lack of a clear link between the target and the disease under investigation4.\n\nTarget selection, the first step in drug discovery programmes, is thus a critical decision point. It has previously been shown that therapeutic targets with a genetic link to the disease under investigation are more likely to progress through the drug discovery pipeline, suggesting that genetics can be used as a tool to prioritise and validate drug targets in early discovery5,6.\n\nOver the last decade, genome-wide association studies (GWASs) have revolutionised the field of human genetics, allowing to survey DNA mutations associated with disease and other complex traits on an unprecedented scale7. Similarly, phenome-wide association studies (PheWAS) are emerging as a complementary methodology to decipher the genetic bases of the human phenome8. While many of these associations might not actually be relevant for the disease aetiology9, these methods hold much promise to guide pharmaceutical scientists towards the next generation of drug targets10.\n\nArguably, one of the biggest challenges in translating findings from GWASs to therapies is that the great majority of single nucleotide polymorphisms (SNPs) associated with disease are found in non-coding regions of the genome, and therefore cannot be easily linked to a target gene11. Many of these SNPs could be regulatory variants, affecting the expression of nearby or distal genes by interfering with the process of transcription (e.g.: binding of transcription factors at promoters or enhancers)12.\n\nThe most established way to map disease-associated regulatory variants to target genes is probably to use expression quantitative trait loci (eQTLs)13, variants that affect the expression of specific genes. Over the last few years, the GTEx consortium assembled a valuable resource by performing large-scale mapping of genome-wide correlations between genetic variants and gene expression across 44 human tissues14.\n\nHowever, depending on the power of the study, it might not be possible to detect all existing regulatory variants as eQTLs. An alternative is to use information on the location of promoters and distal enhancers across the genome and link these regulatory elements to their target genes. Large, multi-centre initiatives such as ENCODE15, Roadmap Epigenomics16 and BLUEPRINT17,18 mapped regulatory elements in the genome by profiling a number of chromatin features, including DNase hypersensitive sites (DHSs), several types of histone marks and binding of chromatin-associated proteins in a large number of cell lines, primary cell types and tissues. Similarly, the FANTOM consortium used cap analysis of gene expression (CAGE) to identify promoters and enhancers across hundreds of cells and tissues19.\n\nKnowing that a certain stretch of DNA is an enhancer is however not informative of the target gene(s). One way to infer links between enhancers and promoters in silico is to identify significant correlations across a large panel of cell types, an approach that was used for distal and promoter DHSs20 as well as for CAGE-defined promoters and enhancers21. Experimental methods to assay interactions between regulatory elements also exist. Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET)22,23 couples chromatin immunoprecipitation with DNA ligation and sequencing to identify regions of DNA that are interacting thanks to the binding of a specific protein. Promoter capture Hi-C24,25 extends chromatin conformation capture by using “baits” to enrich for promoter interactions and increase resolution.\n\nOverall, linking genetic variants to their candidate target genes is not straightforward, not only because of the complexity of the human genome and transcriptional regulation, but also because of the variety of data types and approaches that can be used. To address this, we developed STOPGAP (systematic target opportunity assessment by genetic association predictions), a database of disease variants mapped to their most likely target gene(s) using different types of regulatory genomic data26. The database is currently undergoing a major overhaul and will eventually be superseded by POSTGAP. A similar resource and valid alternative is INFERNO (inferring the molecular mechanisms of noncoding variants)27.\n\n\nWorkflow\n\nIn this workflow, we will explore how regulatory genomic data can be used to connect the genetic and transcriptional layers by providing a framework for the functional annotation of SNPs from GWASs. We will use eQTL data from GTEx14, FANTOM5 correlations between promoters and enhancers21 and promoter capture Hi-C data25.\n\nWe start with a common scenario: we ran a RNA-seq experiment comparing patients with a disease and healthy individuals, and would like to discover key disease genes and potential therapeutic targets by integrating genetic information in our analysis.\n\nR version 3.4.2 and Bioconductor version 3.6 were used for the analysis. The code below will install all required packages and dependencies from Bioconductor and CRAN:\n\n\n\nThe RNA-seq data we will be using comes from blood of patients with systemic lupus erythematosus (SLE) and healthy controls28.\n\nWe are going to use recount29 to obtain gene-level counts:\n\n\n\nOther Bioconductor packages that can be used to access data from gene expression experiments directly in R are GEOquery30 and ArrayExpress31.\n\nSo, we have 117 samples. This is what the data looks like:\n\n\n\nWe note that genes are annotated using the GENCODE32 v25 annotation, which will be useful later on. Let’s look at the metadata to check how we can split samples between cases and controls:\n\n\n\nThe most interesting part of the metadata is contained in the characteristics column, which is a CharacterList object:\n\n\n\nLet’s create some new columns with this information that can be used for the differential expression analysis. We will also make sure that they are encoded as factors and that the correct reference layer is used:\n\n\n\nWe can have a look at the new format:\n\n\n\nIt looks more readable. Let’s now check how many samples we have in each group:\n\n\n\nTo speed up code execution we will limit the number of SLE samples. For simplicity, we select the first 18 (healthy) and the last 18 (SLE) samples from the original RangedSummarizedExperiment object:\n\n\n\nNow we are ready to perform a simple differential gene expression analysis with DESeq233:\n\n\n\nNote that we used an extremely simple model; in the real world you will probably need to account for co-variables, potential confounders and interactions between them. edgeR34 and limma35 are good alternatives to DESEq2 for performing differential expression analyses.\n\nWe can now look at the data in more detail. We use the variance stabilising transformation (VST)36 for visualisation purposes:\n\n\n\nFirst, let’s look at distances between samples to see if we can recover a separation between SLE and healthy samples:\n\n\n\nWe will use the pheatmap and RColorBrewer packages for drawing the heatmap (Figure 1).\n\n\n\nSimilarly, we can perform a principal component analysis (PCA) on the most variable 500 genes (Figure 2).\n\n\n\nThis looks better, we can see some separation of healthy and SLE samples along both PC1 and PC2, though some SLE samples appear very similar to the healthy ones. Next, we select genes that are differentially expressed below a 0.05 adjusted p-value threshold:\n\n\n\nWe can look at a summary of the results:\n\n\n\nWe can also visualise the log fold changes using an MA plot (Figure 3).\n\n\n\nFor convenience, we will save our differentially expressed genes (DEGs) in another object:\n\n\n\nWe also map the GENCODE gene IDs to gene symbols using the annotation in the original RangedSummarizedExperiment object, which is going to be convenient later on:\n\n\n\nWe have more than 3500 genes of interest at this stage. Since we know that therapeutic targets with genetic evidence are more likely to progress through the drug discovery pipeline6, one way to prioritise them could be to check which of these can be genetically linked to SLE. To get hold of relevant GWAS data, we will be using the gwascat Bioconductor package37, which provides an interface to the GWAS catalog38. An alternative is to use the GRASP39 database with the grasp2db40 package.\n\n\n\nsnps is a gwasloc object which is simply a wrapper around a GRanges object, the standard way to express genomic ranges in Bioconductor. We are interested in SNPs associated with SLE:\n\n\n\nWe can visualise these as a Manhattan plot to look at the distribution of GWAS p-values over chromosomes on a negative log scale (Figure 4). Note that p-values lower than 1e-25 are truncated in the figure and that we have to load ggplot241 to modify the look of the plot:\n\n\n\nWe note here that genotyping arrays typically include a very small fraction of all possible SNPs in the human genome, and there is no guarantee that the tag SNPs on the array are the true casual SNPs42. The alleles of other SNPs can be imputed from tag SNPs thanks to the structure of linkage disequilibrium (LD) blocks present in chromosomes. Thus, when linking variants to target genes in a real-world setting, it is important to take into consideration neighbouring SNPs that are in high LD and inherited with the tag SNPs. For simplicity, we will skip this LD expansion step and refer the reader to the Ensembl REST API43, the Ensembl Linkage Disequilibrium Calculator and the Bioconductor packages trio44 and ldblock45 to perform this task.\n\nIn order to annotate these variants, we need a a TxDb object, a reference of where transcripts are located on the genome. We can build this using the GenomicFeatures46 package and the GENCODE v25 gene annotation:\n\n\n\nWe also have to convert the gwasloc object into a standard GRanges object:\n\n\n\nLet’s check if the gwasloc and TxDb object use the same notation for chromosomes:\n\n\n\nOK, they do. Now we can annotate our SNPs to genes using the VariantAnnotation47 package:\n\n\n\nWe lost all the metadata from the original snps object, but we can recover it using the QUERYID column in snps_anno. We will only keep the SNP IDs and GWAS p-values:\n\n\n\nWe can visualise where these SNPs are located with ggplot241 (Figure 5).\n\n\n\nAs expected11, the great majority of SNPs are located within introns and in intergenic regions. For the moment, we will focus on SNPs that are either coding or in promoter and UTR regions, as these can be assigned to target genes rather unambiguously:\n\n\n\n\n\nNow we can check if any of the genes we found to be differentially expressed in SLE is also genetically associated with the disease:\n\n\n\n\n\nSo, we have 7 genes showing differential expression in SLE that are also genetically associated with the disease. While this is an interesting result, these hits are likely to be already well-known as potential SLE targets given their clear genetic association.\n\nWe will store essential information about these hits in a results data.frame:\n\n\n\nBut what about all the SNPs in introns and intergenic regions? Some of those might be regulatory SNPs affecting the expression level of their target gene(s) through a distal enhancer. Let’s create a dataset of candidate regulatory SNPs that are either intronic or intergenic and remove the annotation obtained with VariantAnnotation:\n\n\n\neQTL data. A well-established way to gain insights into target genes of regulatory SNPs is to use eQTL data, where correlations between genetic variants and expression of genes are computed across different tissues or cell types13. We will use blood eQTL data from the GTEx consortium14. To get the data, you will have to register and download the file GTEx_Analysis_v7_eQTL.tar.gz from the GTEx portal to the current working directory:\n\n\n\nWe have to extract the genomic locations of the SNPs from the IDs used by GTEx:\n\n\n\nWe can then convert the data.frame into a GRanges object:\n\n\n\nWe also need to ensure that the chromosome notation is consistent with the previous objects:\n\n\n\nFrom the publication14, we know the genomic coordinates are mapped to genome reference GRCh37, so we will have to uplift them to GRCh38 using rtracklayer48 and a mapping (“chain”) file. The R.utils package is required to extract the gzipped file:\n\n\n\nWe will use the GenomicRanges package46 to compute the overlap between GWAS SNPs and blood eQTLs:\n\n\n\nSo, we have 59 blood eQTL variants that are associated with SLE. We can now check whether any of the genes differentially expressed in SLE is an eGene, a gene whose expression is influenced by an eQTL. We note that gene IDs in GTEx are mapped to GENCODE v1914, while we are using the newer v25 for the DEGs. To match the gene IDs in the two objects, we will simply strip the last bit containing the GENCODE gene version, which effectively gives us Ensembl gene IDs:\n\n\n\nWe can add these 4 genes to our list:\n\n\n\nFANTOM5 data. The FANTOM consortium profiled gene expression across a large panel of tissues and cell types using CAGE19,21. This technology allows mapping of transcription start sites (TSSs) and enhancer RNAs (eRNAs) genome-wide. Correlations between these promoter and enhancer elements across a large panel of tissues and cell types can then be calculated to identify significant promoter - enhancer pairs. In turn, we will use these correlations to map distal regulatory SNPs to target genes.\n\nWe can read in and have a look at the enhancer - promoter correlation data in this way:\n\n\n\nEverything we need is in the fourth column, name: genomic location of the enhancer, gene identifiers, Pearson correlation coefficient and significance. We will use the splitstackshape package to parse it:\n\n\n\nNow we can extract the genomic locations of the enhancers and the correlation values:\n\n\n\nWe can select only the enhancer - promoter pairs with a decent level of correlation and significance and tidy the data at the same time:\n\n\n\nNow we would like to check whether any of our candidate regulatory SNPs are falling in any of these enhancers. To do this, we have to convert the data.frame into a GRanges object:\n\n\n\nSimilar to the GTEx data, the FANTOM5 data is also mapped to GRCh3719, so we will have to uplift the GRCh37 coordinates to GRCh38:\n\n\n\nWe can now compute the overlap between SNPs and enhancers:\n\n\n\nWe note that some of the SNPs are assigned to more than one gene. This is because enhancers are promiscuous and can regulate multiple genes.\n\nWe can now check if any of these genes is differentially expressed in our RNA-seq data:\n\n\n\nWe have identified 2 genes whose putative enhancers contain SLE GWAS SNPs. Let’s add these to our list:\n\n\n\nPromoter Capture Hi-C data. More recently, chromatin interaction data was generated across 17 human primary blood cell types25. More than 30,000 promoter baits were used to capture promoter-interacting regions genome-wide. These regions were then mapped to enhancers based on the Ensembl Regulatory Build49 and can be accessed in the supplementary data of the paper:\n\n\n\nIn this case, we will have to map the promoter baits to genes first. We can do this by converting the baits to a GRanges object and then using the TxDb object we previously built to extract positions of transcription start sites (TSSs):\n\n\n\nNow we can create a GRanges object of the enhancers in the promoter capture Hi-C data with the bait annotation attached:\n\n\n\nNext, we basically repeat the steps we have taken when working with the FANTOM5 data to find SLE GWAS SNPs overlapping with these enhancers:\n\n\n\nWe check if any of these enhancers containing SLE variants are known to putatively regulate genes differentially expressed in SLE:\n\n\n\nAnd finally we add these 3 genes to our list. These are our final results:\n\n\n\n\nConclusions\n\nIn this Bioconductor workflow we have used several packages and datasets to demonstrate how regulatory genomic data can be used to annotate significant hits from GWASs and provide an intermediate layer connecting genetics and transcriptomics. Overall, we identified 17 SLE-associated SNPs that we mapped to 16 genes differentially expressed in SLE, using eQTL data14 and enhancer - promoter relationships from CAGE19 and promoter capture Hi-C experiments25.\n\nWhile simplified, the workflow also demonstrates some real-world challenges encountered when working with genomic data from different sources, such as the use of different genome references and gene annotation conventions, the parsing of files with custom formats into Bioconductor-compatible objects and the mapping of genomic locations to genes.\n\nAs the sample size and power of GWASs and gene expression studies continue to increase, it will become more and more challenging to identify truly significant hits and interpret them. The use of regulatory genomics data as presented here can be an important skill and tool to gain insights into large biomedical datasets and help in the identification of biomarkers and therapeutic targets.\n\n\nData and software availability\n\nDownload links for all datasets are part of the workflow. Software packages required to reproduce the analysis can be installed as part of the workflow. Code is available at https://github.com/enricoferrero/bioconductor-regulatory-genomics-workflow.\n\nArchived code as at time of publication: http://doi.org/10.5281/zenodo.115423550\n\nLicense: CC-BY 4.0",
"appendix": "Competing interests\n\n\n\nEF is a full time employee of GSK.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWaring MJ, Arrowsmith J, Leach AR, et al.: An analysis of the attrition of drug candidates from four major pharmaceutical companies. Nat Rev Drug Discov. 2015; 14(7): 475–86. PubMed Abstract | Publisher Full Text\n\nDiMasi JA, Grabowski HG, Hansen RW: Innovation in the pharmaceutical industry: New estimates of R&D costs. J Health Econ. 2016; 47: 20–33. PubMed Abstract | Publisher Full Text\n\nHarrison RK: Phase II and phase III failures: 2013–2015. Nat Rev Drug Discov. 2016; 15(12): 817–8. PubMed Abstract | Publisher Full Text\n\nCook D, Brown D, Alexander R, et al.: Lessons learned from the fate of AstraZeneca’s drug pipeline: a five-dimensional framework. Nat Rev Drug Discov. 2014; 13(6): 419–31. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nRitchie ME, Phipson B, Wu D, et al.: limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43(7): e47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnders S, Huber W: Differential expression analysis for sequence count data. Genome Biol. 2010; 11(10): R106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarey VJ: Gwascat. 2017. Publisher Full Text\n\nMacArthur J, Bowler E, Cerezo M, et al.: The new NHGRI-EBI Catalog of published genome-wide association studies (GWAS Catalog). Nucleic Acids Res. 2017; 45(D1): D896–901. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEicher JD, Landowski C, Stackhouse B, et al.: GRASP v2.0: an update on the Genome-Wide Repository of Associations between SNPs and phenotypes. Nucleic Acids Res. 2015; 43(Database issue): D799–804. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarey VJ: Grasp2db. 2007. Publisher Full Text\n\nWickham H: Ggplot. New York, NY: Springer New York; 2009. Publisher Full Text\n\nBush WS, Moore JH: Chapter 11: Genome-wide association studies. PLoS Comput Biol. 2012; 8(12): e1002822. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYates A, Beal K, Keenan S, et al.: The Ensembl REST API: Ensembl Data for Any Language. Bioinformatics (Oxford, England). 2015; 31(1): 143–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchwender H, Li Q, Berger P, et al.: trio: testing of SNPs and SNP interactions in case-parent trio studies. 2015. Publisher Full Text\n\nCarey VJ: Ldblock. 2017. Publisher Full Text\n\nLawrence M, Huber W, Pagès H, et al.: Software for computing and annotating genomic ranges. PLoS Comput Biol. 2013; 9(8): e1003118. PubMed Abstract | Publisher Full Text | Free Full Text\n\nObenchain V, Lawrence M, Carey V, et al.: VariantAnnotation: a Bioconductor package for exploration and annotation of genetic variants. Bioinformatics (Oxford, England). 2014; 30(14): 2076–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLawrence M, Gentleman R, Carey V: rtracklayer: an R package for interfacing with genome browsers. Bioinformatics (Oxford, England). 2009; 25(14): 1841–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZerbino DR, Wilder SP, Johnson N, et al.: The ensembl regulatory build. Genome Biol. 2015; 16: 56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFerrero E: enricoferrero/bioconductor-regulatory-genomics-workflow: editorial changes (Version v0.2). Zenodo. 2018. Data Source"
}
|
[
{
"id": "30355",
"date": "05 Feb 2018",
"name": "Aaron T. L. Lun",
"expertise": [
"Reviewer Expertise Computational biology",
"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\nThis workflow is clear, well-written and makes good use of existing resources and Bioconductor software. It addresses an interesting problem in the integration of RNA-seq, GWAS, eQTL and Hi-C data for causal gene discovery in disease contexts. However, it would benefit from some more elaboration in certain sections. I have listed my comments below in more detail, ordered by the location in the workflow they refer to. For most part, I believe they are easily addressed.\n- The final paragraph of the introduction seems out of place; I do not see any reference to POSTGAP, STOPGAP or INFERNO anywhere else in the article. Was the workflow presented here used to identify the candidate genes in these resources?\n- A more comprehensive description of the SLE data set, and the motivation behind using it, would be helpful.\n- There seems to be a typo when loading the SRP062966 dataset; it should be load(file.path(\"SRP062966\", \"rse_gene.Rdata\")), at least on my machine.\n- I don't see why it's desirable to call scale_counts(). Major DE analysis frameworks are easily capable of handling differences in library sizes. Direct scaling would actually be detrimental to NB models like edgeR and DESeq2, as it distorts the mean-variance relationship. In particular, scaled counts can have sub-Poisson variation, which cannot be handled by NB models. It seems better to call read_counts() to obtain the gene-level read counts.\n- rse$FIELD can be used instead rather than colData(rse)$FIELD, which may simplify the code.\n- Some explanation of the other factors (anti-rho, ISM) would be helpful, given that the effort has already been taken to define them.\n- The simplicity of the model used in the DE analysis is probably unhelpful in the context described in the workflow. I would like to see more elaboration on how to handle batch effects and other confounding factors that are almost definitely present in large-scale studies. For example, what happens to the DE genes when additional explanatory factors are added to the model, e.g., anti-rho or ism status? Presumably age and sex are also relevant factors, if that information is available in the data set.\n- Generally, some of the plots could be accompanied by more commentary in text, explaining how to interprete the plot. For example, the MA plot in Figure 3 shows that DE genes are detected in both directions, at a range of abundances. It would be similarly useful to have text for the heatmap in Figure 1 and the Manhattan plot in Figure 4, among others.\n- LD expansion seems like quite an important step, especially when SNPs are being linked to genes based on overlaps to promoters/UTRs. If the LD blocks are large, expansion would result in many more potential causal SNPs and a greater number of overlaps (and thus candidate genes). While I appreciate the attempt to simplify the workflow, skipping this step seems like it would unnecessarily reduce the number of candidate genes.\n- snps seems to have GRCh38 coordinates. Is this also the case for GENCODE 25? It would be helpful to have a cautionary note regarding the need to make sure the same version of the genome is used throughout a workflow. I recognise that this is mentioned later when liftOver() is used, but it is better to be explicit about this where possible.\n- Oscillating between head() and tail() to preview the dataset is unhelpful and confusing.\n- While I don't expect a thorough examination of the set of (7 easy, 4 hard, 3 via Hi-C) candidate genes for SLE, some discussion of the biological significance of the detected genes would be appreciated. It would provide a high-level validation of the workflow and link it back to the drug discovery context.\n- For the promoter Hi-C section, you could consider using the linkOverlaps() method in the InteractionSet package, to link SNPs to gene promoters via the identified Hi-C interactions. This might be simpler than the current code, and possibly faster; the nearest() step in particular takes quite a long time.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "3430",
"date": "23 Feb 2018",
"name": "Enrico Ferrero",
"role": "Author Response",
"response": "Aaron, many thanks for reviewing my paper in depth. In response to your comments: - The final paragraph of the introduction seems out of place; I do not see any reference to POSTGAP, STOPGAP or INFERNO anywhere else in the article. Was the workflow presented here used to identify the candidate genes in these resources? --> I expanded the paragraph to provide more context on STOPGAP, POSTGAP and INFERNO and to clarify why they are mentioned in the introduction but not used in the actual workflow. - A more comprehensive description of the SLE data set, and the motivation behind using it, would be helpful. --> More background and details on the dataset have been added in the second and third paragraph of the section \"Gene expression data and differential gene expression analysis\". - There seems to be a typo when loading the SRP062966 dataset; it should be load(file.path(\"SRP062966\", \"rse_gene.Rdata\")), at least on my machine. --> Fixed, thanks. - I don't see why it's desirable to call scale_counts(). Major DE analysis frameworks are easily capable of handling differences in library sizes. Direct scaling would actually be detrimental to NB models like edgeR and DESeq2, as it distorts the mean-variance relationship. In particular, scaled counts can have sub-Poisson variation, which cannot be handled by NB models. It seems better to call read_counts() to obtain the gene-level read counts. --> For this section, I followed the recount quick start guide [1] and workflow [2]. Both show scaling of the counts with scale_counts() before feeding these to DESeq2. I tried switching to read_counts() but, somewhat counter-intuitively, the function returns values with decimal numbers, which in turn causes an error (\"some values in assay are not integers\") when calling the DESeqDataSet() function. As both scale_counts() and read_counts() seem to be acceptable, but the first one is the preferred approach by the recount developers, I switched back to scale_counts() after encountering the DESeq2 error above. The other option would have been to manually round the numbers returned by read_counts() but that seemed more questionable to me than scaling them. - rse$FIELD can be used instead rather than colData(rse)$FIELD, which may simplify the code. --> Simplified, thanks. - Some explanation of the other factors (anti-rho, ISM) would be helpful, given that the effort has already been taken to define them. --> I added context for these experimental factors in the third paragraph of the \"Gene expression data and differential gene expression analysis\" section and after printing the rse$characteristics object is printed. - The simplicity of the model used in the DE analysis is probably unhelpful in the context described in the workflow. I would like to see more elaboration on how to handle batch effects and other confounding factors that are almost definitely present in large-scale studies. For example, what happens to the DE genes when additional explanatory factors are added to the model, e.g., anti-rho or ism status? Presumably age and sex are also relevant factors, if that information is available in the data set. --> I agree this is not ideal, but there are good reasons why other factors are not included. First, age, gender or other demographics are not available for this dataset. Second, the ISM and anti-Ro factors are disease characteristics and are obviously only measured on the SLE patients (and not on the healthy ones). If either or both of those factors are included in the model, you get the classic \"model matrix is not full rank\" error [3] because they are both collinear with the disease status (all healthy samples are \"control\" for both anti-Ro and ISM). I've been more explicit about these shortcomings in the paragraph following the code chunk where the model is built. - Generally, some of the plots could be accompanied by more commentary in text, explaining how to interprete the plot. For example, the MA plot in Figure 3 shows that DE genes are detected in both directions, at a range of abundances. It would be similarly useful to have text for the heatmap in Figure 1 and the Manhattan plot in Figure 4, among others. --> I expanded the main text and legends for figures 2, 4 and 5 (previously 1, 3 and 4) to include a better description and explanation of the plots. I believe figures 3 and 6 (previously 2 and 5) were already adequately described. I also added 3 new figures (1, 7 and 8) to clarify the steps involved in the workflow and to provide a more in-depth understanding of the final results. - LD expansion seems like quite an important step, especially when SNPs are being linked to genes based on overlaps to promoters/UTRs. If the LD blocks are large, expansion would result in many more potential causal SNPs and a greater number of overlaps (and thus candidate genes). While I appreciate the attempt to simplify the workflow, skipping this step seems like it would unnecessarily reduce the number of candidate genes. --> Unfortunately I can't come up with a good way to perform this step in R as part of the workflow at present. The ldblock package hasn't been updated in a while and its functions rely on downloading the HapMap data from the NCBI website, which was retired in 2016 and is no longer available for download [4]. Even if it was still available, it would require downloading several GBs of data, one chromosome at a time. The previously referenced trio package uses data structures specific to case - parent trio studies which are not compatible with the use case presented in the workflow and are not designed for hundreds of SNPs, and was thus removed. The Ensembl LD Calculator is a web UI with a limit of 20 SNPs per query that can't be integrated in a programmatic workflow, so it was removed too. I guess the Ensembl REST API could be an option, but it would require introducing a few new libraries and a considerable amount of code to interact with the API and parse its output into R/Bioconductor objects, with the risk of distracting the reader from the main purpose of this (Bioconductor) workflow. It would also require performing several hundreds queries in a for loop making compilation of the document extremely long and following the workflow impractical. I modified the text in the manuscript to communicate more clearly the reasons for skipping this step. If you have other suggestions on how to do this, I would be happy to consider them. - snps seems to have GRCh38 coordinates. Is this also the case for GENCODE 25? It would be helpful to have a cautionary note regarding the need to make sure the same version of the genome is used throughout a workflow. I recognise that this is mentioned later when liftOver() is used, but it is better to be explicit about this where possible. --> I added a clarification and a warning about this in the third paragraph of the \"Accessing GWAS data\" section, after importing the GWAS data. - Oscillating between head() and tail() to preview the dataset is unhelpful and confusing. --> I removed all instances of tail() and replaced them with head(). - While I don't expect a thorough examination of the set of (7 easy, 4 hard, 3 via Hi-C) candidate genes for SLE, some discussion of the biological significance of the detected genes would be appreciated. It would provide a high-level validation of the workflow and link it back to the drug discovery context. --> I added a new section \"Functional analysis of prioritised hits\" (and a new figure, 8) where I describe the biological significance and functional relevance of the results, while also discussing some of the hits in more detail from a drug discovery perspective. - For the promoter Hi-C section, you could consider using the linkOverlaps() method in the InteractionSet package, to link SNPs to gene promoters via the identified Hi-C interactions. This might be simpler than the current code, and possibly faster; the nearest() step in particular takes quite a long time. --> Thanks, I had heard of the InteractionSet package but hadn't used it before. I agree it's better to represent the promoter capture Hi-C data in this native structure. I still had to use the nearest() function (which executes almost instantaneously on my laptop) to map promoters to gene IDs though. Also, note that I didn't need the linkOverlaps() function in the end and simply used findOverlaps(..., use.region = \"second\") instead. [1] https://bioconductor.org/packages/3.7/bioc/vignettes/recount/inst/doc/recount-quickstart.html [2] https://f1000research.com/articles/6-1558/v1 [3] http://seqanswers.com/forums/showthread.php?t=33032 [4] https://www.ncbi.nlm.nih.gov/variation/news/NCBI_retiring_HapMap/"
}
]
},
{
"id": "30353",
"date": "07 Feb 2018",
"name": "Vincent J. Carey",
"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 want to preface this long review with some very broad comments. I think this undertaking is very worthwhile from several perspectives. Bioconductor is used along various avenues to create a unifiable analytic process from very diverse data resources: state-of-the-art transcriptomics from recount, current GWAS catalog from EMBL/EBI, variant annotation for SLE GWAS hits from the eponymous package using GENCODE for gene models, eQTL data from GTEx, enhancer annotation from FANTOM5, and promoter capture data whose origins could be better described. This is a tour de force but I feel it should be communicated more clearly and executed more cleanly. The paper is full of \"dumps\" of show events for R objects that impede the narrative flow drastically. A diagram that shows how the various resources combine in a scientifically coherent way would be a huge step forward for the paper and for practitioners. More reckoning of limitations that arise from complexity is also in order. eQTLs are far from simple, and should not be used as 'lists'. Enhancer and promoter 'lists' also need to be used with care.\nWhat then about this paper? It shows the resources and it shows a path. Isn't that enough? I don't think so. If Bioconductor and online publication make it easier to do and to publish complex analyses, then the presentation should be of at least as high a quality as we find in articles that are behind paywalls. In this case I feel the quality would be improved through condensation. The object dumps should be removed and replaced by meaningful tabulations and diagrams. The big picture should be stated more clearly and concisely. The limitations should also be discussed clearly. I would love to see a small set of functions that carry out the salient operations chained together to produce the solution. Then, given the programmatic compactness, we can discuss how to evaluate the robustness of the results of the analysis by carrying out sensitivity analysis. In particular, it would be great to see how the different elements of the system contribute to the ultimate enumeration of targets.\n---\nThe premise of this article is that \"therapeutic targets with a genetic link to the disease under investigation are more likely to progress through the drug discovery pipeline\". GWAS, PheWAS, eQTLs, epigenomic roadmap projects, and other general studies of gene regulation should be harvested to improve capacity to define genetic and genomic origins of disease, with an aim to fostering design of treatments that are focused on the molecular events underlying the disease process. The introduction concludes with mention of STOPGAP, and POSTGAP, and INFERNO, but it is not clear whether the paper is intended to describe how content of STOPGAP is developed from basic data resources like those readily available to Bioconductor users. I feel that the introduction, though well-referenced, is too long and does not clearly state the paper's main goal.\nThere is no discussion of the experimental design underlying the RNA-seq study. Presumably the data were generated from this component of ref 28:\n\"Finally, we tested the levels of Alu transcripts in blood cells of SLE patients and controls(22) using RNAseq (99 active SLE, 18 healthy controls; Fig. S12). RNA-seq reads mapping to Alu elements were found at significantly higher levels in SLE subjects than controls (p=6.5E-6), Fig. 4E). Hierarchical clustering of the most highly expressed Alu RNAs (Fig. S13) segregated Interferon Signature Metric (ISM)-high SLE subjects from control and ISM-low patients\"....\nThere is no discussion of heterogeneity of SLE or the difficulty of learning from a collection of 18 cases. A reference to https://www.ncbi.nlm.nih.gov/pubmed/25102991 may be in order.\n\nEven though online publications are often free from page count limitations, entirely too much space is consumed by long row-broken R print events. On the one hand the recoding of SRA annotation on phenotype is important and should be exposed, on the other hand, the author could carry out the recoding programmatically in a well-parameterized function and simply update the key object by applying this function. The function can go in a package related to the paper/workflow. Instead of printing out a dataframe on p.7, it would be much better to have a contingency table showing the final layout of case and control characteristics.\np.7 \"For simplicity, we select the first 18 (healthy) and the last 18 (SLE) samples from the original RangedSummarizedExperiment object\". Is this essential to the performance of the workflow? Would a more systematic matching be possible? What kind of \"simplicity\" does this arbitrary selection create? I understand that the main purpose of the paper is to illustrate a process, but if this thinning of the data is not essential to the illustration, why do it?\np. 8: \"Note that we used an extremely simple model; in the real world you will probably need to account for co-variables, potential confounders and interactions between them. edgeR and limma are good alternatives to DESEq2 for performing differential expression analyses.\" This suggests that you can't adjust for confounders in DESeq2, is this so? Did you not have access to any relevant cofactors in the SLE data?\np. 9: You are really using 59000 genes after vst to do exploratory visualization of SLE vs control expression patterns? Would gene filtering be helpful? Is there any chance of batch effect or other surrogate variable effect that should be assessed prior to such presentations?\nBy page 12 we have completed a relatively elementary differential expression analysis. It seems to me that the length of this part of the process is excessive, because the real interest is in learning about regulatory elements from other resources.\nAt this point I hope I have made clear how I think the rest of the paper should be revised to make its points more effectively.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\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? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "3429",
"date": "23 Feb 2018",
"name": "Enrico Ferrero",
"role": "Author Response",
"response": "Vincent, many thanks for reviewing my paper in depth. In response to your comments (please note that I took the liberty to format some of your points and omit some parts for readability): - [...] I think this undertaking is very worthwhile from several perspectives [..] and promoter capture data whose origins could be better described. --> Promoter capture Hi-C is indeed briefly introduced as a technique in the introduction. I have now added some more context on the Javierre et al., 2016 dataset and emphasised its relevance for this workflow at the beginning of the \"Promoter capture Hi-C data\" subsection. - [...] The paper is full of \"dumps\" of show events for R objects that impede the narrative flow drastically. --> I didn't realise how annoying this was until you mentioned it. I removed the great majority of dumps, leaving only a few to document the structure of datasets just imported or very final objects. For all dumps, I also ensured that a minimal amount of rows were printed. - A diagram that shows how the various resources combine in a scientifically coherent way would be a huge step forward for the paper and for practitioners. --> I included a diagram providing a schematic overview of the workflow as figure 1 and referenced it in the last paragraph of the introduction. Please note that the diagram is created in R with the DiagrammeR package but the code is hidden as it is not strictly relevant for the purposes of the workflow. - More reckoning of limitations that arise from complexity is also in order. eQTLs are far from simple, and should not be used as 'lists'. Enhancer and promoter 'lists' also need to be used with care. --> I added a few sentences at the beginning of the \"eQTL data\" subsection cautioning on the complexity of GWAS/eQTL integration and provided a short overview of available alternatives which are more methodologically robust. - [...] In particular, it would be great to see how the different elements of the system contribute to the ultimate enumeration of targets. --> I added figure 7 to show the relative contributions of the different approaches to the final results. - [...] The introduction concludes with mention of STOPGAP, and POSTGAP, and INFERNO, but it is not clear whether the paper is intended to describe how content of STOPGAP is developed from basic data resources like those readily available to Bioconductor users. --> I expanded that paragraph to provide more context on STOPGAP, POSTGAP and INFERNO and to clarify the intent of mentioning those resources in the introduction. - I feel that the introduction, though well-referenced, is too long and does not clearly state the paper's main goal. --> I shortened the introduction by removing the paragraph about GWAS and PheWAS and by removing or shortening several other sentences. I added a short, final paragraph stating more clearly the main goals of the workflow. - There is no discussion of the experimental design underlying the RNA-seq study. Presumably the data were generated from this component of ref 28: [...] --> That's correct. I added more context on the original study, including an overview of the experimental design, in the third paragraph of the \"Gene expression data and differential gene expression analysis\" section. - There is no discussion of heterogeneity of SLE or the difficulty of learning from a collection of 18 cases. A reference to https://www.ncbi.nlm.nih.gov/pubmed/25102991 may be in order. --> I addressed this point with a better introduction to SLE and its heterogeneity in the second paragraph of the \"Gene expression data and differential gene expression analysis\" section. - [...] Instead of printing out a dataframe on p.7, it would be much better to have a contingency table showing the final layout of case and control characteristics. --> I agree this is a more effective way to summarise the data. I removed the dataframe printing and included a contingency table showing features of case and control samples. - p.7 \"For simplicity, we select the first 18 (healthy) and the last 18 (SLE) samples from the original RangedSummarizedExperiment object\". Is this essential to the performance of the workflow? Would a more systematic matching be possible? What kind of \"simplicity\" does this arbitrary selection create? I understand that the main purpose of the paper is to illustrate a process, but if this thinning of the data is not essential to the illustration, why do it? --> Indeed, this was mostly done to speed up execution while compiling the document. I removed that chunk and all 117 samples are now used in the analysis. - p. 8: \"Note that we used an extremely simple model; in the real world you will probably need to account for co-variables, potential confounders and interactions between them. edgeR and limma are good alternatives to DESEq2 for performing differential expression analyses.\" This suggests that you can't adjust for confounders in DESeq2, is this so? Did you not have access to any relevant cofactors in the SLE data? --> I reworded that sentence to clarify that DESEq2 is equivalent to edgeR and limma when it comes to multiple cofactors in the model. I also included a better description of the metadata available for this dataset and explained why it is not possible to include demographic statistics (unavailable) or other experimental factors (collinear with disease status) in the model. - p. 9: You are really using 59000 genes after vst to do exploratory visualization of SLE vs control expression patterns? Would gene filtering be helpful? Is there any chance of batch effect or other surrogate variable effect that should be assessed prior to such presentations? --> I have now applied a simple filter to remove genes with extremely low counts directly on the dds object and ahead of VST, as documented in the DESeq2 vignette [1] and the Bioconductor RNA-seq workflow [2]. This reduces the number of genes considerably, helping to speed up code execution too. I also clarified in the \"Gene expression data and differential gene expression analysis\" section that one of the aims of the hierarchical clustering and PCA in figure 2 and 3 is indeed to assess presence of batch effects or surrogate variables. Note that all available experimental variables are now included as annotation in the heatmap in figure 1. - By page 12 we have completed a relatively elementary differential expression analysis. It seems to me that the length of this part of the process is excessive, because the real interest is in learning about regulatory elements from other resources. --> The \"Gene expression data and differential gene expression analysis\" has now been considerably condensed by removing superfluous object dumps, merging code chunks and reducing the text to a minimum. One could go as far as removing the exploratory data analysis and figures, but I'd rather keep them to provide some context and a minimal differential expression analysis to be used as the starting point for the integration of the GWAS data. - At this point I hope I have made clear how I think the rest of the paper should be revised to make its points more effectively. --> Indeed. The workflow was largely redacted, condensed and improved by limiting R object dumps, providing more context on the features of the datasets used and more insights into the methodology and results of the analysis through the use of visualisations and data summaries. [1] https://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html [2] https://www.bioconductor.org/help/workflows/rnaseqGene/"
}
]
}
] | 1
|
https://f1000research.com/articles/7-121
|
https://f1000research.com/articles/7-218/v1
|
22 Feb 18
|
{
"type": "Research Article",
"title": "Can work-related stress and job satisfaction affect job commitment among nurses? A cross-sectional study",
"authors": [
"Mahdi Eskandari",
"Mohammad Ali Heidari Gorji",
"Mahdi Eskandari"
],
"abstract": "Background: Considering the significant role of nurses in health care and the importance of commitment in public health, the aim of this study was to examine the association between work-related stress, and job satisfaction and commitment among nurses. Methods: This cross-sectional study has been performed among 100 nurses working in a teaching hospital affiliated to Mazandaran University of Medical Sciences, northern Iran, in 2015. The participants were assessed by standard questionnaires on work-related stress, and job satisfaction and commitment. Descriptive indexes were analysed via regression and correlation. Results: The participants were aged between 25 and 45 years. In total, 85% of nurses held a bachelor degree and the rest were postgraduates. Most of the nurses (77%) had experience in their job for more than 10 years. Total score of mean job commitment showed a high score among nurses (102.9±8.9); job satisfaction also showed a mean of (261.6±27.44). Total score of work-related stress was (112.0±11.99). The results indicated that job satisfaction and work-related stress explained 54% of variance in job commitment. The overall job satisfaction (Beta = 0.471, p<0.05) and overall work stress (Beta = -0.635, p<0.102) influenced job commitment. Conclusion: The results of the present study showed that work stress and job satisfaction affects job commitment among nurses. Therefore, it is advisable to pay more attention to job satisfaction and stress in these sensitive occupations, to promote commitment and job efficacy.",
"keywords": [
"nurse",
"work-related stress",
"job satisfaction",
"commitment",
"management"
],
"content": "Introduction\n\nHospital units are the most stressful places in hospitals and hospital successful management depends on multiple collaboration of disciplines, such as nurses1,2. Job performance and outcomes are inter-related to some psychological and social factors, such as job environment and leadership, positive attitude and efficacy, satisfaction, and commitment3–6. A strong relationship between job satisfaction and job commitment has been previously revealed7,8. Moreover, a relationship between job commitment, job satisfaction, work experience, organizational collaboration and organizational justice has also been found. For example, Hoogendoorn et al. believed workers undertake duties with higher responsibility when they have commitment to the job9.\n\nJob commitment, its determinants and consequences have received significant attention in the literature, since commitment is related to many organizational indicators. Wang et al. pointed out that organizational commitment is “the comparative power of a person’s identification with and participation in a specific organization”10. Organizational commitment can be found in the literature very frequently, with three fundamental components related to its definition: affective (employees are emotionally attached to organization), continuance (employees' commitment), and normative (employees’ feeling to remain in the organization)11–13. Some studied show that mortality and morbidity of most fire fighters are related both directly or indirectly to their stressful work condition14,15. Moreover, according to Moran, emergency service organizations workers, including the ambulance service and rescue squads as well as the fire brigade, are exposed to both everyday stressors common to many work environments and extreme stressors of emergency events, including traumatic accidents or disasters16. Results from Bennett et al. on 78 United Kingdom fire fighters indicated a considerable amount of job stress7. Bowron also found similar results among emergency service workers17.\n\nLusa and colleagues assessed male rescue workers and fire-fighters and from fire brigades of Finland. They have reported high stress among these workers18. Murphy et al. indicated a relationship between work-based morale and job satisfaction of fire-fighters and paramedics. Conflict with managers was the job stressor that powerfully related to reports of poor work morale and low job satisfaction19. It has been revealed that low commitment among emergency working women was related with income, marital status, age, education and job satisfaction; singles, highly educated and low job experience were related with low job commitments20. A low mental health and higher job burnout among emergency workers than the general population has been revealed21,22. Cicei found a relationship between work stress and the three dimensions of organizational commitment23, while Li et al. revealed a relationship between work satisfaction and job stress24.\n\nReviewing previous studies indicates that they are mostly focused on teachers and there are very few studies conducted regarding commitment-related factors among domestic health care professionals25,26. Performances in these kinds of jobs are critically related to life or death of people. Therefore, the aim of this study was to investigate the role of job satisfaction and work stress in organizational commitment among nurses at a hospital in Iran.\n\n\nMethods\n\nThis was a cross-sectional, correlational study aimed at investigating role of job satisfaction and work-related stress in organizational commitment among nurses working in Imam Teaching Hospital affiliated to Mazandaran University of Medical Sciences, Sari, Iran in June to September 2015. Approval from the Ethics Committee of Mazandaran University of Medical Sciences for the study, as well as written informed consent from nurses for participation, was obtained (approval number: IR.Mazums.rec.95.2350).\n\nThe study population included all 138 nurses who worked in hospitals, of them 100 nurses gave consent to participate in this study and completed the questionnaires. In order to keep confidence of samples, names were not recorded and the overall results were reported to the organization without any personal information. The researcher contacted the hospital and explained the aim of study and after agreement of supervisors, nurses were invited to participate. The inclusion criteria were having at least a Bachelor of Science degree in nursing and one year job experience. The participants were insured that their results will be kept confidently and there is no need to fill their name on the questionnaires.\n\nOrganizational commitment. Allen and Meyers’s Organizational Commitment scale includes sub-scales such as Affective Organizational Commitment (AOC), Continuance Organizational Commitment (COC) and Normative Organizational Commitment (NOC). This questionnaire comprises 24 items scoring in 7 options from absolutely agree to absolutely disagree27. Reliability and validity of the scale has been approved in several studies in Iran12,28. A study reported Cronbach’s alpha ranging from 0.74 to 0.83 and inter correlation for AOS is 0.49 (p<0.05), for COS is 0.22 (p<0.05) and 0.12 (p<0.05) is for NOS27. The participants filled out the questionnaires themselves during their work hours when they had free time to feel comfortable in the nurse rest room. Please see Supplementary File 1 for a Farsi translation used in the present study.\n\nJob satisfaction. Job satisfaction was evaluated using the Job Descriptive Index (JDI) introduced by Smith et al. The JDI was selected as it was proved to be reliable and a valid measure of job satisfaction by many studies29,30. It was invented to evaluate satisfaction through 6 aspects: the work itself, pay, promotion, supervision, environment and co-workers. Participants were asked to describe a particular aspect of their job with a word or phrase. The validity of the above mentioned questionnaire was proved through content validity and factor analysis, which showed an acceptable level of validity. Reliability also was high r=0.93 (p=0.01)31,32. Please see Supplementary File 2 for a Farsi translation used in the present study.\n\nWork-related stress. The HSE work-related stress questionnaire contains 35 items that enquire about ‘working conditions’ that are the potential causes of work related stress24. The working conditions relate to the 6 stressors of Management Standards. The answers of employees were based on how they feel about these aspects of their work: demand, control, support of authorities, supporting co-workers, relationship, role, changes. The questionnaire was developed by the Health and Safety Executive (HSE) and in China, each item has 5 options (never=1, seldom=2, sometimes=3, most of the time=4, always=5). Low scores indicate higher health and safety in aspect of stress, and higher scores means high stress. In order to survey the validity and reliability of this questionnaire, a previous study surveyed 749 military staff, selected via cluster sampling, from whole country. Please see Supplementary File 3 for a Farsi translation used in the present study.\n\nParticipants also completed the mental health questionnaire of Goldberg33. Reliability of this questionnaire in Cronbach alpha and split-half was 0.78 and 0.65, respectively. This result indicates appropriate reliability and validity for work stress questionnaire24.\n\nData analysis. Statistical Package for Social Sciences (SPSS; IBM, USA) version 16 was used to analyse the date. Descriptive indexes (frequencies) were analysed using ANOVA and regression with correlation applied to find out correlations between variables.\n\n\nResults\n\nThe participants’ demographic characteristics are shown in Table 1.\n\nTotal mean score of job commitment was high among nurses (102.9±8.9). Moreover, mean total score of job satisfaction and work stresses were 261.6±27.44 and 112.0±11.99, respectively. Table 2 shows the regression between job commitment, work stress and job satisfaction.\n\nTable 3 shows the adjusted R² of our model is 0.245 with the R² = 0.245, which means that the linear regression explains 24.5% of the variances of job commitment of samples. It means that the liner relationship between the variables (in other words R²=0.245), with F = 15.745 is significantly high, thus it can be assumed that there is a linear relationship between work stress, job satisfaction and job commitment variables in our model.\n\nIn addition, overall job satisfaction (Beta = 0.471, p< 0.05) and total work stress (Beta = -0.635, p< 0.102) had an influence on job commitment. Therefore, job satisfaction and work stress significantly predicts job commitment.\n\n\nDiscussion\n\nConsidering previous theories and study results, in an organization where employees have low job satisfaction and high work stress, levels of production and productivity will be critically low34. In such conditions of job commitment and job turnover35, less innovation and higher employee turnover rate of employees are seen frequently34. Asegid et al. stated that high work stress is a predictor of job dissatisfaction and intention to low job commitment36.\n\nHigh job satisfaction and proper performance leads to a better work environment, higher cooperation among co-workers and commitment to the organization. Individuals who feel a of in work in line with various attitudes, such as salary or other compensation plans, may have low satisfaction and try to leave the organization37–39.\n\nIt has been stated that there is a significant relationship between job satisfaction and the stress of medical emergency work places. High job stress and low job security in work places result in reduced job satisfaction40. Job stress leads to reduced job satisfaction, increased desertion and reduction quality of nursing care. Identifying nurses’ job problems and the solutions of removing stressful situations can lead to increased job satisfaction and reduced desertion and absence in work places41. In addition, the more stress leads to lower satisfaction and mental health status of nurses. The higher the nurses job stress, the more they experience work place accidents42.\n\nIn our study among nurses, the employees’ commitment is high; they were more likely to remain and work with the organization rather than leave the organization. Thus, organizational commitment is negatively correlated to affect turnover intention in our study environment. It has been advocated that employees who are emotionally associated to their organization are likely to remain with the organization past the age that would benefit them the most by retiring17. Although nursing is the second top stressful job position in the world, it seems satisfaction with work and commitment decreased their stress in this study. The finding of the present study is similar to the study by Seston et al., who found that job commitment is closely related to job satisfaction42.\n\n\nConclusions\n\nThe findings of the present study can help rescue-related organizations, such as health care professionals, in designing and developing strategies to enhance organizational commitment of the employees, which directly links with organizational performance, effectiveness and productivity. It may become one of the tools and guidance for further actions of management. In order to increase job commitment, leaders should provide enough facilities for staff, give appropriate benefits for those who perform well, and provide friendly and close relationships with staff. The organization also needs to understand the needs of the staff to provide social and mental support.\n\n\nData availability\n\nDataset 1: Raw data for all variables collected in the present study, including demographic variables, and results of the job commitment, job satisfaction and work-related stress questionnaires. Coding as follows: Marital Status- 1=Single, 2=Married, 3=Divorced; Education- 1=Bachelor, 2=Post-graduate; Record (work duration in year)- 1=1–5, 2=6–10, 3= 11–15, 4=16–20, 5=21–25, 6= 26–30; Commitment (Organizational Commitment)- 1= completely disagree, 2= very disagree, 3= disagree, 4= no opinion, 5=agree, 6=very agree, 7=completely agree; JDI(Job Descriptive Index)- 1=completely agree, 2=agree, 3= no opinion, 4= disagree, 5= completely disagree; WS (Work-related Stress)- 1=never, 2= seldom, 3= sometimes, 4=often, 5= always. DOI, 10.5256/f1000research.12595.d19251843",
"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: Farsi translation of Allen and Meyers’s Organizational Commitment scale used in the present study.\n\nClick here to access the data.\n\nSupplementary File 2: Farsi translation of Job Descriptive Index used in the present study.\n\nClick here to access the data.\n\nSupplementary File 3: Farsi translation of HSE work-related stress questionnaire used in the present study.\n\nClick here to access the data.\n\n\nReferences\n\nArdalan A, Mesdaghinia A, Masoumi G, et al.: Higher education initiatives for disaster and emergency health in iran. Iran J Public Health. 2013; 42(6): 635–8. PubMed Abstract | Free Full Text\n\nAhanchian MR, EmamiZeydi A, Armat MR: Conflict management styles among Iranian critical care nursing staff: a cross-sectional study. DimensCrit Care Nurs. 2015; 34(3): 140–5. PubMed Abstract | Publisher Full Text\n\nNegussie N, Berehe C: Factors affecting performance of public hospital nurses in Addis Ababa region, Ethiopia. J Egypt Public Health Assoc. 2016; 91(1): 26–30. PubMed Abstract | Publisher Full Text\n\nMiarkolaei H: An investigation on relationship between employees’ job satisfaction and organizational commitment. Management Science Letters. 2014; 4(4): 669–678. Publisher Full Text\n\nNejati A, Rodiek S, Shepley M: The implications of high-quality staff break areas for nurses' health, performance, job satisfaction and retention. J Nurs Manag. 2016; 24(4): 512–23. PubMed Abstract | Publisher Full Text\n\nDall'Ora C, Ball J, Recio-Saucedo A, et al.: Characteristics of shift work and their impact on employee performance and wellbeing: A literature review. Int J Nurs Stud. 2016; 57: 12–27. PubMed Abstract | Publisher Full Text\n\nBennett P, Williams Y, Page N, et al.: Levels of mental health problems among UK emergency ambulance workers. Emerg Med J. 2004; 21(2): 235–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang X, Chontawan R, Nantsupawat R: Transformational leadership: effect on the job satisfaction of Registered Nurses in a hospital in China. J Adv Nurs. 2012; 68(2): 444–51. PubMed Abstract | Publisher Full Text\n\nHoogendoorn WE, Bongers PM, de Vet HC, et al.: High physical work load and low job satisfaction increase the risk of sickness absence due to low back pain: results of a prospective cohort study. Occup Environ Med. 2002; 59(5): 323–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang L, Tao H, Ellenbecker CH, et al.: Job satisfaction, occupational commitment and intent to stay among Chinese nurses: a cross-sectional questionnaire survey. J Adv Nurs. 2012; 68(3): 539–49. PubMed Abstract | Publisher Full Text\n\nVagharseyyedin SA: An integrative review of literature on determinants of nurses' organizational commitment. Iran J Nurs Midwifery Res. 2016; 21(2): 107–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBahrami MA, Barati O, Ghoroghchian MS, et al.: Role of Organizational Climate in Organizational Commitment: The Case of Teaching Hospitals. Osong Public Health Res Perspect. 2016; 7(2): 96–100. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMohamadzadeh Nojehdehi M, Ashgholi Farahani M, Rafii F, et al.: A Comparison of Organizational Climate and Nurses' Intention to Leave Among Excellence Awarded Hospitals and Other Hospitals in 2013. Iran Red Crescent Med J. 2015; 17(5): e19000. PubMed Abstract | Free Full Text\n\nSaijo Y, Ueno T, Hashimoto Y: Twenty-four-hour shift work, depressive symptoms, and job dissatisfaction among Japanese firefighters. Am J Ind Med. 2008; 51(5): 380–91. PubMed Abstract | Publisher Full Text\n\nMurphy SA, Beaton RD, Pike KC, et al.: Firefighters and paramedics: years of service, job aspirations, and burnout. AAOHN J. 1994; 42(11): 534–40. PubMed Abstract\n\nMoran C: Personal predictions of stress and stress reactions in fire fighter recruits. Disaster Prev Manag. 2001; 10(5): 356–365. Publisher Full Text\n\nBowron JS, Todd KH: Job stressors and job satisfaction in a major metropolitan public EMS service. Prehosp Disaster Med. 1999; 14(4): 236–9. PubMed Abstract | Publisher Full Text\n\nLusa S, Häkkänen M, Luukkonen R, et al.: Perceived physical work capacity, stress, sleep disturbance and occupational accidents among firefighters working during a strike. Work Stress. 2002; 16(3): 264–274. Publisher Full Text\n\nGholipour Baradari A, Hoseini SH, Zamani Kiasari A, et al.: Effect of zinc supplement on job stress of ICU nurses. J Babol Univ Med Sci. 2013; 15( 1): 38–45. Reference Source\n\nFaraji O, Ramazani AA, Hedaiati P, et al.: Relationship Between Job Characteristics and Organizational Commitment: A Descriptive Analytical Study. Iran Red Crescent Med J. 2015; 17(11): e19815. PubMed Abstract | Free Full Text\n\nEscribà-Agüir V, Martín-Baena D, Pérez-Hoyos S: Psychosocial work environment and burnout among emergency medical and nursing staff. Int Arch Occup Environ Health. 2006; 80(2): 127–33. PubMed Abstract | Publisher Full Text\n\nAbdo SA, El-Sallamy RM, El-Sherbiny AA, et al.: Burnout among physicians and nursing staff working in the emergency hospital of Tanta University, Egypt. East Mediterr Health J. 2016; 21(12): 906–15. PubMed Abstract | Publisher Full Text\n\nCicei CC: Occupational stress and organizational commitment in Romanian public organizations. Procedia Soc Behav Sci. 2012; 33: 1077–1081. Publisher Full Text\n\nLi L, Hu H, Zhou H, et al.: Work stress, work motivation and their effects on job satisfaction in community health workers: a cross-sectional survey in China. BMJ Open. 2014; 4(6): e004897. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavis J, Wilson SM: Principals' efforts to empower teachers: Effects on teacher motivation and job satisfaction and stress. The Clearing House. 2000; 73(6): 349–53. Publisher Full Text\n\nKlassen RM, Chiu MM: Effects on teachers' self-efficacy and job satisfaction: Teacher gender, years of experience, and job stress. J Educ Psychol. 2010; 102(3): 741–756. Publisher Full Text\n\nAllen NJ, Meyer JP: Affective, Continuance, and Normative Commitment to the Organization: An Examination of Construct Validity. J Vocat Behav. 1996; 49(3): 252–76. PubMed Abstract | Publisher Full Text\n\nNasiri pour A, Raeissi P, Omrani A, et al.: The Relationship between Nurses’ Organizational Commitment and Services Quality. Journal of Client-Centered Nursing Care. 2015; 1(3): 133–138. Publisher Full Text\n\nSmith PC, Balzer WK, Brannick M, et al.: The revised JDI: A facelift for an old friend. The Industrial-Organizational Psychologist. 1987; 24: 31–33.\n\nSmith PC, Kendall LM, Hulin CC: The measurement of satisfaction in work and retirement. Chicago, IL: Rand McNally. 1969. Reference Source\n\nSchumm WR, Gade PA, Bell DB: Dimensionality of military job satisfaction items: an exploratory factor analysis of data from the spring 1996 Sample Survey of Military Personnel. Psychol Rep. 2003; 92(3 Pt 1): 809–19. PubMed Abstract | Publisher Full Text\n\nKinicki AJ, Mckee-Ryan FM, Schriesheim CA, et al.: Assessing the construct validity of the job descriptive index: a review and meta-analysis. J Appl Psychol. 2002; 87(1): 14–32. PubMed Abstract | Publisher Full Text\n\nGoldberg D, Williams P: A User’s Guide to the General Health Questionnaire. Windsor: NFER-Nelson, 1998.\n\nKhamisa N, Oldenburg B, Peltzer K, et al.: Work related stress, burnout, job satisfaction and general health of nurses. Int J Environ Res Public Health. 2015; 12(1): 652–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGolbasi Z, Kelleci M, Dogan S: Relationships between coping strategies, individual characteristics and job satisfaction in a sample of hospital nurses: cross-sectional questionnaire survey. Int J Nurs Stud. 2008; 45(12): 1800–6. PubMed Abstract | Publisher Full Text\n\nAsegid A, Belachew T, Yimam E: Factors Influencing Job Satisfaction and Anticipated Turnover among Nurses in Sidama Zone Public Health Facilities, South Ethiopia. Nurs Res Pract. 2014; 2014: 909768. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMosadeghrad AM, Ferdosi M: Leadership, job satisfaction and organizational commitment in healthcare sector: proposing and testing a model. Mater Sociomed. 2013; 25(2): 121–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPackard JS, Motowidlo SJ: Subjective stress, job satisfaction, and job performance of hospital nurses. Res Nurs Health. 1987; 10(4): 253–61. PubMed Abstract | Publisher Full Text\n\nMurrells T, Robinson S, Griffiths P: Job satisfaction trends during nurses' early career. BMC Nurs. 2008; 7: 7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPanayiotis T, Paragiotis R, Charalambos P: The Effect of Job Related Stress on Employees’ Satisfaction: A Survey in Health Care. Procedia Soc Behav Sci. 2013; 73: 718–726. Publisher Full Text\n\nTorshizi L, Ahmadi F: Job stressors from clinical nurses perspective. Iran J Nurs. 2011; 24(70): 49–60. Reference Source\n\nSeston E, Hassell K, Ferguson J, et al.: Exploring the relationship between pharmacists' job satisfaction, intention to quit the profession, and actual quitting. Res Social Adm Pharm. 2009; 5(2): 121–32. PubMed Abstract | Publisher Full Text\n\nEskandari M, Heidari Gorji MA: Dataset 1 in: Can work-related stress and job satisfaction affect job commitment among nurses? A cross-sectional study. F1000Research. 2018. Data Source"
}
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[
{
"id": "31147",
"date": "05 Mar 2018",
"name": "Ghahraman Mahmoudi",
"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 should revise the major comments below:\nAbstract: the problem in first paragraph was not accurate.\nThe methods section: this should mention how sample size was done or based on which statistical formula? Also what was the method used for sample?\nThe section of introduction: this should state the opposing and agreeing research and the gaps between this from the current literature. This needs to be stated to show how the study here differentiates from existing and accomplished works.\nThe section of discussion was poor and the main results need to be supported by other research that agrees and disagrees with your results. This section should also mention researchers’ point of view.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "32940",
"date": "27 Apr 2018",
"name": "Marcello Campagna",
"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 study authors aims to investigate the role of job satisfaction and work related stress in organizational commitment among nurses of a Hospital in Sari, Iran. In particular, authors collected data on organizational commitment, job satisfaction and work related stress by means of validated self-administered questionnaires in 100 nurses from 138 overall workforce, who give consent to participate at the study. Methods used in this study appear appropriate, but lacking in some aspects. Statistical analysis does not need an extensive revision, but needs some specifications. The article don’t need a revision of the English language. Below authors can find some specific observation:\nIntroduction: literature cited about work related stress mainly take into consideration studies about fire fighters and emergency service workers, but few studies about work related stress, job satisfaction and commitment in nurses. Why don’t you considerate literature about work related stress, job satisfaction and commitment in nurses? Please review the literature about this aspect and cite papers focused on hospital nurses.\n\nMethods: In the first part of the section “study design” you repeated the aim of the study, already mentioned in the last part of introduction. Please, specify the hypothesis of the present study Study population: exclusion criteria was not mentioned. Please explicit acceptance rate for study participation. Authors reported “all 138 nurses who worked in hospitals”. Do they represent the total workforce? Please explain precisely the recruitment method. The HSE questionnaire should be administered for homogenous groups. Have you recruited nurses from various hospital wards? Please specify. If yes, authors should report which wards were included, how many nurses from each ward and at last verify the homogeneity of response rates. Complexly, it is not clear how you arrived at the sample of 100 nurses from the sample of 139 at initial phase. Authors should specify all passages of recruitment strategy, without any omission. Please report the characteristics of excluded nurses. Questionnaires: please report Crombach’s alpha for each subscale of organizational commitment, job satisfaction and work related stress.\n\nJob satisfaction: “reliability also was high” How did you measure reliability? Why don’t you report Crombach’s alpha? Work related stress: You stated that “low scores indicate higher health and safety in aspect of stress and higher scores means high stress” but this is not always true. In fact, following HSE method, job control and job support are positive factors and higher scores for this aspects generally represent a better situation. Did you overturn questions (revers items) about job control and job support to bypass this problem? If no, you cannot consider a total higher score of work stress as negative. Authors should better explain how you evaluated work stress. The Crombach’s alpha for work related stress has to be considered separately for each subscale. Results: table 1. Add information about nurses’ gender and ward provenience. Are they all female? They performed shift work and if yes, what shift work modality was performed? The mean scores of job commitment, work stress and job satisfaction are not indicative. Authors should report results for each subscale. Table 3. Authors should report the dependent variable and indicate other covariates in the model (age, work duration, marital status ecc). Have you considered those variables in the regression model?\n\nDiscussion: discussion is too short and maybe it would be better to in depth discuss the results. Authors should do some comparisons between results of the present paper and those obtained in other similar study, also giving an interpretation. The lack of comparisons limits the global extensions of those findings to other work settings. Authors should also give an interpretation of results. Authors should discuss limitations of the study (e.g. small sample, possible sources of bias and the lack of some information about home stress that could influence results of the present study). Authors should also explicit what this paper adds to knowledge of this argument and the possible contribution to the scientific community.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "31149",
"date": "11 Jun 2018",
"name": "Amir Emami Zeydi",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for allowing me to have the opportunity to review the manuscript entitled “Can work-related stress and job satisfaction affect job commitment among nurses? A cross-sectional study”. In a cross sectional study, the authors evaluate the association between work-related stress, and job satisfaction and commitment among nurses who worked in a teaching hospital affiliated to Mazandaran University of Medical Sciences, northern Iran. The results indicated that job satisfaction and work-related stress explained 54% of variance in job commitment. The overall job and overall work stress influenced job commitment. I congratulate the authors for their interesting study in the field of health services management. I believe that this paper would have a moderate to high level of interest for the health care managers and administrators, as well as readers of this journal. Overall the manuscript has been well written. However, I’d like the authors to address few issues in order to improve their manuscript. Abstract: For a study to receive the respect it deserves, the abstract should be as well written as possible. In the background section of the abstract, It's better to state a bit about the importance of job stress for nurses, as a sentence. The questionnaires that have been used in the study should be stated in the method section of the abstract. Only stating that the standard questionnaires have been used is not enough. Reporting the mean job satisfaction and work-related stress scores alone is not informative. What do these scores mean? Please clarify. All keywords should be MeSH headings and should be checked against this list http://www.nlm.nih.gov/mesh/ Introduction: One of the limitations of the introduction of this manuscript is the lack of attention and the reporting the prevalence of job stress and job commitment among nurses in general and Iranian nurses in particular. Please refer to the related published studies in this regard and address the issue in the introduction. Please clarify. I believed that the below mentioned studies are appropriate and can be used:\nGheshlagh RG, Parizad N, Dalvand S, Zarei M, Farajzadeh M, Karami M, Sayehmiri K. The prevalence of job stress among nurses in Iran: A meta-analysis study. Nurs Midwifery Stud 2017;6:143-81. Dagget T, Molla A, Belachew T. Job related stress among nurses working in Jimma Zone public hospitals, South West Ethiopia: a cross sectional study. BMC Nurs. 2016 Jun 16;15:39[re-2]. Najimi A, Goudarzi AM, Sharifirad G. Causes of job stress in nurses: A cross-sectional study. Iran J Nurs Midwifery Res. 2012 May;17(4):301-53 Veličković VM, Višnjić A, Jović S, Radulović O, Šargić Č, Mihajlović J, Mladenović J. Organizational commitment and job satisfaction among nurses in Serbia: a factor analysis. Nurs Outlook. 2014 Nov-Dec;62(6):415-274. Khosravani M, Khosravani M, Rafiei F, Mohsenpour M. Organizational commitment and its dimensions in nurses working in Arak’s hospitals. Med Ethics J 2017; 11(39): 37-445.\nMethods: What were the exclusion criteria? Please clarify. How was the sample size calculated? Please clarify. Please explain how to interpret the results of the Organizational commitment and also Job satisfaction questionnaires, in the study methodology. You should explain each of your abbreviations the first time it appears in the text. You should write the full name first, followed by the abbreviation in parentheses. Please revise it for ANOVA (in the data analysis section)! Discussion: Ideally, the discussion section should have the following broad heading; a brief summary of the study and results, comparison of results with the existing literature, clinical evaluation of the work, importance of the findings, strengths and weaknesses of the study. At the end of the discussion readers must know whether the research hypothesis has been proved or not. Please revise the discussion section according the abovementioned style, as much as possible. What is the limitation of the study? Please clarify The below mentioned studies can be used in the discussion section for enriching this section:\nNaghneh MHK, Tafreshi MZ, Naderi M, Shakeri N, Bolourchifard F, Goyaghaj NS. The relationship between organizational commitment and nursing care behavior. Electron Physician. 2017 Jul 25;9(7):4835-48406. Ding X, Yang Y, Su D, Zhang T, Li L, Li H. Can Job Control Ameliorate Work-family Conflict and Enhance Job Satisfaction among Chinese Registered Nurses? A Mediation Model. Int J Occup Environ Med. 2018 Apr;9(2):97-1057. Sepahvand F, Atashzadeh-Shoorideh F, Parvizy S, Tafreshi MZ. The relationship between some demographic characteristics and organizational commitment of nurses working in the Social Security Hospital of Khorramabad. Electron Physician. 2017 Jun 25;9(6):4503-45098. Al-Aameri AS. Job satisfaction and organizational commitment for nurses. Saudi Med J. 2000 Jun;21(6):531-59. Ingersoll GL, Olsan T, Drew-Cates J, DeVinney BC, Davies J. Nurses' job satisfaction, organizational commitment, and career intent. J Nurs Adm. 2002 May;32(5):250-6310.\nConclusion: Conclusions must be based on the results of this study. New idea or concepts should not be introduced in conclusion section. Please revise the conclusion section.\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? Partly",
"responses": []
}
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https://f1000research.com/articles/7-218
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https://f1000research.com/articles/7-109/v1
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25 Jan 18
|
{
"type": "Correspondence",
"title": "Improving the biomedical research literature: insights from authors’ editors can help journal editors define and refine their core competencies",
"authors": [
"Valerie Matarese",
"Karen Shashok",
"Valerie Matarese"
],
"abstract": "A team of stakeholders in biomedical publishing recently proposed a set of core competencies for journal editors, as a resource that can inform training programs for editors and ultimately improve the quality of the biomedical research literature. This initiative, still in its early stages, would benefit from additional sources of expert information. Based on our experiences as authors’ editors, we offer two suggestions on how to strengthen these competencies so that they better respond to the needs of readers and authors – the main users of and contributors to research journals. First, journal editors should be able to ensure that authors are given useful feedback on the language and writing in submitted manuscripts, beyond a (possibly incorrect) blanket judgement of whether the English is “acceptable” or not. Second, journal editors should be able to deal effectively with inappropriate text re-use and plagiarism. These additional competencies would, we believe, be valued by other stakeholders in biomedical research publication as markers of editorial quality.",
"keywords": [
"Authors’ editor",
"Citation",
"Core competencies",
"Decision-making editor",
"English language",
"Journal policies",
"Plagiarism",
"Scientific editor"
],
"content": "Correspondence\n\nJournal editing cannot be learned in higher education, and alternative training opportunities are not readily available. To guide such training and ultimately improve the quality of published research, Moher and colleagues defined core competencies (CC) for editors of biomedical journals. They did a literature review1, surveyed 148 journal editors2, and used a Delphi-like process to rank different competencies2, resulting in a consensus statement signed by 30 stakeholders in research publishing3. We commend this initiative to help journal editors work responsibly and accountably, and offer suggestions on areas that might benefit from additional input.\n\nMoher et al. do not use the term “journal editor” but rather “scientific editor”, defined as someone “who make[s] decisions on the content and policies of a journal — including editors-in-chief and associate/academic editors”2. This definition excludes other editors who contribute to the quality of research publications, in contrast to the broader meaning of “science editor” used by two stakeholder groups in the consensus initiative (Council of Science Editors, European Association of Science Editors). To avoid confusion regarding who the CC are intended for, a term such as “decision-making editor” might be helpful. For simplicity’s sake, here we use “journal editor” to refer to the type of editor we assume the CC are intended for.\n\nAs Moher et al. concede, “time and resource constraints … limited inclusion of perspectives of other relevant groups (e.g. authors, readers, peer reviewers)” in developing the CC3. We believe input from authors is fundamental to efforts to define competencies of journal editors, and suggest that insights into authors’ (sometimes less than satisfactory) experiences with journals can be provided by another type of editor, namely authors’ editors4–10. These editors help researcher-authors prepare manuscripts for publication by reading drafts and suggesting changes to structure and content (substantive editing), language and style (language editing), and appearance and format (e.g. compliance with journals’ instructions)9,11. In addition, many authors’ editors train researchers in publication skills12–17 and help authors navigate editorial processes18–21. Authors’ editors’ knowledge of the publication process and their close interactions with the producers, distributors and consumers of research information make them qualified to help define CC for journal editors and identify deficiencies in current practices8.\n\nAuthors’ editors are often more familiar with researchers’ local circumstances and challenges than journal editors are. Although the writers of the consensus statement and their informants are themselves researchers and therefore authors, they were perhaps not representative of the wider population of “real-world” researchers who have limited contact with English-speaking opinion leaders in biomedical publishing. In contrast, many authors’ editors work with researchers whose first language is not English or who are based outside the global North and West. Familiarity with other languages and cultures gives authors’ editors insights into the types of competencies researchers from diverse geographical, cultural and linguistic backgrounds would value in journal editors.\n\nLike journal editors, authors’ editors are taking steps to critically evaluate and improve their working methods. A growing body of literature9 facilitates knowledge transfer to colleagues in different settings. PhD degrees have been awarded to authors’ editors for applied linguistics research based on their work practices in the Netherlands22, Spain23 and China (Luo24 and unpublished; available upon request). Continuing professional development for authors’ editors is available through national and international associations (Table 1). Authors’ editors in these associations can provide valuable information about journal editor CC that researchers would value. As authors’ editors ourselves, we offer suggestions on how to improve the CC based on insights we and our colleagues gain about researchers’ experiences with peer review.\n\na Country and continent in which most members live\n\nIn our experience, reviewers and journal editors increasingly cite “problems with the English” as a reason for rejection, even of manuscripts free from language errors. Meanwhile, biomedical journals publish an ever-increasing proportion of articles that were judged by reviewers to have “acceptable English” but which contain awkwardly worded statements that defy comprehension and undermine reproducibility2(pp5–9). To avoid these problems, journal editors should be able to either provide authors with useful feedback on the language (e.g. by endorsing or overruling reviewers’ complaints) or delegate this responsibility to an appropriately skilled reviewer or editorial staffer. Relying solely on blanket “acceptable/unacceptable” assessments of the writing contributes, in our experience, to cynicism among authors regarding the fairness and quality of peer review, and to the proliferation of poorly written articles. Although we realize that skills in “dealing with language issues” and knowledge about the “fundamentals of editing” were considered but then excluded from the CC2, we believe that inclusion of a competency in this area would be welcomed and perceived as a marker of editorial quality.\n\nAnother competency researchers would appreciate is the ability of journal editors to deal effectively with inappropriate text re-use. This omission from the CC is surprising, especially since plagiarism featured in two of the 23 highly ranked statements in the Delphi process2. While working with authors on manuscripts, authors’ editors sometimes encounter re-used text and inadequate citation, and use these opportunities to explain why these practices may be inappropriate and how to avoid them25. But these individual efforts are not enough to stop the global spread of plagiarism in published research, which journal editors may inadvertently facilitate if they do not check manuscripts carefully enough before publication. Journal editors should be able to interpret the results of “plagiarism-detection” software and deal sensitively with the manuscripts these tools single out (as proposed by the Committee on Publications Ethics, publicationethics.org/files/u2/02A_Plagiarism_Submitted.pdf). Setting a maximum allowable percentage of text overlap, without considering the context of the non-original text, may send inconsistent messages about appropriate and inappropriate text re-use. Manuscript rejection based solely on the percentage of non-original text can, in our experience, alienate well-meaning authors from journals that use this criterion.\n\nThese are just two of the areas where authors’ editors can provide valuable input for future efforts to define and refine CC for biomedical journal editors. Alongside earlier efforts to support professional and ethical practices26–28 (see also: publicationethics.org/files/editable-bean/COPE_Core_Practices_0.pdf, and www.wame.org/about/syllabus-for-prospective-and-newly-appointed), the CC may indeed help gatekeepers meet researchers’ and readers’ expectations for editorial practices that ultimately improve the quality of published research.",
"appendix": "Competing interests\n\n\n\nVM and KS are both self-employed authors’ editors and realize that this article might attract clients. VM is the author of the book Editing Research and realizes that this article might affect sales. VM and KS are both long-time members of Mediterranean Editors and Translators, and have been unpaid speakers at CPD events run by this organization. KS was Vice Chair of Mediterranean Editors and Translators during 2006. KS is a long-time member of Asetrad and was an unpaid speaker at a CPD event run by this organization in 2017.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Marije de Jager for constructive feedback on an earlier draft of the manuscript.\n\n\nReferences\n\nGalipeau J, Barbour V, Baskin P, et al.: A scoping review of competencies for scientific editors of biomedical journals. BMC Med. 2016; 14(1): 16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGalipeau J, Cobey KD, Barbour V, et al.: An international survey and modified Delphi process revealed editors’ perceptions, training needs, and ratings of competency-related statements for the development of core competencies for scientific editors of biomedical journals [version 1; references: 2 approved]. F1000Res. 2017; 6: 1634. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoher D, Galipeau J, Alam S, et al.: Core competencies for scientific editors of biomedical journals: consensus statement. BMC Med. 2017; 15(1): 167. PubMed Abstract | Publisher Full Text | Free Full Text\n\nApplewhite LB: The author’s editor. Med Communications. 1973; 1: 16–20.\n\nTacker MM: Author’s editors: catalysts of scientific publishing. CBE Views. 1980; 3: 3–11. Reference Source\n\nEastwood S: The author’s editor in the setting of a university or research center. J Res Communication Studies. 1981; 3: 211–6.\n\nMcNab SM: The ethics of author’s editing. In: Weeks RA, Kinser DL, editors. Editing the refereed scientific journal: practical, political and ethical issues. New York: IEEE; 1994; 167–77.\n\nShashok K: Author’s editors: facilitators of science information transfer. Learn Publ. 2001; 14(2): 113–21. Publisher Full Text\n\nMatarese V: Editing research: the author editing approach to providing effective support to writers of research papers. Medford: Information Today; 2016. Reference Source\n\nBurrough-Boenisch J: Editing non-native English: reflections from a Netherlands-based editor on those who do it and the skills they should have. The 21st Century Text. 2013; 13(1). Reference Source\n\nBurrough-Boenisch J, Matarese V: The authors’ editor: working with authors to make drafts fit for purpose. In: Matarese V, editor. Supporting research writing: roles and challenges in multilingual settings. Oxford: Chandos; 2013; 173–89. Publisher Full Text\n\nEastwood S, Derish PA, Berger MS: Biomedical publication for neurosurgery residents: a program and guide. Neurosurgery. 2000; 47(3): 739–48; discussion 748–9. PubMed Abstract | Publisher Full Text\n\nDerish PA, Maa J, Ascher NL, et al.: Enhancing the mission of academic surgery by promoting scientific writing skills. J Surg Res. 2007; 140(2): 177–83. PubMed Abstract | Publisher Full Text\n\nCameron C, Deming SP, Notzon B, et al.: Scientific writing training for academic physicians of diverse language backgrounds. Acad Med. 2009; 84(4): 505–10. PubMed Abstract | Publisher Full Text\n\nCameron C, Chang S, Pagel W: Scientific English: a program for addressing linguistic barriers of international research trainees in the United States. J Cancer Educ. 2011; 26(1): 72–8. PubMed Abstract | Publisher Full Text\n\nDornhoffer MK: Beyond editing: an experience in mentoring provided by an academic health care center’s office of grants and scientific publications. AMWA J. 2012; 27(4): 147–51.\n\nMatarese V: Collaborative research writing: developmental editing with an underlying educational vein. In: Matarese V, editor. Supporting research writing: roles and challenges in multilingual settings. Oxford: Chandos; 2013; 221–35. Publisher Full Text\n\nShashok K: Authors’ editors in the 21st century: promoters of publication quality and efficiency. European Science Editing. 2014; 40(3): 60–2. Reference Source\n\nBreugelmans R, Barron JP: The role of in-house medical communications centers in medical institutions in nonnative English-speaking countries. Chest. 2008; 134(4): 883–5. PubMed Abstract | Publisher Full Text\n\nAribisala W: How are non-native-English-speaking authors coping with the requirements to publish in English-language journals? Science Editor. 2010; 33(6): 189. Reference Source\n\nShashok K: Speaking virtually the same language: coding and decoding messages between authors and editors.European Science Editing.1994; 53: 5–7.\n\nBurrough-Boenisch J: Culture and conventions: writing and reading Dutch scientific English. Utrecht: Netherlands Graduate School of Linguistics; 2002; LOT no. 59. Reference Source\n\nShaw O: From process to product: a text-based ethnographic study of ten biomedical research articles by Spanish authors. Doctoral thesis, Universidad de Zaragoza; 2017. Reference Source\n\nLuo N, Hyland K: Intervention and revision: expertise and interaction in text mediation. Written Communication. 2017; 34(4): 414–40. Publisher Full Text\n\nKerans ME, de Jager M: Handling plagiarism at the manuscript editor’s desk. European Science Editing. 2010; 36(3): 62–6. Reference Source\n\nBard AJ: Ethics of editors. In: Weeks RA, Kinser DL, editors. Editing the refereed scientific journal: practical, political and ethical issues. New York: IEEE; 1994; 147–55.\n\nKleinert S, Wager E: Responsible research publication: international standards for editors. A position statement developed at the 2nd World Conference on Research Integrity, Singapore, July 22–24, 2010. In: Mayer T, Steineck N, editors.Promoting research integrity in a global environment. Singapore: World Scientific Publishing; 2012; 317–28. Reference Source\n\nSmart P, Maisonneuve H, Polderman A, editors: Science editors’ handbook, 2nd ed. Redruth: European Association of Science Editors; 2013."
}
|
[
{
"id": "30325",
"date": "05 Feb 2018",
"name": "Joy Burrough-Boenisch",
"expertise": [
"Reviewer Expertise I am an experienced authors' editor myself",
"though not in the field of biomedicine. I have published on editing",
"particularly on the editing of non-native English."
],
"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\nWritten by two long-standing, respected professional biomedical authors’ editors, this article responds to the proposal to create core competencies (CC) “for scientific editors of biomedical journals”. Although commending the proposal, Matarese and Shashok point out that Moher et al.’s use of the term “scientific editor”, instead of “journal editor” or “decision-making editor\", might raise confusion about the type of editor to which the CC apply. They also argue that when refining the CC, authors’ experiences with journals provide useful insights into the CC needed by journal editors.\nMatarese and Shashok point out that some scientific editors do not work for a publisher or journal but instead work closely with researcher-authors, helping them to produce publishable papers – a service particularly helpful to authors writing in their second (or third) language or living in the Global South. These “authors’ editors” can also help their author clients to deal with reviewer comments and journal-related correspondence, thereby accumulating valuable information on authors’ experiences with journal editors. Authors’ editors are thus well-placed to contribute to formulating certain CC, especially in relation to assessing the quality of the English and providing clear, constructive feedback to authors. Drawing on their extensive knowledge of journal–author intercommunication, Matarese and Shashok plead for reversal of the decision not to include competence to deal with language issues and knowledge of the “fundamentals of editing” from the CC. They also express surprise at another omission: competency in plagiarism detection. Again, they point out relevant insights that authors’ editors can supply.\nMatarese and Shashok’s case for expanding and refining the CC with the help of authors’ editors is convincingly argued. They have underpinned it with references to relevant publications by authors’ editors that are probably unfamiliar to editors of science journals. The table of editors’ associations indicates that there are professional associations providing CPD that committed, principled authors’ editors may join. All this helps broadcast the actual and potential usefulness of authors’ editors to researcher-authors, journals, journal reviewers and journal editors. However, to avoid creating the impression that all authors’ editors are suitably trained, optimally competent in all the editing they undertake, involved members of appropriate associations and proactive in undertaking CPD, Matarese and Shashok should acknowledge that there are no CC for authors’ editors, nor are authors’ editors required to have certain academic or professional qualifications. Matarese and Shashok are currently among the most respected and high-profile biomedical authors’ editors; their plea to involve authors’ editors in developing CC for journal editors should be placed in that context.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes",
"responses": [
{
"c_id": "3441",
"date": "21 Feb 2018",
"name": "Karen Shashok",
"role": "Author Response",
"response": "We appreciate Dr. Burrough-Boenisch’s cautionary note on the variations in authors’ editors’ skills, practices and commitment to continuing education, which are to be expected for any profession that does not have defined entry qualifications or agreed-upon CC. The author editing literature contains several proposals for key skills and tasks (see, for example, Lang 1999; Burrough-Boenisch 2013; Chapter 7 in Matarese 2016), but although they overlap to some extent, they also tend to reflect the particular circumstances of different authors’ editors’ professional settings, clients and disciplinary focus. Although several professional associations offer formal certification, these programs do not cover all facets of author editing, such as the interpersonal aspects of working with researchers and the value of multilingualism. Like journal editors, authors’ editors with different skills, strengths and specialist knowledge can provide valuable services in some areas, but may have shortcomings in other areas. Our impression is that authors’ editors are likely to be transparent about their strengths and limitations, in order to help researchers decide whether they are able to provide the right types of support. We have noted the lack of universally agreed-upon professional qualifications for authors’ editors in version 2. Burrough-Boenisch J: Editing non-native English: reflections from a Netherlands-based editor on those who do it and the skills they should have. The 21st Century Text. July 30, 2013. Lang TA: A curriculum for biomedical writing and editing: a second volley. CBE Views. 1999; 22(1): 3–5. Matarese V: Editing research: the author editing approach to providing effective support to writers of research papers. Medford: Information Today; 2016."
}
]
},
{
"id": "30328",
"date": "12 Feb 2018",
"name": "Na Luo",
"expertise": [
"Reviewer Expertise I am a researcher on the activities of authors’ editors. I have published on authors’ editing."
],
"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 correspondence, two well-known European authors’ editors, Dr. Valerie Matarese and Ms. Karen Shashok, raise two very interesting points to enhance the core competencies (CC) list for biomed journal editors in Moher et al (2017). They aptly argue that the perspective from authors is essential to define journal editor CC and thus inappropriate to be overlooked. Then they indicate that authors’ editors can give insights on authors’ behalf when reliable author opinion is not readily available by calling attention to the work of authors’ editors and their advantage over journal editors in familiarity with authors in different contexts.\nThe two points they suggest to be added to the CC list of Moher et al (2017) are not only insightful but also very caring for most EAL (English as an additional language) scholars who often struggle to publish in a language they do not have much control. The first one is that the CC list should re-include journal editors’ competence to give useful feedback on language and writing to authors instead of relying on standardized reviewer reports where this aspect is simply judged “acceptable” or “unacceptable”. In a highly globalized academic community where biomedical journals are at the very forefront of globalization, it is unrealistic to expect most reviewers to give concrete language and writing feedback to researchers, particularly EAL ones. Therefore, it seems incumbent on journal editors to possess this as a core competence. What I am thinking here is that journal editors may also need more knowledge of authors’ editors. This is because EAL researchers need to be referred to the right authors’ editors for support when fixing the language and writing problems goes beyond their own capacity.\nThe second competence Matarese and Shashok suggest to add is that journal editors should be able to interpret results of plagiarism-detection software sensibly and sensitively rather than simplistically relying on arbitrarily set maximum-text-overlap percentages. As is known to writing researchers and authors’ editors, text re-use is an important, if not the most, way for EAL scholars to learn academic writing and compose papers in English. Journal editors’ lack of ability to tell the difference between plagiarism and well-meant text re-use can render them to punish the innocent and alienate EAL scholars from their journals, as convincingly argued by the two authors of this piece.\nOverall, I find the suggestions of Matarese and Shashok very useful to refine the CC list for journal editors, as given in Moher et al (2017), in a well-crafted text. Their effort to support EAL scholars in ways beyond working on manuscripts should be commended. Therefore, I recommend that this correspondence be indexed as soon as possible. If they would like to add that journal editors also need to have the ability to refer EAL scholars to proper authors’ editors, I would appreciate this piece even more.",
"responses": [
{
"c_id": "3440",
"date": "21 Feb 2018",
"name": "Karen Shashok",
"role": "Author Response",
"response": "We concur with Dr. Luo’s understanding of language and writing in the current globalized context of research publishing, including some EAL scholars’ recourse to text re-use to produce acceptable English. We also agree that suggestions by journal editors on where authors could obtain help is a competency that researchers are likely to value, and have noted this in version 2. Many journals already list commercial editing service providers on their websites, but the scope and quality of these services vary. Journal editors have differing criteria for “acceptable” English or writing, but as the ultimate arbiters of when the language and writing are “good enough” for publication in their journal, they should, for the sake of transparency and fairness, let authors know what criteria they use to decide which texts are publishable and which need improvement."
}
]
}
] | 1
|
https://f1000research.com/articles/7-109
|
https://f1000research.com/articles/7-211/v1
|
21 Feb 18
|
{
"type": "Brief Report",
"title": "Epigenetic silencing of lncRNA MORT in 16 TCGA cancer types",
"authors": [
"Lukas Vrba",
"Bernard W. Futscher",
"Bernard W. Futscher"
],
"abstract": "We have previously described a hominid-specific long non-coding RNA, MORT (also known as ZNF667-AS1, Gene ID: 100128252), which is expressed in all normal cell types, but epigenetically silenced during cancer-associated immortalization of human mammary epithelial cells. Initial analysis of The Cancer Genome Atlas (TCGA) showed that 15 of 17 cancer types, which represent the 10 most common cancers in women and men, display DNA methylation associated MORT silencing in a large fraction of their tumors. In this study we analyzed MORT expression and DNA methylation state in the remaining 16 TCGA cancer types not previously reported. Seven of the 16 cancer types showed DNA methylation linked MORT silencing in a large fraction of their tumors. These are carcinomas (cervical cancer, and cancers of esophagus, stomach, and bile duct), and the non-epithelial tumors mesothelioma, sarcoma, and uterine carcinosarcoma. Together with the findings from our previous report, MORT expression is silenced by aberrant DNA methylation in 22 of 33 of TCGA cancer types. These 22 cancers include most carcinoma types, blood derived cancers and sarcomas. In conclusion, results suggest that the MORT gene is one of the most common epigenetic aberrations seen in human cancer. Coupled with the timing of MORT gene silencing during in vitro epithelial cell immortalization and its occurrence early in the temporal arc of human carcinogenesis, this provides strong circumstantial evidence for a tumor suppressor role for MORT.",
"keywords": [
"DNA Methylation",
"Gene Silencing",
"lncRNA",
"ncRNA",
"lincRNA",
"MORT",
"ZNF667-AS1",
"Epigenetics"
],
"content": "Introduction\n\nMORT was originally found as a transcript silenced during in vitro immortalization of human mammary epithelial cells1. Like a significant majority of lncRNAs, MORT’s molecular function remains enigmatic. The MORT gene is specific to higher primates, is expressed in all normal human cell types, and MORT RNA is located predominantly in the cytoplasm1. Analysis of MORT expression and the DNA methylation state of its promoter in 17 cancer types from The Cancer Genome Atlas (TCGA)2, which represent the 10 most frequent cancers in males and females, showed MORT is epigenetically silenced in 15 of 17 these cancers1. Based on the data from the original in vitro study1, we predicted epigenetic MORT silencing occurs early in human carcinogenesis and therefore could be seen in premalignant lesions, such as ductal carcinoma in situ of the breast and colonic adenomas. We used data from clinical samples from published genomic data sets3–8 to address this possibility, and indeed, MORT loss occurs prior to or at the stage of pre-malignancy and not thereafter9. Taken together these facts suggest that MORT transcript has a tumor suppressive role and is not simply an epigenetic “passenger error.”\n\nSince our previous analysis of MORT in TCGA datasets was not exhaustive and only reported on 17 out of 33 TCGA cancer types, the goal of this short study was to extend our earlier work and complete the analysis of MORT DNA methylation associated gene silencing in the final 16 TCGA cancer types.\n\n\nMethods\n\nWe integrated the MORT expression level and the DNA methylation state of its promoter region using TCGA data as described before1. The Illumina HiSeq RNA-seq and HumanMethylation450 DNA methylation data for samples of 16 TCGA cancer types listed in Table 1 were downloaded from the GDC data portal. The data were analyzed in the R programming environment, version 3.4.210. The mean RNA-Seq rpkm values for the two exons constituting the MORT RNA were plotted against the mean DNA methylation beta value of the 7 CpGs from the MORT promoter region for the individual samples of each cancer type. The Spearman correlation coefficient rho between the MORT RNA level and the DNA methylation of MORT promoter was calculated using the function cor.test.\n\nThe numbers of primary tumor and normal samples for which both the MORT RNA expression and the MORT promoter DNA methylation data were available are listed. *DNA methylation data from HumanMethylation27 platform that covers 2 CpGs out of 7 CpGs covered by HumanMethylation450 were used.\n\n\nResults and discussion\n\nSeven of sixteen analyzed cancer types (CESC, CHOL, ESCA, MESO, SARC, STAD, and UCS) show strong MORT silencing by DNA methylation (Figure 1). The negative correlation rho between MORT expression and DNA methylation in these cancers is below -0.5; the DNA methylation level in some tumor samples of these cancers exceeds 0.5 beta (> 50% DNA methylation), and a large fraction of the tumor samples in these cancer types have very low to no MORT expression level (Figure 1). The correlation of MORT expression and promoter DNA methylation in the remaining nine cancer types is also negative; however, the maximum level of the DNA methylation of MORT promoter in some of these cancers is either very low (UVM), or a very few tumor samples have MORT silenced (ACC, KICH, OV, and THYM), and some of the cancer types (GBM, LGG, PCPG, and TGCT) do not appear to display MORT gene silencing (Figure 1).\n\nThe x-axis shows the MORT expression level according to RNA-seq and y-axis shows the level of MORT promoter DNA methylation according to Illumina HumanMethylation450 microarray. The correlation coefficient rho between the MORT expression and the DNA methylation of MORT promoter for each tumor type is displayed. The OV has a very low number (10) of samples analyzed by the HumanMethylation450 platform, therefore the data from the HumanMethylation27 platform that covers 2 CpGs out of 7 CpGs covered by HumanMethylation450 were used.\n\nThe analysis presented shows DNA methylation associated MORT gene silencing in 7 of 16 TCGA cancer types. Compared to the 17 TCGA cancer types presented in our original study,1 most the 16 cancer types presented here lack their respective normal tissues samples and some of them have lower amounts of tumor samples (Table 1). Nevertheless, the distribution of MORT expression and DNA methylation data in tumor samples clearly indicates MORT silencing in multiple cancer types (Figure 1).\n\nCervical tumors (CESC) have high proportion of MORT silencing (Figure 1); more than 75% of 304 cervical tumor samples have MORT promoter DNA hypermethylated and MORT silenced. Using TCGA data, a recent study found MORT downregulated in cervical cancer11, but surprisingly did not report on or hypothesize potential mechanisms for this transcriptional repression. Here we confirm and extend their initial analysis of MORT silencing in cervical cancer and show further that this silencing is strongly linked to aberrant DNA methylation of the MORT promoter.\n\nCombined together with the findings from our previous report1, Table 2 shows MORT is silenced by DNA methylation in a super majority of TCGA cancer types (22 of 33). MORT loss occurs predominantly due to epigenetic silencing and increased DNA methylation of its promoter in breast cancer9. This could likely be extended to all 22 cancer types with the high fraction of MORT negative samples and the high correlation between MORT RNA level and MORT promoter DNA methylation, where MORT likely plays a tumor suppressive role. The other 11 cancer types, with a little to no MORT silencing, might have tumor suppressive pathway, where MORT is involved, interrupted elsewhere and/or MORT may play some additional vital role in tissues these tumors originate from - e.g. prostate, thyroid, brain, testes, or ovary - since these tissues typically have the highest levels of MORT RNA1.\n\nThe cancer types with MORT silencing in a large fraction of tumor samples are indicated. Results from this study are indicated (*), results from our previous report (ref 1) are indicated (**).\n\nIn summary, our findings show that the MORT gene is one of the most common epigenetic aberrations seen in human cancer. Coupled together with MORT silencing occurring early in the temporal arc of human carcinogenesis it strongly supports a tumor suppressive role for MORT.\n\n\nData availability\n\nIllumina HiSeq RNA-seq and HumanMethylation450 DNA methylation data for TCGA cancer types used in the present study can be downloaded from the GDC data portal.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Maynard Chair in Breast Cancer Epigenomics at the University of Arizona Cancer Center and the Cancer Center Support Grant (P30 CA023074).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe results shown here are based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/.\n\n\nReferences\n\nVrba L, Garbe JC, Stampfer MR, et al.: A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers. Epigenetics. 2015; 10(11): 1074–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNCI, NHGRI, NIH: The Cancer Genome Atlas. NIH, National Cancer Institute, National Human Genome Research Institute, 2016. Reference Source\n\nAbba MC, Gong T, Lu Y, et al.: A Molecular Portrait of High-Grade Ductal Carcinoma In Situ. Cancer Res. 2015; 75(18): 3980–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFleischer T, Frigessi A, Johnson KC, et al.: Genome-wide DNA methylation profiles in progression to in situ and invasive carcinoma of the breast with impact on gene transcription and prognosis. Genome Biol. 2014; 15(8): 435. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson KC, Koestler DC, Fleischer T, et al.: DNA methylation in ductal carcinoma in situ related with future development of invasive breast cancer. Clin Epigenetics. 2015; 7: 75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuo Y, Wong CJ, Kaz AM, et al.: Differences in DNA methylation signatures reveal multiple pathways of progression from adenoma to colorectal cancer. Gastroenterology. 2014; 147(2): 418–29.e8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQu X, Sandmann T, Frierson H Jr, et al.: Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter. Oncogene. 2016; 35(50): 6403–6415. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReyngold M, Turcan S, Giri D, et al.: Remodeling of the methylation landscape in breast cancer metastasis. PLoS One. 2014; 9(8): e103896. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVrba L, Futscher BW: Epigenetic Silencing of MORT Is an Early Event in Cancer and Is Associated with Luminal, Receptor Positive Breast Tumor Subtypes. J Breast Cancer. 2017; 20(2): 198–202. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTeam RC: R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing, 2017. Reference Source\n\nZhao LP, Li RH, Han DM, et al.: Independent prognostic Factor of low-expressed LncRNA ZNF667-AS1 for cervical cancer and inhibitory function on the proliferation of cervical cancer. Eur Rev Med Pharmacol Sci. 2017; 21(23): 5353–60. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "31096",
"date": "01 Mar 2018",
"name": "Adam R Karpf",
"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 a straightforward study of DNA methylation and RNA expression of the lncRNA MORT in 16 TCGA cancer types not previously reported. Most of the paper is clearly presented and the conclusions are reasonable. Suggestions for improvement are presented below.\n\n1) Statements are made regarding MORT being “one of the most common” epigenetic aberrations seen in human cancer. No support for this statement is presented. 2) The classification of some tumor types as showing “strong” MORT silencing by DNA methylation, and other tumor types as not showing this, is arbitrary. A quantitative definition is needed. 3) Fig 1 might be clearer if presented in two panels, subdivided by whether the quantitative definition of “strong silencing” is met. The same is true for Table 2, which could be subdivided into two parts. 4) Since normal samples were not available for many of the tumors profiled, a more accurate statement might be “DNA methylation regulation” rather than “DNA methylation silencing,” as reduced expression in tumors as compared to normal tissues was not shown. 5) A diagram showing the MORT gene and the position of the methylation sites examined by the TCGA would be helpful. 6) Can the authors reference any data, e.g. from other publications, showing that DNA methylation actively suppresses MORT expression, by for example turning on MORT expression using treatment with a DNA methyltransferase inhibitor? 7) In the introduction, the statement “prior to or at the stage of pre-malignancy and not thereafter” is confusing, and gives the impression that MORT becomes hypomethylated, or its expression is elevated, in tumors as compared to pre-malignant lesions. A better way to phrase this would be to say e.g. that “MORT is repressed in pre-malignant lesions and remains repressed in tumors.” 8) The final sentence in the discussion uses the wording “strongly supports” when “suggests” would be more accurate, given the absence of functional data showing tumor suppression by MORT.",
"responses": []
},
{
"id": "31980",
"date": "03 Apr 2018",
"name": "Gwen Lomberk",
"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\nSTRENGTHS: The authors utilize the TCGA database to extend their previous studies on MORT that has been primarily characterized in breast cancer, but also observed to undergo silencing in 15 out of the 17 most common cancers. The current work takes this analysis deeper into the 33 TCGA cancer types and perform a more thorough analysis of the DNA methylation associated with the 16 cancer types evaluated here.\n\nSUGGESTIONS FOR IMPROVEMENT:\n\n-The authors make a bold statement that “the MORT gene is one of the most common epigenetic aberrations seen in human cancer”. This statement is not supported by the data presented. In order to make a statement of this level, the authors would have to provide comparisons to other documented genes that undergo a high frequency of epigenetic alteration. -This statement becomes more difficult to make when considering that several of the tumors present do not have normal counterparts for comparison. We therefore do not know if those tissue types whether the DNA methylation is abnormal or typical for the tissue type.\n\n-Authors should consider revising some of the scales on the graphs in Figure 1 to highlight the lack of methylation (for example, in testicular germ cell tumors). Similarly, the cutoff for methylation could be shown by a dotted line or similar feature.\n\n-The location of methylation relative to the MORT gene would be informative. Of the tumor types that are methylated, are the same regions methylated across types?\n\n-The authors should be cautious not to overstate their conclusions throughout the text since the conclusions are speculative without experiments to support their statements. Nevertheless, these correlations present interesting speculation and avenues for further investigations.",
"responses": []
},
{
"id": "31592",
"date": "03 Apr 2018",
"name": "Alexander Dobrovic",
"expertise": [
"Reviewer Expertise DNA methylation biomarkers",
"liquid biopsies",
"digital PCR"
],
"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 brief article provides an analysis of MORT epigenetic silencing across 16 TCGA tumour types not previously reported by the authors enabling assessment across all TCGA tumour types. MORT silencing seems to be a key early event in carcinogenesis. This indicates that further studies of MORT biology are required as well as investigation of MORT methylation as a potential biomarker.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-211
|
https://f1000research.com/articles/7-209/v1
|
20 Feb 18
|
{
"type": "Brief Report",
"title": "Cryopreservation of orchid seeds through rapid and step freezing methods",
"authors": [
"Marco Cerna",
"Paulina Valdivieso",
"Rino Cella",
"Bence Mátyás",
"Cristina Aucapiña",
"Marco Cerna",
"Paulina Valdivieso",
"Rino Cella",
"Cristina Aucapiña"
],
"abstract": "Ecuador has a great variety of climatic regions that potentiate biodiversity. The family Orchidaceae constitutes one of the most important of the country, having identified about 4032 species with a high degree of endemism, therefore the development and research of alternative methods of storage and conservation of species is a strategy of primary interest for researchers and for society in general. In cryopreservation, temperatures reach below -190°C in order to paralyze the chemical reactions and keep the plant material viable for long periods. The present research focuses on the development of protocols for cryopreservation of seeds, aimed at the preservation of biodiversity, focusing on the family Orchidaceae, for the subsequent generation of a seed bank. The assays were performed on seeds of Epidendrum quitensium, Sobralia rosea, and Epidendrum anderssonii. Two freezing rates were tested: rapid freezing at -196°C; and step freezing at -22°C, -60°C to 196°C, further analyzed four combinations from Dimethylsulfoxide DMSO, glycerol and sucrose (DMSO 1M; DMSO 1M + glycerol 1M; DMSO 1M + sucrose 1M; DMSO 1M + glycerol 0,5M + sucrose 0,5M). The best results were obtained both in rapid and stepped freezing without the use of cryo-protective substances, by introducing the seeds directly into liquid nitrogen. Species of the genus Epidendrum presented a more efficient response in comparison to Sobralia. The viability of the seeds was evaluated by the tetrazolium test.",
"keywords": [
"Orchidaceae",
"Epidendrum",
"Sobralia",
"seeds",
"cryoconservation",
"liquid nitrogen",
"Tetrazolium."
],
"content": "Introduction\n\nThe Republic of Ecuador is located on the South American continent. From north to south the country is crossed by the Andes mountain range and has four climatic regions: Coast, Andes, Amazon and the Insular region1. Its position in the middle of the world, the luminous intensity, the ocean currents and the different altitudes produce 82 types of ecosystems (see Ministry of Environment document on ecosystems in Ecuador) There is a great variety of climatic regions that have an important effect in the diversification of plant formations2. Concerning the Orchidaceae family, in Ecuador as of 2010, 4032 species of orchids have been identified, of which 1714 (42.5%) are endemic3; 4.5% of the orchids of the planet are found in Ecuador. Seed banks allow the conservation of the biodiversity ex situ and prioritize species used for food, medicine and those in danger of extinction. Orchidaceae is a large family with many endangered species and all of them are included in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) I and II4. Cryopreservation is an efficient strategy to safeguard these species, but unfortunately, orchid seeds have short lifetimes5; the longevity depends on the moisture content and storage temperature, so it is necessary to experiment with efficient storage systems for each species5. The advantages of cryopreservation are: storage for an indefinite period, genetic stability of the individuals, reduced infrastructure, can have independent energy and the stored genetic material does not require manipulation6.\n\nTherefore, the objective of this research was to define protocols for cryopreservation of orchid seeds, in order to install a seed bank that promotes the conservation of vulnerable species.\n\n\nMethods\n\nThe collection of plant material was made through the authorization of the Ministry of Environment of Ecuador No. 17-2011- Investigación-B- DPMS/MAE,FloraX, N0. 08−2013−0869−I CFAU−F LO−DAPI −UNO−MAE and the Botanical Garden \"Orquídeas de Sarina\" patent No. 006-2015- FLO-DPAP- MA.\n\nThe cryopreservation tests were developed with the seeds of 3 species: Epidendrum quitensium Rchb.f., Sobralia rosea Poepp. & Endl. and Epidendrum anderssonii Hágsater & Dodson (Figure 1). The cryopreservation tests were developed with 3 species:\n\n• 2392 Epidendrum quitensium Rchb.f., (0° 17’52.1\"N 78° 22’33.3\"W 3200 msnm)\n\n• 2420 Sobralia rosea Poepp.& Endl. (0° 52’11.8\"N 78° 26’53.8\"W 600 msnm)\n\n• 2706 Epidendrum anderssonii Hágsater&Dodson (0° 50’36.2\"N 78° 25’01.5\"W 1200 msnm)\n\nA) Epidendrum quitensium, B) Sobralia rosea, C) Epidendrum anderssonii.\n\nThe species pertain to three different altitudes and were selected from many sources and have capsules with viable seeds. The seeds collected from the forest were stored in an absorbant paper bag with respective codes for the plant, after they were stored in a Ziplock bag with rice of 12% humidity.\n\nTwo types of freezing were tested, suggested according to Mroginski et al7. The sample units had 0.2 g of seeds stored in cryo tubes (091.11.102, ˙ISOLAB, Wertheim, Germany) of 2 ml. Steps of freezing: freezing was carried out in the following sequence, 0°C for 1 hour by placing the samples in an refrigerator (Electrolux, Stockholm, Sweden), -22°C for 1 hour placing the seeds in a freezer (Selecta Templow, Barcelona, Spain), - 60°C for 1 hour inserting the seeds in an ultra low temperature freezer (New Brunswick Scientific, Edision, NJ, USA), then the seeds were held at 196°C by submerging the samples in liquid nitrogen contained in a thermal container. Finally the samples were placed in racks and stored in a thermal tank (STATEBOURNE biorack 5400, Washington, UK). Rapid freezing: the samples were placed directly in liquid nitrogen at 196°C by immersion using a procedure similar to that used in steps of freezing. In addition, four combinations of cryo preservatives were analyzed: 1- DMSO 1M (Fisher Scientific, Hampton, NH, USA); 2- DMSO 1M (Fisher) – glycerol 1M; 3- DMSO 1M (Fisher) – sucrose 1M; 4-DMSO 1M (Fisher) – glycerol 0.5M – sucrose 0.5M (Fisher) (Table 1).\n\nM: molar.\n\nSeed viability was tested after freezing. Briefly, 5mg of seeds was added to 1.5 ml of 10% sucrose solution and left at 25° C for 24 hours, the seeds were washed with water and 1ml of triphenyl tetrazolium chloride solution (TTC, 1%) (Sigma-Aldrich, St Louis, MI, USA) was added, and then incubated at 40° C for 24 hours. Finally, the seeds were washed with sterilised water and observed under the microscope with a 4x lens (MC100Led, MI-CROS, St. Veit/Glan, Austria). The process for calculating the TTC method was carried out as follows: -Observe the seeds in microscope using lense 4X. -Identify viable seeds and non viable seeds. -Use cross multiplication to determine the average of viability of all seeds.\n\nThe experimental design 2x5 with three repetitions was applied to analyse the freezing methods (Table 2). The results were analyzed by unidirectional ANOVA with 95% confidence. To determine the best treatments the Duncan test was used. This analysis was carried out with RStudio 3.1 (package: Agricolae).\n\n\nResults\n\nThe seeds were considered viable when red coloration of the embryo was observed8 (Figure 2).\n\nViable seeds (dark red embryos) and non-viable (pale embryos). A) Epidendrum quitensium, B) Sobralia rosea, C) Epidendrum anderssonii.\n\nAccording to the data obtained (Table 3, Figure 3), there is a significant difference in the results when comparing the data between the species and between the treatments. According to the Duncan test, the best treatments were rapid freezing and step freezing without the use of cryopreservatives. The least efficient treatment was step freezing with the use of DMSO as a cryopreservant (Table 4). The species Epidendrum quitensium and Epidendrum anderssonii showed better results (Figure 4).\n\nResults obtained using the Tukey test.\n\nValues represent percentage of viability assessed by the TTC method, N: cryo preservative; D: DMSO; S: sucrose; G: glycerol.\n\nResults are given as orchid seed viability percentage; a, b, c and d, indicate groups with statistical significance. Classification was made under an alpha of 0.01, and 78 degrees of freedom for error. Symbols (treatment): N: none, D: DMSO, G: glycerol, S: sucrose. Symbols (types of freezing) P: Freeze steps, R: Rapid.\n\n\nDiscussion\n\nCurrently, cryopreservation is a safe and cost-effective option for the conservation of endangered species9. In the present investigation, a protocol was developed for cryopreservation of orchid seeds that provides a high percentage of viability, is easy to apply and economical. The seeds of orchids frozen at -196°C can be kept alive with a moisture content of 12% and do not require cryo-protective substances, confirming what is described by Iriondo et al. and others10,11. The use of cryopreservatives is recommended for seeds with a high moisture content, as stated by Reed and others12–14. Furthermore, Harding15 states that it is necessary to demonstrate the genetic stability of plants regenerated from cryopreserved plant material to approve their release and reintroduction into the environment; but to date, there have been no reports showing changes at the phenotypic, biochemical, chromosomal or molecular levels attributed to storage systems by cryoconservation14. The cryoconservation method that gave the best results was the “Rapid” freezing without the addition of any cryopreservative substance.\n\n\nData availability\n\nDataset 1: TTC-stained seeds subjected to the “Rapid” cryopreservation process: Epidendrum quitensium 10.5256/f1000research.13622.d19423315\n\nDataset 2: TTC-stained seeds subjected to the “Rapid” cryopreservation process: Sobralia rosea 10.5256/f1000research.13622.d19423416\n\nDataset 3: TTC-stained seeds subjected to the “Rapid” cryopreservation process: Epidendrum anderssonii. 10.5256/f1000research.13622.d19423517\n\nDataset 4: Percentage for seed viability calculations 10.5256/f1000research.13622.d19423618",
"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\nNeill D: Geografía. En Jørgensen P, & P. & S.L.-Y. (Ed.), Catalogue of the vascular plants of Ecuador. Missouri Bot. Gard. 1999; 1181. Reference Source\n\nEndara L, Jost L: Orchidaceae. En León-Yánez S, Valencia R, Pitman N, Endara L, Ulloa C, Navarrete H, El libro rojo de las especies endémicas del Ecuador. (Segunda edición ed.). Quito, Ecuador: Herbario QCA, Pontificia Universidad Católica del Ecuador. 2010.\n\nCerna M, Aucapiña C, López, P: Definición de protocolos para el uso de fitohormonas en el crecimiento de orquídeas a nivel in vitro. (Olmedo GF, Ed.) ESPE: Revista Congreso de Ciencia y Tecnología, 2016; 11. : 12–19. Reference Source\n\nWolfgang S: Orchid seeds– Nature’s tiny treasures. London: Kew Royal Botanical Garden. Recuperado el 12 de Enero de 2015, de, 2013. Reference Source\n\nHine A, Vargas P, Abdelnour A: Crioconservación de semillas de teca (Tectona grandis L.f). Agronomía Costarricence. 2013; 37(1): 51–60. Reference Source\n\nMroginski L, Roca W, Kartha K: Crioconservación del germoplansma. En Roca W & L M, Cultivos de tejidos en la agricultura: Fundamentos y aplicaciones. Cali, Colombia: CIAT. 1991; 715–730Reference Source\n\nMachado-Neto N, Custódio C, Hosomi S, et al.: Orchid seed Stores for Sustainable Use: Fast protocol for Tetrazolium test in orchid seeds. OSSSU.ORG. 2009. Reference Source\n\nGonzáles M, Engelmann F: Crioconservación de plantas en América Latina y el Caribe. San José, Costa Rica: IICA. 2013. Reference Source\n\nIriondo J, Pérez C, Pérez-García F: Effect of seed storage in liquid nitrogen on germination of several crop and wild species. Seed Sci Technol. 1992; 20: 165–171. Reference Source\n\nAbdelnour A, Alvarado C: Crioconservación de semillas y protocormos de especies de la familia Orchidaceae en peligro de extinción. Costa Rica: Instituto Tecnológico de Costa Rica. 2013. Reference Source\n\nReed BM: Implementing cryogenic storage of clonally propagated plants. Cryo Letters. 2001; 22(2): 97–104. PubMed Abstract\n\nGonzales MT, Engelmann F: Cryopreservation of plant germplasm using the encapsulation-dehydration technique: review and case study on sugarcane. Cryo Letters. 2006; 27(3): 155–168. PubMed Abstract\n\nEngelmann F: Integration of cryopreservation in plant genetic resource conservation strategies in France. Cryo Letters. 2010; 31–82.\n\nHarding K: Genetic integrity of cryopreserved plant cells: a review. Cryo Letters. 2004; 25(1): 3–22. PubMed Abstract\n\nCerna M, Valdiviezo P, Cella R, et al.: Dataset 1 in: Cryopreservation of orchid seeds through rapid and step freezing methods. F1000Research. 2018. Data Source\n\nCerna M, Valdiviezo P, Cella R, et al.: Dataset 2 in: Cryopreservation of orchid seeds through rapid and step freezing methods. F1000Research. 2018. Data Source\n\nCerna M, Valdiviezo P, Cella R, et al.: Dataset 3 in: Cryopreservation of orchid seeds through rapid and step freezing methods. F1000Research. 2018. Data Source\n\nCerna M, Valdiviezo P, Cella R, et al.: Dataset 4 in: Cryopreservation of orchid seeds through rapid and step freezing methods. F1000Research. 2018. Data Source"
}
|
[
{
"id": "31828",
"date": "28 Mar 2018",
"name": "Alzbeta Novotna",
"expertise": [
"Reviewer Expertise Isolation",
"molecular determination and cryopreservation of orchid mycorrhiza fungi and associated bacteria. Experienced with orchid seed banking techniques."
],
"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 in the presented manuscript titled “Cryopreservation of orchid seeds through rapid and step freezing methods” a new protocol for cryopreservation of seeds of three tropical orchids in liquid nitrogen (LN). They tested rapid and progressive cooling with the controlled temperature at 0°C, -22°C, -60°C and -196°C. They also included the application of three different cryoprotectants such as DMSO, glycerol and sucrose. The viability of tested seeds was checked using tetrazolim test. Red coloured embryos were considered as the main indicator of the preserved viability of the tested seeds. I suppose that this work brings a novelty to a cryoscience research, especially due to the geographical origin of the studied material. Neotropical Orchidaceae should be prioritized in various research studies for their ecological vulnerability and/or a shortage of knowledge. Especially, the research achievements from such country as Ecuador (the orchid biodiversity hotspot), are important for science. However, numerous modifications and a deep correction of the text should be accomplished.\nEnglish correction is strongly recommended throughout the whole text. There are many errors in English grammar in the current form of the manuscript, which cause huge difficulties in text reading. Abstract: The authors should be focused on the topic of cryopreservation. The first two sentences in the abstract are out of topic. Moreover, the number of the orchid species (provided number 4032) is not correct. The authors should consider to check the work of D. Neill published in 2015 (¿Cuantas especies nativas de plantas vasculares hay en Ecuador?; Revista Amazónica Ciencia y Tecnología) and Cataloque of the Vascular Plants of Ecuador available on www.tropicos.org, for example. Abstract, sentence “In cryopreservation, temperatures reach below -190°C” should be rephrased because the cryostorage is a long-lasting preserving of studied material at temperature of -80°C, -130°C upto -196°C, which is the temperature of the liquid nitrogen. Abstract, the authors should rephrase the sentence “The present research focuses on……” because of repetition of the verb to focus. Instead of “…for a subsequent generation of a seed bank” to use “for future seed collection” for example. Abstract, “the use of cryo-protective substances”, better to use the term „cryoprotectants“. Introduction; The first half of the abstract is out of the topic of cryopreservation. The authors should be focused on this topic. Moreover, the authors should provide much more citations regarding this topic and discuss shortly the history of cryopreservation of seeds with more interest in orchid seeds. There are many available papers regarding this topic. Methods; The first paragraph should be included in “Acknowledgement” at the end of the paper, not in methods. However, each part of the Methods should be provided with a number, e.g. 1. 1 Collection of the seed material. The sentence “The cryopreservation test were developed with 3 species” is repetition of the first sentence of this paragraph. The authors should use \"a.s.l.\"as the abbreviation for above sea level; the provided abbreviation \"msnm\" is not understandable. The part of the sentence” The species pertain to three different altitudes….. „ is a repetition of the information written before. Authors write “…..were selected from many sources“, what does it mean? The authors should provide more detailed information about the sources. Then, the part of the sentence …“and have capsules with viable seeds” does not bring the clear information. Were the capsules opened during collection or not? When did the authors collect the seeds (the date)? Methods; Freezing speed. Better to rewrite the title, e.g. Cryopreservation of the seeds This part should be shorten. Besides, the citation of the first sentence Mroginski et al. does not correspond with the number 7. Totally different citation is written under this number. The authors should check the citations of provided literature in the text. Moreover, the authors should provide the information about duration of cryostorage. This very important information is missing. In the paragraph “Freezing speed” the authors provide information about placing the tested orchid seeds under the temperature 0°C in a refrigerator. However, this fact is not written in the abstract. The authors should eliminate “…., contained in a thermal container”. The sentence “Finally the samples were places in racks and store in thermal tansk……” should be eliminated, because it is not understandable. Freezing speed; In addition,….should be eliminated because the application of cryoprotectants is one of the main objectives of this research paper. It is not something additional. Why did the authors not test application of sucrose and glycerol individually? The authors should clearly demonstrate, which treatment is considered as a control. Table 1 should be deleted, because all the information given in this table is written in the text. Figure 1; Title should be improved, e.g. “Orchid species used in this study”. The paragraph of “Seed viability” should be eliminated. Instead just one sentence with a citation of the work, where this method was used as first, should be placed at the end of the previous paragraph. Statistical analysis; The authors should explain better of the meaning “The experimental design 2x5”. It is not clear from the written text. Results; The sentence “The seeds were considered viable when red…..” is actually the part of Material and methods. Results; this part should be re-written. The information should be provided more clearly. Table 3; What is CI? Better to use term “Gradual” instead of “step”. The abbreviations of each treatment are already provided in Material and Methods. Figure 3; Authors should provide information about the meaning of “E” (in e.g. ED, EDG) and “R” (in e.g. RD, RDG). Figure 4; Authors should provide information about the meaning of sp2392, sp2420 and sp2706. It is not clear. Discussion; First, the obtained main result should be provided and discussed with much more available literature. Especially, the authors should compare their findings with achievements in other studies. The provided references are very limited. The works dealing with cryostorage of seeds of other plant families should be included. The given number of cited paper in discussion does not correspond to the number provided in the References.",
"responses": []
},
{
"id": "32906",
"date": "09 Apr 2018",
"name": "Song-Jun Zeng",
"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 described a protocol for cryopreservation of seeds of three tropical orchids in liquid nitrogen (LN). They tested rapid and progressive cooling with the controlled temperature at 0°C, -22°C, -60°C and -196°C. They also included the application of three different cryoprotectants such as DMSO, glycerol and sucrose. The viability of tested seeds was checked using tetrazolim test. The protocol might have potential for the cryopreservation of these orchids. However, the manuscript cannot be accepted to publish at present and need major revision for the following main reasons:.\nAbstract: Authors could not bring the conclusion ‘Species of the genus Epidendrum presented a more efficient response in comparison to Sobralia.’, because only one species was be tested in the this manuscript.\n\nIntroduction: Orchidaceae is a large family with many endangered species and all of them are included in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) I and II. And should be revised as ‘or’. Current research of orchid Cryopreservation should be introduced in the section.\n\nMethods: DPMS/MAE,FloraX the behind of ‘,’ should have a blank.\n\nEpidendrum quitensium Rchb.f., Sobralia rosea Poepp. & Endl. and Epidendrum anderssonii Hágsater & Dodson Rchb.f., Poepp. & Endl. and Hágsater & Dodson should not be italic.\n\n2392 Epidendrum quitensium Rchb.f., 2420 Sobralia rosea Poepp.& Endl., Epidendrum anderssonii Hágsater & Dodson Epidendrum quitensium, Sobralia rosea, Epidendrum anderssonii should be italic.\n\nFigure 1: Epidendrum quitensium, Sobralia rosea, Epidendrum anderssonii should be italic. Figure 2: A) Epidendrum quitensium, B) Sobralia rosea, C) Epidendrum anderssonii A, B, C should be labelled in Photos.\n\nFigure 3: What is ED, EDD……what is the meaning of “E”?\n\nsp2706 (T57.2), sp2392 (T 57.03), sp2420(T39.37), should be replaced by Latin name of orchids species.\n\nDiscussion: The discussion should be rewritten. There were few relevant literatures of orchid Cryopreservation were cited in this manuscript. There have lots of related literatures for orchid Cryopreservation at present, which should be discussed and what is the innovation of the manuscript?\n\nAll the source data should be executed by statistical analysis.",
"responses": []
},
{
"id": "33998",
"date": "11 Jul 2018",
"name": "Éva Borbélyné Hunyadi",
"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 paper entitled \"Cryopreservation of orchid seeds through rapid and step freezing methods\" written by Marco Cerna et al. two types of freezing technique were tested by the seeds of 3 species: Epidendrum quitensium Rchb.f., Sobralia rosea Poepp. & Endl. and Epidendrum anderssonii Hágsater & Dodson.\nThe authors present step freezing and rapid freezing methods for cryopreservation purposes. There was a significant difference in the results between the species and between the treatments. The cryoconservation method that provided the best results was the \"Rapid\" freezing, without the addition of any substance.\n\nThe introduction is well structured, helping the readers to understand the issue the method tries to solve. Considering that it is a research note the methodology is very detailed.\n\nNevertheless, in my opinion it is important to highlight the viability of the developed method on a wider spectrum if possible. In the current form of the Discussion, only general statements can be found regarding the viability of the developed method.\nA more concrete concluding sentence is missing that could answer the following question:\nFor what kind of other plants, could this method be useful?\nIf the developed method can be applied \"only\" for orchid seeds, a concluding sentence should be placed in the Discussion, stating the limiting factors (in terms of species) of the method. Although in this study only 3 species were examined, I believe that its important to inform the readers about the authors' recommendation regarding the potential usefulness in other species.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-209
|
https://f1000research.com/articles/7-208/v1
|
20 Feb 18
|
{
"type": "Research Article",
"title": "Cross sectional study to determine chloroquine resistance among Plasmodium falciparum clinical isolates from Khartoum, Sudan",
"authors": [
"Walaa Salah Abdulla Mohammed",
"Kyakonye Yasin",
"N.S. Mahgoub",
"Muzamil Mahdi Abdel Hamid",
"Walaa Salah Abdulla Mohammed",
"Kyakonye Yasin",
"N.S. Mahgoub"
],
"abstract": "Background: Malaria continues to present a global health threat; the World Health Organization (WHO) reported 214 million cases of malaria by the year 2015 with a death rate of 438000. Sudan is endemic to malaria with over 95% of malaria cases due to Plasmodium falciparum. Chloroquine is a well-established drug in the treatment of P. falciparum malaria although its use has declined since its introduction as the drug of choice in treatment of malaria in Sudan. The mechanism of resistance has been attributed to mutations in P. falciparum Chloroquine resistance transporter gene coding for a key food vacuole proteins. In current study we aimed at verifying the genetic cause of resistance to Chloroquine in field isolates of P. falciparum. Methods: Twenty P. falciparum cases were diagnosed from East Nile hospital in Khartoum and recruited in the investigation. Nested PCR was conducted to isolate mutation region in the PfCRT gene and the amplicons were sequenced using Sanger sequencing technique (Macrogen, Soule Korea). Results: 16/20 (80%) of the field isolates contained base pair mutation of codon 76 in the pfcrt gene thus being resistant to chloroquine treatment and only 4/20 (20%) did not contain such mutation. Conclusions: High treatment failures associated with Chloroquine treatment is evident of the high prevalence of mutant strains of P. falciparum field isolates thus suggesting the reduced relevance of Chloroquine as a treatment choice in the management of P. falciparum malaria.",
"keywords": [
"Chloroquine",
"resistant",
"sensitive",
"P. falciparum",
"pfcrt"
],
"content": "Introduction\n\nThe use of chloroquine in low middle income countries (LMICs) has helped to reduce mortality and morbidity. In 1940, sixteen years after its discovery, Chloroquine had been used as a first drug of choice for malaria treatment due to its high efficacy, especially in highly endemic areas in Africa1. It has been used as the main treatment for malaria in Africa for over 50 years. Since the late 1980s, resistance to chloroquine has rapidly increased across much of Eastern and Southern Africa2.\n\nDrug resistance of Plasmodium falciparum represents one of the greatest public health challenges in Africa. Tropical Africa accounts for more than 90% of the 300–500 million clinical cases of malaria occurring each year3. Malaria is transmitted by the female anopheles mosquito with P. falciparum being most prevalent; however recent studies in most parts Sudan have indicated a high prevalence of P. vivax infection4.\n\nChloroquine resistance is associated with a parasitic digestive vacuole, in which the toxic free heme is converted into an insoluble non-toxic crystalline form called hemozoin5. In sensitive parasites, Chloroquine diffuses across the food vacuole membrane and accumulates inside it to inhibit hemozoin crystallization, which leads to formation of another highly toxic-dimeric hematin complex1,5. Resistant strains fail to accumulate sufficient Chloroquine inside their food vacuoles as a result of mutations in the FV membrane proteins5. Mutation in P. falciparum Chloroquine resistance transporter gene Pfcrt, which codes for one of the food vacuole proteins is considered the strongest predictor for Chloroquine resistance5. This mutation leads to the substitution of the amino acid lysine with threonine at position 76 (K76T)5.\n\nBy 2004, the National Malaria Control Programme of Sudan updated the policy of malaria treatment due to high prevalence of Chloroquine resistant cases. In this policy, Artesunate + Sulfadoxine-Pyrimethamine (AS + SP) was adopted as the first-line treatment for uncomplicated P. falciparum malaria, and Artemether-Lumefantrine (AL) was adopted as a second-line treatment6.\n\nHowever, until now, Chloroquine is still considered as one of the cheapest and safest drugs ever used for malaria treatment. Besides, recent clinical observations show a decreasing trend in prevalence of P. falciparum Chloroquine resistance, which has brought Chloroquine reintroduction back into the discussion about the management of malaria7,8.\n\nHence, in this pilot study, we aimed to assess the prevalence of Chloroquine resistant P. falciparum from clinically isolated samples by sequencing the Pfcrt gene in patients with uncomplicated malaria in Khartoum, Sudan.\n\n\nMethods\n\nThis study was reviewed and approved by Institute Of Endemic Diseases Research and Ethics Committee (ethical approval number 6/2016). Oral informed consent was obtained from participating patients or from parents or guardians for the case of minors (under 19 years). Oral consent was obtained over written consent, since the majority of the patients included in this study were illiterate.\n\nA cross sectional study was conducted between December 2015 and January 2016 within Khartoum East Nile Hospital. According to Isaac and Michael (1995) suggestion of pilot study sample size, twenty patients, between age 4–55 years, have been randomly selected from the hospital attendance sheet9. After getting positive results of uncomplicated malaria diagnostic tests, the selected patients were immediately asked to be a part of the present study. Thirteen of the patients were women and seven were men. Demographic data was obtained from participating patients including ethnicity and recent history of malaria infection. Only patients confirmed with P. falciparum monoinfection were included in further studies. Pregnant or lactating women and patients presenting with signs or symptoms of severe malaria or having a history of taking antimalarials during the previous month or another febrile disease requiring treatment were excluded.\n\n1ml blood sample was collected from each patient and stored in a labeled EDTA tube, thick and thin blood films were prepared for microscopical examination to detect and isolate Plasmodium species and estimate of parasitemia level.\n\n1ml blood sample, for mutation molecular detection, was collected from each patient and stored in a labeled EDTA tube. A mixture of 1ml of Saponin (0.5%) (Sigma-Aldrich) and 150 µL of each blood sample was incubated overnight at 4°C, and then centrifuged at 6000 rpm speed. 1ml of phosphate-buffered saline (PBS) (Sigma-Aldrich) was added and the mixture was centrifuged, at 6000 speed, for four consecutive times. 70 µL of chelex (Bio-Rad) and 30 µL of water were added to the precipitant and the mixture heated at 95°C for 20 minutes in 4 rounds (5 min/round). After every round the mixture was vortexed for 1 minute. DNA was collected and stored at -20°C until a PCR reaction was conducted.\n\nFor round 1 of nested PCR, 2µL of extracted DNA was multiplied using 0.5µL of Taq DNA polymerase, 2µL buffer, 2µL dNTPs, 10.5µL nuclease-free PCR water, 1.5µL of forward primer (CRTP1; Macrogen), and 1.5µL reverse primer (CRTP2; Macrogen). Primer details are provided in Table 1. The PCR initial denaturation was conducted at 95°C for 3min, 94°C for 30 s for the successive denaturation steps, DNA annealing was set at 56°C for 30 s and elongation at 60°C for 1 min, a total of 45 cycles were conducted and final extension at 72°C for 5min.\n\nFor round 2 of nested PCR, 1 µL of round 1 product was used with CRTD1 (Macrogen), as forward primer and CRTD2 (Macrogen) as reverse primer, with the PCR profile as summarized below.\n\nInitial denaturation at 95°C for 5 min and 92°C for 30 s for succeeding denaturation steps, annealing was conducted at 48°C for 30 s, elongation at 65°C for 30 s. Final elongation was conducted at 65°C for 3 min. The DNA amplicons were run in 1.5% agarose gel stained with 2% ethidium bromide electrophoresis gel and finally bands were visualized under UV light. The amplicons were then sequenced using Sanger sequencing techniques at Macrogen (Soul Korea). All sequences were obtained and compared to the nucleotide sequence of Plasmodium falciparum 3D7 reference strain provided by Biomedical Primate Research Centre (BPRC, Netherlands).\n\nSequence cleaning and blast analysis were conducted using FinchTV software version 1.4.0. Visualization of nucleotide sequences and alignment with wild type sequences was conducted using Bioedit software version 7.2.5.\n\n\nResults\n\nAll samples from 20 patients were successfully amplified. Nucleotide sequences were obtained and analyzed using bioinformatics analysis. Nucleotide sequences were aligned and compared to standard sequence of Plasmodium falciparum 3D7 reference strain. 16/20 (80%) of the sequenced DNA contained ACA triplet codon, which codes for Threonine at the Apol 1 restriction enzyme cutting region. Only 4/20 (20%) samples contained the AAA triple codon which codes for Lysin in the wild type Pfcrt gene, as shown in Figure 1.\n\n(a) Multiple sequence alignment highlighting the Pfcrt gene at the Apol 1 cutting region. (b and c) Highlight point mutation in Pfcrt gene where the A nucleotide in a wild-type is substituted with C nucleotide in the mutant strain, (d and e) the nucleotide sequence chromatogram of wild type and mutant strains respectively. (f and g) The translated amino acid in wild and mutant strains, respectively.\n\n\nDiscussion\n\nChloroquine has been widely used in many malaria endemic regions of many sub-Sahara African regions for its cost benefit and cost effective effects; and has therefore been considered as the golden choice in the treatment of malaria10. In the current study, Chloroquine resistance showed an increase to 80% from the previously documented figure of 72.7% in 2007 in Sudan8. This evidence differs from that documented from surveys in Malawi, Tanzania and Mozambique as in such regions there has been a decreasing trend of Chloroquine resistance prior to withdrawal of Chloroquine from the treatment guidelines11.\n\nIn Malawi, resistant strains of P. falciparum (Pfcrt- T76) had declined from >t;85% to 0% within 13 years following Chloroquine withdrawal from treatment guidelines. In Tanzania P. falciparum resistance reduced from >80% to <10% in ten years, and in Mozambique statistics showed a decline of Chloroquine resistance from >95% to 20% within five years of Chloroquine withdrawal11. However, studies from Kenya indicated a slow decline in Chloroquine resistant P. falciparum from 95% to 60% between 1993 to 2006 following malaria policy changes12. The results of the present study are similar to studies from Uganda as the frequency of Pfcrt- T76 was between 100 and 98.7% in 2008, about eight years post-Chloroquine replacement11.\n\nIn present study, the small sample size represents a limitation and the final result shows obvious persistence of P. falciparum chloroquine resistant strains in endemic areas where Chloroquine use was continued following the World Health Organization's declaration of sidelining Chloroquine as a first treatment choice for malaria.\n\n\nConclusions\n\nPfcrt K76T mutation still persists in Sudan, which makes it impossible for the reintroduction of Chloroquine as an antimalarial treatment choice on the current National clinical guidelines of Sudan. However for a better implementation of this policy further studies need to be conducted with a larger representative sample from all regions of Sudan as this was a primary survey that involved a narrow sample.\n\n\nData availability\n\nDataset 1: Raw sequences for the 20 patients included in the present study. Files included: sequence in pdf format, sequence for opening in FinchTV software (.ab1 file), and sequence for opening in Notepad (TXT file). DOI, 10.5256/f1000research.13273.d19297813",
"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\nZhou R, Zhang H, Yang C, et al.: Molecular mutation profile of pfcrt in Plasmodium falciparum isolates imported from Africa in Henan province. Malar J. BioMed Central; 2016; 15(1): 265. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWinstanley P, Ward S, Snow R, et al.: Therapy of falciparum malaria in sub-saharan Africa: from molecule to policy. Clin Microbiol Rev. 2004; 17(3): 612–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBloland PB, Kazembe PN, Oloo AJ, et al.: Chloroquine in Africa: critical assessment and recommendations for monitoring and evaluating chloroquine therapy efficacy in sub-Saharan Africa. Trop Med Int Health. 1998; 3(7): 543–52. PubMed Abstract | Publisher Full Text\n\nSuliman MMA, Hamad BM, Albasheer MMA, et al.: Reemergence of chloroquine (CQ) as multi-targeting antimalarial agents: 2016. 2016.\n\nMushtaque M, Shahjahan S: Reemergence of chloroquine (CQ) analogs as multi-targeting antimalarial agents: a review. Eur J Med Chem. Elsevier Ltd; 2015; 90: 280–95. PubMed Abstract | Publisher Full Text\n\nAdeel AA, Elnour FA, Elmardi KA, et al.: High efficacy of artemether-lumefantrine and declining efficacy of artesunate + sulfadoxine-pyrimethamine against Plasmodium falciparum in Sudan (2010–2015): evidence from in vivo and molecular marker studies. Malar J. BioMed Central; 2016; 15(1): 285. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang X, Mu J, Li G, et al.: Decreased prevalence of the Plasmodium falciparum chloroquine resistance transporter 76T marker associated with cessation of chloroquine use against P. falciparum malaria in Hainan, People’s Republic of China. Am J Trop Med Hyg. 2005; 72(4): 410–4. PubMed Abstract\n\nMenegon M, Talha AA, Severini C, et al.: Frequency distribution of antimalarial drug resistance alleles among Plasmodium falciparum isolates from Gezira State, central Sudan, and Gedarif State, eastern Sudan. Am J Trop Med Hyg. 2010; 83(2): 250–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohanson GA, Brooks GP: Initial scale development: sample size for pilot studies. Educ Psychol Meas. 2010; 70(3): 394–400. Publisher Full Text\n\nTajebe A, Aemero M, Francis K, et al.: Identification of chloroquine resistance Pfcrt-K76T and determination of Pfmdr1-N86Y copy number by SYBR Green I qPCR. Asian Pac J Trop Biomed. Hainan Medical University; 2015; 5(3): 208–20. Publisher Full Text\n\nMohammed A, Ndaro A, Kalinga A, et al.: Trends in chloroquine resistance marker, Pfcrt-K76T mutation ten years after chloroquine withdrawal in Tanzania. Malar J. 2013; 12(1): 415. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMwai L, Ochong E, Abdirahman A, et al.: Chloroquine resistance before and after its withdrawal in Kenya. Malar J. 2009; 8: 106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbdulla Mohammed WS, Kyakonye Y, Mahgoub NS, et al.: Dataset 1 in: Cross sectional study to determine chloroquine resistance among clinically isolated samples of Plasmodium falciparum from Khartoum, Sudan. F1000Research. 2018. Data Source"
}
|
[
{
"id": "31876",
"date": "03 Apr 2018",
"name": "Jagadish Mahanta",
"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 concept is old and methodology is well established, however geography specific information still has the relevance and the results of such studies have policy implications in the programme. In this context, the investigators should give elaborate justification for revisiting resistance pattern at molecular level by including the clinical experience of physicians or in-vitro drug sensitivity using CHQ across the country.\n\nAs the studies of such nature have geographical relevance, the coverage should have been wide rather than limiting in a small area.\n\nAs stated in the text, drug policy has been changed since 2004 and chloroquine is no longer in use, therefore sampling should have been from different areas of the country and detail description of the patient’s therapeutic history, residential status and travel detail prior to infection should have been included.\n\nInvestigator should elaborate on site selection, patient selection and randomization detail during sampling.\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?\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": []
},
{
"id": "33752",
"date": "08 May 2018",
"name": "Satish K. Dhingra",
"expertise": [
"Reviewer Expertise P. falciparum drug resistance"
],
"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. The premise of this study uses PfCRT K76T as a molecular maker of chloroquine resistance. However, a recent study from French Guiana in South America has shown that despite the presence of PfCRT K76T parasites can be sensitive to chloroquine (Pelleau et al. 2015, PNAS). So stating that parasite population in Sudan are clinically resistance to CQ based only upon the status of position 76 is incorrect. The authors need to sequence full length pfcrt in order to draw that conclusion.\n2. Overall the sample size is small and not very diverse.\n3. Authors fail to cite key publications, as an example Fidock et al. 2000, Mol Cell.\n\n4. First sentence, second paragraph in discussion is missing a citation.\n5. The authors need to also elaborate of the prevalence of P. vivax in the area and whether reintroduction of CQ is for targeting this species and how this can impact the population structure of the P. falciparum.\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": []
},
{
"id": "31878",
"date": "16 May 2018",
"name": "Gabriel N. Magoma",
"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 for the research on the determination of prevalence chloroquine resistance among clinical isolates is generally sound given the significance of the Malaria disease.\nThe statement that there chloroquine has been in used despite the ban is not clearly articulated. The argument supporting the reintroduction of chloroquine is not well articulated. The sample size is also quite small and the selection criteria of study participants is not well supported. There is an implication of inclusion of children statement that there were 13 women and 7 men contradict the inclusion of children.\nThe methods are not sufficiently detailed since the gel photos of the amplification products are missing and there is lack of explanation as to how the products were treated before sequencing. If this are articulated it is possible to ensure that the methods are fully reproducible.\nThe analysis of the presence of the mutation, the interpretation and subsequent conclusions are generally adequate.\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?\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/7-208
|
https://f1000research.com/articles/6-1136/v1
|
17 Jul 17
|
{
"type": "Method Article",
"title": "Virtual-screening workflow tutorials and prospective results from the Teach-Discover-Treat competition 2014 against malaria",
"authors": [
"Sereina Riniker",
"Gregory A. Landrum",
"Floriane Montanari",
"Santiago D. Villalba",
"Julie Maier",
"Johanna M. Jansen",
"W. Patrick Walters",
"Anang A. Shelat",
"Gregory A. Landrum",
"Floriane Montanari",
"Santiago D. Villalba",
"Julie Maier",
"Johanna M. Jansen",
"W. Patrick Walters"
],
"abstract": "The first challenge in the 2014 competition launched by the Teach-Discover-Treat (TDT) initiative asked for the development of a tutorial for ligand-based virtual screening, based on data from a primary phenotypic high-throughput screen (HTS) against malaria. The resulting Workflows were applied to select compounds from a commercial database, and a subset of those were purchased and tested experimentally for anti-malaria activity. Here, we present the two most successful Workflows, both using machine-learning approaches, and report the results for the 114 compounds tested in the follow-up screen. Excluding the two known anti-malarials quinidine and amodiaquine and 31 compounds already present in the primary HTS, a high hit rate of 57% was found.",
"keywords": [
"Malaria",
"neglected diseases",
"virtual screening",
"machine learning"
],
"content": "Introduction\n\nTeach-Discover-Treat (TDT) is an initiative that aims to provide high-quality tutorials of important tasks in computer-aided drug discovery, in order to impact education and drug discovery for neglected diseases1. The TDT steering committee consists of computational chemists from both academia and industry. To encourage the creation of high-quality tutorials by the computational chemistry community, competitions are launched with a series of different challenges, and the results/tutorials are made available through the website of the initiative (http://www.tdtproject.org). The competitions are open to everybody. After the first successful competition in 20122, a second competition was launched in 2014, with four challenges. In this study, we focus on Challenge 1: ligand-based virtual screening (VS) against malaria. The goal was to build a predictive model for anti-malaria activity based on a phenotypic high-throughput screen (HTS), and to use that model subsequently to select the next set of compounds for screening. In a ligand-based VS, typically no structural information of the target is available, and thus the prediction of potentially active compounds is based on the principle that similar molecules exhibit similar activity3. The challenge is thereby to find an appropriate molecular description for similarity, which can depend heavily on the compound selection and/or target4–7. In recent years, machine-learning (ML) methods have emerged as an attractive tool to boost the predictive power of ligand-based VS approaches8–12.\n\nMalaria is caused in humans by several species of the protozoan parasite Plasmodium. The most lethal species is Plasmodium falciparum (Pf), which causes organ failure and accumulates in the brain capillaries if left untreated. Malaria is still one of the most prevalent and deadly diseases in Africa, Asia and the Americas, with an estimate of 198 million cases in 2013 leading to approximately 584,000 deaths according to the 2014 world malaria report of the World Health Organization (WHO)13. Recent advances in malaria research and drug discovery have been reviewed14–19. The anti-malaria drugs can be broadly classified into three groups: (i) compounds that interfere with the heme detoxification, (ii) compounds that target folate metabolism, and (iii) compounds that inhibit mitochondrial electron transport. The current standard of care for uncomplicated malaria is artemisinin-based combination therapies. Artemisinins belong to the third group of anti-malaria drugs and rapidly kill all the blood stages of the parasite, however, they are also cleared in a short time20. Unfortunately, the emergence of resistant strains has become a major problem in recent years21,22, requiring the development of new and possibly orthogonal drugs. In the past, whole-cell phenotypic screening campaigns against Pf have been successful in identifying new lead compounds23.\n\nChallenge 1 of the 2014 TDT competition involved three tasks: (i) analysis of the data from a single-concentration phenotypic HTS of 305,568 compounds, including hit-list triaging and selection of compounds for a follow-up screen with EC50 measurement, (ii) building and validation of a predictive anti-malaria activity model, including a held-out test-set of 1056 compounds, and (iii) follow-up hit finding by applying the predictive model to rank-order a large dataset of commercially available compounds. The top 1000 molecules of this ranked list were considered further for experimental testing. For training, the challenge provided results for 305,568 compounds from the primary HTS, as well as EC50 data from a follow-up confirmatory screen for a subset of the compounds.\n\nIn this study, we present the results of two Workflows. Workflow 1 was the overall winner of the competition, and Workflow 2 showed the best performance on the held-out test set measured in the phenotypic Pf screen. Note that the two Workflows interpreted the challenge differently. In Workflow 1, only data from the primary HTS was used in the training of the predictive model in order to mimic the early phase of a drug discovery campaign. In Workflow 2, the EC50 data from the confirmatory assay was taken into account in order to improve the labelling of the training set. Each Workflow provided a ranked list of the top 1000 molecules, from which a total of 114 compounds (80 from Workflow 1 and 38 from Workflow 2, four were in common) were selected based on vendor availability for screening in a Pf phenotypic assay. Excluding the two known anti-malarials quinidine and amodiaquine and the 31 compounds already present in the primary HTS, 46 of 81 compounds were found to be active in the follow-up assay, which corresponds to a hit rate of 57%.\n\n\nMethods\n\nThe basis for the virtual screening workflows was a phenotypic high-throughput screen against Pf with 305,568 compounds, together with a confirmatory dose-response screen for 1524 compounds, which are reported in 23. The data is deposited in ChEMBL as part of the Neglected Tropical Diseases set (ChEMBL-NTD)24. The data is also available on the TDT website (http://www.tdtproject.org/challenge-1---malaria-hts.html). In addition, an external held-out test set with 1056 molecules was provided for comparison of submissions25. This dataset was generated in the laboratory of R. K. Guy in 2014, following the same procedure as described in 23, at the time of the TDT competition. Results for this held-out set are given in the Supplementary material.\n\n\nWorkflow 1\n\nThe tutorial was written in the form of an IPython notebook and a series of Python scripts for the computationally demanding tasks to be executed separately. The tutorial is available on the TDT website (http://www.tdtproject.org) and on GitHub (https://github.com/sriniker/TDT-tutorial-2014). The tutorial makes use of a number of open-source Python libraries: the cheminformatics toolkit RDKit version 2013.09 (http://www.rdkit.org), the machine-learning toolkit scikit-learn version 0.13 (http://scikit-learn.org), pandas for working with data tables, and libraries for scientific computing, numpy version 1.6.2 and scipy version 0.9.0. Figures are plotted using matplotlib version 1.1.0. The components of the Workflow are shown schematically in Figure 1.\n\nThe input for the workflow was the hit list from the phenotypic HT screen, with a classification into ‘active’, ‘inactive’, and ‘ambiguous’ compounds23. From the original 305,568 compounds tested in the screen, 1528 were found to be active and 293,608 inactive. The 10,432 molecules with an ambiguous outcome were discarded.\n\nTo triage the hit list in the first task, property filters (Table 1) based on previously described filters26,27 were applied for in silico post-processing of the primary HTS data, which resulted in 1512 remaining active compounds.\n\nThese filters are used in Workflow 1.\n\nNext, the active molecules were checked for potentially problematic substructures using the PAINS filters described in 28. 1225 molecules passed these filters. From these, 500 molecules had to be picked for testing in a confirmatory assay. While making this selection, a balance between the desire to have a good sampling of the chemical space covered by the primary actives and the desire to get some structure-activity relationship (SAR) information from the confirmatory assay had to be found. The compounds were therefore clustered using the Butina algorithm29 based on Tanimoto similarity with a cutoff = 0.5. The Tanimoto similarity was calculated using RDKit fingerprints (a subgraph-based fingerprint similar to the Daylight fingerprint), with a maximum path length of five. 304 clusters were found, with only 40 clusters having more than five members. The cluster centers provide a set of diverse seeds. To ensure the chance to obtain information about SAR, molecules around the cluster centers were selected: Starting with the largest cluster, the five molecules most similar to the cluster center (or 50% of the cluster members if the cluster contained less than 5 molecules) were picked.\n\nThree different machine-learning (ML) models together with three different molecular fingerprints were tested for the predictive model in task 2. The ML methods were random forest (RF)30, Naive Bayes (NB) and logistic regression (LR), which showed a good performance in a previous benchmarking study13. The RF models were built using 100 trees, a maximum depth of 100, and minimum one sample in a leaf. For NB and LR, the default parameters in scikit-learn were used. The fingerprints were atom pairs (AP)31, RDKit fingerprint with a maximum path length of five (RDK5) and Morgan fingerprint with a radius of 2 (Morgan2)32, and are described in more detail in 8. In the version of the Workflow submitted in the competition, the AP and RDK5 fingerprints were hashed to 2048 bits, and the Morgan2 fingerprints to 1024 bits. Later on we found that a fingerprint size of 4096 bits resulted in better performances due to fewer collisions. To determine which ML method/fingerprint combinations performed best and should therefore be combined using heterogeneous classifier fusion13, a retrospective evaluation was performed using the primary HTS data. Here, all data points from the primary screen were used (i.e. none of the property-/substructure-filters discussed above were applied) as some filters may be too strict and the ML methods are rather robust to noise. The data points were randomly split 50 times into a training set (90%) and a test set (10%). A ML model was built using the training set and the molecules in the test set were ranked based on the predicted probability to be active. From the ranked list, the receiver operating characteristic (ROC) curve was calculated and subsequently the area under the ROC curve (AUC) was determined. In addition, the enrichment factor at 5% was determined. A detailed discussion of the different evaluation methods is given in 8. The results from the retrospective evaluation, averaged over the 50 repetitions, are listed in Table 2. Based on these results and the analysis of the diversity in the active molecules that were identified, a classifier fusion model was proposed based on RF with RDK5, RF with Morgan2 and LR with RDK5 (Table 2). As a last step, a fusion model was trained using all data points of the primary HTS in order to obtain predictions for the held-out test set and for task 3.\n\nThe random selection was repeated 50 times and the results were averaged over the repetitions. The maximum possible EF5% value is 20.0. Fingerprints with 4096 bits were used.\n\nIn task 3, the goal was to select a list of 1000 compounds from the eMolecules (https://www.emolecules.com) catalog, with nearly 5.5 million commercially available compounds. As a first step, the molecules were filtered using the property filters described in Table 1 except logP. logP was not applied at this stage to reduce the computational cost. This resulted in approximately 4.4 million compounds. For these, molecular fingerprints (RDK5 and Morgan2) were generated with 4096 bits and the anti-malaria activity was predicted using the fusion model trained on the primary HTS in task 2. The top ranked 10,000 compounds were taken for further selection. The logP filter (see Table 1) and PAINS substructure filters were applied at this point. Filtering resulted in 7955 compounds. To select 1000 molecules from these, the following procedure was applied:\n\nThe highest-ranked molecule is selected as first cluster center.\n\nTaking the next lower molecule, the similarity to the first molecule is calculated:\n\n☐ If the similarity is below 0.5, the molecule is selected as a new cluster center.\n\n☐ If the similarity is above 0.85 and the cluster does not contain 6 molecules yet (including the cluster center), the molecule is selected and added to the cluster.\n\n☐ Else the molecule is discarded.\n\nThe procedure was continued until 1000 compounds were selected. Unfortunately, a bug in the selection step of the original tutorial resulted in the 1000 compounds being randomly selected from the top ranked 10,000 compounds. In addition, compounds already in the primary HTS used for training were not explicitly removed from the eMolecules catalog. A corrected version of the tutorial is provided on GitHub (https://github.com/sriniker/TDT-tutorial-2014).\n\n\nWorkflow 2\n\nThe tutorial is available on the TDT website (http://www.tdtproject.org) and on GitHub (https://github.com/sdvillal/tdt-malaria-followup). RDKit version 2013_09_2 (http://www.rdkit.org) was used to read the SMILES strings, compute descriptors and fingerprints. Scikit-learn version 0.14 (http://scikit-learn.org) was used to build the models.\n\nThe input was again the original primary HTS data23 with 1528 active compounds, 293,608 inactive compounds and 10,432 molecules with an ambiguous outcome. In addition, pEC50 data from a dose-response confirmatory screen for 1524 compounds23 was taken into account. Compounds were relabeled using, when available, the confirmatory pEC50 data. Any compound with a pEC50 of at least 5 was considered positive for anti-malarial activity independent of the original classification. As a result, 296 molecules were relabeled from positive to negative; 192 molecules were relabeled from ambiguous to negative; 52 molecules relabeled from ambiguous to positive; 4 molecules were relabeled from negative to positive. The final dataset contained 1288 compounds labeled as positives, 294,092 as negatives, and 10,188 as ambiguous. Ambiguous compounds were not considered for modeling.\n\nTo describe the chemical structures of the compounds, the 196 RDKit descriptors available by default were computed. This first set will be referred to as “RDKit descriptors” set. Morgan fingerprints of both extended connectivity (ECFP) and functional class (FCFP) types32 were computed with a radius of up to 200 (meaning that all possible substructures are enumerated for each compound). Typically, circular fingerprints are hashed and folded to a fixed size, but this may lead to collisions, i.e. two different substructures are hashed to the same bit in the folded fingerprint. To avoid this problem, hashing or folding was not used in Workflow 2. All the existing substructures were saved as SMILES strings and uniquely encoded by a large bitset containing all substructures occurring in the training set. The unfolded ECFP and FCFP fingerprints were appended together in one vector.\n\nRandom forests29 and extremely randomized trees33 of 10, 20, 50, 100, 500, 1000, 2000, 4000 and 6000 trees were computed on the RDKit descriptors set, using multiple random seeds. Both methods use bagging to select instances for building each tree. As a result, for each individual tree, some instances were not used for training and are referred to as “out-of-bag”. These instances can be used for an unbiased estimate of the prediction error, instead of performing a computationally expensive cross-validation. Therefore, the out-of-bag scores were used as a measure of the quality of the models, and AUC, accuracy and enrichment at 5% were computed from these scores. The ensemble of trees with 6000 trees gave the best results and was therefore selected for deployment (i.e. used for the computation of the final scores for the unlabeled datasets).\n\nAfter a first exploration of multiple parameters for logistic regression on the fingerprint set by cross-validation, the following parameters for building the models were chosen: a penalty of l1 or l2, a regularization parameter C of 1 or 5, a default tolerance of 0.0001, and the fingerprints were kept unfolded. Cross-validation was computed for 3, 5, 7 or 10 folds with five different seeds each. For each fold, the AUC and enrichment at 5% were computed. When a fold reached an AUC below 0.88, then the rest of the cross-validation was skipped and the next model was built.\n\nThe best models among the many logistic regressions models for which all folds could be completed were the ones with a penalty of l1 and C of 1 and an average AUC over all folds over 0.92; as well as those with a penalty of l2 and C of 5 and an average AUC over all folds over 0.93. These particular models were selected for deployment (i.e. used for the computation of the final scores for the three tasks).\n\nThe first task involved the selection of 500 molecules from the primary HTS set with promising activity for follow-up confirmatory measurements. For this, the predictions of the deployment models were combined by plain averaging of the model scores. Note that this corresponds to model fitting scores, since the screening set is the training set used for building the deployment models. The 500 molecules with the highest average scores were selected for the follow-up testing.\n\nIn 1992, Wolpert introduced the concept of stacked generalization34 to combine different models and boost the predictive power of the resulting ensemble. Here, feature-weighted linear stacking was used to combine our deployment models35. For this, a linear regression was trained using the out-of-bag scores (for the ensemble of trees models) and cross-validation scores (for the logistic regression models) as independent variable, and antimalarial activity as dependent variable. The resulting linear combination of models was applied to obtain the final score for the 1056 compounds of the held-out test set.\n\nFor the selection of new candidates, the same feature-weighted linear stacking as described for Task 2 was used. The resulting linear combination of individual model scores was applied to obtain the final score for the compounds of the eMolecules catalog (https://www.emolecules.com). The 1000 top-scoring compounds were selected as new candidates for further anti-malaria screening. Compounds already present in the primary HTS and the confirmatory screen used for training were not explicitly removed from the eMolecules catalog.\n\n\nFinal selection process\n\nFrom the two lists of 1000 new candidates, 114 molecules were selected for testing in a follow-up assay based on availability at vendors who agreed to be TDT sponsors. The set included two known anti-malarials quinidine (proposed by Workflow 1) and amodiaquine (proposed by Workflow 2). Compounds that were already in the primary HTS and the confirmatory screen provided by the TDT challenge were not removed.\n\n\nExperimental procedures\n\nThe potency of new candidates was determined as reported earlier23. Plasmodium falciparum strain 3D7 was acquired from the Malaria Research and Reference Reagent Resource Center (MR4, catalog #MRA-102). Briefly, asynchronous parasites were maintained in culture based on the method of Trager36. Parasites were grown in presence of fresh group O-positive erythrocytes (Key Biologics, LLC, Memphis, TN) in Petri dishes at a hematocrite of 4-6% in RPMI based media (RPMI 1640 supplemented with 0.5% AlbuMAX II, 25 mM HEPES, 25 mM NaHCO3 (pH 7.3), 100 µg/mL hypoxanthine, and 5 µg/mL gentamycin). Cultures were incubated at 37°C in a gas mixture of 90% N2, 5% O2, 5% CO2. For IC50 determinations, 20 µl of RPMI 1640 with 5 µg/ml gentamycin were dispensed per well in an assay plate (Corning 384-well microtiter plate, clear bottom, tissue culture treated, catalog no. 8807BC). An amount of 60 nl of compound, previously serial diluted in a separate 384-well white polypropylene plate (Corning, catalog no. 8748BC), was dispensed to the assay plate by hydrodynamic pin transfer (FP1S50H, V&P Scientific Pin Head) and then an amount of 20 µl of a synchronized culture suspension (1% rings, 4% hematocrite) was added per well, thus making a final hematocrite and parasitemia of 2% and 1%, respectively. Assay plates were incubated for 72 h, and the parasitemia was determined by a method previously described37. An amount of 10 µl of the following solution in PBS (10X Sybr Green I, 0.5% v/v triton, 0.5 mg/ml saponin) was added per well. Assay plates were shaken for 1 min, incubated in the dark for 90 min, then read with the Envision spectrophotomer at Ex/Em of 485 nm/535 nm.\n\nEC50 values were calculated using a four-parameter logistic equation as described previously23. Compounds were arrayed in ten concentrations, varying from approximately 10 µM to 5 nM, and the R drc package was used to fit the observed response to the four-parameter Hill equation38. The purity of all compounds was determined by UPLC (UV and ELSD purity average) and results from any compound with a purity below 95% were not reported.\n\nMorgan2 fingerprints32 and Tanimoto similarities were calculated using the RDKit. The scaffolds in the set of newly tested compounds were determined using the Bemis-Murcko algorithm39.\n\n\nResults\n\nThe external held-out test set of the TDT challenge consisted of 101 actives and 955 inactives. The performances of the ML models of Workflow 1 and Workflow 2 on the held-out test set (1056 molecules) are given in Table 3. For Workflow 1, the results using fingerprints with 1024/2048 bits or with 4096 bits are reported. Note that the maximum possible EF5% for the held-out test set is 10.5 (as the fraction χ = 0.05 is smaller than the ratio of actives to inactives8), whereas it is 20.0 for the primary HTS dataset. Workflow 2 gave the best performance for the held-out test set from all five submissions to this TDT challenge. For Workflow 1, the version using 1024/2048 bits was the one submitted to the TDT challenge. Later, it was found that a substantial amount of collisions due to hashing occurred in the short fingerprints, which affected the performance. Using longer fingerprints (i.e. 4096 bits), the performance could be improved and was found to be similar to that of Workflow 2. This highlights the resistance to noise of the ML methods used, since in Workflow 1 the false positives in the primary data were included. In Workflow 2, these false positives were corrected using the information from the confirmatory screen.\n\nPredictions were obtained using the fusion models of Workflow 1 and the linear combination of model scores of Workflow 2. The maximum possible EF5% value is 10.5.\n\nFor both Workflows, the AUC and the EF5% values were found to be substantially lower for the held-out test set compared to the values for the 10%-test split in Table 2. Although the size distribution and flexibility of the compounds in the different sets were similar (Table 4) and the similarities within and across the datasets were generally low (left panel in Figure 2), there are slightly more highly similar compounds among the actives of the primary HTS (as in the original classification23) than between those and the actives in the held-out test set (right panel in Figure 2). In addition, there were some highly similar compounds between the actives in the primary HTS and the inactives in the held-out test set.\n\nThe compounds in the primary HTS were split into 1528 actives and 293,606 inactives. The compounds in the held-out test set were split into 101 actives and 955 inactives. For the primary screen, the original classification into actives and inactives was used23. For the held-out test set, a cutoff of 10 μM was employed.\n\nNormalized Tanimoto similarity distribution using a Morgan2 fingerprint32 within the actives in the primary HTS (blue), and between them and the actives (green) and inactives (red) of the held-out test set. The full distributions (left) and the slice between 0.8 and 1.0 similarity (right) are shown. For the primary screen, the original classification into actives and inactives was used23. For the held-out test set, a cutoff of 10 μM was employed.\n\nFrom the combined set of 2000 candidates predicted by Workflow 1 and Workflow 2, 114 were tested in a follow-up assay (80 from Workflow 1 and 38 from Workflow 2, four compounds were predicted by both Workflows). The identifiers, SMILES, EC50 values and raw data for all 114 compounds are given in the Supplementary material. Of these, two were known anti-malarials (quinidine and amodiaquine) selected as positive control. In addition, 31 compounds (six from Workflow 1 and 28 from Workflow 2, three were in common) were already present in the primary HTS and confirmatory screen provided by the TDT challenge, as such molecules were not explicitly removed from the eMolecules catalog before the virtual screen (Supplementary Table S1). One of these compounds, SJ000154494 (Figure 3, EC50 = 0.44 µM as measured in this study) was found inactive in the previous primary screen and confirmatory screen23, which was likely a false negative in the latter screen because dose-response testing immediately following the primary screen was done using compounds from stock solutions ranging in age, whereas the current experiment was performed on fresh powder.\n\nThe results for the remaining 81 new compounds and the two known anti-malarials are listed in Table 5. A list of all 114 compounds, including SMILES is provided as a separate file in the Supplementary material. Partially active or single-point active molecules were counted as inactives. As the list of 1000 compounds in Workflow 1 was randomly selected from the top 10,000 ranked compounds in the eMolecules database, the ranks in the latter list are also reported in Table 5. From the nine molecules proposed by Workflow 2, only two were not in the top 10,000 list from Workflow 1, indicating that the two approaches pick generally similar features but do not score them in the same manner. Of the 81 new compounds, 46 were found to be active, resulting in an overall hit rate of 57%. In more detail, Workflow 1 gave a hit rate of 52% and Workflow 2 a hit rate of 100%. Due to the small number of compounds tested, we cannot judge if this difference in hit rate is significant. As the TDT initiative relies on contributions of compounds, a more systematic assessment is outside the scope of this effort. Interestingly, the most active compounds were ranked rather low in the top-1000 list of Workflow 2 and the top-10,000 list of Workflow 1 compared to the other molecules tested, which emphasizes again that it is important in ligand-based VS to pick the compounds for follow-up testing relatively broadly from the top fraction.\n\nThe columns are as follows: EC50 values, the final scores (active or inactive), and the ranks in the Workflows 1 and 2. Partially active or single-point active compounds were considered inactives (marked by italic font). ChEMBL-NTD datasets: Novartis-GNF Malaria Box (N)40, St. Jude Children's Research Hospital Dataset (J)24, GSK TCAMS (G)41, DNDi HAT set (D). Compounds marked with (P) were tested in PubChem assays.\n\nFor Workflow 1, six of the 73 new compounds were tested previously in anti-malaria activity assays found in ChEMBL-NTD (https://www.ebi.ac.uk/chemblntd/) and PubChem (https://pubchem.ncbi.nlm.nih.gov) and three of them were found to be active. Three main scaffolds covered 25 of the 73 compounds: thiazolidin-4-one-type, 8-hydroxyquinoline-type, and aminopyrimidine-type scaffolds (Table 6). The compounds with the thiazolidin-4-one-type scaffold were the largest group. The scaffold can be seen as a variation of compound SJ000154494 (Figure 3), but the compounds in this group were mostly inactive. In addition, the scaffold may be a potential PAINS substructure due to its similarity with rhodanine, although it is currently not part of the filters28. The 8-hydroxyquinoline scaffold is a phenolic Mannich base, which is a PAINS substructure. The most interesting scaffold is the aminopyrimidine-type with a second N-alkyl substituent instead of a known N-aryl substituent. The most active compound of this series, SJ000866807, exhibits a good ligand efficiency with an EC50 of 0.2 μM and a molecular weight of only 266 g/mol. From this series of compounds only one (SJ000866811) was listed in PubChem, but this was in an assay for anti-cancer activity (AID 743276). However, similar compounds were previously reported in the Novartis-GNF Malaria Box40 (Figure 4).\n\nChEMBL-NTD datasets: Novartis-GNF Malaria Box (N)40, St. Jude Children's Research Hospital Dataset (J)24, GSK TCAMS (G)41, DNDi HAT set (D). Compounds marked with (P) were tested in PubChem assays.\n\nThese compounds are similar to the group of compounds predicted by Workflow 1 with the same scaffold (Table 6).\n\nThe nine new compounds proposed by Workflow 2 are shown in Figure 5. Five of them had been tested active previously in one of the ChEMBL-NTD assays or in PubChem assays for anti-malaria activity. Two compounds (SJ000866810 and SJ000866799) have the same 8-hydroxyquinoline-type scaffold as in Workflow 1, and one compound (SJ000866764) has a similar aminopyrimidine-type scaffold. Among the most active compounds predicted by both Workflows was a series of molecules with a benzothiazole scaffold (Figure 6). Compounds with a similar scaffold were tested previously in PubChem assays for anti-malaria activity or are part of the ChEMBL-NTD datasets. Compound SJ000040830 showed also high anti-leishmanial activity23. There may be, however, potential PAINS issues with this scaffold, although not covered by the current PAINS filters, as the extended π-system may act as Michael-like acceptor.\n\nThe molecules are ordered by decreasing activity.\n\n(Top): Compounds predicted by Workflow 1 and Workflow 2. (Bottom): Compounds that are actives from PubChem, Novartis-GNF Malaria Box40 and St. Jude Children's Research Hospital24.\n\nThe use of ligand-based VS based on results from a primary HTS to select new, potentially active compounds for testing is a common task in drug discovery. Here, we presented two detailed Workflows using open-source tools for educational purposes, and report the application of these Workflows for the identification of anti-malarial compounds as part of the 2014 TDT challenge. Information from a previous primary HTS performed at the St. Jude Children's Research Hospital (and a confirmatory screen in case of Workflow 2) was used for training. Of the 2000 compounds proposed by the Workflows, 114 were selected for follow-up testing based on availability. Excluding the two known anti-malarials quinidine and amodiaquine and the 31 compounds already present in the primary screen, 46 out of 81 new compounds were found to be active, which corresponds to a high hit rate of 57% and shows that the machine-learning methods in the presented Workflows both successfully identified scaffolds with anti-malaria activity. There was a good agreement between the two Workflows in the general scaffolds that were identified, even though the exact compounds and rankings were not the same. The most interesting group of compounds in the tested set contains an aminopyrimidine-type scaffold with a second N-alkyl substituent instead of a known N-aryl substituent. In particular, the most active compound SJ000866807 of this series shows good ligand efficiency.\n\nThe tutorials are available on the TDT website (http://www.tdtproject.org/2012-competition--tutorials.html) and on GitHub (https://github.com/sriniker/TDT-tutorial-2014 and https://github.com/sdvillal/tdt-malaria-followup). Both tutorials use only freely available software as specified above. The data from the primary HTS and confirmatory dose-response assay used in the TDT competition are available on the TDT website (http://www.tdtproject.org/challenge-1---malaria-hts.html) and are also deposited in ChEMBL, as part of the Neglected Tropical Diseases set (ChEMBL-NTD). The identifiers, SMILES, EC50 values and raw data for the held-out test set25, as well as for the 114 compounds tested in this study, are given in the Supplementary material.",
"appendix": "Author contributions\n\n\n\nSR and GL have created and applied Workflow 1. FM and SV have created and applied workflow 2. AS and JM have performed the follow-up assay. JJ and PW are members of the TDT steering committee and have organized the acquisition of chemical substances from vendors for testing in the follow-up assay.\n\n\nCompeting interests\n\n\n\nThe authors declare no competing financial interests.\n\n\nGrant information\n\nSR thanks the Novartis Institutes for BioMedical Research education office for a Presidential Postdoctoral Fellowship.\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 eMolecules as TDT partner in the compound acquisition, and ChemBridge and Enamine for providing compounds. The authors also thank the other sponsors of TDT (http://www.tdtproject.org/partners-and-sponsors.html) and the TDT steering committee (http://www.tdtproject.org/steering-committee.html).\n\n\nSupplementary material\n\nSupplementary Table S1: Identifiers, EC50values, final scores and ranks in the Workflows 1 and 2 for the 31 tested compounds that were part of the primary HTS screen.\n\nClick here to access the data.\n\nSupplementary Table S2: Identifiers, SMILES, EC50 values and raw data for the 1056 molecules in the external held-out test set.\n\nClick here to access the data.\n\nSupplementary Table S3: Identifiers, SMILES, EC50 values and raw data for the 114 molecules tested in this study.\n\nClick here to access the data.\n\n\nReferences\n\nJansen JM, Cornell W, Tseng YJ, et al.: Teach-Discover-Treat (TDT): collaborative computational drug discovery for neglected diseases. J Mol Graph Model. 2012; 38: 360–362. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nHastings IM: The origins of antimalarial drug resistance. Trends Parasitol. 2004; 20(11): 512–518. PubMed Abstract | Publisher Full Text\n\nKlein EY: Antimalarial drug resistance: a review of the biology and strategies to delay emergence and spread. Int J Antimicrob Agents. 2013; 41(4): 311–317. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuiguemde WA, Shelat AA, Bouck D, et al.: Chemical genetics of Plasmodium falciparum. Nature. 2010; 465(7296): 311–315. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuiguemde WA, Shelat AA, Garcia-Bustos JF, et al.: Global Phenotypic Screening for Antimalarials. Chem Biol. 2012; 19(1): 116–129. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmithson DC, Clark J, Connelly M, et al.: Held-out test set with 1056 molecules, to be published. 2012.\n\nWalters WP, Namchuk M: Designing screens: how to make your hits a hit. Nat Rev Drug Discov. 2003; 2(4): 259–266. PubMed Abstract | Publisher Full Text\n\nDavies JW, Glick M, Jenkins JL: Streamlining lead discovery by aligning in silico and high-throughput screening. Curr Opin Chem Biol. 2006; 10(4): 343–351. PubMed Abstract | Publisher Full Text\n\nBaell JB, Holloway GA: New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem. 2010; 53(7): 2719–2740. PubMed Abstract | Publisher Full Text\n\nButina D: Unsupervised Data Base Clustering Based on Daylight's Fingerprint and Tanimoto Similarity: A Fast and Automated Way To Cluster Small and Large Data Sets. J Chem Inf Comput Sci. 1999; 39(4): 747–750. Publisher Full Text\n\nBreiman L: Random forests. Mach Learn. 2001; 45(1): 5–32. Publisher Full Text\n\nCarhart RE, Smith DH, Venkataraghavan R: Atom Pairs as Molecular Features in Structure-Activity Studies: Definition and Applications. J Chem Inf Comput Sci. 1985; 25(2): 64–73. Publisher Full Text\n\nRogers D, Hahn M: Extended-connectivity fingerprints. J Chem Inf Model. 2010; 50(5): 742–754. PubMed Abstract | Publisher Full Text\n\nGeurts P, Ernst D, Wehenkel L: Extremely Randomized Trees. Mach Learn. 2006; 63(1): 3–42. Publisher Full Text\n\nWolpert DH: Stacked Generalization. Neural Netw. 1992; 5(2): 241–259. Publisher Full Text\n\nSill J, Takacs G, Mackey L, et al.: Feature-Weighted Linear Stacking. ArXiv e-print, 0911.0460. 2009. Reference Source\n\nTrager W, Jensen JB: Human malaria parasites in continuous culture. Science. 1976; 193(4254): 673–675. PubMed Abstract | Publisher Full Text\n\nSmilkstein M, Sriwilaijaroen N, Kelly JX, et al.: Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrob Agents Chemother. 2004; 48(5): 1803–1806. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRitz C, Streibig JC: Bioassay Analysis using R. J Stat Softw. 2005; 12(5): 22. Publisher Full Text\n\nBemis GW, Murcko MA: The properties of known drugs. 1. Molecular frameworks. J Med Chem. 1996; 39(15): 2887–2893. PubMed Abstract | Publisher Full Text\n\nNovartis-GNF Malaria Box, Gagaring K, Borboa R, et al.: Genomics Institute of the Novartis Research Foundation (GNF), 10675 John Jay Hopkins Drive, San Diego CA 92121, USA and Novartis Institute for Tropical Disease, 10 Biopolis Road, Chromos # 05-01, 138 670 Singapore.\n\nGamo FJ, Sanz LM, Vidal J, et al.: Thousands of chemical starting points for antimalarial lead identification. Nature. 2010; 465(7296): 305–310. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "24285",
"date": "02 Aug 2017",
"name": "David Ryan Koes",
"expertise": [
"Reviewer Expertise computational drug discovery"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe report describes the two winning ligand-based virtual screening methods of the 2014 Teach-Discover-Treat exercise. Both workflows and the supporting data are available in their entirety online (behind a request for the user's email address) and are adequately described in the manuscript. The report is of general interest to the community and a useful resource to practitioners in ligand-based drug discovery.\nI have a few minor suggestions for strengthening the manuscript.\nPage 4. A few sentences describing \"heterogeneous classifier fusion\" would be appreciated.\nPage 5. Descriptors. I would be interested in knowing the number of bits (i.e. unique ECFP/FCFP fragments) required to represent the full dataset (that is, the number of features in the input, which I suspect is actually larger than the number of examples?).\nPage 5. Task 2. The weights for the two models found by the linear regression would be interesting to report (is one model favored more heavily than the other?).\nTable 4. This would be a bit more informative if variance was reported as well.\nFigure 2. It isn't clear to me exactly what this is reporting. Is this the distribution of all possible pairs between the two sets? Please clarify.\nTable 5. My understanding is that the Rank (1000) numbers are essentially meaningless as the compounds were (accidentally) randomly selected. Can the corrected top 1000 ranks be provided as well (or instead) and clearly labeled as such (realizing that not all compounds will have such a rank).\n\nIt's also hard to get a sense of enrichment from these numbers since only 114 compounds were tested but the ranks have a much larger span. For example, the workflow 2 active compounds have poor ranks (>500), but this is misleading since there were no highly ranked (novel) compounds tested. I would really appreciate some visualization of enrichment relative to ranking (e.g. ROC curve) for 114/81 compounds tested for workflow 1.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "3423",
"date": "14 Feb 2018",
"name": "Sereina Riniker",
"role": "Author Response",
"response": "We follow the order of the points raised by the reviewer. We will add a sentence about \"heterogeneous classifier fusion\" in the revised version, including a reference to Ref. 13, which contains a detailed discussion of this concept. Descriptors in Workflow 1: The actives in the primary HTS set yield 5935 unique Morgan2 fragments. Together with the inactives there are 56'351 unique fragments. The number of 4096 bits used for the folded Morgan2 fingerprints in Workflow 1 is clearly much smaller than the number of unique fragments, however, a balance must be found between the size of the fingerprint (and associated computational cost) and the number of collisions. In our case, we found that a size of 4096 bits presents a good compromise. Workflow 1, Task 2: The weights of all models in the classifier fusion of Workflow 1 were the same. The MAX rank was used. We will add the standard deviation to Table 4. Figure 2 reports the distribution of all possible pairs between the two sets. We will adapt the legend of Figure 2 to make it clearer. Table 5: As the list with ranks served as the input for compound selection, the rankings are used in Table 5 to mark which molecules came from Workflow 1. We will replace column \"Rank (1000) Workflow 1\" with a column \"Proposed by Workflow\" to provide the same information. We will also add a separate file in the Supplementary Material with the 1000 molecules selected by the correct Workflow 1. Both enrichment factors and ROC curves compare the ranking of active/inactive molecules against random distribution. The number of actives is exceptionally high in the set of 81 compounds (i.e. 58 %), because these were selected among the highest-ranked compounds from the 5.5 millions in eMolecules. Due to this high percentage of actives in the list, the calculation of enrichment factors or ROC curves does not make much sense in the present case. We thank the reviewer for carefully reading the manuscript and for the constructive and insightful feedback."
}
]
},
{
"id": "24675",
"date": "16 Aug 2017",
"name": "Matthew P. Baumgartner",
"expertise": [
"Reviewer Expertise computational chemistry"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report on their participation in the 2014 Teach-Discover-Treat (TDT) initiative. The goal of the TDT is to encourage the creation of practical tutorials for computational chemistry. The authors present the two workflow tutorials that they developed for Challenge 1 of the competition. The challenge involved three tasks: analyzing single-point phenotypic HTS results and follow-up dose-response data for a subset of the compounds, building a predictive model of the anti-malaria activity and using that predictive model to select compounds from a set of commercially available compounds for prospective testing. The first workflow presented by the authors only used the HTS data for its predictions and the second used both the HTS and dose-response data.\n\nOverall I think that the paper is a thorough and easy-to-follow description of the methods and results of the two workflows, but there a few items that I feel require revisions.\n\nPage 5, a brief description of what \"heterogeneous classifier fusion\" is would be appreciated\nPage 6. The authors should list the total number of features that they use as descriptors in workflow 2.\n\nPage 6. When building the random forests and extremely randomized trees of varying sizes, the ensembles of trees with 6000 trees (the largest number tested) were shown to preform best. The authors should explain why they did not try a higher number of trees.\n\nPage 6. In the \"Task 2...\" paragraph. The authors should state what the resulting linear combination of the models was. The ratio would be interesting to know.\nPage 8, in the paragraph starting \"The results for the remaining...\". It states in the text that there were 9 compounds predicted by workflow 2 that tested, but in Table 5, there are 10 compounds from workflow 2 listed. This should be corrected or clarified.\n\nPage 8 and Table 5. As the compounds from Workflow 1 were selected randomly due to an error, is it meaningful to list their rankings at all?\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "3424",
"date": "14 Feb 2018",
"name": "Sereina Riniker",
"role": "Author Response",
"response": "We follow the order of the points raised by the reviewer. We will add a sentence about \"heterogeneous classifier fusion\" in the revised paper, including a reference to Ref. 13, which contains a detailed discussion of this concept. Descriptors in Workflow 2: The RDKit descriptor set consists of 196 features and the unfolded fingerprints set, after removal of redundant features, 1'265'410 different substructures. We will add this information in the revised version. Workflow 2: Larger numbers of trees were not investigated due to limited computer resources. However, a plateau in the performance curve was observed after 2000 trees, thus only small improvement can be expected for models with more than 6000 trees. We will add a figure to the Supplementary Material. Workflow 2, Task 2: The linear combination in Workflow 2 placed substantially more weight on the tree models (coefficient 1.07) than on the logistic regression models (coefficient 0.07). We found later that this weighting was not optimal for the prediction of the held-out test set. Logistic regression models alone would have performed better than the original submission from Workflow 2. We will add a table with this information in the Supplementary Material and a comment in the main text. There were nine new compounds and one known anti-malarial amodiaguine, i.e. together ten compounds. We will add the term \"new\" in one sentence of the corresponding paragraph to make it clearer. Table 5: We agree that they are not true rankings anymore, but because this list served as the input for compound selection the rankings are used in Table 5 to mark which molecules came from Workflow 1. We will replace column \"Rank (1000) Workflow 1\" with a column \"Proposed by Workflow\" to provide the same information. We thank the reviewer for carefully reading the manuscript and for the constructive and insightful feedback."
}
]
},
{
"id": "24288",
"date": "24 Oct 2017",
"name": "Andrea Volkamer",
"expertise": [
"Reviewer Expertise Structural bioinformatics/computational chemistry"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors introduce the two winning workflows (WFs) from the Teach-Discover-Treat competition 2014 featuring ligand-based virtual screening pipelines for anti-malaria compounds together with results from an experimental follow-up study. The workflows show hit rates of 57% (52% WF1, 100% WF2) in a prospective study on a rather small sample size due to compound availability and funding reasons (72 WF1/ 9 WF2 novel compounds). The article is well written, easy to understand and the study showed promising results in finding new active compounds. Furthermore, both workflows are available to the community.\nMinor comments that could be addressed to improve the manuscript:\nMethods/Workflows: - More detail on the more advanced ML techniques, number of features, and especially feature importances would be helpful for the reader. - The data set is highly unbalanced (~1.5K actives vs. 290K inactives). Could one expect a boost in performance when using under-/oversampling methods? - This may have been addressed in the competition itself, but it would be interesting to see how a simple model performs on the data, for example simply ranking by similarity to known actives?\nEvaluation: - The authors admit the little flaw in the original WF1 that the top 1000 molecules accidently represent a random selection from the top 10K. It’s hard to compare the results now that the selection and testing phase is over, but it would be nice to see some evidence that the intended selection strategy would actually have been superior. E.g. the positions of the intended ranking could be included in Table 5, the prospectively tested set is small but a trend may become apparent? - Since 31 of the selected 114 compounds (~30%) were present in the HTS, I’m wondering how many of the HTS compounds were present in eMolecules in total? Because they have been used for training, taking them out of the evaluation would be more convincing and would probably also improve the rankings of the tested compounds.\n\n- The authors claim that the two methods pick generally similar compounds, which can be somehow expected from the design of the two WFs (similar MLs and fingerprints). Nevertheless, this trend is not obvious to me from the few mentioned values, e.g., 7 compounds selected from WF2 are in top 10K from WF1 (page 8). It would be more meaningful to calculate the overlap of the top 1000 compounds between the methods or the similarity between these compounds (also with respect to different top 1000 selections in WF1, see point above).\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "3425",
"date": "14 Feb 2018",
"name": "Sereina Riniker",
"role": "Author Response",
"response": "We follow the order of the points raised by the reviewer. For both workflows, the code is freely available on GitHub (URLs are given in the paper). Dataset imbalance: Workflow 1 uses an undersampling method for the random forest to deal with this issue. We will add a sentence describing this in the revised version. No over- or undersampling method was used for the models in Workflow 2. However, the logistic regression models did include an instance weighting scheme that gives more weight to instances of the minority class (i.e. C is modified per sample proportionally to the proportion of its class in the dataset). We will add in the Supplementary Material to show that class imbalance correction did not influence the model performance in Workflow 2. To our knowledge this comparison has not been done in the competition itself for the external held-out set. In Task 2 of Workflow 1, the performance of the ML models is compared to simple ranking by similarity (see Table 2). The latter showed already a good baseline performance such that only random forest models were able to outperform it. We will add a sentence discussing this. The question is what the comparison would be to define superiority without results for all compounds. Ideally, the enrichment in the selected 1000 compounds versus in the direct top 1000 should be compared but that is not possible anymore at this stage. It is important to stress that the selection procedure was aimed at improving the SAR information in the selected 1000 compounds not necessarily at increasing the hit rate, because the top 10'000 are already the highest ranked compounds among 5.5 millions in eMolecules. We will add a sentence regarding this in the revised version. We will replace column \"Rank (1000) Workflow 1\" in Table 5 with a column \"Proposed by Workflow\" to provide the information about which workflow proposed which molecules. Further, we will add a separate file in the Supplementary Material with the 1000 molecules selected by the correct Workflow 1. We agree with the reviewer that removing the HTS compounds from the eMolecules catalogue prior to ranking should have been done, but for the present work it is unfortunately too late. 52% of the (true) top 1000 compounds of Workflow 1 have a similar compound in the top 1000 of Workflow 2, using Morgan2 fingerprints (radius = 2, 4096 bits) and a Tanimoto similarity cut-off = 0.8. We will add a figure with the distribution of the similarity value between each compound of Workflow 1 and its most similar compound in Workflow 2 in the Supplementary Material together with a short discussion in the main text. We thank the reviewer for carefully reading the manuscript and for the constructive and insightful feedback."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1136
|
https://f1000research.com/articles/7-204/v1
|
19 Feb 18
|
{
"type": "Systematic Review",
"title": "The relationship of cigarette smoking in Japan to lung cancer, COPD, ischemic heart disease and stroke: A systematic review",
"authors": [
"Peter N. Lee",
"Barbara A. Forey",
"Alison J. Thornton",
"Katharine J. Coombs",
"Barbara A. Forey",
"Alison J. Thornton",
"Katharine J. Coombs"
],
"abstract": "Background: To present up-to-date meta-analyses of evidence from Japan relating smoking to major smoking-related diseases. Methods: We restricted attention to lung cancer, chronic obstructive pulmonary disease (COPD), ischemic heart disease (IHD) and stroke, considering relative risks (RRs) for current and ex-smokers relative to never smokers. Evidence by amount smoked and time quit was also considered. For IHD and stroke only, studies had to provide age-adjusted RRs, with age-specific results considered. For each disease we extended earlier published databases to include more recent studies. Meta-analyses were conducted, with random-effects RRs and tests of heterogeneity presented. Results: Of 40 studies, 26 reported results for lung cancer and 7 to 9 for each other disease. For current smoking, RRs (95%CIs) were lung cancer 3.59 (3.25-3.96), COPD 3.57 (2.72-4.70), IHD 2.21 (1.96-2.50) and stroke 1.40 (1.25-1.57). Ex-smoking RRs were lower. Data for lung cancer and IHD showed a clear tendency for RRs to rise with increasing amount smoked and decrease with increasing time quit. Dose-response data were unavailable for COPD and unclear for stroke, where the association was weaker. Conclusions: Compared to studies in other Asian and Western countries, current smoking RRs were quite similar for IHD and stroke. The comparison is not clear for COPD, where the Japanese data, mainly from cross-sectional studies, is limited. For lung cancer, the RRs are similar to those in other Asian countries, but substantially lower than in Western countries. Explanations for this are unclear, but less accurate reporting of smoking by Japanese may contribute to the difference.",
"keywords": [
"Smoking",
"lung cancer",
"COPD",
"heart disease",
"stroke"
],
"content": "Introduction\n\nIt is well established that the relative risk (RR) of lung cancer from smoking is lower in Japan than in Western populations1–5. However, studies of smoking and lung cancer in Japan have proliferated in recent years, and there have been no meta-analyses in the last 10 years, except for a review of prospective studies reported up to 20086. While that review also considered other smoking-related diseases, there have been no other recent comprehensive meta-analyses of the relationship of smoking to chronic obstructive lung disease (COPD) or cardiovascular disease (CVD), based on studies in Japan.\n\nHere we summarize results of Japanese studies relating smoking to lung cancer, COPD, ischaemic (or coronary) heart disease (IHD) and stroke, limiting attention to comparison with never smokers, and considering estimates for current smokers, overall and by amount smoked, and for ex-smokers, overall and by time quit. As we earlier published comprehensive reviews of the evidence for lung cancer4 and COPD7 we extend our meta-analyses to include later papers. For IHD and stroke we extend meta-analyses based on studies published from 19908, not attempting to include earlier publications.\n\nApart from presenting the meta-analysis results, we also briefly compare and contrast the results for Japan with those for other regions, commenting on possible reasons for differences seen.\n\n\nMethods\n\nThis systematic review was conducted according to PRISMA guidelines9. A completed PRISMA checklist can be found in Supplementary File 1. Throughout this paper we use the RR to include its various estimates, including the odds ratio and the hazard ratio. Where results are referred to as “significant” without further detail, this implies p<0.05.\n\nWe sought studies providing data on RRs for current smokers and/or ex-smokers compared to never smokers for one or more of the diseases lung cancer, COPD, IHD and stroke. Studies providing data from which RRs could be calculated were accepted, as well as those giving the estimates directly.\n\nFor lung cancer, attention was restricted to epidemiological prospective or case-control studies involving 100 lung cancers or more, extending our earlier review4 of studies published in the 1900s. That review considered specific histological types of lung cancer, but here we restrict attention to overall lung cancer.\n\nFor COPD, cross-sectional studies were also considered, and there was no restriction on number of cases, thus extending an earlier review7 of studies published before 2007. That review also considered chronic bronchitis and emphysema, but we limit attention to studies using the definitions of COPD described there. We also follow the exclusion criteria given in that review.\n\nFor IHD and stroke, we extend an earlier review, described in a supplementary file to a recent publication8, which considered studies of prospective or case-control design which involved at least 100 CVD cases and were published between 1990 and 2010. We restricted attention to studies providing data for IHD, acceptable disease definitions including coronary heart disease and acute myocardial infarction, and/or for stroke. Studies providing results only for specific disease subtypes, or only for combined CVD were not included.\n\nWhile for lung cancer and COPD we accepted studies only providing RRs for the sexes combined, for IHD and stroke the studies had to provide sex-specific RRs. Also for IHD and stroke, studies had to provide RRs adjusted at least for age.\n\nStudies satisfying the specific criteria were first sought from the three earlier reviews4,7,8. Additional papers were also sought from recent reviews of the evidence relating quitting smoking to these diseases10–13. Finally Medline searches were conducted to update the evidence considered. The searches were conducted along the lines considered in the three earlier reviews4,7,8 but restricted to Japan and to a later publication date range – from 2000 for lung cancer, from 2007 for COPD, and from 2010 for IHD and stroke, all searches being conducted in early 2017. No attempt was made to consider studies on IHD and stroke published before 1990. In all the searches, abstracts were first examined, with potentially relevant papers then being obtained and examined in detail.\n\nThe earlier reviews4,7,8,10–13 had already allocated relevant papers to studies, noting multiple papers on the same study, and papers reporting on multiple studies. Similar procedures were carried out to continue this process, with some new publications providing updated information on existing studies. As previously, potential overlaps between studies were noted.\n\nWe extended existing databases to include the additional RRs for current or ex-smoking. All estimates considered were for smoking cigarettes only, cigarettes undefined, or any product. The never smoking denominator could include those who never smoked anything, or never smoked cigarettes. RRs were included, where available, for current smoking overall and for sets of RRs grouped by amount smoked, and for ex-smoking overall and for sets of RRs grouped by time quit. As previously, near-equivalent definitions were accepted when exact definitions were not available (e.g. never smokers could include long-term ex-smokers and recent quitters could be treated as current rather than ex-smokers). Given a choice, the RR adjusted for the most potential confounding variables was selected. Sexes-combined RRs were entered only if sex-specific estimates were unavailable. Age-specific RRs were entered, where available, for IHD and stroke, but not for lung cancer or COPD.\n\nWhere necessary RRs were derived from data provided using standard methods, as described elsewhere4.\n\nFixed-effect and random-effects meta-analyses were calculated using standard methods14 with between estimated heterogeneity quantified by H, the ratio of the heterogeneity chisquared to its degrees of freedom. This is directly related to the I-squared statistic15 by the formula I2 = 100 (H – 1)/H. For all meta-analyses, Egger’s test of publication bias16 was also included. Analyses were conducted for current smoking overall and for ex-smoking overall, preferring cigarette only smoking versus never smoking of anything where there was a choice of definition, and also for about 5, 20 and 45 cigarettes currently smoked and about 12, 7 and 3 years quit for ex-smokers. For an RR to be included in these two dose-response analyses, the grouped level had to include the stated value, but not either of the other two. For IHD and stroke, 20 was replaced by 19 in the above scheme for amount smoked so as to maximise usage of the available data.\n\nFor current smoking overall and for ex-smoking overall, meta-analyses were conducted separately by sex with the significance of the between sex difference also estimated. Where sufficient data were available, we also conducted tests of variation by levels of other factors, which varied by disease.\n\nWe did not attempt to derive study-specific scores based on study quality and risk of bias, as the relative importance of different sources of bias or poor quality cannot be reliably assessed. Instead we carried out some meta-analyses showing how estimates varied by aspects of study quality and bias, including study size, number of adjustment variables, and study type. We also considered factors affecting quality and bias in the discussion section.\n\n\nResults\n\nFor lung cancer, the earlier review4 included 19 studies conducted in Japan, of which 11 provided relevant data. Three additional relevant studies were reported in the quitting review11 with a further 12 found from the updated Medline search.\n\nFor COPD, the earlier review7 included four studies in Japan, one rejected as having no relevant data and the results from another later being found to be superseded by a more recent publication. No additional studies were identified in the quitting review13 but the updated Medline search found four further relevant studies.\n\nFor IHD and stroke, the earlier review8 included four relevant publications, three describing individual studies and one a pooled analysis of three studies. One additional study was identified in the quitting reviews10,12, and four additional relevant publications were identified from the Medline search, three describing individual studies and one a pooled analysis of ten studies.\n\nSupplementary File 2 provides fuller details of the literature searches.\n\nTable 1 gives details of the 40 studies included in the analyses, presented in order of the date of the publication reporting the relevant results. Of these, the numbers giving results for lung cancer, COPD, IHD and stroke are, respectively, 26, seven, nine and seven, some studies reporting on more than one of these diseases. The table provides information for each study on the study type, the location, the years in which it was conducted, the population considered, and the number of cases of each disease it considered. Two publications based on the Japan Collaborative Cohort (JACC) are treated as separate studies in the table as the publications relate to different diseases and periods of follow-up. The same is true for three publications based on the Japan Public Health Center (JPHC) study.\n\na C = case-control study, CS = cross sectional study, P = prospective study\n\nb * = unknown. Values in brackets are approximate, based on one year before the first publication. For prospective studies, baseline year(s)/final follow-up year.\n\nc Unless shown otherwise in this column, the study specified no major inclusion or exclusion criteria.\n\nd In whole study.\n\ne Combined results from three prospective studies; the JPHC and JACC studies had wide national coverage, and the TPCS was conducted in three prefectures (Miyagi, Aichi, Osaka). The first reference cited gives the lung cancer findings and the second the cardiovascular findings.\n\nf Combined results from ten prospective studies, each with at least 10 years follow-up. These included the JACC study.\n\nTwo pooled analyses of results are treated as single studies in Table 1. The pooled analysis of three studies reported by Wakai et al41 for lung cancer and by Honjo et al42 for CVD included results from the JACC, JPHC and MARUG1 studies. It may have some overlap of results for lung cancer with the findings from JACC (OZASA), JPHC (SOBUE and SHIMAZU) and MARUG1 and for CVD with the findings from JACC (MATSUNAGA) and JPHC (ESHAK). The pooled analysis of ten studies on CVD by Nakamura et al49 may have some overlap of results with the findings from UESHIM and JACC (MATSUNAGA), but is predominantly based on studies not considered elsewhere.\n\nOf the 40 studies, 18 are case-control and 6 of cross-sectional design (all of COPD), with the rest prospective. Ten of the studies were published before 2000, although 20 had been completed by then. The largest study was HIRAYA, which involved 1917 lung cancer cases, 3,548 cases of IHD and 12,732 of stroke, though the SOBUE2 study involved somewhat more lung cancer cases, 2,083.\n\nTable 2 gives the RRs for current and ex-smoking while Supplementary File 3 gives them by amount smoked and time quit. As seen in Table 2, most lung cancer estimates are adjusted for age plus at most one other potential confounding variable, while nearly all COPD estimates are unadjusted, even for age. All the IHD and stroke estimates are (as required) adjusted for age, and most of these also for a number of additional variables.\n\naEx-smoker data are from an earlier reference56\n\nMeta-analysis results (random effects estimates) are shown for current smoking in Table 3, for amount smoked by current smokers in Table 4, for ex-smoking in Table 5 and for time quit by ex-smokers in Table 6. Supplementary File 4 gives some additional results for current and ex-smoking for lung cancer, IHD and stroke. Below we summarize the results by disease risk.\n\na n = number of estimates combined, R = random-effects meta-analysis RR (95% CI), H = heterogeneity chisquared per degree of freedom, PH = probability value for heterogeneity expressed as p<0.001, p<0.01, p<0.05, p<0.1 or NS (p≥0.1). PB = probability value for between level comparison similarly expressed.\n\na n = Number of estimates combined, R = random effects meta-analysis RR (95% CI).\n\nb Number of sets of RRs available for the key value analyses, where the dose for comparison is never smoked. See also Supplementary File 3 for details.\n\nc Base for comparison is never smoked. For lung cancer and COPD; the first category for which results are provided includes 5 cigs/day, but does not include 20 cigs/day; the second includes 20 cigs/day, but does not include 5 or 45 cigs/day; and the third includes 45 cigs/day, but does not include 20 cigs/day. For IHD and stroke; 20 cigs/day is replaced by 19.\n\na n = number of estimates combined, R = random-effects meta-analysis RR (95% CI), H = heterogeneity chisquared per degree of freedom, PH = probability value for heterogeneity expressed as p<0.001, p<0.01, p<0.05, p<0.1 or NS (p≥0.1). PB = probability value for between level comparison similarly expressed.\n\na n = Number of estimates combined, R = random effects meta-analysis RR (95% CI).\n\nb Number of sets of RRs available for the key value analyses, where the comparison is with never smoked. See also Supplementary File 3 for details.\n\nc One study reported RRs (95% CIs) of 2.08 (1.08–4.00) for \"early quitters\" and 2.42 (1.11–5.25) for \"late quitters\", early quitters having reported current smoking in 1994 but not in 1999 or 2006, and late quitters having reported current smoking in 1994 and 1999 but not in 2006.\n\nd Base for comparison is never smoked. The first category for which results are provided includes quit 12 years ago but does not include quit 7 years ago; the second includes quit 7 years ago but does not include quit 3 or 12 years ago; and the third includes quit 3 years ago but does not include quit 7 years ago.\n\nFor current smoking, the overall RR shown in Table 3 is 3.59 (95%CI 3.25–3.96), based on 39 estimates. As shown in Table 2, the RRs range from 1.22 to 6.76, with all but two statistically significant. While the estimates are heterogeneous, no single factor is responsible for this, though (see also Supplementary File 4) there is evidence that RRs are higher in males and where adjusted for more variables. Table 4 shows that the RRs increase steadily with amount smoked, rising from 2.89 (2.44–3.43) for “about 5 cigs/day” to 6.42 (5.14–8.02) for “about 45 cigs/day”.\n\nCompared to current smoking the RR for ex-smoking (Table 5) of 2.26 (2.03–2.52) is lower and shows less heterogeneity, with the only factor showing significant variation being publication year, with RRs higher in older (pre-1980) studies. RRs clearly reduced with increasing time of quit, evident both in the individual data sets for each study and the summary analysis in Table 6.\n\nFor current smoking, the overall RR shown in Table 3 is 3.57 (95%CI 2.72–4.70), based on 10 estimates. However, as Table 2 shows, the two RRs from HIRAY2 are atypically high, and excluding these results substantially reduced the heterogeneity and reduced the RR to 3.10 (2.57–3.75). There is no significant variation by sex. Analyses by further subgroups were not attempted, due to the limited number of estimates for which results are available. There are no available results by amount smoked.\n\nFor ex-smoking (see Table 5) the overall RR was 3.03 (2.00–4.57). This reduced to 2.16 (1.68–2.77) after excluding the high results from HIRAY2 (Table 2). A single study reported similar RRs for late quitters and early quitters, as shown in the footnote to Table 6.\n\nFor current smoking, the overall RR (Table 3) is 2.21 (95%CI 1.96–2.50). However, the estimates are clearly heterogeneous, with RRs somewhat higher in females than males. The additional results in Supplementary File 4 show that RRs tend to be greater in more recently published studies, but did not vary significantly by age or by the number of variables adjusted for. There was variation by study size, but this did not show any systematic trend. As shown in Table 4, the RRs increase steadily with amount smoked, rising from 1.71 (1.50–1.94) for “about 5 cigs/day” to 2.70 (2.16–3.39) for “about 45 cigs/day”.\n\nFor ex-smoking, the overall RR (Table 5) of 1.46 (1.24–1.71) is clearly lower than that for current smoking. RRs tended to be higher in females, and in less adjusted estimates. In those who had quit for “about 12 years” the RR at 1.22 (0.81–1.84) is not significantly increased, but those for shorter quit times are both elevated to a similar extent (Table 6).\n\nFor current smoking, the overall RR (Table 3) is 1.40 (95%CI 1.25–1.57), less elevated for the other diseases. There is clear heterogeneity, RRs tending to be higher in females, for those aged <65, in more recently published studies, and in studies involving fewer cases (see also Supplementary File 4).\n\nThere is no clear relationship of risk of stroke to amount smoked (Table 4) though the largest estimate is for the highest dose.\n\nOverall, risk is not significantly elevated in former smokers (Table 5) with the RR estimated as 1.05 (0.96–1.15). However, the analyses show some increase in females, and in short-term quitters (Table 6).\n\nEach meta-analysis included a test of publication bias (detailed results not shown).\n\nFor lung cancer, there is no evidence of publication bias for current smoking and only marginal evidence (p<0.05) for ex-smoking, where RRs were somewhat greater in smaller studies. For CVD, the strongest evidence of publication bias (p = 0.003) is for the analysis of current smoking RRs for stroke. This corresponds with the evidence of higher risks in smaller studies. No evidence of publication bias is seen for COPD for either current or ex smoking.\n\nThe meta-analyses reported include all available data, accepting some overlap of results between studies. Some additional analyses were conducted for current smoking either omitting results from the publications reporting combined analyses (3 STUDIES, 10 STUDIES) or omitting results from studies considered in these analyses (JPHC, JACC, MARUG1). For lung cancer, compared to the original estimate of 3.59 (95%CI 3.25–3.96), the first omission gave 3.55 (3.20–3.93) while the second gave 3.46 (3.06–3.91). For IHD, the original RR of 2.21 (1.96–2.50) became 2.01 (1.76–2.29) for the first omission and 2.20 (1.88–2.57) for the second. For stroke 1.40 (1.25–1.57) became 1.22 (1.11–1.34) and 1.44 (1.25–1.65). For CVD it should be noted that omitting the 10 STUDIES results lost relevant information as many of its individual studies were not included elsewhere.\n\n\nDiscussion\n\nThe results presented show some increased risk of all four diseases with current smoking, and a lesser increase with ex-smoking, that with stroke not being clearly significant. The evidence for COPD is clearly the thinnest being based largely on cross-sectional studies and on unadjusted RRs, and providing little or no data for amount smoked or time quit. The other diseases do show a tendency for RRs to increase with amount smoked and to decline with increasing time quit, though again the associations are less clear for stroke, the disease most weakly associated with smoking.\n\nIn considering these results, various aspects of the data require comment.\n\nSmoking of tobacco products other than cigarettes, such as cigars or pipes, is rare in Japan57 and whether authors related results to unspecified smoking, to cigarette smoking or to cigarette only smoking would be of little practical relevance. Similarly the precise definition of the comparison group, never smokers, is unlikely to be important.\n\nFor lung cancer, there is no evidence that RRs differ between prospective and case-control studies. Since all the RRs for CVD came from prospective studies, and virtually all those for COPD came from cross-sectional studies, variation by study type could not usefully be examined for these diseases.\n\nIt was beyond the scope of the study to investigate variation by disease subtypes, though we note that, for lung cancer, some studies (e.g. AKIBA, ITO, MARUG2, JPHC(SOBUE)) present evidence consistent with there being higher RRs for squamous cell carcinoma than for adenocarcinoma.\n\nIt has previously been established that the variation in RR by age is much greater for cardiovascular disease than for lung cancer or COPD4,7,8. For this reason we only considered age-specific data for IHD and stroke. The results generally confirmed the higher RRs in younger individuals.\n\nIn order to limit the scope of the project, attention was restricted to RRs adjusted for the most potential confounding variables where there was a choice. For lung cancer, there was some evidence that more adjusted RRs were higher, but for cardiovascular disease no such trend was seen. RRs for COPD were generally unadjusted.\n\nFormal tests for outliers were not attempted, but it was evident from inspection of Table 2 that the very large RRs for the HIRAY2 study were inconsistent with the rest of the available results, and removal of the results from the meta-analysis materially reduced the RRs for both current and ex-smoking. Otherwise there seemed to be no clear outliers, unusually low or high RRs typically having a very wide 95%CI, being based on limited data.\n\nImprecision of the effect estimates could have resulted from errors in diagnosis of disease or errors in determining smoking habits. It was notable that mortality studies generally did not rely on autopsy-confirmed diagnosis, and that smoking habits recorded were usually based on self-report by the individual with no confirmation of non-smoking status by measurement of biomarkers such as cotinine.\n\nTable 7 presents meta-analysis relative risks for current smoking by region from this study, from reviews of ours4,7,8 and from other selected recent reviews2,6,58–60 chosen as they provided RR estimates for the sexes combined by region. It was clear for IHD that there is little evidence of a material difference in RR between estimates from Japanese studies and those from studies in other Asian countries or Western countries. In all cases the RR is quite close to 2. The pattern is broadly similar for stroke, with the RR for stroke, typically about 1.4, less than that for IHD, with the minor exception of Scandinavia, where the RR is based on only two estimates. For COPD, the available data are limited, but provide some suggestion that, compared to Japan, RRs are somewhat higher for North America though similar for Europe.\n\nEvidence of international variation in current smoking RRs is much clearer for lung cancer, where the meta-analysis RRs reported for Japan and other Asian countries range from 2.46 to 3.64, while those for North America, Europe and Australia/New Zealand are substantially higher, ranging from 7.53 to 12.55. The explanation for this difference has been discussed in a number of previous publications (e.g.1–3,35) without any clear explanation being offered. An international case-control study involving populations in the USA and Japan3 found no substantial international differences in average daily consumption or mean duration of smoking, but noted that US cases began smoking 2.5 years earlier than Japanese cases. They suggested that possible explanations for the higher smoking risk in the US study may “include a more toxic cigarette formulation of American manufactured cigarettes as evidenced by higher concentrations of tobacco-specific nitrosamines in both tobacco and mainstream smoke, the much wider use of activated charcoal in the filters of Japanese than in American cigarettes, as well as documented differences in genetic susceptibility and lifestyle factors other than smoking.” Other authors1,2,35 have referred to the severe shortage of cigarettes in Japan during and shortly after World War II, the higher incidence of lung cancer in nonsmokers in Japan due to indoor air pollutants (including environmental tobacco smoke), the low fat intake and high intake of several phytochemicals in the Japanese diet, and the lower indoor radon concentrations in Japan than in the USA.\n\na Estimated from data for sexes separately\n\nWhether lung cancer risk in nonsmokers in Japan is higher than in Western countries is in any case open to question. A recent publication61 that indirectly estimated absolute lung cancer mortality rates by smoking status based on a systematic review, found that they were quite similar in Japan to those in most Western countries. For age 70–74 years, mortality rates (per 100,000 per year) in those who had never smoked were estimated as 42.5 (95% CI 34.5–52.4) in Japan based on n = 14 estimates, as compared, for example, to 37.6 (32.6–43.3, n = 54) for the USA, 61.5 (46.8–80.8, n = 26) for the UK, 29.6 (21.9–40.0, n = 20) for Scandinavia, 38.2 (29.3–49.8, n = 31) in other countries in Western Europe, and 32.3 (22.3–46.8, n = 11) for Eastern Europe. It was China, not Japan, that had a markedly higher lung cancer rate of 99.1 (90.2–108.8, n = 38) in never smokers.\n\nOne potential explanation for the difference in the relative risk of lung cancer between Asian and Western populations may lie in differences in the accuracy of reporting smoking habits. We are currently involved in a separate project to review accuracy of reporting smoking habits, using cotinine to validate self-reported smoking habits. We are aware of five studies in Asian populations, three in Japan62–64, one in Korea65 and one of South-East Asians resident in the USA66, which report results separately for never, ex and current smokers and by sex. All five give results for women, and four do so for men, and the proportion of true current smokers in self-reported never or ex-smokers (as judged by high cotinine levels) in women (range 12.3% to 61.6%, overall 45.8%) is much higher than it is men (range 0.4% to 6.0%, overall 3.4%). The proportion is also much higher than in 13 data sets (five in males, five in females, three in sexes combined) reported in six publications67–72 describing studies in Western populations (England, Finland, Germany, USA) involving large numbers (>2000) of subjects. Here percentages range from 0.4% to 6.1%, with the overall estimates 1.6% for males, 3.2% for females, and 2.3% for the whole sample.\n\nAlthough the difference is impressive, the percentage that affects the relative risk for current versus never smokers is the proportion of the current smokers in self-reported never smokers. Here the overall percentages are 7.8% in Asian females, 5.5% in Asian males, 1.4% in Western females and 2.3% in Western males. If one assumes that the true RR for current smoking and lung cancer is X, the observed RR based on self-reported data will be X / (1 + (X − 1) p) where p is the proportion of true current smokers among self-reported never smokers. Thus if X = 10, the observed RRs would be 5.9 in Asian females, 6.7 in Asian males, 8.9 in Western females and 8.3 in Western males, based on the data sets investigated. Although there are difficulties in interpreting these results for various reasons, including between-study variation in the body fluids and cut-offs used to determine true smokers, and the possibility that self-reported never smokers who are considered to be current smokers may smoke less than current smokers who admit smoking, we feel that these results suggest that different levels of misclassification of smoking habits between Asian and Western populations may contribute to the lower observed current smoker RRs in Asian populations.\n\nThis review is concerned with the effects of active smoking in Japan on the four diseases concerned. Recent reviews by ourselves73–76 and others77 have found that evidence in Japan on passive smoking is very sparse, except for lung cancer. For IHD, our recent review75 cites only the Hirayama study78 as reporting a non-significant relative risk of 1.16 (95% CI 0.94–1.43), while our review of passive smoking and stroke74 cites the Hirayama study as finding “no significant trend” and a study by Nishino et al79 as giving a relative risk of 0.75 (0.80–1.12). Our review of passive smoking and COPD76 again cited only the relative risk from the Hirayama study of 1.38 (0.86–2.21), though one very recently published study by Ukawa et al80 did report significantly increased RRs of 2.40 (1.39–4.15) and 2.88 (1.68–4.93) for passive smoking at home for ≤4 days per week and almost every day, as compared to none.\n\nFor lung cancer, the evidence is much more extensive and two recent reviews73,77 reported very similar overall relative risks for spousal or at home smoking of 1.26 (1.11–1.45) and 1.28 (1.10–1.48) based on 13 or 12 individual estimates, although our review73 suggested that most, if not all, of the ETS/lung cancer association might be explained by inadequate adjustment for potential confounding by diet and education and by bias due to misclassification of some true smokers as nonsmokers. Even were this association a causal result of exposure to passive smoking it could not explain the substantial difference in active smoking RRs between Asian and Western populations. Not only do the RRs for passive smoking not vary significantly by location73, but even if passive smoking exposure were particularly common in Japanese nonsmokers, the relatively weak association of passive smoking with lung cancer risk could not possibly explain why active smoking relative risks are two-fold or more higher in Western than in Asian populations.\n\n\nConclusions\n\nIn Japanese studies, smoking is related to an increased risk of all four diseases studied, though the increase is relatively weak for stroke, and the evidence is limited for COPD. For IHD, the estimated RR for current smoking, of 2.21 (95%CI 1.96–2.50) is similar to that reported in other Asian and in Western populations and is dose-related, increasing with amount smoked and reducing with years quit. For lung cancer, the estimated RR for current smoking of 3.59 (3.25–3.96), which is also clearly dose-related, is similar to that in other Asian populations but substantially less than in Western populations. The explanation of this difference is unclear but high rates of denial of cigarette smoking may contribute.\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": "Author contributions\n\n\n\nPNL decided on the methodology, checked the literature searches, studied the derivation of the relative risks, carried out the meta-analyses for IHD and stroke, checked the meta-analyses for lung cancer and COPD, and drafted the manuscript. KJC carried out the literature searches and derived the relative risks and checked the meta-analyses for IHD and stroke. AJT derived the relative risks for lung cancer and COPD. BAF carried out the meta-analyses for lung cancer and COPD, and provided detailed comments on early drafts of the paper. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nP N Lee Statistics and Computing Ltd, for whom PNL and KJC are directors, and BAF and AJT are consultants, have for many years carried out work for many different tobacco companies and organizations, including Philip Morris International, the sponsors of this study. The work described here has been carried out independently of the sponsors.\n\n\nGrant information\n\nFunding was provided by Philip Morris International. The only role they had in the design of the study and collection, analysis, and interpretation of data was to suggest that the review should be conducted and to provide a brief comment on a near final draft of the paper, suggesting only that we refer to our recent publication on indirectly estimated lung cancer rates by smoking status, reference 61 of the submitted paper.\n\n\nAcknowledgements\n\nWe thank Dr John Fry for statistical advice, and Yvonne Cooper and Diana Morris for assistance in preparing the various drafts of this publication. We also thank the sponsors for their financial support.\n\n\nSupplementary material\n\nSupplementary File 1: Completed PRISMA checklist.\n\nClick here to access the data.\n\nSupplementary File 2: Searches conducted. This document gives more details on the searches conducted.\n\nClick here to access the data.\n\nSupplementary File 3: Relative risks by amount smoked and duration of quitting.\n\nClick here to access the data.\n\nSupplementary File 4: More detailed meta-analysis results for cardiovascular disease and lung cancer.\n\nClick here to access the data.\n\n\nReferences\n\nSobue T, Yamamoto S, Hara M, et al.: Cigarette smoking and subsequent risk of lung cancer by histologic type in middle-aged Japanese men and women: the JPHC study. Int J Cancer. 2002; 99(2): 245–251. PubMed Abstract | Publisher Full Text\n\nWakai K, Inoue M, Mizoue T, et al.: Tobacco smoking and lung cancer risk: an evaluation based on a systematic review of epidemiological evidence among the Japanese population. Jpn J Clin Oncol. 2006; 36(5): 309–324. 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PubMed Abstract | Publisher Full Text\n\nHatanaka Y, Tamakoshi A, Tsushita K: [Risk factors for ischemic heart disease in males in the prime of life: An eight-year follow-up study]. Sangyo Eiseigake Zasshi. 2015; 57(3): 67–76. PubMed Abstract | Publisher Full Text\n\nMatsunaga M, Yatsuya H, Iso H, et al.: Similarities and differences between coronary heart disease and stroke in the associations with cardiovascular risk factors: The Japan Collaborative Cohort Study. Atherosclerosis. 2017; 261: 124–130. PubMed Abstract | Publisher Full Text\n\nAkiba S: Analysis of cancer risk related to longitudinal information on smoking habits. Environ Health Perspect. 1994; 102(Suppl 8): 15–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nForey B, Hamling J, Hamling J, et al.: International Smoking Statistics. A collection of worldwide historical data. Web edition. Sutton, Surrey: P N Lee Statistics and Computing Ltd, 2006–2016. Reference Source\n\nHuxley R, Jamrozik K, Lam TH, et al.: Impact of smoking and smoking cessation on lung cancer mortality in the Asia-Pacific region. Am J Epidemiol. 2007; 165(11): 1280–1286. PubMed Abstract | Publisher Full Text\n\nWoodward M, Lam TH, Barzi F, et al.: Smoking, quitting, and the risk of cardiovascular disease among women and men in the Asia-Pacific region. Int J Epidemiol. 2005; 34(5): 1036–1045. PubMed Abstract | Publisher Full Text\n\nWang J, Ye DQ, Wang K: [Meta-analysis on the stroke with overweight or obesity, smoking and alcohol drinking in Chinese residents]. Zhonghua Yu Fang Yi Xue Za Zhi. 2008; 42(2): 115–118. PubMed Abstract | Publisher Full Text\n\nLee PN, Forey BA: Indirectly estimated absolute lung cancer mortality rates by smoking status and histological type based on a systematic review. BMC Cancer. 2013; 13: 189. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAkiyama Y, Ohkawa Y, Matsuki H, et al.: Misclassification of smoking status: comparison of questionnaire data and urinary cotinine analysis. In: Leslie GB, Leslie KJ, Huang J and Qin Y, editors. Indoor air quality in Asia. Proceedings of the Second international conference on indoor air quality in Asia; 18–20 October 1994; Beijing. Rothenfluh, Switzerland: Indoor Air International: 1994; 319–323.\n\nLee PN: \"Marriage to a smoker\" may not be a valid marker of exposure in studies relating environmental tobacco smoke to risk of lung cancer in Japanese non-smoking women. Int Arch Occup Environ Health. 1995; 67(5): 287–294. PubMed Abstract | Publisher Full Text\n\nTsutsumi A, Kagawa J, Yamano Y, et al.: Relation between cotinine in the urine and indices based on self-declared smoking habits. Environ Health Prev Med. 2002; 6(4): 240–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJung-Choi KH, Khang YH, Cho HJ: Hidden female smokers in Asia: a comparison of self-reported with cotinine-verified smoking prevalence rates in representative national data from an Asian population. Tob Control. 2012; 21(6): 536–542. PubMed Abstract | Publisher Full Text\n\nWewers ME, Dhatt RK, Moeschberger ML, et al.: Misclassification of smoking status among Southeast Asian adult immigrants. Am J Respir Crit Care Med. 1995; 152(6 Pt 1): 1917–1921. PubMed Abstract | Publisher Full Text\n\nOffice of Population Censuses and Surveys: Health survey for England 1993. Series HS no. 3. Vol London: HMSO, 1995; 498.\n\nJarvis MJ, Feyerabend C, Bryant A, et al.: Passive smoking in the home: plasma cotinine concentrations in non-smokers with smoking partners. Tob Control. 2001; 10(4): 368–374. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVartiainen E, Seppälä T, Lillsunde P, et al.: Validation of self reported smoking by serum cotinine measurement in a community-based study. J Epidemiol Community Health. 2002; 56(3): 167–170. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWagenknecht LE, Burke GL, Perkins LL, et al.: Misclassification of smoking status in the CARDIA study: a comparison of self-report with serum cotinine levels. Am J Public Health. 1992; 82(1): 33–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWallner-Liebmann SJ, Grammer TB, Siekmeier R, et al.: Smoking denial in cardiovascular disease studies. Adv Exp Med Biol. 2013; 788: 35–38. PubMed Abstract | Publisher Full Text\n\nHeller WD, Sennewald E, Gostomzyk JG, et al.: Validation of ETS exposure in a representative population in Southern Germany. Proceedings of the Proceedings of Indoor Air '93; 4-8 July 1993; Helsinki. 1993; 3: 361–365.\n\nLee PN, Fry JS, Forey B, et al.: Environmental tobacco smoke exposure and lung cancer: a systematic review. World J Metaanal. 2016; 4: 10–43. Publisher Full Text\n\nLee PN, Thornton AJ, Forey BA, et al.: Environmental Tobacco Smoke Exposure and Risk of Stroke in Never Smokers: An Updated Review with Meta-Analysis. J Stroke Cerebrovasc Dis. 2017; 26: 204–216. PubMed Abstract | Publisher Full Text\n\nLee PN, Forey BA, Hamling JS, et al.: Environmental tobacco smoke exposure and heart disease: A systematic review. World J Metaanal. 2017; 5(2): 14–40. Publisher Full Text\n\nLee PN, Forey BA, Coombs KJ, et al.: Epidemiological evidence relating environmental smoke to COPD in lifelong non-smokers: a systematic review [version 1; referees: awaiting peer review]. F1000Research. 2018; 146. Publisher Full Text\n\nHori M, Tanaka H, Wakai K, et al.: Secondhand smoke exposure and risk of lung cancer in Japan: a systematic review and meta-analysis of epidemiologic studies. Jpn J Clin Oncol. 2016; 46(10): 942–951. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHirayama T: Lung cancer in Japan: effects of nutrition and passive smoking. In: Mizell M and Correa P, editors. Lung cancer: causes and prevention. Proceedings of the International Lung Cancer Update Conference; March 3-5, 1983; New Orleans, Louisiana. Deerfield Beach, Florida: Verlag Chemie International, Inc, 1984; 175–195. Data clarifications appear in Passive smoking [Letter]. N Z Med J.1990; 103(883): 54. PubMed Abstract\n\nNishino Y, Minami Y, Kawai M, et al.: Cigarette smoking and breast cancer risk in relation to joint estrogen and progesterone receptor status: a case-control study in Japan. Springerplus. 2014; 3: 65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUkawa S, Tamakoshi A, Yatsuya H, et al.: Passive smoking and chronic obstructive pulmonary disease mortality: findings from the Japan collaborative cohort study. Int J Public Health. 2017; 62(4): 489–494. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "31116",
"date": "16 Mar 2018",
"name": "Joshua E. Muscat",
"expertise": [
"Reviewer Expertise 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\nThese authors have vast experience in MA and the analysis is carefully done and interpreted. The methods conform to appropriate MA methodology.\nThis is a useful paper as differences in smoking-related risks between Japan and Western countries have important implications with regard to potential cigarette toxicity or risk moderators. The conclusions are quite reasonable.\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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": [
{
"c_id": "3514",
"date": "19 Mar 2018",
"name": "Peter Lee",
"role": "Author Response",
"response": "I thank Dr Muscat for his kind comments."
}
]
},
{
"id": "38476",
"date": "06 Feb 2019",
"name": "Wolf-Dieter Heller",
"expertise": [
"Reviewer Expertise Statistics",
"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\nThe authors report the results of meta-analyses based on studies in Japan relating cigarette smoking to the risk of the four major smoking-related diseases. Data are summarized for current and former smokers and in relation to amount smoked and time quit. The relative risks reported for Japan are compared with those for other Asian and Western studies reported in published meta-analyses.\nThe results shown are mostly quite similar to those reported elsewhere, with relative risks for current smoking elevated for all four diseases, relative risks for former smoking somewhat lower, and evidence of a dose response with increasing amount smoked and decreasing time quit. While current smoking relative risks for heart disease and stroke for Japan are similar to those in other countries, it is notable that, for lung cancer, the relative risks for Japan are much lower than seen in Western countries, though similar to those seen in other Asian countries. The paper includes an interesting discussion about the reasons why this might be so.\nGenerally, the paper is very well structured and clearly written, and the material presented provides considerable reassurance about the completeness of the searches and the accuracy of the meta-analysis results presented. Over the last decades Peter Lee and his co-authors have published a broad range of papers dealing with the risk of smoking in general. Monitoring these publications over many years I was always impressed how clearly the papers are written, especially the MA ones.\nThe results of the present publication are convincing, clear and concise. Concerning the lung cancer results this paper again points towards an important aspect in public health which definitely needs further research: the substantially lower RR in Japan compared to western countries.\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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": [
{
"c_id": "4411",
"date": "15 May 2019",
"name": "Peter Lee",
"role": "Author Response",
"response": "I thank Dr Heller for his kind comments and the time he spent on the review."
}
]
}
] | 1
|
https://f1000research.com/articles/7-204
|
https://f1000research.com/articles/6-2122/v1
|
12 Dec 17
|
{
"type": "Opinion Article",
"title": "Retract p < 0.005 and propose using JASP, instead",
"authors": [
"Jose D. Perezgonzalez",
"M. Dolores Frías-Navarro",
"M. Dolores Frías-Navarro"
],
"abstract": "Seeking to address the lack of research reproducibility in science, including psychology and the life sciences, a pragmatic solution has been raised recently: to use a stricter p < 0.005 standard for statistical significance when claiming evidence of new discoveries. Notwithstanding its potential impact, the proposal has motivated a large mass of authors to dispute it from different philosophical and methodological angles. This article reflects on the original argument and the consequent counterarguments, and concludes with a simpler and better-suited alternative that the authors of the proposal knew about and, perhaps, should have made from their Jeffresian perspective: to use a Bayes factors analysis in parallel (e.g., via JASP) in order to learn more about frequentist error statistics and about Bayesian prior and posterior beliefs without having to mix inconsistent research philosophies.",
"keywords": [
"practical significance",
"statistical significance",
"research evidence",
"p-values",
"Bayes factors",
"reproducibility",
"replicability"
],
"content": "Argument\n\nSeeking to address the lack of research reproducibility due to the high rate of false positives in the literature, Benjamin et al. (2017a); Benjamin et al. (2017b) propose a pragmatic solution which “aligns with the training undertaken by many researchers, and might quickly achieve broad acceptance” (also Savehn, 2017): to use a stricter p < 0.005 standard for statistical significance when claiming evidence of new discoveries.\n\nThe proposal is subject to several constrains in its application: (1) to claims of discovery of new effects (thus, not necessarily to replication studies); (2) when using null hypothesis significance testing (arguably Fisher’s approach, perhaps even Neyman-Pearson’s, but excluding other p-value-generating approaches such as resampling); (3) in fields with too flexible standards (namely 5% or above); (4) when the prior odds of alternative-to-null hypothesis is in the range 1-to-5, to 1-to-40 (stricter standards are required with lower odds); (5) for researcher’s consumption (thus, not a standard for journal rejection, although “journals can help transition to the new statistical significance threshold”; also, “journals editors and funding institutions could easily enforce the proposal”, Wagenmakers, 2017; and “its implementation only requires journal editors to agree on the new threshold”, Machery, 2017); (6) while still keeping findings with probability up to 5% as suggestive (and meriting publication if so “properly labelled”); (7) despite many of the proponents believing that the proposal is nonsense, anyway (that is, it is a quick fix, not a credible one; also Ioannidis in Easwaran, 2017; Resnick, 2017; Wagenmakers, 2017; Wagenmakers & Gronau, 2017).\n\nThe main analyses supporting the proposal were the following: (a) a calibration of p-values to Bayes factors under certain plausible alternatives (their Figure 1, Benjamin et al., 2017b, p. 2); (b) an estimated false positive rate under certain plausible prior odds of alternative-to-null hypotheses as a function of power (their Figure 2, Benjamin et al., 2017b, p. 3); and (c) a “critical mass of researchers now endors[ing] this change” (a.k.a., the 72 co-authors). Meanwhile, the proposal downplays the costs due to increasing sample size in order to ensure enough research power, the potential increase in false negatives and misconduct, and the existence of alternative solutions well known by, at least, some of the authors (despite Machery’s, 2017, claim to the contrary).\n\nNotwithstanding its potential impact, Benjamin et al.’s (2017a); Benjamin et al.’s (2017b) proposal is not recognizant of the entire engine of science (Figure 1), which has motivated an even larger mass of authors to offer their counter-arguments.\n\n\nCounter-arguments\n\nFrom Philosophy of science, Mayo (2017a), in particular, provides important counter-arguments against the philosophical standing of Benjamin et al.’s proposal. Quoting Greenland et al. (2016), Mayo asserts that whether p-values exaggerate the evidence against the null hypothesis depends on the philosophical background within which such claim is made: It may seem so from a Bayesian perspective, but both from a frequentist and an error statistics perspective, it is the Bayes factor which exaggerates the evidence (also 2017e). “I find it especially troubling”—she continues—“to spoze an error statistician…ought to use a Bayes Factor as the future gold standard for measuring his error statistical tool…even though Bayes Factors don’t control or measure error probabilities” (2017b). Furthermore, Mayo pinpoints the (old) fallacy of transposing the conditional, whereby the (error) probability of a test is confused with the (posterior) probability of a belief (also Trafimow et al., 2017). And despite “60 years (sic) old…demonstrations [showing] that with reasonable tests and reasonable prior probabilities, the disparity vanishes…they still mean different things” (2017c). In particular, “in an adequate account [of severity testing], the improbability of a claim must be distinguished from its having been poorly tested. (You need to be able to say things like, ‘it’s plausible, but that’s a lousy test of it.’)” (2017d). And “the method for such checking is significance tests!” (2017b).\n\nFrom Methodology, a large number of critics have faulted Benjamin et al.’s disregard of what is really affecting replication in order to focus on a relatively minor issue. Mayo, for example, lists biasing selection effects, cherry-picking, multiple testing, hunting for significance, optional stopping, violated statistical assumptions, missing or irrelevant statistical-substantive links, and questionable measurements as the main culprits in flawed findings and lack of reproducibility (2017a–e; also Gelman & McShane, 2017; Gelman, 2017b). Lakens et al. (2017) add lack of experimental redundancy, logical traps, research opacity, and poor accounting of sources of error, as well as the risks of reduced generalisability and research breadth were Benjamin et al.’s proposal to succeed. Methodological concerns were also raised by Amrhein & Greenland (2017); Black (2017); Byrd (2017); Chapman (2017); Crane (2017); Ferreira & Henderson (2017); Greenland (2017); Hamlin (2017); Kong (2017); Lew (2017); Llewelyn (2017); Martin (2017); McShane et al. (2017); Passin (2017); Steltenpohl (2017); Trafimow et al. (2017); Young (2017); Zollman (2017); and Morey (2017). Some researchers even propose the use of preregistration as a way of minimizing above problems (Hamlin, 2017; Llewelyn, 2017; van der Zee, 2017)\n\nThe pseudoscientific NHST element is the cornerstone of Benjamin et al.’s proposal, as it mixes Fisher’s tests of significance with Neyman-Pearson’s alternative hypotheses, Type II errors and power estimation, and with Bayesian prior probabilities, in order to argue about the false positive rate as the culprit for the lack of replicability. Indeed, the false positive rate argument has been heavily criticized by several authors: Mayo (2017b; also Byrd, 2017) chiefly derides as questionable the prior probabilities given to the hypotheses; Colquhoun (2017) denounces the credibility of Benjamin et al.’s false positive rate calculation for some of the prior probabilities used, which he sees as a rather liberal rate; Chapman (2017; also Byrd, 2017) claims that the prior probability ratios for the hypotheses are not realistic; Llewelyn (2017) raises an argument on prior probabilities and their combination with accumulated evidence in order to estimate the probability of future replication, not finding a more restrictive level of significance an adequate antecedent for increasing such probability; Krueger (2017) adds that, under the new proposal, the proportion of false negatives rises faster than the speed at which false positives drops, leading Phaf (2017) to wonder whether, because of it, “it makes more sense to increase, rather than reduce, the significance level for novel findings”; Kong (2017) states that the false positive rate formula is misleading and finds the expected false positive rate when testing the conventional 5% threshold.\n\nFurthermore, Perezgonzalez (2017) argued that the misinterpretation of p-values as evidence in favour or against a hypothesis has more to do with the pseudoscientific use of NHST than with frequentist testing proper (also Mayo, 2017b; McShane et al., 2017; Amrhein & Greenland, 2017). As Benjamin et al.’s proposal is made within the pseudo-philosophical argument of NHST (e.g., confusing statistical and substantive significance; Mayo, 2017c), a lower threshold of significance does not improve such ‘magical thinking’ (also Argamon, 2017; Diebold, 2017; Greenland, 2017; Krueger, 2017; Phaf, 2017). Lakens et al. (2017; also Morey, 2017; O’Rourke, 2017) equally warn that the proposal exaggerates the focus on single p-values in scientific practice, education, decision making and publication.\n\nUnsupported conclusions are another consequence of pseudoscientific thinking, and McShane et al. (2017) and Amrhein & Greenland (2017) highlight the risks of studies published under the new banner exaggerating effect sizes and overconfidence in significant results while discounting non-significant findings. De Brigard (2017) argues, instead, for the need to be humbler and not generalize to populations, irrespective of statistical significance, effects that may only be present in the sample.\n\nThe descriptive data analysis element was briefly touched upon by Mayo (2017a)—who recommends abandoning significance testing in favour of inferring the magnitudes that are well (or poorly) indicated by the data (a.k.a., CIs)—and Lew (2017), Phaf (2017), and McShane et al. (2017)—whom argue for the need to interpret evidence in the context of other findings and theoretical models in lieu of NHST.\n\nRegarding data testing, Mayo (2017a); Mayo (2017b) and Perezgonzalez (2017) have no particular problem with a lower level of significance although both reckon Benjamin et al.’s proposal does not address anything in particular with frequentist testing proper. Mayo equally sees such lowering unnecessary as “you might not want to be too demanding before claiming evidence of a [null] model violation” (2017b), while the “lack of replication is effectively uncovered thanks to statistical significance tests” (2017e). Young (2017) reminds us of the appropriate use of significance tests in experimental contexts. McShane et al. (2017) also argues the credibility of uniformly most powerful priors and tests and the loss of its connection to Bayesianism as for justifying Benjamin et al.’s proposal (but see Wagenmakers & Gronau’s counter-argument, 2017).\n\nOther authors recommend continuing using p-values either as part of frequentist tests proper (Bates, 2017) or without significance testing, the latter including Colquhoun (2017), Mayo (2017b; who also proposes estimating false positive rates), Greenland (2017, who proposes transforming them to bits of information against the null hypothesis), Diebold (2017); Funder (2017); Lakens et al. (2017, who recommend justifying whatever thresholds may be used at the design stage), McShane et al. (2017, who recommend using them as descriptives among other neglected factors—but see Wagenmakers & Gronau’s, 2017, counter-argument, and Gelman’s, 2017a, counter-counter-argument), and Amrhein & Greenland (2017). Argamon (2017) suggests substituting Bayesian statistics, instead.\n\nAddressing replication directly, Chapman (2017) and Trafimow et al. (2017) point out that the problem with replication is not too many false positives but insufficient power. Krueger (2017; also McShane et al., 2017, Trafimow et al., 2017) chides Benjamin et al. for the incoherence of considering replication as order-dependent and inverting the exploratory-confirmatory nature of replication by proposing to make the former more difficult to achieve and the latter more liberal. He further chides them on whether they would disallow their own past findings at the 5% threshold. Lakens et al. (2017; also Crane, 2017) found biases in the analyses done by Benjamin et al., and conclude that there is no [Bayesian] evidence that a lower level of significance improves replicability. They also warn of the risks of fewer availability of replication studies were the proposal to succeed (also Greenland, 2017). Gelman (2017b); Hamlin (2017); Ickes (2017); Kong (2017); Roberts (2017), and Trafimow et al. (2017) propose to stop the proverbial beating around the bushes and perform direct replications as a straightforward measure of replicability.\n\nThe hypothesis testing element (namely full Bayesian hypothesis testing) has been seldom touched upon but briefly by Llewelyn (2017), who extended the need for prior probabilities and even more accumulated evidence as pre-requisite for estimating the probability of hypotheses being ‘true’.\n\nIn conclusion, despite Benjamin et al.’s best intentions, their proposal only reinforces the NHST pseudoscientific approach with a stricter bright-line threshold for claiming evidence it cannot possibly claim—irrespective of how well it calibrates with Bayes factors under certain conditions—while dismissing potential consequences such as the stifling of research and its publication, an increase in false negatives, an increase in misbehaviour, and a continuation of pseudoscientific practices. Furthermore, theirs is a frequentist solution many of their Bayesian proponents do not even believe in, born out of a false belief of lacking equally simple but better-suited alternatives (Machery, 2017).\n\nIn reality, an equally simple and better suited alternative exists, perhaps the only one the authors could be entitled to make from their Jeffresian perspective, hence our own recommendation: Use JASP, the stand-alone, free-to-download, R-based statistical software with user-friendly interface, developed by Wagenmakers’ team (https://jasp-stats.org1). JASP allows for analysing the same dataset using frequentist (Fisher’s tests of significance) and Bayesian tools (Jeffreys’s Bayes factors) without necessarily forcing a mishmash of philosophies of science. JASP allows for the Popperian (also Meehl’s, Mayo’s) approach to severely testing a null hypothesis but also to ascertain the credibility of tests based on the observed data. JASP allows researchers to qualitatively calibrate their significance tests to Bayes factors but also the possibility of calibrating Bayes factors to frequentist tests so as to ascertain their error probabilities. JASP is an existing simple and easy application that shows two interpretations of the same data, which aligns well with the training undertaken by frequentist and Jeffreysian researchers alike while allowing them the opportunity to learn the alternative philosophy—irrespective of which they will choose for publication—and may as well help diminish the misinterpretation of results and the reaching of unsupported conclusions. In so doing, it may even help improve replicability.\n\n\nNotes\n\n1Worthy alternatives are Morey’s R package for Bayes factors (http://bayesfactorpcl.r-forge.r-project.org), Morey’s Bayes factor calculator (http://pcl.missouri.edu/bayesfactor), and Dienes’s Bayes factor calculator (http://www.lifesci.sussex.ac.uk/home/Zoltan_Dienes/inference/Bayes.htm). Even better would be to skip comparing models altogether and go for full Bayesian hypothesis testing (e.g., Kruschke, 2011). Yet none of those alternatives surpass, at present, JASP’s interface and its flexibility for parallel analysis.",
"appendix": "Competing interests\n\n\n\nNo competing interests.\n\n\nGrant information\n\nThis publication was financially supported by Massey University Research Fund (MURF) Mini Grant 2017 for Open Access publication, Massey University, New Zealand.\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\nAmrhein V, Greenland S: Remove, rather than redefine, statistical significance. Nat Hum Behav. 2017. Publisher Full Text\n\nArgamon SE: New \"p < 0.005\" standard considered harmful [Web log comment]. 2017. Reference Source\n\nBates T: Changing the default p-value threshold for statistical significance ought not be done, and is the least of our problems [Web log post]. 2017. Reference Source\n\nBenjamin DJ, Berger JO, Johannesson M, et al.: Redefine statistical significance. PsyArXiv Preprints. 2017a. Publisher Full Text\n\nBenjamin DJ, Berger JO, Johannesson M, et al.: Redefine statistical significance. Nat Hum Behav. 2017b; 1: 0189. Publisher Full Text\n\nBlack J: Thresholds [Web log comment]. 2017. Reference Source\n\nByrd J: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017. Reference Source\n\nChapman P: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017. Reference Source\n\nColquhoun D: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017. Reference Source\n\nCrane H: Why \"Redefining Statistical Significance\" will not improve reproducibility and could make the replication crisis worse. PsyArXiv Preprints. 2017. Publisher Full Text\n\nDe Brigard F: Should we redefine statistical significance. A brains blog roundatble [Web log comment]. 2017. Reference Source\n\nDiebold FX: New p-value thresholds for statistical significance [Web log post]. 2017. Reference Source\n\nEaswaran K: Should we redefine statistical significance. A brains blog roundatble [Web log comment]. 2017. Reference Source\n\nFerreira F, Henderson JM: Defending .05: It’s not enough to be suggestive [Web log post]. 2017. Reference Source\n\nFunder D: Thresholds [Web log post]. 2017. Reference Source\n\nGelman A: Response to some comments on “Abandon Statistical Significance” [Web log post]. 2017a. Reference Source\n\nGelman A: When considering proposals for redefining or abandoning statistical significance, remember that their effects on science will only be indirect! [Web log post]. 2017b. Reference Source\n\nGelman A, McShane B: Should we redefine statistical significance. A brains blog roundatble [Web log comment]. 2017. Reference Source\n\nGreenland S: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017. Reference Source\n\nGreenland S, Senn SJ, Rothman KJ, et al.: Statistical tests, p values, confidence intervals, and power: a guide to misinterpretations. Eur J Epidemiol. 2016; 31(4): 337–350. Publisher Full Text\n\nHamlin K: Should we redefine statistical significance? A brains blog roundtable [Web log comment]. 2017. Reference Source\n\nIckes W: Thresholds [Web log comment]. 2017. Reference Source\n\nKong X: Redefine statistical significance? Let's just do science in a scientific way [Web log post]. 2017. Reference Source\n\nKrueger JI: Fear of false positives [Web log post]. 2017. Reference Source\n\nKruschke JK: Doing Bayesian data analysis. A tutorial with R and BUGS. Amsterdam, The Netherlands: Academic Press. 2011. Reference Source\n\nLakens D, Adolfi FG, Albers CJ, et al.: Justify your alpha: A response to “Redefine statistical significance”. PsyArXiv Preprints. 2017. Publisher Full Text\n\nLew M: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017. Reference Source\n\nLlewelyn H: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017. Reference Source\n\nMachery E: Should we redefine statistical significance. A brains blog roundatble [Web log comment]. 2017. Reference Source\n\nMartin S: Response to some comments on “Abandon Statistical Significance” [Web log comment]. 2017. Reference Source\n\nMayo D: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log post]. 2017a. Reference Source\n\nMayo D: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017b. Reference Source\n\nMayo D: Should we redefine statistical significance? A brains blog roundtable [Web log comment]. 2017c. Reference Source\n\nMayo D: New venues for the statistics wars [Web log post]. 2017d. Reference Source\n\nMayo D: Going round and round again: a roundtable on reproducibility & lowering p-values [Web log post]. 2017e. Reference Source\n\nMcShane BB, Gal D, Gelman A, et al.: Abandon statistical significance. 2017. Reference Source\n\nMorey RD: When the statistical tail wags the scientific dog [Web log post]. 2017. Reference Source\n\nO’Rourke K: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017. Reference Source\n\nPassin T: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017. Reference Source\n\nPerezgonzalez JD: Better Science - The call for significance of 5‰ (0.005) [Video file]. 2017. Reference Source\n\nPhaf RH: Comment on redefine statistical significance [Web log post]. 2017. Reference Source\n\nResnick B: What a nerdy debate about p-values shows about science — and how to fix it [Web log post]. 2017. Reference Source\n\nRoberts B: Thresholds [Web log comment]. 2017. Reference Source\n\nSavehn T: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017. Reference Source\n\nSteltenpohl CN: The littlest p: redefining statistical significance [Web log post]. 2017. Reference Source\n\nTrafimow D, Amrhein V, Areshenkoff CN, et al.: Manipulating the alpha level cannot cure significance testing. Comments on “Redefine statistical significance”. PeerJ Preprints. 2017; 5: e3411v1. Publisher Full Text\n\nvan der Zee T: Arguing for all the wrong reforms [Web log post]. 2017. Reference Source\n\nWagenmakers EJ: Redefine statistical significance Part I: Sleep trolls & red herrings [Web log post]. 2017. Reference Source\n\nWagenmakers EJ, Gronau Q: Redefine statistical significance Part IX: Gelman and Robert join the fray, but are quickly chased by two kangaroos [Web log post]. 2017. Reference Source\n\nYoung S: “A megateam of reproducibility-minded scientists” look to lowering the p-value [Web log comment]. 2017. Reference Source\n\nZollman K: Should we redefine statistical significance? A brains blog roundtable [Web log comment]. 2017. Reference Source"
}
|
[
{
"id": "28961",
"date": "14 Dec 2017",
"name": "Patrizio Tressoldi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a timely and well organized synthesis of the debate ignited by Benjamin et al.’s, 2017 influential paper which is still going on. My only suggestions are the followings: - to add a legend to the acronyms in Figure 1, e.g. ES, CI, etc.; - to correct the statement that the JASP software allows only a Jeffreys’s Bayes Factor given that in the present .8.4.0 version, the user can also choose informed priors based on Cauchy, Normal or t distributions; - I think that a final synoptic table related to the information offered by the different statistical indices, i.e., p value, CI, Bayes Factor H1/H0, Effect Size, etc., could help the readers acknowledge the different informational value among them and consequently to choose those ones which answer better the questions they ask their data.\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": "3301",
"date": "28 Dec 2017",
"name": "Jose Perezgonzalez",
"role": "Author Response",
"response": "Thank you very much for your prompt review. I will upload a new version incorporating two of the suggestions made (plus proper acknowledgement). I will append an example to address the third suggestion as soon as a current research using a double frequentist/Jeffresian is finished."
}
]
},
{
"id": "28966",
"date": "21 Dec 2017",
"name": "Juan Carro Ramos",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors discuss the article by Benjamin et al., 2017 and their proposal of p <.005 as a possible solution to the lack of research reproducibility due to the high rate of false positives in the literature. The article reviews the current debate topics on NHST and contributes to the reflection on how to advance towards a Science that allows accumulating valid scientific knowledge. Suggestions: Write down at the end of the article a simple example where the result is interpreted with NHST and with the Bayes factor and the utility of JASP is presented.\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": "3302",
"date": "28 Dec 2017",
"name": "Jose Perezgonzalez",
"role": "Author Response",
"response": "Thank you very much for your prompt review. I will append an example to address your suggestion as soon as a current research using a double frequentist/Jeffresian approach is concluded."
}
]
},
{
"id": "28963",
"date": "12 Jan 2018",
"name": "Stephen Senn",
"expertise": [
"Reviewer Expertise Biostatistics",
"Drug Development",
"Statistical Inference"
],
"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 consider this is a valid contribution to the debate on this topic but like many such contributions it reflects a particular viewpoint, my own contributions1 in this field are no exception, and like many contributors, and, again, I am probably also guilty, there is minimal recognition that their own views are not reasonable from other perspectives. In particular (see below) they are over-fond of the adjective pseudoscientific and their use of it without further justification is, in my view, a form of pseudo-argumentation.\nParticular objections that I have are the following.\nThey state: \"The pseudoscientific NHST element is the cornerstone of Benjamin et al.’s proposal, as it mixes Fisher’s tests of significance with Neyman-Pearson’s alternative hypotheses.\" This, in my opinion, is an unsubstantiated and misleading jibe. First, although this claim is often made, it is not correct that NHST is some sort of monstrous hybrid of Neyman-Pearson (N-P) and Fisherian views. It very naturally uses common elements that belong in both.(2) For instance, Fisher, although usually associated with P-values, introduced the habit of tabulating percentage points in Statistical Methods for Research Workers. Secondly, as Lehmann, a classic proponent of the N-P view explained in Testing Statistical Hypotheses, P-values are a practical way of allowing scientist who may not agree on appropriate Type I error rates to share results efficiently. Furthermore, although I have little enthusiasm for what Benjamin et al are doing, I think that fact that NHST seems to represent some sort of a fusion of Fisher and NP (a point that is much exaggerated and of little relevance) is the least important aspect of what they are doing. If they had rejected everything the Fisher proposed and described what they were doing as trying to convince diehard N-P acolytes that the common 5% level was better changed to 0.5%, it would have zero technical effect on what they are proposing and would not make it either better or worse. Whatever problems or virtues there are with the Benjamin et al proposal, the Fisher, N-P fusion is a complete red herring. Will JASP and Bayes Factors (BF) really save statistical inference? I doubt it. One of the problems with the Jeffreys approach is the huge influence that the lump of probability on the 'null' has on the posterior inference. NHST is far less dependent on this and, in a context in which I work, that of drug development, the practical challenge is to avoid recommending a treatment that is worse. it is a strength of NHST that one does not have to worry too much about whether one is talking about precise(or point hypotheses) on the one hand or dividing ones on the other. Of course, Bayesians could regard this as not a strength but a weakness, on the lines that if it does make a difference to Bayesian posterior statements (and the difference can be enormous) it ought to in the NHST context. (It is a very common habit of Bayesians to abrogate the right of judging other systems by theirs.) However, I also note that amongst the Bayesian commentators on the recent ASA statement, some dismissed precise hypotheses as being completely irrelevant. Like many commentators, they appear to accept much of the replication crisis at face value. However, there are four aspects here that are important. i) Gaming can effect any system (as they recognise) ii) Much of the discussion has taken it as obvious that failure to replicate is a problem of the original study only, rather than a shared problem. However, it can only be the former if the second study is perfect and that includes not being subject to sampling variation itself. What thus becomes important is to judge how well studies of infinite size are predicted, not those that just happen to be the same size as the original (2). iii) It assumes that what is important is the extent to which the P-value predicts the P-value but there is no claim under NHST that this is so. What is relevant is the extent to which it predicts the sign of the true difference Furthermore, to shackle the Bayesian approach with the same standard (what's sauce for the goose is sauce for the gander) would require that a high BF predicted a further high BF (using data from the subsequent study only) with high probability. JASP on its own will not deliver this. iv) Finally, if the only replication that matters is failure to replicate 'significance' then the proposal of Benjamin et al is nowhere near radical enough. 'Significance' must never be granted. On the other hand, if false negatives are also a problem, and not just false positives, then the solution has to be rethought from the beginning. (To be fair to the authors, they do cite Krueger's, 2017 warning about this but it is unclear to me to what extent they take it on board in what they are proposing.)\nHowever, putting aside my objections to some of the polemics of the article, the idea of supplementing P-values by other inferential statistics strikes my as being sensible and I certainly approve their suggestion of keeping Bayes factors separate from P-values. In fact, I have no objection to Bayes Factors provided that their very tentative nature is recognised and I certainly sign up to the idea of the utility of having different ways of looking at data34.\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": "3438",
"date": "19 Feb 2018",
"name": "Jose Perezgonzalez",
"role": "Author Response",
"response": "Thank you very much for your prompt review. Indeed, we should have been more explicit on the labels used. The 'pseudoscientific' emphasis follows on a particular rhetoric in a previous article, mainly based on the philosophical incompatibility between Bayes's and frequentist philosophies (e.g., Gigerenzer, 2004; Mayo, 2010). I have amended this now by making such reference explicit. On the other hand, that rhetoric is also used to differentiate NHST from Fisher's and Neyman-Pearson's approach. The latter two are technically similar (in fact, N-P simply perform a significance test after controlling for power, something that Fisher could not do because he didn't contemplate a specific alternative hypothesis). Substituting NHST for either approach is alright if we know what we are talking about, which seems to be your point regarding the 'red herring', actually. Quite often, however, NHST is used to confuse both, e.g., to talk about power or Type II error in theory when they are not even controlled in practice, something we see quite often in Statistics textbooks for Psychology, for example. In a manner of speaking, Benjamin et al., also blame Fisher for things that pertains better to Neyman-Pearson's approach (power analysis). We also agree with your point about Bayes Factors: it will not save statistical inference, and the lump on the point null begs the question, as well. Thus, our recommendation: Benjamin et al., should have recommended, at most, a parallel analysis, not a demotion of p-values. Such parallel analysis could actually help learn more about the data, and in recent research, we used BF to learn more about our significant as well as about our nonsignificant results, something rather difficult to do with a significant test alone (in this case, it is \"easier\" to justify the lump on the null as a way of learning how close our nonsignificant data is to such 'artificial' model of a pure '0' effect size). I have linked to an example using such double frequentist/Jeffresian approach, as Supplementary Data."
}
]
}
] | 1
|
https://f1000research.com/articles/6-2122
|
https://f1000research.com/articles/7-103/v1
|
23 Jan 18
|
{
"type": "Research Article",
"title": "Positive Mental Health from the perspective of Iranian society: A qualitative study",
"authors": [
"Arash Mirabzadeh",
"Monir Baradaran Eftekhari",
"Katayoun Falahat",
"Homeira Sajjadi",
"Meroe Vameghi",
"Gholamreza Ghaedamini Harouni",
"Arash Mirabzadeh",
"Monir Baradaran Eftekhari",
"Homeira Sajjadi",
"Meroe Vameghi",
"Gholamreza Ghaedamini Harouni"
],
"abstract": "Background: According to the World Health Organization, mental health relates, not only to the absence of mental disorder, but also to Positive Mental Health. Studies have shown that promoting positive mental health, not only reduces the prevalence and incidence of mental disorders, but also affects the process of treatment and reduces related burden. However, this concept has different interpretations in different cultures, and in many societies, mental health is still considered the absence of mental illness. Thus, the present study was conducted to provide an in-depth understanding of Iranian adults` perspective towards the concept of positive mental health. Materials and Methods: In the present qualitative study, eight focus group discussions (6 to 8 adults in each session) were held consisting of 30 to 60 year-old men and women from Tehran. Data were analyzed in \"DeDoose\" qualitative software using content analysis. Results: According to the data obtained, participants found no difference between positive mental health and mental health, mostly equating it to the absence of mental disorders and having positive energy, peace in and satisfaction with life. According to the results, positive mental health has four domains of emotional/psychological, spiritual, social, and life skills. Conclusion: Understanding an individual’s positive mental health concepts culturally and providing appropriate community based programs can significantly promote the mental health of the community.",
"keywords": [
"Positive Mental Health",
"qualitative study",
"focus group discussion"
],
"content": "Introduction\n\nAccording to the World Health Organization (WHO), mental health is more than just the absence of mental illness and it is also considered a state of well-being where individuals realize their skills, cope with normal life stresses, can work fruitfully and productively, and are able to make a contribution to their community (see WHO statement on mental health promotion). Based on the results obtained in a study by Keyes et al., complete mental health is a function of two interrelated independent entities, with positive mental health (PMH) as one axis, and the absence of mental disorder as the other1–3. The complete mental health approach covers previous approaches such as the pathogenic approach, which defines mental health as the absence of mental disorders, and the salutogenic approach, that focuses on the positive aspects of human abilities and functioning in emotions and behaviors1.\n\nMany studies on mental disorders and related diseases have been conducted so far throughout the world. A systematic review study by Steel et al., reported that worldwide one in five adults had experienced a mental disorder during the previous 12 months and 29.2% during their lifetime (lifetime prevalence) in 20144. These disorders occur at younger ages, and are chronic, and thus have many adverse effects on various aspects of life, including educational performance, employment, income, personal relationships and social participation5. Thus, there is a need for further, more effective investment into prevention, treatment, and rehabilitation of these disorders.\n\nDespite the advances made in medical and psychiatric therapies, the prevalence and burden of mental diseases has not decreased6. Therefore, community-based interventions should be conducted to prevent the incidence of new cases, and thus promote mental health at a community level7. Using the slogan \"mental health for all\", many countries have implemented extensive policies to promote mental health with an emphasis on its positive aspects8. In fact, the concept of PMH includes personal capabilities in psychological, emotional, and social dimensions and promoting PMH leads to a reduction in psychological disorders, a decrease in the probability of all-cause mortality, prevention of suicidal behaviors, and impairment in academic performance9–12. Many studies suggest that PMH, as a key factor, will lead to positive cognition, positive behavior, and increased cognitive capabilities12,13.\n\nAccording to the literature, PMH or mental well-being has two traditional approaches: Hedonic or positive feeling/affects that relies on feeling good (subjective wellbeing, life satisfaction and happiness), and Eudemonic, or positive functioning, with emphasis on optimal social and psychological functioning (engagement, fulfillment, sense of meaning and social wellbeing)3,14. PMH is formed by the combination of these two approaches, and is conceptualized by the presence of emotional, psychological, and social well-being15. Since this concept is affected by demographic, biological, cultural, socioeconomic and psychological factors etc.16,17, it occurs very differently in different countries. For instance, the high level of PMH (flourishing) is 11.6% in South Korea, but 76.9% in Canada18. A study conducted by Nosratabadi on Iranian students showed that 16% of students were classified as flourishing19, and a study by Joshanloo et al. reported the level of PMH among Iranian students was lower than those in the Netherlands and South Africa20. Understanding and determining the concept of PMH based on cultural-social differences is essential for promoting PMH and preventing psychological disorders. Furthermore, no study has yet addressed this subject in Iranian culture. Therefore, it was decided to conduct a qualitative study to carefully explore the concept of PMH and its domains in the Iranian society.\n\n\nMethods\n\nThe present study was conducted using a qualitative approach, which has proven useful in cultural, psychological, and psychiatric research21. The reason for choosing a qualitative approach was its ability to provide an in-depth understanding of the concept of PMH from the perspective of the society21.\n\nData were collected from May to August 2017. Participants were selected by purposive sampling technique22 and with maximum diversity from 30 to 60 years-old Iranian men and women living in different districts of Tehran, capital of Iran. According to purposive sampling and with the aim of obtaining maximum variation of participants` experiences regarding PMH, different characteristics were considered in sampling. These characteristics were gender, age, marital status, level of education, occupation and the social class.\n\nData was collected using focus group discussions (FGD). This method was chosen for its flexibility, and ability to boost constructive interactions amongst individuals, while increasing the understanding of the concepts being studied21. The number of FGDs was decided according to data saturation, and discussions were performed in single gender groups (men and women separately). Each FGD lasted between 60 and 90 minutes on average, and ended when no new concept could be extracted. Each session was conducted with the participation of 6 to 8 Iranian adults. The locations for group discussions were chosen by interviewees (workplace, tailor shop, municipality health home etc.). In each FGD, first the moderator (corresponding author) introduced herself and her colleague (note taker) and explained the study objectives. A guide questionnaire was used to conduct and direct FGDs. This guide questionnaire had been designed by the research team members using a review of the literature and then validated by group discussion with experts (Supplementary File 1). After the introduction, FGD began with a general question “What characteristics do you think a person with mental health has? Please explain”. Then, the FGD continued with other questions such as” In your opinion does mental health have any domains?” The concept of PMH and its domains were also asked and debated. During discussions, participants were encouraged to express openly about their opinions and experiences, and probing words such as where, when, how, why, etc. were used to ensure proper understanding of concepts referred to by participants, with the intention of exploring new concepts. The FGD ended when no new topics were expressed.\n\nAll discussions (conducted in Persian) were recorded using a digital voice recorder. Each FGD was transcribed and all field notes and contextual details were added immediately after the session. Data were analyzed using directed content analysis23. By using the Dedoose qualitative software Version 7.6.6, all transcribed discussions were imported into the software as a Microsoft Word template. Discussions were read line-by-line and appropriate codes were assigned by the software. Then, coding was performed according to the study framework and the concept of PMH and its domains. Themes and subthemes extracted were used as the basis for creating new codes or modifying previous ones. Inductive codes were identified according to categories already found. Various aspects of trustworthiness were observed. All results obtained from each group discussion were shared with participants and checked by them (respondent validation). Data analysis was performed by two experts in the research team (dependability). Details of methodology, including collection of data, analysis, coding, etc. were clearly recorded (transferability). Team consistency was observed by the research team members during the process of analysis24.\n\n\nEthics and consent\n\nAll participants completed informed consent forms. They were assured that their identities and other related information and responses would remain confidential throughout the survey and after.\n\nParticipation was on a voluntary basis, meaning they could leave the FGD at any time during the session. Permission to record the discussions was obtained from participants for appropriate and precise analysis. The study protocol was approved by the ethical committee of the University of Social Welfare and Rehabilitation Sciences (Code: IR.USWR.REC.1396.204).\n\n\nResults\n\nIn the present study, eight FGDs were conducted with individuals living in Tehran (4 FGDs with men and women separately). A total of 51 adults (29 women and 22 men) took part in these FGDs. The mean age of participants was 46.1. About one fifth of participants were unemployed and 47% had at least one university education. In this study, all individuals who had university education were considered as educated and those with high school certificate and lower degrees were deemed uneducated persons. Demographic variables of FGD participants are shown in Table 1.\n\nIn our qualitative investigation, two main themes were extracted; namely, general concept of positive mental health and domains of positive mental health, and four subthemes namely a) emotional/psychological domain, b) spiritual domain, c) life skills domain, and d) social domain (see Table 2).\n\nThe majority of participants found no difference between PMH and mental health, and in fact considered them absolutely the same. Some believed that PMH meant getting positive energy from family and social environments. In fact, in providing a general definition of PMH, the focus was on positive energies, with both internal and external origins.\n\n\"PMH means absorbing all positive energies…\"(An uneducated woman).”PMH means having control over your behaviors and solving your problems\"(An educated woman). \"PMH means energizing yourself and enjoying your life …\"(An educated man).\n\nIn view of most participants, PMH is a personal attribute that initially begins with being happy and living a happy life, and continues with satisfaction, flexibility, peace, and altruism.\n\n\"I believe it is better to be happy in every situation. Laughing is the treatment of all incurable ailments…\"(An educated man).\n\nSome considered happiness as the logical outcome of being in control of the situation. “Happiness is brought about by control over different situations. We behave correctly when we are in control” (An educated man), and women believed: “PMH rather meant enjoying what you have and being satisfied with your circumstances”.\n\nThe view of many participants was, PMH has social dimensions, as well. Interpersonal relationships, respect for the rights of others, and observing social values are among dimensions that make up PMH.\n\n\"You must behave in such a way to respect the personality of every single person in the community\"(An educated man). \"A mentally healthy person does not do anything that is against the custom\" (An educated woman).\n\nHaving different skills was cited to complement the definition of PMH, and it was separately proposed as one of the dimensions of PMH due to its particular importance.\n\nAccording to participants’ views, we identified four domains of PMH in Iran, namely emotional/psychological, spiritual, social, and life skills in Iranian culture. Overall, these domains consisted of 29 subthemes covering different characteristics of an individual with PMH. Emotional/psychological is the main domain that covers many interlinked attributes of a mentally healthy individual. As an Islamic Asian country, where Iranian culture and Islamic beliefs are intertwined, spirituality and religiousness are considered as an essential element of PMH, which are presented as a separated domain. It also should be noted that developing life skills was mentioned separately due to the importance that participants placed on it (Table 2).\n\nEmotional/psychological domain. All FGD participants said that emotions and personal characteristics are essential elements of mentally healthy people. They described emotions such as love, kindness, happiness etc., as the software of a human being. One must have suitable and harmonized software and hardware to be defined as a healthy person.\n\n- Satisfaction and calmness: According to participants’ views, satisfaction was one of the most important attributes of those with PMH. Both gender groups believed that satisfaction was a unique attribute that protects an individual against adversities and brings calmness.\" It is not important what our financial status is; it is important seizing the moment and always feeling satisfied….\"(An educated woman). \"Financial issues are important; they don't bring happiness although some people lose their mental stability when they become poor….\" (An uneducated woman).\n\n- Affection was another attribute that was proposed in the emotional domain of PMH. Affection included loving oneself, family, friends, and society leads to kindness and power to support and assist others. \"To Love yourself, others, family, colleagues, and neighbors without any expectation is very effective. One with PMH should be in love, really in love with others …. “(An uneducated man).\n\n- Empathy which was derived from feeling of love cited by participants. Participants believed in it as an inseparable trait of people with good mental health despite of religion or ethnicity “An emotionally healthy person cannot rest when he sees someone in trouble; saddens by sadness of others, cheers up with other people's happiness. Such a person cannot be indifferent, and is affected by other people's feelings and emotion…“(An educated woman).\n\n- Expressing feelings and emotions: Mostly women pointed to this characteristic as a coping mechanism encountering unpleasant situations. Men cited to be mentally healthy one should consider logic in his emotion. “Anyone who does not consider emotion will lose his/her mental health…no one likes an apathetic person…” (An uneducated woman).\n\n- Mutual understanding: Most of participants noted it is an essential element in a relationship, especially in family relationships. One with good mental health understands others emotionally and psychologically, and can respect and support them more appropriately.\n\n- Optimism: Many participants agreed that positive thinking and positive feeling creates trust, hope and progress in activities and that is the reason why people interested in optimistic people \"Being positive generates hope, even if it is faked…\" (An educated women).\n\n- Personality stability is another issue in the psychological dimension of PMH. Participants expressed mentally healthy people never change their manner easily and are reliable. In normal situations, everyone has a balanced character while he/she may experience different levels of instability in adversity “…if each person has a steady personality in the face with miseries, and maintains his/her balance, that person is a mentally healthy… ”(Men`s group).\n\n- Feeling good: This characteristic was mentioned in both male and female FGDs \"feeling good is important, and everyone should find out what makes her/him well. Some feel good by laughing and some by crying. It depends on personal characteristics…\" (An uneducated woman).\n\n- Patience This dimension also included patience in adversity in all life conditions. A patient person has less expectation and more contentment. “The threshold of patience and tolerance is very high in a mentally healthy person and he is patient in all adversities …\"(Men`s group).\n\n- Autonomy: \"Autonomy of personality is very important. Dependent people are very vulnerable…\" (Men`s group).\n\n- Flexibility: Participants believed that flexible individuals experience less stress. \"Mentally healthy people accept life problems and have a better position to come to terms with them…\" (Women’s group).\n\nSpiritual domain. In people's view, spirituality is another of PMH’s personal dimension and emerged as a component of it. Spirituality was perceived to be essential in encountering adverse events. Participants mentioned a wide range of virtues based on their religious beliefs and values. They did not differentiate between spirituality and religiousness, although most of them stated their religious beliefs and practices.\n\n- Connection with God: Most women believed that this dimension has a role in the definition of PMH because it strengthens being thankful, contented, and patient, which were recalled as flexibility. \"When you are connected with God, everything is perfect. People, who are connected with God, can better accept difficult circumstances of life…” \"Anyone who seeks help only from God and has a strong connection with God, it means she is perfect….”\n\n- Faith in God and belief in the other world (Judgment day): Another issue relating to the spiritual domain of PMH is faith which brings forgiveness, feeling of gratitude and peace. \"Belief in the judgment day and worship are attributes of mentally healthy person…” (An uneducated woman). “Being thankful and having faith is the core key. Having faith makes the individual peaceful, and a peaceful person is mentally healthy….\" (An educated man).\n\n- Trust in God: Another issue in this domain is trust in God. Most participants believed that people, who trust in a solid support like God, attain peace and tranquility that guarantees their mental health.\n\n- Moral values: Many moral qualities and values considered attributes of a mentally healthy person were presented by participants in all FGDs like truthfulness, kindness, forgiveness, charity, devotion, thankfulness, composure, helpfulness, etc.\n\nSocial domain. All participants believed that individuals with PMH are social people. They enjoy their relationship with others and exchange positive energy. A mentally healthy person can be considered as a supportive resource. Participants expressed different elements of social domain.\n\n- Social relations and interactions: The majority of participants believed this issue should be considered as part of the definition of PMH. Having good interactions and possessing such skills was mentioned in all FGDs. Also, proper relationships were announced as key to providing support. “Mentally healthy individuals communicate so well that they are said to have ‘the charm’; everybody (young and old) appreciates them, and they can communicate to all age groups. They can always fascinate everybody ….\"(An uneducated woman). \"A mentally healthy person communicates to others pleasantly… everybody can have his support…\" (An educated man).\n\n- Respect other people's rights: This characteristic was addressed only by the men’s groups. \"One should respect other people's rights, and be able to accept fellow human beings as they are, and not express superiority over others, meaning that one should not be narcissistic…\"\n\n- Acceptance of social norms \"In my view, a mentally healthy person respects law and lives according to the society's established norms and customs…\" (Women`s group).\n\n- Abiding and respecting law was another issue proposed in the social dimension, which was cited in the definition of PMH from participants' perspective.\n\n- Standing up against injustice was another issue proposed in the social dimension of PMH, which was mostly suggested by men. \"A person is said to have PMH if they stand for their rights in community as much as they can…”\n\nLife skills domain. In most participants’ views, PMH means having certain life skills which are necessary and help people cope with challenging situations, think positively, and reach their goals in a better way.\n\n- Stress management: The overwhelming majority of participants experienced stress especially in their daily life and considered stress management, or having control over life adversities, as an inseparable part of PMH. \"In my view, a person is said to be healthy if they can have proper control over life problems or stresses that may occur, whichever side the stress is from…\"( An educated woman).”In dealing with problems, people should try to find the best solution, but those with the poor mentality cannot accept and overcome the problems and become indifferent…”( Men`s group).\n\n- Anger control: Another life skill recalled in providing PMH was anger control. In the view of the participants, anyone that can control anger in adversity and react appropriately has sound mental health.\n\n- Financial management: An essential life skill considered by majority participants in order to maintain PMH was to control income and expenses. They believed all people experience different types of financial instable situations. The one who has the power of financial management to control and manage these instabilities in his life more efficiently has good mental health. “…mentally healthy individuals can manage their personal life in such a way so as to make their family feel fortunate despite financial difficulties…”( Men’s group)\n\n- Accountability was also cited as an important skill in the definition of PMH. \"Dutifulness, however defined by the individual is respectable, and a sign of good mental health…. \" (Women`s group).\n\n- Time management: It was mostly referred to by women as another life skill that affects PMH. “A mentally healthy person uses her/his time well ….and saves time to help others …”\n\n- Problem-solving: This skill is among the most important skills pointed out by most participants. “A healthy person is a kind and sincere person who solves their problems calmly and does not react with haste…. (Men`s group).\n\n\nDiscussion\n\nThe main objective of this present study was to explore PMH definition and its domains in accordance with the Iranian culture. Although this element of mental health seems to be the same in different regions, it is obvious that the concept of PMH is interpreted differently in diverse cultures with respect to its expression and significance25.\n\nThe obtained results showed that Iranian participants considered the absence of mental disorder as a prerequisite for mental health and no one considered absence of mental disorder and PMH as two distinguished notions. According to Keyes et al., mental health has two separate but correlating axis – one referring to presence or absence of mental disorders, and the other expressing a high level of PMH to a low level of PMH - and existence of one does not contradict the presence of the other. It means despite having mental disorder, an individual can have some level of PMH or vice versa1,10,13. The results of the study implemented by Gilmour in a Canadian community supported Keyes` two continua model18. This theory was not announced by any of the Iranian individuals participating in FGDs. Whereas Laidlaw and his challenges explored in their qualitative study that Scottish people paid attention to PMH and mental health separately26.\n\nIn fact, Iranian participants generally considered PMH as getting positive energy, living happily, satisfaction and having peace and flexibility, with four main dimensions of emotional/psychological, spiritual, social, and life skills. Despite the difference in the perception of PMH in different cultures14, our findings appear to comply with the PMH concepts in the literature, especially the WHO description5.\n\nResults of the study revealed four interrelated domains as emotional/psychological, spiritual, social, and life skills in Iranian culture. The findings support Vaingankar`s qualitative research in Singapore, another Asian country with a diverse cultural context, that described PMH domains as personal growth and characteristics, coping strategies, spiritual beliefs and relationships25. Previous studies implemented by Keyes and his colleagues described PMH as salutogenic approaches of subjective wellbeing: with positive feeling defined as emotional wellbeing and positive functioning reflected as psychological wellbeing and social wellbeing1,3.\n\nThe role and importance of satisfaction reasonably creating and maintaining PMH were mentioned in the emotional/psychological dimension. According to the present study results, in the emotional domain, attributes such as satisfaction, love, empathy, understanding feelings and emotions, living happily, and positive thinking were suggested by participants. Review of the literature is also indicative of the importance of satisfaction in the maintenance and promotion of PMH in different cultural countries (see WHO Summary Report on PMH)25. Studies conducted by Monlina and Lyubomirsky indicated the importance of happiness, happy living and other positive emotions in increasing mental/physical well-being27,28. According to a study conducted in Singapore, positive thinking is able to affect promotion of PMH through interaction with people's personality attributes25.\n\nIn the spiritual domain, virtues such as faith in God and trust in a supernatural force, and also belief in the hereafter, were highlighted by the Iranian participants as with other Asian religious regions25,29,30. It seems that spirituality is a notable way of obtaining wellbeing and there is a widespread belief that people with spiritual beliefs and faith have better mental wellbeing31,32. Although religiosity is considered as a main variable in defining PMH, Ganga and his challenge showed that people with different religions experience diverse level of PMH30. It is important to realize more about interpersonal variations. Previous studies have shown that the most common mechanisms through which religion promotes well-being include social support networks, coping resources and positive emotions33,34. The spiritual dimension also increases creativity, patience, hope and vitality through communication and coping strategies35.\n\nIn the social domain, proper social relations, respect for the rights of others, acceptance of social norms, and lawfulness were cited by Iranian participants. The literature contains evidence on the significant role of proper communication with others and high level of mental well-being36. Close relationships provide support when needed as well as mutual cooperation25. Social support networks have a considerable relationship with mood, performance and the quality of life (see WHO statement on mental health promotion). New theories of well-being also point to the social aspects which correlate with the present study results37.\n\nRegarding life skills, which were explored as one of the main dimensions of PMH; stress management, accountability, anger control, problem solving, financial management, and time management were proposed in the present study. Stress coping skills appear to be one of the most important skills for finding peace and promoting well-being. In fact, stress coping mechanisms are not only important as factors affecting PMH, but also as PMH outcomes38. Studies showed there is a strong relationship between financial management and satisfaction, and mental wellbeing39. Also, existing evidence demonstrates a positive relationship between problem solving skills and social competencies40. Generally, life skills empower people to cope with stresses and emotions, think critically, and are capable of making proper decisions.\n\nThe present study has certain limitations. There is lack of generalizability and representativeness since the study was conducted using a qualitative method, and only its process and methodology can be generalized. The findings need more research attempts to be applicable to the broader Iranian adult population due to the multi ethnicity context of the country.\n\nIn this study, we interpreted the concept of PMH and its aspects according to Iranian adults’ experiences. The results highlighted the significance and influences of emotional, psychological traits, spiritual and religious beliefs, social aspects and life skills on PMH. Although PMH is considered a well-defined concept, sited as top priority and measured annually in various national mental health surveys in most developed countries, it is a new subject in our nations and needs more investigations and research so it can be introduced as a culturally and linguistically multidimensional concept. Furthermore, a proper understating of how mental health professionals conceptualize the concept of PMH is essential in policy making and implementing proper community-based interventions in order to promote and protect mental health.\n\n\nData availability\n\nUsing Persian language as the national language of Iran during the process of data gathering, all raw data including quotes are available in Persian. Translation and availability of complete raw data will be done upon the request and the permission of Ethical Committee of University of Social Welfare and Rehabilitation Sciences in order to maintain the participants’ confidentiality. Anyone wishing to access the data should first contact the corresponding author who will facilitate contact with the ethical review board (http://www.uswr.ac.ir Persian site, http://en.uswr.ac.ir/ English site. Contact email international_affairs@uswr.ac.ir)",
"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\nAcknowledgment\n\nThis study is derived from PhD dissertation regarding Positive mental health and its social determinants that has been approved by research council of University of Social welfare and Rehabilitation Sciences. The authors appreciate all participants who contributed the study and shared their experiences regarding Positive mental health.\n\n\nSupplementary material\n\nSupplementary File 1: Guide questionnaire in English and Persian.\n\nClick here to access the data.\n\n\nReferences\n\nKeyes CL: Mental health as a complete state: How the salutogenic perspective completes the picture. Bridging Occupational, Organizational and Public Health. Springer, 2014; 179–92. Publisher Full Text\n\nKeyes CL: Promoting and protecting mental health as flourishing: a complementary strategy for improving national mental health. Am Psychol. 2007; 62(2): 95–108. PubMed Abstract | Publisher Full Text\n\nKeyes CL: Mental illness and/or mental health? Investigating axioms of the complete state model of health. J Consult Clin Psychol. 2005; 73(3): 539–48. PubMed Abstract | Publisher Full Text\n\nSteel Z, Marnane C, Iranpour C, et al.: The global prevalence of common mental disorders: a systematic review and meta-analysis 1980–2013. Int J epidemiol. 2014; 43(2): 476–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarry MM: Addressing the determinants of positive mental health: concepts, evidence and practice. Int J Ment Health Promotion. 2009; 11(3): 4–17. Publisher Full Text\n\nWhiteford HA, Degenhardt L, Rehm J, et al.: Global burden of disease attributable to mental and substance use disorders: findings from the Global Burden of Disease Study 2010. Lancet. 2013; 382(9904): 1575–86. PubMed Abstract | Publisher Full Text\n\nHuppert FA: Psychological Well-being: Evidence Regarding its Causes and Consequences†. Appl Psychol: Health and Well-Being. 2009; 1(2): 137–64. Publisher Full Text\n\nAllen J, Balfour R, Bell R, et al.: Social determinants of mental health. Int Rev Psychiatry. 2014; 26(4): 392–407. PubMed Abstract | Publisher Full Text\n\nHuppert FA, Whittington JE: Evidence for the independence of positive and negative well-being: implications for quality of life assessment. Br J Health Psychol. 2003; 8(Pt 1): 107–22. PubMed Abstract | Publisher Full Text\n\nKeyes CL, Eisenberg D, Perry GS, et al.: The relationship of level of positive mental health with current mental disorders in predicting suicidal behavior and academic impairment in college students. J Am Coll Health. 2012; 60(2): 126–33. PubMed Abstract | Publisher Full Text\n\nKeyes CL, Simoes EJ: To flourish or not: Positive mental health and all-cause mortality. Am J Public Health. 2012; 102(11): 2164–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWood AM, Joseph S: The absence of positive psychological (eudemonic) well-being as a risk factor for depression: A ten year cohort study. J Affect Disord. 2010; 122(3): 213–7. PubMed Abstract | Publisher Full Text\n\nKeyes CL, Dhingra SS, Simoes EJ: Change in level of positive mental health as a predictor of future risk of mental illness. Am J Public Health. 2010; 100(12): 2366–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFriedli L: Mental health, resilience and inequalities. WHO Regional Office for Europe Copenhagen. 2009. Reference Source\n\nHerrman H, Jané-Llopis E: The status of mental health promotion. Public Health Rev. 2012; 34: 6. Publisher Full Text\n\nHuppert FA, So TT: Flourishing Across Europe: Application of a New Conceptual Framework for Defining Well-Being. Soc Indic Res. 2013; 110(3): 837–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarlsson H: Problems in the definitions of positive mental health. World psychiatry. 2012; 11(2): 106-7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGilmour H: Positive mental health and mental illness. Health Rep. 2014; 25(9): 3–9. PubMed Abstract\n\nNosratabasi M, Joshanloo M, Mohammadi F, et al.: Are Iranian Students Flourishing? Dev Psychol. 2010; 7(25): 83–94. Reference Source\n\nJoshanloo M, Wissing MP, Khumalo IP, et al.: Measurement invariance of the Mental Health Continuum-Short Form (MHC-SF) across three cultural groups. Pers Individ Dif. 2013; 55(7): 755–9. Publisher Full Text\n\nHolloway I, Galvin K: Qualitative research in nursing and healthcare. John Wiley & Sons; 2016. Reference Source\n\nRobinson OC: Sampling in interview-based qualitative research: A theoretical and practical guide. Qual Res Psychol. 2014; 11(1): 25–41. Publisher Full Text\n\nHsieh HF, Shannon SE: Three approaches to qualitative content analysis. Qual Health Res. 2005; 15(9): 1277–88. PubMed Abstract | Publisher Full Text\n\nRolfe G: Validity, trustworthiness and rigour: quality and the idea of qualitative research. J Adv Nurs. 2006; 53(3): 304–10. PubMed Abstract | Publisher Full Text\n\nVaingankar JA, Subramaiam M, Lim YW, et al.: From well-being to positive mental health: conceptualization and qualitative development of an instrument in Singapore. Qual Life Res. 2012; 21(10): 1785–94. PubMed Abstract | Publisher Full Text\n\nLaidlaw A, McLellan J, Ozakinci G: Understanding undergraduate student perceptions of mental health, mental well-being and help-seeking behaviour. Stud High Edu. 2016; 41(12): 2156–68. Publisher Full Text\n\nMolina FT: Happiness: Lessons from a new science. Sociologias. SciELO Brasil. 2015; 17(40). Publisher Full Text\n\nLyubomirsky S, King L, Diener E: The benefits of frequent positive affect: does happiness lead to success? Psychol Bull. 2005; 131(6): 803–55. PubMed Abstract | Publisher Full Text\n\nAbdel-Khalek AM: Islam and mental health: A few speculations. Ment Health, Religion & Culture. 2011; 14(2): 87–92. Publisher Full Text\n\nGanga NS, Kutty VR: Influence of religion, religiosity and spirituality on positive mental health of young people. Ment Health, Religion & Culture. 2013; 16(4): 435–43. Publisher Full Text\n\nGilbert P, Nicollus V: Inspiring hope: Recognising the importance of spirituality in a whole person approach to mental health. NIMHE; 2003. Reference Source\n\nMacGillivray PS, Sumsion T, Wicks-Nicholls J: Critical elements of spirituality as identified by adolescent mental health clients. Can J Occup Ther. 2006; 73(5): 295–302. PubMed Abstract | Publisher Full Text\n\nJu C, Zhang B, You X, et al.: Religiousness, social support and subjective well-being: An exploratory study among adolescents in an Asian atheist country. Int J Psychol. 2016. PubMed Abstract | Publisher Full Text\n\nRyu S, Lee OEK: Faith, spirituality, and values among Asian-American older adults: an exploratory factor analysis of the Multidimensional Measures of Religion and Spirituality. Ment Health, Religion & Culture. 2016; 19(8): 920–31. Publisher Full Text\n\nKoenig HG: Research on religion, spirituality, and mental health: a review. Can J Psychiatry. 2009; 54(5): 283–91. PubMed Abstract | Publisher Full Text\n\nBarry MM: Promoting positive mental health and well-being: Practice and policy. Mental Well-Being. Springer, 2013; 355–84. Publisher Full Text\n\nBelgrave FZ, Berry BM: Community Approaches to Promoting Positive Mental Health and Psychosocial Well-Being. Handbook of Mental Health in African American Youth. 2016: 121–140. Publisher Full Text\n\nHumphreys K, Mankowski ES, Moos RH, et al.: Do enhanced friendship networks and active coping mediate the effect of self-help groups on substance abuse? Ann Behav Med. 1999; 21(1): 54–60. PubMed Abstract | Publisher Full Text\n\nCollard S, Hayes D: Financial Wellbeing In Later Life: Evidence And Policy. 2014. Reference Source\n\nPatel A: Need For Life Skills Education among Tribal and Non Tribal Students. Int J Indian Psychol. 2014; 2(1): 17–28. Publisher Full Text"
}
|
[
{
"id": "30134",
"date": "05 Feb 2018",
"name": "Maryam Tehranizadeh",
"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\nThanks for sharing this manuscript with me. Present study focus on positive mental health from the perspective of Iranian society. It is a qualitative investigation, conducted through focus group discussions.\nThe methods section write carefully and extracted results present in appropriate format. Only as minor comments please follow bellow points:\nReferring to related reference, present more details on Dedoose qualitative software Version 7.6.6 applications Add more detail about development the guide questionnaire Add the main strengths and limitation to the end part of discussion\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": []
},
{
"id": "30133",
"date": "05 Feb 2018",
"name": "Mitra Modirian",
"expertise": [
"Reviewer Expertise Non-communicable 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\nPresent paper revealed a well-designed study entitled “Positive Mental Health from the perspective of Iranian society: A qualitative study” through which benefitting from a qualitative design, data gathered by focus group discussions.\nRequirements for qualitative studies have been followed completely and results and extracted conclusion presented in practical format.\nSome notes: Sampling should be explained more with details of referred technique. Discussion: the participants were residents of capital city of Iran, so the society of participants was different from other Iranians which should be notified. Also the negative points and limitation of study should be clearly more explained, there are more social and individual factors (social category and class, financial support, family of each participants, awareness of quality and quantity of insurance-services for which may be focused due to interruption with results of present study, but there was no notification of them in discussion or conclusion.\n\nThese findings also could be used for future complementary researches.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/7-103
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https://f1000research.com/articles/6-445/v1
|
07 Apr 17
|
{
"type": "Research Article",
"title": "Evaluation of hearing level in patients on long term aspirin therapy",
"authors": [
"Apar Pokharel",
"Sangita Bhandary",
"Sangita Bhandary"
],
"abstract": "Introduction: Aspirin is a routinely prescribed drug, most notably for cardiovascular diseases, such as myocardial ischemia. This cross sectional, comparative study study aims to explore differences in hearing status between the cardiovascular disease patients on aspirin therapy and age matched healthy controls. Methods: The study population consisted of 182 patients with heart disease taking long term aspirin (i.e., for more than one year). The control population consisted of 221 age matched healthy controls who were not taking aspirin. Results: Not aspirin, but the age of the patient was found to be the important risk factor for hearing loss. Conclusions: When confounding factors like age of the patient, hypertension and diabetes were taken into account, aspirin in its antiplatelet dose was not found to be the cause of any audiological problems like tinnitus and hearing loss.",
"keywords": [
"aspirin",
"sensorineural hearing loss",
"cardiovascular diseases"
],
"content": "Introduction\n\nThe great industrial and technological revolutions of the past two centuries have resulted in changes in the causes of illnesses and death. Infections and malnutrition were the most common cause of death before 1900; nowadays in developed countries, because of improved nutrition and public health measures, the greatest cause of morbidity has been cardiovascular disease (CVD) and cancer. As lifestyle changes have also been observed in developing countries, morbidity and mortality rates due to cardiovascular conditions are also becoming more common in these countries. This is known as the epidemiological transition because this shift is highly correlated with changes in personal and collective wealth (the economic transition), social structure (the social transition) and demographics (the demographic transition)1.\n\nAspirin is a routinely prescribed drugs, most notably for cardiovascular diseases such as, myocardial ischemia. Myocardial ischemia or ischemic heart disease (IHD) is a disease where there is a decreased blood supply to the heart muscle due to coronary artery disease (CAD)2. The ototoxic effects of high doses (several grams per day) of salicylates, reversible hearing loss and tinnitus, are well documented3.\n\nNo studies have been done in the past to assess hearing loss in patients with cardiovascular diseases on aspirin therapy in Nepal. The present study was done to explore the differences in hearing status between patients with cardiovascular disease on aspirin therapy and age matched healthy controls. It also aims to analyze correlation of hearing loss with the age of patients and presence of co-morbid illnesses.\n\n\nMethods\n\nThe study was conducted in the department of Otorhinolaryngology and Head & Neck Surgery, BPKIHS, Dharan. The duration of the study was one year (from 1st Feb, 2011 to 1st Feb, 2012). The objective of this cross sectional comparative study was to evaluate hearing level in patients on long term aspirin therapy (75mg, once a day) and to assess whether there was correlation between the severity of hearing loss and duration of aspirin therapy.\n\nThe study population consisted of cases of patients with heart disease taking long term aspirin (i.e., for more than one year). They were informed about the design and purpose of the study and requested to visit the ear, nose and throat outpatient department (ENT OPD) to take part in the study voluntarily. The control population consisted of age matched healthy controls who were not taking aspirin. No major, active interventions were carried out other than those routinely required for diagnosis. Ethical approval was obtained from the institutional ethical review board of B.P. Koirala Institute of Health Sciences (87/2070/071). Written informed consent was obtained from both study and control populations prior to the study. The current study did not involve any invasive procedure and did not cause any physical or mental harm to the patients. No other interventions were carried out. There was no financial burden to the patients during study. The inclusion criteria was patients of age group 15–75 years who were taking aspirin for heart disease. The exclusion criteria was patients with hearing loss after trauma i.e. after explosion, head injury, ear trauma, or perforation of tympanic membrane, and patients with positive family history of hearing loss, or with CNS disease.\n\nAll study participants visited ENT OPD so that their clinical history could be assessed according to the proforma and so that they could undergo clinical and otological examination. General physical examination and detailed otological examination of ear, nose and throat was carried out according to proforma to exclude middle ear pathology and conductive hearing loss. All the study participants had normal ears during ear examinations. The Heine Mini 3000 otoscope was used in all cases for the examination of the external auditory canal and the tympanic membrane.\n\nThe study population was asked about regular use of aspirin which was defined as daily intake of 75mg4. The study population was interviewed regarding hearing loss and tinnitus and any past history of ear disease was excluded. Tinnitus is a recurrent ringing, roaring or buzzing sensation lasting for five minutes or longer5.\n\nComplete birth and developmental history was taken to exclude congenital and other causes of acquired hearing loss. Detailed drug history was taken, looking especially for ototoxic drugs. Past history of ear trauma and head injury was noted to rule out prior hearing loss. Systemic causes of hearing loss e.g. Diabetes mellitus, hypertension, and chronic kidney disease, also had to be ruled out.\n\nTuning fork tests (Rinne’s, Weber’s) were carried out with 256, 512, 1024 Hz tuning fork. Lower frequency fork e.g. 128 was not used because of difficulty in interpretation. Rinne and Weber tests were done to find the better hearing cochlea. Each participant underwent a hearing test by pure tone audiometry (PTA) in a sound-proof room. The Madsen Electronics Orbiter 922 Version 2 clinical audiometer was chosen for the study, and tests were performed by a trained audiometrician. The audiometric testing was done by a single person to ensure test-retest reliability. The extent of hearing loss was determined in all subjects. An average of two pure tone responses was calculated in cases of doubt. The results were documented as low (250–500 Hz), middle (1000–2000KHz), and high (4000–8000Hz).The value of the worst ear was taken when there was slight difference in the hearing loss value between the two ears. Hearing thresholds of the study group were compared with those of control group. All findings were noted and categorized on the basis of a proforma data collection sheet. A graph was plotted with the PTA findings. The data were entered in Excel and analysis was carried out using SPSS version 16.0. The Chi-squared test was done to compare hearing loss between case and control, across different age groups and at different durations of intake of aspirin.\n\n\nResults\n\nThe study population consisted of 182 patients with heart disease taking long term aspirin (i.e., for more than one year). The control population consisted of 221 age matched healthy controls who were not taking aspirin. 45.3% of the study population were male and 54.7% were female. Among controls, 48.1% of the cases were male and 51.9% of the cases were female.\n\nBoth study and control populations were categorized into three groups according to age. The first group ranged between 15–50 years of age, the second 51–59 and the third, 60–75. The mean age was 63.7 years for the study population and 64.2 years for controls. 47.6% of the study population was in the 60–75 year age group; in the control group the number of participants in the 60–75 age group which was higher, with 48.4%.\n\n64.3% of the study population were of Indo-Aryan origin and 35.7% were of East Asian origin. Among the controls, 60.1% were of Indo-Aryan origin and 39.9% were of East Asian origin.\n\nThe occupation of participants was more or less equally distributed, with housewives dominating in both cases and controls. Careful history was taken to rule out noise induced hearing loss in each occupation as seen in Figure 1.\n\nOut of 182 study population, only 115 presented with tinnitus. Among 221 controls, 124 presented with tinnitus. The result was statistically insignificant as in Figure 2.\n\nOut of 182 study population, only 120 presented with sensorineural hearing loss (SNHL). Among 221 controls, 126 presented with SNHL. The result was not statistically significant (p value= 0.68) implying that aspirin was not the cause of hearing loss in study population as seen in Figure 3.\n\nThe data obtained from bivariate analysis was followed by binary logistic regression analysis with backward conditional method using SPSS version 16.0 in order to adjust and explore the significance of explanatory variables. Hearing loss was considered the dependent variable. The independent variables which had p value of 20% or less in the bivariate analysis were then included for binary logistic regression analysis. The variables subjected to the model were age group, duration of aspirin use, hypertension and diabetes mellitus.\n\nWhen logistic regression was applied among the study population it was found that age of the patient was most significantly associated with hearing loss. Patients aged 51–59 were 14.258 times more prone to develop hearing loss and patient aged 60–75 were 61.389 times more prone to develop hearing loss than patients aged less than 50 years of age (Table 1).\n\nIt was also found that only age of the study population was significantly associated with tinnitus. Duration of aspirin use, hypertension and diabetes were not found to be associated with tinnitus. People less than 50 years of age were 32.612 less likely to develop tinnitus than people between 60–75 years of age. Similarly patients between 51–59 years of age were 2.799 times less likely to develop tinnitus than people aged between 60–75 years (Table 2).\n\n\nDiscussion\n\nLogistic regression analysis demonstrated that- other factors like duration of aspirin intake, and other comorbidities like hypertension and diabetes were not found to produce any significant effect on the hearing status of the patient. Our results oppose Curhan et al’s, which had shown that aspirin given for its anti-platelet effect in cardiovascular patients is responsible for causing hearing loss even in low doses. Hearing loss was seen as one of the complications of regular analgesic use. The risk was seen greatest among men below the age of 60 years. Above the age of 60 years, no relation was seen between hearing loss and aspirin use4. In the present study, the mean age of the patient was 63.7 years. As most patients on regular aspirin were above the age of 60, aspirin was not seen as the principal cause of sensorineural hearing loss in our study.\n\nWith increasing age, the prevalence of the hearing loss also increases6. Studies have shown that after the age of 60, hearing thresholds worsen on average by 1 dB per year, but the rate of decline of hearing loss may be even greater in men aged 48–59 years7,8. The relationship between regular use of aspirin and hearing loss was observed as strongest in men younger than 60 years of age. A possible explanation for this might be that after the age of 60 the cumulative effects of age and other comorbidities will add up in the causation of hearing loss. Bainbridge et al showed similar impact of age and diabetes on hearing loss9.\n\nHigh doses of salicylates can have ototoxic effects which include reversible hearing loss and tinnitus. Animal models have shown that salicylate is responsible for abnormal function of the outer hair cell and decreased blood flow in the cochlea3. Membrane conductance of the outer hair cell changes because of biochemical and electrophysiological alteration induced by salicylates. Salicylates also cause auditory microvasculature vasoconstriction, most probably caused by their antiprostaglandin activity10,11.\n\nHistopathologic studies of human temporal bones and animals on salicylate administration show degeneration of the strial microvasculature12,13. Microvascular vasoconstriction leads to strial degeneration. However, strial vascularis degeneration is also one of the characteristic features of age-related hearing loss14. Animal studies have shown that strial degeneration leads to its atrophy and to capillary loss, with basement membrane thickening and deposition of laminin and immunoglobulin in the strial vasculature15–17.\n\nInflammatory mediators like TNF-α affects microvascular tone, thereby reducing cochlear blood flow18. Other inflammatory biomarkers like white blood cell count, neutrophil count, IL-6 and CRP are associated with hearing loss in older people19. Over a 10 year period, older people with higher levels of CRP were two times more likely to develop hearing loss than normal20. Aspirin inhibits platelet enzyme cyclo-oxygenase (COX). Increased expression of genes for IL- 1β, IL-6, TNF-α, and COX-2 occurs during prostaglandin biosynthesis, and this effect is inhibited by aspirin21. Aspirin is also responsible for the synthesis of anti-inflammatory compounds called ‘aspirin-triggered 15-epi-lipoxins’. This is responsible for the drug's anti-inflammatory effect even at low doses22. Aspirin also decreases chronic inflammatory biomarkers responsible for the aging process23. A randomized controlled trial where 100 mg of aspirin was given for acute coronary syndrome showed decrease in the level of both CRP and TNF-α24. These studies show that that aspirin in low doses may have a protective effect on hearing instead of a deteriorating effect. Recently, a clinical trial is going on to see whether aspirin in low doses can decrease the progression of age-related hearing loss25.\n\nThis study has certain limitations. Aspirin in low dose is an over the counter medication and is used by a large proportion of the population. To find out whether aspirin is responsible for causing hearing loss or not, sample size needs to be huge, and the samples should be followed up for a long period of time. Because of time constraints, this was not possible for this study. Hearing evaluation before the patient began aspirin treatment was also not carried out.\n\n\nConclusions\n\nHearing impairment is an important public health issue. Aspirin is one of the most common medications used now-a-days owing to the increased prevalence of cardiovascular diseases. The present study shows that long term use of aspirin doesn’t cause any hearing loss. The relationship between long term use of low dose aspirin and hearing loss is still a debatable subject. Other factors like age of the patient, hypertension and diabetes mellitus must also be looked at if a person on aspirin develops symptoms of hearing loss.\n\n\nData availability\n\nDataset 1: Raw data collected from the study population.\n\nDOI, 10.5256/f1000research.11131.d15696026\n\nDataset 2: Raw data collected from the control population.\n\nDOI, 10.5256/f1000research.11131.d15696127",
"appendix": "Author contributions\n\n\n\nThe study was conducted at the B.P. Koirala Institute of Health Sciences, Dharan, Nepal. AP was responsible for the data collection and analysis, and for writing up the article. SB was responsible for the concept of the study and for monitoring its progress.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to acknowledge Dr. Pranil Pradhan for helping us with the statistics of this study.\n\n\nReferences\n\nOmran AR: The epidemiologic transition: a theory of the epidemiology of population change. 1971. Milbank Q. 2005; 83(4): 731–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnthony SF, Braunwald E, Kasper DL, et al.: Harrison’s Principles of Internal Medicine. 16th Edition. New York: McGraw-Hill, Medical Pub. Division. 2008; 1514–1544.\n\nJung TT, Rhee CK, Lee CS, et al.: Ototoxicity of salicylate, nonsteroidal antiinflammatory drugs, and quinine. Otolaryngol Clin North Am. 1993; 26(5): 791–810. PubMed Abstract\n\nCurhan SG, Eavey R, Shargorodsky J, et al.: Analgesic use and the risk of hearing loss in men. Am J Med. 2010; 123(3): 231–237. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHalla JT, Hardin JG: Salicylate ototoxicity in patients with rheumatoid arthritis: a controlled study. Ann Rheum Dis. 1988; 47(2): 134–137. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCruickshanks KJ, Wiley TL, Tweed TS, et al.: Prevalence of hearing loss in older adults in Beaver Dam Wisconsin. the epidemiology of hearing loss study. Am J Epidemiol. 1998; 148(9): 879–886. PubMed Abstract | Publisher Full Text\n\nLee FS, Matthews LJ, Dubno JR, et al.: Longitudinal study of pure-tone thresholds in older persons. Ear Hear. 2005; 26(1): 1–11. PubMed Abstract | Publisher Full Text\n\nWiley TL, Chappell R, Carmichael L, et al.: Changes in hearing thresholds over 10 years in older adults. J Am Acad Audiol. 2008; 19(4): 281–292, quiz 371. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBainbridge KE, Hoffman HJ, Cowie CC: Diabetes and hearing impairment in the United States: audiometric evidence from the National Health and Nutrition Examination Survey, 1999 to 2004. Ann Intern Med. 2008; 149(1): 1–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStypulkowski PH: Mechanisms of salicylate ototoxicity. Hear Res. 1990; 46(1–2): 113–45. PubMed Abstract | Publisher Full Text\n\nBrien JA: Ototoxicity associated with salicylates. A brief review. Drug Saf. 1993; 9(2): 143–8. PubMed Abstract | Publisher Full Text\n\nNelson EG, Hinojosa R: Presbycusis: a human temporal bone study of individuals with downward sloping audiometric patterns of hearing loss and review of the literature. Laryngoscope. 2006; 116(9 Pt 3 Suppl 112): 1–12. PubMed Abstract | Publisher Full Text\n\nGratton MA, Schulte BA: Alterations in microvasculature are associated with atrophy of the stria vascularis in quiet-aged gerbils. Hear Res. 1995; 82(1): 44–52. PubMed Abstract | Publisher Full Text\n\nSpicer SS, Schulte BA: Spiral ligament pathology in quiet-aged gerbils. Hear Res. 2002; 172(1–2): 172–85. PubMed Abstract | Publisher Full Text\n\nOhlemiller KK: Mechanisms and genes in human strial presbycusis from animal models. Brain Res. 2009; 1277: 70–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGates GA, Mills JH: Presbycusis. Lancet. 2005; 366(9491): 1111–20. PubMed Abstract | Publisher Full Text\n\nSakaguchi N, Spicer SS, Thomopoulos GN, et al.: Immunoglobulin deposition in thickened basement membranes of aging strial capillaries. Hear Res. 1997; 109(1–2): 83–91. PubMed Abstract | Publisher Full Text\n\nScherer EQ, Yang J, Canis M, et al.: Tumor necrosis factor-α enhances microvascular tone and reduces blood flow in the cochlea via enhanced sphingosine-1-phosphate signaling. Stroke. 2010; 41(11): 2618–2624. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVerschuur CA, Dowell A, Syddall HE, et al.: Markers of inflammatory status are associated with hearing threshold in older people: findings from the Hertfordshire Ageing Study. Age Ageing. 2012; 41(1): 92–97. PubMed Abstract | Publisher Full Text\n\nNash SD, Cruickshanks KJ, Klein R, et al.: Long-term variability of inflammatory markers and associated factors in a population-based cohort. J Am Geriatr Soc. 2013; 61(8): 1269–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChung HY, Cesari M, Anton S, et al.: Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev. 2009; 8(1): 18–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrancaleone V, Gobbetti T, Cenac N, et al.: A vasculo-protective circuit centered on lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 operative in murine microcirculation. Blood. 2013; 122(4): 608–617. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingh T, Newman AB: Inflammatory markers in population studies of aging. Ageing Res Rev. 2011; 10(3): 319–29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen Y, Xu F, Zhang Y, et al.: Effect of aspirin plus clopidogrel on inflammatory markers in patients with non-ST-segment elevation acute coronary syndrome. Chin Med J (Engl). 2006; 119(1): 32–6. PubMed Abstract\n\nLowthian JA, Britt CJ, Rance G, et al.: Slowing the progression of age-related hearing loss: Rationale and study design of the ASPIRIN in HEARING, retinal vessels imaging and neurocognition in older generations (ASPREE-HEARING) trial. Contemp Clin Trials. 2016; 46: 60–66. PubMed Abstract | Publisher Full Text\n\nPokharel A, Bhandary S: Dataset 1 in: Evaluation of hearing level in patients on long term aspirin therapy. F1000Research. 2017. Data Source\n\nPokharel A, Bhandary S: Dataset 2 in: Evaluation of hearing level in patients on long term aspirin therapy. F1000Research. 2017. Data Source"
}
|
[
{
"id": "21658",
"date": "19 Apr 2017",
"name": "Adam Sheppard",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere are typographic errors included a repeated word three lines into the abstract.\nThe author’s state that pure tone audiometric evaluations were performed but do not include graphical data in the manuscript, nor do they indicate frequency specific thresholds in the raw data attached, only PTAs. Air conduction and bone conduction information needs to be included in order to ascertain the impact of standing hearing loss has on the included participants, especially since many occupations of participants included are readily exposed to noise such as farmers, labourers, and drivers. Furthermore, ototoxic events primarily initiate at ultra-high frequencies > 8 kHz, which was not even measured in the present study and can have significant impact on conclusions.\nThe use of tuning fork is not exactly clear if pure tone audiometry was performed. Unless pure tone audiometry was only performed on the “better hearing cochlea”, in which case the authors should have performed these studies binaurally.\nThe studies included do not fully address the impact that salicylate may have on hearing capability. Animal models have shown that salicylate can impact not only DPOAEs but permanently damage spiral ganglion neurons as well. Which could result in recoverable DPOAE’s and thresholds but have a permanent impact on word recognition capabilities. While these observations have been made with significantly higher dosage of salicylate, and may not be present at lower dosages, details should still be included in the manuscript.\nThere are many claims in the text which are written as though supported by research. While I believe the text included in the manuscript, proper references need to be included for readers. This event is widespread throughout the manuscript.\nThe authors claim their data opposes Curhan et al’s research. While findings were different, they need to include the significant differences between the two studies, one primary difference being the number of participants. This is addressed as the limitation, but should be also be interpreted in regards to difference in study results with Curhan et al. Despite the lower number of participants in the present study, the participants were well controlled age and gender. However, the youngest age group is inappropriately large. Dramatic differences in hearing are expected between the ages of 15-50 and should not be included in the same group.\nThe definition of tinnitus in the text is incorrect. The authors reference a previous studies classification used in that research paradigm (performed in 1988), but is not generally accepted definition of tinnitus. This likely has resulted in the inaccurately inflated occurrence of tinnitus in used participants. Typically, tinnitus can be present in ~15-20% of the general public, but the ~63% reported in the control group in the present study is much higher than expected.\nOverall, the findings of the study could potentially be of interest. However, significant changes need to be made with clarity of writing, appropriate citations, divulging specific raw data to include frequency specific air and bone conduction results, better classification of tinnitus and labeling of such in raw data, along with other methodological changes to specifically address the question at hand.\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": [
{
"c_id": "2671",
"date": "25 Apr 2017",
"name": "Apar Pokharel",
"role": "Author Response",
"response": "Thank you for reviewing my article. I want to add few explanations for your review comments. 1. I have included noise induced hearing loss as my exclusion criteria. For ototoxic medications, I have taken detailed history before recruiting the study population and have tried to exclude such patients. 2. PTA was done for both ears. 3. DPOAE was not done this facility is not available in my institution. 4. In my study the percentage of tinnitus has come higher, because both in my study population and control, I have included patients with comorbidities like DM, HTN which themselves are responsible for causing hearing loss. Thanks for your review."
}
]
},
{
"id": "22554",
"date": "08 May 2017",
"name": "Elsdon Storey",
"expertise": [
"Reviewer Expertise Neurology",
"large drug trials in the elderly (including hearing/aspirin)"
],
"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:\nIt is well-known that large doses of aspirin may produce reversible tinnitus and hearing loss. The authors set out to test whether prolonged use of low dose aspirin for heart disease (presumably mostly ischaemic in nature) has a deleterious effect on hearing. Unfortunately, there are a number of problems with this study.\n\nSubjects:\nThe authors are correct in their acknowledgement of vascular risk factors such as hypertension and diabetes for age-related hearing loss. (This age-related loss is presumably predominant in this study, judging from the increase in deafness by age group.) The design of the study does not allow ischaemic heart disease – itself also a risk factor for age-related hearing loss – to be accounted for, as all the aspirin-taking group were on aspirin for heart disease, whereas none of the control group had this risk factor. This confounding factor can only be addressed in a blinded, randomised prospective trial of aspirin in healthy individuals, as the authors acknowledge.\n\nThere is some confusion regarding the risk factors diabetes and hypertension in the manuscript.\na) What criteria were used for these diagnoses? Furthermore, were the subjects screened for these risk factors as part of the study, or was there reliance on medical record review or on patient self-description?\nb) These risk factors “ ... had to be ruled out” (Methods, para 5, page 2), but these factors were included in the regression analysis (Results, para 7, page 3). Were those with hypertension and diabetes indeed excluded from the study?\n\nWhile there was adjustment for age and sex, other known risk factors for age-related hearing loss such as smoking and educational level (Stevens G, et al. 2013 1; Agrawal Y, et al. 2008 2) were not reported. Were there any group differences in these factors?\n\nThe age range stratification (15-50; 51-59; 60-75) seem rather arbitrary and uneven. Were these divisions chosen to equalise numbers? The grouping of 15 year olds with 50 year olds seems inappropriate, and presumably the heart disease for which the 15 year olds were on aspirin was rather different. I think that the study would have been stronger with the exclusion of this paediatric group (presumably small). A histogram of the age distribution of subjects and controls by year of age (not age group) would be illuminating.\nMethods:\nFigure 1: The definition of hearing impairment is unclear. Why was the WHO classification not used? This averages the loss in dB at 4 frequencies: 0.5, 1, 2 and 4 kHz in the better ear, with mild loss being diagnosed at > 25 dB and moderate at > 40 dB. I presume that a dichotomous classification of hearing impairment was applied given that chi-squared tests were applied to the data, but the definition of hearing loss is not actually stated.\nIn a related matter, the results were documented as low, middle, high frequency loss (Methods, para 6, page 2), but this stratification is not subsequently referred to in the Results section.\nNoise-related deafness was excluded on history (“careful history was taken to rule out noise-induced hearing loss in each occupation” – Results, para 4, page 3). Was the presence of a prominent notch on PTA at 4 kHz used to complement the history of noise exposure as a cause of exclusionary noise-related deafness? (Incidentally, this exclusionary statement should appear in Methods rather than Results.)\nConductive loss was excluded on clinical examination and tuning fork tests. Was there a reason that a bone-ear conduction gap on audiometry was not used to complement this exclusionary point?\n\nResults:\nThe results are tabulated with inappropriate precision (often to 4 or 5 significant figures).\n\nDiscussion:\nAge-related hearing impairment is presumably the major cause of deafness in this study, as the relative risks of the older age bands suggest. There is animal evidence for microvascular involvement in age-related hearing loss (Prazma J, et al. 19903; Shi X. 20114). There is evidence from humans linking inflammatory states to age-related hearing loss by correlation of markers of mild systemic inflammatory states (raised IL-6 and CRP levels) with hearing loss (Verschuur C, et al. 2012 5). In other words, a reasonable hypothesis would be that low dose aspirin is actually protective against age-related hearing loss. In this context the study by Curhan et al. cited by the authors deserves further discussion. It found greater rates of hearing loss with prolonged regular use of aspirin, NSAIDs, and paracetamol (acetaminophen). There are two problems with that study, however: firstly, it relied on self-report of symptoms of deafness, with confirmation by a professional again self-reported. A second and potentially greater problem is confounding by indication, in that the reason for taking these medications in the first place (e.g. mild inflammatory state) may itself have been responsible for some or all of the hearing impairment.\n\nConclusion:\nAs it stands, this submission requires considerably more work to reach a suitable level. However, the primary data is presumably still available, and the submission could be re-written and re-analysed, perhaps along the lines suggested above, to improve its suitability.\n\nA minor comment is that the English expression requires some revision, although it is largely understandable in its current form.\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? Partly",
"responses": [
{
"c_id": "2704",
"date": "15 May 2017",
"name": "Apar Pokharel",
"role": "Author Response",
"response": "Thank you for your review. There are few queries raised which I want to address. The patients were all taken from the Department of Internal Medicine. There all patients are screened for blood sugar level and bood pressure. It is very difficult to find pateints with heart disease and having neither hypertension nor diabetes. So there were included in the study and the risk of hearing loss was calculated using regression analysis. Smoking was taken into account for the hearing loss. To minimize its effect as a confounding factor, in the control group population, smokers were also included. Education level was not seen in the study as the range of age was very high i.e., 15-65 years. The age grouping was done as 15-50yrs, 51-59yrs, 60-75 yrs because, it is very difficult to get cases with heart diseases on aspirin therapy in age less than 50 years. This age classification is also similar to article by Sharon G. Curhan, MD called “Analgesic Use and the Risk of Hearing Loss in Men “ published in Am J Med. 2010 March ; 123(3): 231–237. Noise induced Hearing Loss was excluded from history taking as well as from pure tone audiogram findings."
}
]
},
{
"id": "21660",
"date": "19 May 2017",
"name": "Ajoy Mathew Varghese",
"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\nResearch question: The authors have aimed to look at the effect of prolonged use of low dose aspirin on hearing. The question is relevant as most of the previous studies on the subject have looked at the effect of high dose aspirin.\n\nAim and objectives stated in abstract and introduction differ from the ones stated in the Methods section. These need to be stated clearly and consistently.\n\nMethods\n\nStudy design: The authors have done a cross sectional comparative study between patients with cardiac disease on aspirin, and controls without cardiac disease and not on aspirin.\n\nControls cannot be termed ‘healthy’. They were age-matched subjects without heart disease, and not on aspirin therapy.\n\nIf age matched controls were selected, why is there a difference in the number of cases and controls? Were there any drop outs from the study?\n\nWhat were the criteria for diagnosing hearing loss? Since patients did not have a baseline audiological test, how were hearing loss and progression of hearing loss defined? Were questionnaires used? Where they standardized?\n\nSystemic risk factors for hearing loss e.g. Diabetes mellitus, hypertension, and chronic kidney disease, also had to be ruled out.’ (para 5 in Methods section) These factors were present both in cases and controls (see Datasets). These factors were not ruled out. They were noted as potential confounders in both groups, and addressed in the logistic regression analysis.\n\nChronic kidney disease has been mentioned along with diabetes and hypertension as a confounding factor in the beginning of the manuscript. However, there is no mention of chronic kidney disease in the rest of the manuscript and data sets, and it has not been addressed in the logistic regression model. Please clarify.\n\nResults\n\nBaseline demographic data should be put in a table to ensure clarity.\n\nPlease depict the tinnitus and hearing loss in cases and controls, with p-values, as tables.\n\nCKD was not included as part of the logistic regression. Why not? Please clarify. The textual interpretation of this could also be better worded.\n\nAudiometric data need to be added both in the data set and the results.\n\nDiscussion\n\nThe discussion needs to be clearer, more concise, and should proceed in a logical sequence. In the discussion, the authors should cite studies that have shown that aspirin could cause hearing loss, and how the current study differs from them, and possible reasons as to why. Then they should discuss studies and theories to the contrary, that agree with the results of the current study. This should be done in a logical and concise manner. Please avoid unnecessary and confusing details of various theories that have no direct implication on the subject of research.\n\nThe ‘prevalence’ of hearing loss in both cases and controls is far above the prevalence quoted in existing literature. Is there an explanation for this?\n\nPlease give reference number for Curhan et al’s paper at the end of the first citation in the manuscript. (para 1 in discussion)\n\nThe comparison of the present study with Curhan et al’s work is not very clear. Are the authors trying to imply that their results conflict with those of this work because the majority of their study population (both cases and controls) was above the age of 60? Please make this clear.\n\nConclusion\n\nConclusion should correspond with the aim and objectives of the study. The present study found no relationship between long term, low dose aspirin use and hearing loss. Moreover, the study did not find any correlation between hearing loss and systemic diseases; hence the last line in the conclusion is not supported by the results of the study, and therefore, may be deleted.\n\nGeneral comments\n\nCorrection of grammar needs to be done throughout the manuscript.\n\nTerminology such as ‘cases’ and ‘controls,’ and ‘incidence’ and ‘prevalence’ has been used loosely and interchangeably. This is not acceptable.\n\nOn the whole, the paper addresses a relevant research question, and may be indexed after making the suggested corrections.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-445
|
https://f1000research.com/articles/7-199/v1
|
16 Feb 18
|
{
"type": "Review",
"title": "Progress and challenges in TB vaccine development",
"authors": [
"Gerald Voss",
"Danilo Casimiro",
"Olivier Neyrolles",
"Ann Williams",
"Stefan H.E. Kaufmann",
"Helen McShane",
"Mark Hatherill",
"Helen A Fletcher",
"Gerald Voss",
"Danilo Casimiro",
"Olivier Neyrolles",
"Ann Williams",
"Stefan H.E. Kaufmann",
"Helen McShane",
"Mark Hatherill"
],
"abstract": "The Bacille Calmette Guerin (BCG) vaccine can provide decades of protection against tuberculosis (TB) disease, and although imperfect, BCG is proof that vaccine mediated protection against TB is a possibility. A new TB vaccine is, therefore, an inevitability; the question is how long will it take us to get there? We have made substantial progress in the development of vaccine platforms, in the identification of antigens and of immune correlates of risk of TB disease. We have also standardized animal models to enable head-to-head comparison and selection of candidate TB vaccines for further development. To extend our understanding of the safety and immunogenicity of TB vaccines we have performed experimental medicine studies to explore route of administration and have begun to develop controlled human infection models. Driven by a desire to reduce the length and cost of human efficacy trials we have applied novel approaches to later stage clinical development, exploring alternative clinical endpoints to prevention of disease outcomes. Here, global leaders in TB vaccine development discuss the progress made and the challenges that remain. What emerges is that, despite scientific progress, few vaccine candidates have entered clinical trials in the last 5 years and few vaccines in clinical trials have progressed to efficacy trials. Crucially, we have undervalued the knowledge gained from our “failed” trials and fostered a culture of risk aversion that has limited new funding for clinical TB vaccine development. The unintended consequence of this abundance of caution is lack of diversity of new TB vaccine candidates and stagnation of the clinical pipeline. We have a variety of new vaccine platform technologies, mycobacterial antigens and animal and human models. However, we will not encourage progression of vaccine candidates into clinical trials unless we evaluate and embrace risk in pursuit of vaccine development.",
"keywords": [
"Tuberculosis",
"vaccine",
"BCG",
"vaccine development",
"clinical trials"
],
"content": "Introduction\n\nTuberculosis (TB) is the leading infectious cause of death world-wide (WHO TB report 2017). Current measures used for TB control are effective but insufficient. The decline in global TB rates remains incremental and the propensity of Mycobacterium tuberculosis (MTB) to develop drug resistance is a serious threat to our ability to control this disease with the currently available tools. Vaccination can be an effective strategy for TB control and it is estimated that Bacille Calmette Guerin (BCG) prevents 120,000 childhood deaths each year1. A TB vaccine that could enhance protection in infancy, or extend protection into adulthood, would have a significant impact on global TB rates2,3.\n\nIn 2012 the TB vaccine community, led by the TuBerculosis Vaccine Initiative (TBVI) and Aeras, published a Blueprint for TB Vaccine Development: a global, integrated strategy, outlining major scientific challenges, critical activities and crucial questions4. The Blueprint summarized the current state of TB vaccine development and identified key areas of research critical for the development of a new, effective TB vaccine (Box 1).\n\n1) Technologies and discovery: A need for better understanding of TB disease, natural resistance, innate, T-cell and antibody responses and diversity in antigen discovery\n\n2) Preclinical models: A need for better models to predict efficacy in humans, for standardization, for comparability and a need to publish experimental failure\n\n3) Biomarkers and immune correlates: A need to predict vaccine efficacy, for use of new technologies, to understand the role of IFN-γ in protection and for longitudinal studies of correlates of risks\n\n4) Clinical trials and harmonization: A need for capacity strengthening of clinical trial sites, to determine appropriate endpoints, to address regulatory and ethical issues and plans for post licensure sustainability.\n\nIt was envisioned that the recommendations would guide the next decade of TB vaccine development. This paper summarizes the major advances and achievements since the publication of the Blueprint in 2012 and updates the critical activities and recommendations for accelerating TB vaccine development today.\n\n\nTB vaccine technologies\n\nA new and more effective TB vaccine is an inevitability. In TB there is no doubt that immunity can prevent disease and no doubt that protective immunity can be induced by vaccination. Evidence for this includes the long lasting immune protection found following immunisation with BCG1,5,6 and natural immunity found in those latently infected with MTB7 and in those who either clear infection or resist disease8. The critical question is when a new TB vaccine will be achieved. The simple answer is that the more we invest in TB vaccine development the sooner we will have an effective TB vaccine.\n\nReflecting on the TB vaccine pipeline over the last 5 years, we see a small number of candidates that have failed at an early clinical stage, and some new candidates (Figure 1). Very few pre-clinical candidates have entered the TB vaccine pipeline and those in the pipeline have moved slowly through the stages of vaccine development or have not progressed at all (Figure 1). A more diverse and dynamic pipeline is needed to accelerate towards our goal of a new TB vaccine. We need to test a broader range of vaccine technologies against a broader range of antigens and we need to move vaccine candidates more rapidly through the pipeline.\n\nVaccine technologies that have shown promise in pre-clinical studies include the cytomegalovirus (CMV) vector, which is a live, attenuated, persistent viral vector able to express multiple MTB antigens. It has been shown that the engineering of the CMV vector leads to constant, low-level replication of the virus, giving sustained antigen expression and long-term immunity, making this technology highly attractive for TB vaccine development9,10. Vaccine technologies under development for the pandemic flu response could also be applied to TB. One such approach uses mRNA as a vaccine vector11. These mRNA vaccines yield high level in vivo Ag expression and are relatively simple to manufacture enabling them to be tested against multiple pathogens with relative ease. Antibody inducing vaccines are underrepresented in the current TB vaccine pipeline and although there are many technologies available there is a need for more basic research in this area. It has been shown in a series of non-human primate (NHP) experiments that mucosal or intravenous vaccination with whole cell mycobacterial vaccines provides better protection than parenteral vaccination, by inducing more T helper (TH) TH17 cells12, more resident memory T cells13, and more effector T cells14.\n\nThe challenge ahead is to increase the rate at which candidate vaccines enter the pipeline and the rate at which candidates move through the pipeline (Box 2). There are promising new approaches, although obstacles need to be overcome for the use of intravenous inoculation or viral vectors which integrate into the host genome. Transparent and robust criteria for moving vaccines from one stage of vaccine development are currently being reviewed and will be used to increase the pace of TB vaccine development.\n\nTB vaccine technologies\n\nThe global TB vaccine community should unite to maintain a dynamic vaccine candidate pipeline from discovery to late stage.\n\nThere are currently no unanimously agreed criteria for advancing vaccine candidates.\n\nTB vaccine research and development\n\nFuture discovery efforts should include investigation of immune quality and vaccine efficacy in response to combinations of vaccine platform, antigen and adjuvant.\n\nThe evaluation of host factors impacting protection should be included in future scientific investigation.\n\nWhole cell mycobacterial vaccines should continue to be central to TB vaccine development.\n\nThe role of animal models\n\nAnimal models and clinical studies should progress in parallel and may offer opportunities for cross-validation.\n\nThe most appropriate animal models should be selected based on evidence and the underlying question(s) to be answered.\n\nThe use of multiple different animal models can have a cumulative value in assessing vaccine candidates or answering pathogenesis questions.\n\nThere is an opportunity for the funders to encourage further standardization of models.\n\nAn obligation to publish animal studies regardless of the outcome (as it is the case for clinical trials) should be encouraged and would facilitate vaccine development.\n\nBiomarkers, Systems Biology and immune correlates\n\nThe approach to biomarkers should remain broad, looking at correlates of safety, risk of stable infection or disease, and vaccine efficacy.\n\nObservational studies will help to identify biomarkers of risk of infection or disease.\n\nInterventional (vaccine) studies and observational studies should be used to create and expand biobank repositories.\n\nExperimental medicine and human challenge\n\nA space for clinical research studies needs to be maintained and expanded.\n\nA favourable regulatory environment is critical for the conduct of clinical research studies and should be advocated for.\n\nInvestment into the establishment of controlled human TB challenge models needs to continue. Learnings from the malaria field should be integrated in this process and synergies with biomarker research needs to be created.\n\nClinical and late stage development\n\nWe need to keep (pipeline) diversity at all levels since we still wait for a clear efficacy signal.\n\nDe-risking candidates through gating criteria does not mean being risk-adverse.\n\nWe need to evaluate and accept some risk but prepare carefully and perform high quality studies which can advance the field even in the absence of an efficacy signal.\n\n\nTB vaccine research and development\n\nNovel vaccine platform technologies alone will not lead us to a new TB vaccine. A key activity in research and development is the identification of target antigens for insertion into vaccine candidates. In the last five years we have further developed the concept that MTB has distinct phases of growth, which may be associated with active mycobacterial replication, persistence and dormancy15. Antigens associated with active bacterial replication include the early secreted antigens, such as the Ag85 family, ESAT-6 and CFP-10. These antigens have been used extensively in TB vaccine development as they are highly immunogenic and have shown protection in animal models. Antigens in the DosR regulon, however, are associated with dormancy and their use offers the possibility of designing vaccines to more specifically target latent MTB infection (LTBI). Most platform approaches used in vaccine development to date predominantly induce a TH1 cluster of differentiation (CD) CD4+ T cell response. However, in large scale screening experiments antigens that induce a CD8+ T cell response have been identified and can be used with CD8+ T cell inducing vectors to specifically boost a CD8+ T cell response16,17. There are also platforms and antigens that promote an antibody response or an unconventional T cell response10,18,19. The broader range in antigen choice has been matched with the development of novel adjuvants, including synthetic and bio-inspired molecules, which mimic naturally occurring cellular processes for more efficient delivery of vaccine components to the cell. The selected adjuvant can direct the immune response induced and the vaccine developer now has the option of driving immunity towards TH1, TH2, TH17 with the adjuvant selected20.\n\nWe now have a greater ability than ever to manipulate the vaccine induced immune response and this can be done at at least three different levels: 1) Choice of vaccine technology; 2) Choice of antigen; 3) Choice of adjuvant. The next question is what type of immune response should be induced?\n\nOur knowledge of protective immunity has greatly increased in the last 5 years and has broadened our awareness of the importance of the interplay between the innate and adaptive immune responses in TB. Correlates of risk studies have identified TypeI/II interferon (IFN) as a risk factor for progression to TB disease in latently infected adolescents21 and bulk T cell activation has been identified as a risk factor for TB in infants and adolescents22. These correlates of risk studies have shown that the underlying host immune environment plays a dominant role in TB disease risk and the impact of this environment on vaccine induced immunity needs to be explored. We also have a greater appreciation that quality rather than quantity of T cells is important for function23. In addition to T cells, it is becoming more apparent that B cells play an important part in immunity to TB24. In particular B cell function is impaired during TB and LTBI, which impacts cellular immunity25. Exploiting antibody-mediated protection against TB, and MTB dissemination in particular, has already shown promise in animal models26.\n\nDespite the emergence of new technologies, whole cell mycobacterial vaccines remain central in TB vaccine development. Complexity of their antigen components including proteins, lipids and glycolipids allows for interaction with innate immune cells and induction of conventional and unconventional T cell responses as well as antibody responses. It has also emerged that BCG itself can manipulate the host immune, environment. BCG can induce epigenetic changes in monocytes, which enhance their capacity for microbial control, not only of mycobacteria but also against unrelated pathogens. This concept of “trained immunity” is thought to result in upregulation of toll-like receptors and CD14 on monocytes27.\n\nHow the quality of the immune response is influenced by host environment, route of delivery, vaccine platform, antigen or adjuvant remains largely unexplored. Now that we have the tools to manipulate the vaccine induced immune response, we need to generate data sets exploring how these tools influence immune quality and vaccine efficacy to efficiently select the optimum combinations of vaccine platform, antigen and adjuvant for TB vaccine development (Box 2).\n\n\nThe role of animal models\n\nThere are several different animal models that are used in the TB vaccine development pipeline and these are useful from the discovery phase right through to advanced pre-clinical development. Animal models are currently perceived as key for demonstration of safety during all stages of development and immunogenicity during early screening, and informative for demonstration of a protective effect against MTB challenge.\n\nSystematic screening in animal models can thus be used to select vaccine candidates that achieve a threshold of safety, immunogenicity and efficacy. There is also the ability to perform comparative head-to-head testing in independent laboratories, for prioritization of the most promising vaccine candidates. Mice, guinea pigs and NHP are the most commonly used species for vaccine testing and study designs vary depending on the animal species, the type of vaccine and the rationale for demonstrating efficacy (e.g. reducing bacterial burden, prolonging survival or preventing reactivation). This complexity in species and study design attempts to reflect complexity of human disease, but it means that there is no single, harmonized animal model that could be used for clear ‘go / no-go’ decisions for candidate TB vaccines. However, there is greater confidence in data that show the efficacy of a candidate in multiple in vivo systems, particularly when those studies are conducted in independent laboratories.\n\nThere has been considerable progress in animal models since the 2012 blueprint. There is an improved understanding of the strengths and weaknesses of the different models28–30 and greater recognition of the need for ‘fit-for-purpose’ study designs to achieve robust, quantifiable measures of efficacy. Animal data are now available for many TB vaccine candidates, some of which are undergoing clinical testing. In all cases the degree of protection of the novel vaccines relative to the controls (unvaccinated or BCG), although statistically significant, is not substantial. More clinical efficacy data are needed to know whether this level of protection in animals is predictive of an efficacy signal in humans; this information is needed urgently to provide biological validity of the animal models and to establish whether existing animal models and study designs need to be refined28. There are also efforts to develop models reflective of the more complex environment of target populations. These include infant animal models for neonatal vaccines; post-MTB exposure vaccination; models of co-morbidities, such as diabetes and HIV infection; and models that involve natural transmission of the pathogen.\n\nData from animal models have become increasingly important to the Stage Gating processes, which aim to assist developers and funders to accelerate candidates from discovery through pre-clinical development. However, negative results are not always reported in the public domain, and therefore the full value of these data to enable lessons to be learnt, has not been realized. Some funders have set requirements for efficacy in NHP to be demonstrated before clinical testing, which highlights the need for stringent thresholds and harmonization in terms of read-outs of vaccine efficacy. An absolute requirement for statistically robust efficacy in NHP is, however, costly and difficult due to limitations of space and animal availability and must be balanced against the cost of collecting data in humans.\n\n\nBiomarkers, Systems Biology and immune correlates\n\nSuccess in studies of individuals with LTBI and active TB patients has led to host biomarkers becoming an integral part of future TB control. Proof of principle has been given that biomarkers can distinguish between active TB disease and LTBI, and evidence is accumulating that biomarkers can predict progression to active TB31. Thus, it has been demonstrated that small-sized biosignatures comprising 3-4 transcripts are capable of reliably discriminating TB disease from LTBI and medium-sized biosignatures comprising 16 transcripts or less allow prediction of active TB by diagnosing incipient, subclinical TB21.\n\nBeyond their application for diagnosis, biomarkers can provide important contributions to the clinical development of vaccines against TB (Figure 2)32. First, individuals with subclinical TB may benefit from preventive TB drug treatment. Second, such biosignatures allow stratification of individuals with subclinical TB for clinical vaccine trials to reduce participant numbers and shorten trial duration. Moreover, such biosignatures will serve as valuable tools for monitoring of clinical trial participants. Although they will not replace the clinical endpoints, early recognition of progression to active TB will provide valuable information.\n\nThis figure has been reproduced with permission from Kaufmann, Evans, Hanekom, Science Transl. Med., 201532.\n\nWhilst most biosignatures defined thus far were derived from observational studies on contacts and TB patients, future studies must focus on biosignatures of vaccine efficacy, although these can only be derived when we have a vaccine which demonstrates efficacy in clinical trials. In the meantime, information can be obtained from observational studies on BCG vaccination in infants. It has, for example, been shown that there is a lower risk of progression to TB disease in BCG vaccinated infants with either a higher INF-γ ELISpot response against mycobacterial antigens or higher Ag85A IgG antibody levels22.\n\nDesign of biosignatures of vaccine efficacy needs to consider the following groups: individuals who develop active TB despite being vaccinated (vaccine failure), individuals who remain healthy because of vaccination, individuals who remain healthy due to natural resistance (independent of vaccination).\n\nFinally, biosignatures can inform about the mechanisms underlying pathogenesis and protection, paving the way for in-depth analysis of the biological functions of differentially regulated biomarkers. For example, correlates of risk studies, performed using samples collected during the MVA85A vaccine efficacy trial, have revealed that T cell activation and CMV infection are associated with future risk of developing TB disease22,33. Deeper understanding of the factors that drive TB risk will facilitate the design of next-generation vaccine candidates.\n\nIntegration of biosignatures into clinical trial design will add cost; however, it is critical that we take every opportunity to add value to clinical studies. Biosignatures will be of great value for refining the vaccine candidate tested and for developing alternative vaccine types and modes of immunization.\n\n\nExperimental medicine and human challenge\n\nClinical trials are an essential part of the product development pathway for TB vaccine development. However, restricting the conduct of clinical trials to product development ignores the utility of experimental medicine studies to generate novel scientific insights. Experimental medicine can be defined as ‘an investigation undertaken in humans, relating where appropriate to model systems, to identify mechanisms of pathophysiology or disease, or to demonstrate proof-of-concept evidence of the validity and importance of new discoveries or treatments’. Experimental medicine and product development are not mutually exclusive. A vaccine could be tested to both address a proof-of-concept experimental medicine question and in parallel be a critical step in a product development pathway. An example of this is the first-in-class testing of an attenuated strain of MTB as a potential vaccine candidate34. Furthermore, candidate vaccines could move between experimental medicine and product development, this flexibility is important as we are still at the frontiers of knowledge in TB vaccine clinical testing. There are many examples of small scale, phase I experimental medicine studies which have provided valuable information on safety and immunogenicity, such as the testing of combination vaccine approaches35 and the testing of novel routes of delivery, e.g. aerosol36.\n\nHuman challenge models are the ultimate in experimental medicine studies. In such studies, healthy vaccinated volunteers are intentionally inoculated with the pathogen in question, to allow the efficacy of a candidate vaccine to be evaluated in a small-scale study prior to embarking on expensive field efficacy trials. Such controlled human challenge models have been game changing in malaria vaccine development37. However, unlike malaria, and other human challenge models in clinical use, we cannot deliberately infect human subjects with virulent MTB for obvious ethical reasons. Efforts to develop a controlled human mycobacterial challenge model using BCG, or attenuated strains of MTB, are underway38,39. Ultimately, human challenge models need validation against field efficacy studies. However, they can also be corroborated against a known vaccine effect in preclinical animal models38.\n\nThere is an underexploited role for experimental medicine in TB vaccine development, in parallel with product development, in early and late (efficacy) trials. We need innovative ways to demonstrate an efficacy signal in humans with TB vaccine candidates. Controlled human mycobacterial infection studies offer a potential way to achieve this. Such studies, if they were to demonstrate a biological signal, would allow the prioritization of candidates for progression to prevention of disease studies. The predictive value of human immunology, animal models, and these surrogate efficacy endpoints can only be determined by progressing some candidates to field efficacy studies. There is no substitute for human efficacy testing against disease in the development of an effective TB vaccine. Considerable information can be gained from efficacy trials regardless of the efficacy results22. Repeated cycles of iteration between animal and human studies will yield important insights and advance the development of an effective vaccine.\n\n\nClinical and late stage development\n\nExperimental medicine studies are not confined to early phase clinical vaccine trials and human challenge studies, they are also being used to make TB vaccine efficacy trials faster, shorter and more cost-effective. This is achieved by leveraging the much higher incidence rates of MTB infection as measured by interferon-γ release assays (IGRA) conversion for prevention of infection studies (POI) and measurement of TB recurrence after treatment for prevention of recurrence studies (POR). The incidence rates of infection and recurrence are much higher when compared to community-based incident TB disease so clinical trials with POI and POR endpoints can be smaller and faster than those with a disease endpoint (POD). POI and POR trials have rapidly gained acceptance as a pathway to demonstrate proof-of-concept prior to large-scale efficacy trials40,41. In South Africa, annual IGRA conversion rates of 6–7% in infants42 and up to 14% in adolescents43 have been reported; and TB disease recurrence rates are estimated at 2–5% for standard of care TB treatment arms in a clinical trial setting. These rates are very compelling, in terms of endpoint accrual, compared to the 0.78% annual incidence of TB disease estimated among South African adults by the World Health Organization. As a result, POI trials have been initiated to test the booster vaccines H4 (NCT02075203) and DAR-901 (NCT02712424).\n\nThe VPM1002 vaccine is being tested in a phase II/III POR trial (NCT03152903) in adults with cured TB and expects a 10% rate of recurrence (relapse or reinfection) over a 12 month period of follow-up.\n\nBridging from e.g. a POI trial to POD trial in the same population presents a very real challenge. Using the high TB incidence South African example to illustrate the most cost-effective clinical trial scenario, average incidence of microbiologically confirmed TB disease in adolescents (incidence 0.43%) is almost half that in adults44. Therefore, a POD trial for the identical population in whom POI might be demonstrated as proof of concept would be large, long and costly. Curiously, even though childhood TB is notoriously paucibacillary, it might be more efficient to conduct a POD trial in newborn infants, the only IGRA- population that is not also exposed to other mycobacteria, including BCG, even if the TB disease endpoint were limited to microbiologically confirmed disease (incidence 0.7%)42.\n\nTB vaccine development efforts are increasingly focused on prevention of pulmonary TB disease in adolescents and adults, to block the MTB transmission cycle. The majority of adults in TB endemic countries like South Africa are IGRA+45, making POD trials in IGRA+ adults more feasible since rates of disease are higher, yet based on our knowledge of historical BCG trials, IGRA+ (and previously BCG vaccinated) adult populations are likely to pose the biggest challenge to demonstrate additional vaccine efficacy46.\n\nProgression of a TB vaccine candidate to POD or a proof-of-concept POI or POR trial is therefore not simply contingent upon application of product development Stage Gate criteria, but a complex consideration of vaccine target population, endpoint accrual, operational efficiency - and most importantly – cost. If we consider the TB vaccine pipeline in 2012 (Figure 1), which included four vaccines in Phase 1, six in Phase 2, and three in efficacy trials, compared to the five vaccines in Phase 1, four in Phase 2, and four in efficacy trials in 2017 (Figure 1), the pipeline appears healthy. However, with few exceptions, most candidates have not progressed through the pipeline in the last five years. It is notable that of the seven candidates now in Phase 2, current and planned clinical trial activity includes three POI and three (pilot safety and immunogenicity or efficacy) POR trials, which raises the question of why experimental medicine strategies intended to supplement the traditional product development pathway have instead replaced traditional safety and immunogenicity, and safety and efficacy trials, to such a large extent. We speculate that the wholesale shift towards experimental medicine strategies is a manifestation of a limited funding environment, which has forced developers to adopt more cost-effective approaches to vaccine testing. One major disadvantage to cost-effective experimental medicine approaches is that we do not know if prevention of infection will result in prevention of disease. Reciprocally, it is possible that prevention of disease could be achieved with a vaccine that had no impact on infection and POI trials would triage out such candidates. Success of a vaccine candidate in a POI or POR trial would likely accelerate clinical development, but should failure in an experimental medicine trial halt progression in the traditional development pathway?\n\nIt also appears that the number and diversity of new TB vaccine candidates entering clinical trials has become increasingly limited, which might severely restrict the options to develop new TB vaccines aimed at a wide target spectrum of age (infants, adolescents, adults or the elderly), MTB exposure status (IGRA+, IGRA-), and indication (POD, POR and therapeutic). No matter how promising an individual candidate may appear in pre-clinical studies, it would be high risk to commit resources only to development of a single candidate, which inevitably carries some risk of failure when safety, immunogenicity and efficacy are tested in humans. This consideration is perhaps most relevant to the highest priority of protecting previously BCG vaccinated, MTB-infected adults against progression to active TB disease.\n\nTherefore, we propose that diversity is an essential quality of a healthy TB vaccine pipeline that will ultimately lead to a successful vaccine, or vaccines, that meet the needs of a variety of susceptible populations, including adults, children and HIV-infected persons living in TB endemic countries. If we accept this premise, it follows that we must accept the inevitable possibility of failure of individual candidates, or specific trial designs, to meet acceptable standards when tested for safety, immunogenicity or efficacy in human populations. We want to accept risk, after serious evaluation of all issues including biological, medical, ethical and legal aspects. We need not fear failure, since progression in our field depends on our willingness to evaluate and accept risk, provided that we learn from each clinical trial, and collect and store sufficient data and samples to improve future chances of success. Perhaps the most illustrative example of ‘failing well’ is the infant trial of the MVA85A candidate, which conclusively failed to demonstrate added protection to that provided by newborn BCG vaccination, but taught invaluable lessons about conduct of infant vaccine trials42,47–49, pre-clinical animal models28, community engagement47, endpoint determination50,51, and biomarkers of risk for TB22,52.\n\nWe propose that logical application of Stage Gate criteria to de-risk and progress a number of diverse vaccine candidates through human trials, in the knowledge that most will fail, is a necessary and efficient strategy to achieve the ultimate goal of a successful TB vaccine. This approach does carry the responsibility, not only to learn iteratively from past failures, but to be prepared to adapt rapidly and decisively in the face of new data. We advocate for empiric advancement of promising candidates into clinical trials, in parallel with iterative studies to better understand risk for and protection against TB. Conversely, we propose that application of our field’s limited global resources to development of only a small number of very carefully selected candidates, no matter how promising, would lead to a shrinking, less diverse pipeline, increasingly vulnerable to the consequences of failure. In that context, excessive risk-aversion is itself a high-risk strategy, given the urgency for a new vaccine to impact on global burden of disease.\n\n\nConclusions\n\nThis paper reviewed progress made in TB vaccine development since the initial Blueprint for the field was published in 2012. Then and now, a TB vaccine is inevitable to put an end to TB. As for many other infectious diseases, a safe, efficacious and affordable vaccine is an essential part of the solution. Getting there will not be easy, and tremendous progress has been made during the last five years. The TB vaccine community now has at its disposal a broad portfolio of platform technologies, vaccine antigens and insight into immunological mechanisms that can be leveraged to expedite TB vaccine development. However, the global TB vaccine pipeline has progressed much less than desired in recent years, and we hope that the new tools and insights from technologies and discovery research will feed into a rich and diverse pipeline in the future. Animal models will be vital to swiftly advance novel vaccine candidates into clinical trials. While the predictive value of animal models can ultimately only be validated by human efficacy data, the evaluation of vaccine candidates in a combination of rationally selected animal models provides an early gauge and Stage Gate. Similarly helpful in moving vaccines through the development path are biomarkers and immune correlates. Biomarker technology has matured enormously over the last five years, yielding tests that are ready to support pre-clinical and clinical development. Further validation of promising biomarkers could come from experimental medicine studies (including a human mycobacterial challenge model) and from larger clinical trials with an efficacy endpoint. Continued clinical research as well as advanced large scale field studies will be critical to validate animal models and biomarkers, establish proof of scientific concepts, and will ultimately yield an efficacy signal that the entire field is waiting for. We cannot afford to relent in this effort and must be willing to invest wisely, knowing that many trials will fail. Acceptance of risk and failure is an integral part of developing vaccines and the potential global public health impact of an effective vaccine should encourage us to continue to invest our intellectual and financial capital in TB vaccine 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.",
"appendix": "Competing interests\n\n\n\nSHEK is co-inventor and co-patent holder of the TB vaccine VPM1002, which has been licensed to Vakzine Projekt Management and sublicensed to Serum Institute of India.\n\nScientific meeting; This paper summarises a discussion convened by TBVI of leading experts in the TB vaccine field at the Watertower meeting held on May 10, 2017, Brussels, Belgium.\n\n\nGrant information\n\nThis work was supported by the Tuberculosis Vaccine Initiative (TBVI).\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\nTBVI for leadership in this overall initiative to update the Tuberculosis Vaccine Blueprint.\n\n\nReferences\n\nHarris RC, Dodd PJ, White RG: The potential impact of BCG vaccine supply shortages on global paediatric tuberculosis mortality. BMC Med. 2016; 14(1): 138. 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PubMed Abstract | Publisher Full Text\n\nLewinsohn DA, Swarbrick GM, Park B, et al.: Comprehensive definition of human immunodominant CD8 antigens in tuberculosis. NPJ Vaccines. 2017; 2: pii: 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBelay M, Legesse M, Mihret A, et al.: IFN-γ and IgA against non-methylated heparin-binding hemagglutinin as markers of protective immunity and latent tuberculosis: Results of a longitudinal study from an endemic setting. J Infect. 2016; 72(2): 189–200. PubMed Abstract | Publisher Full Text\n\nHarriff MJ, Wolfe LM, Swarbrick G, et al.: HLA-E Presents Glycopeptides from the Mycobacterium tuberculosis Protein MPT32 to Human CD8+ T cells. Sci Rep. 2017; 7(1): 4622. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnudsen NP, Olsen A, Buonsanti C, et al.: Different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens. Sci Rep. 2016; 6: 19570. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZak DE, Penn-Nicholson A, Scriba TJ, et al.: A blood RNA signature for tuberculosis disease risk: a prospective cohort study. Lancet. 2016; 387(10035): 2312–2322. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFletcher HA, Snowden MA, Landry B, et al.: T-cell activation is an immune correlate of risk in BCG vaccinated infants. Nat Commun. 2016; 7: 11290. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrme IM, Robinson RT, Cooper AM: The balance between protective and pathogenic immune responses in the TB-infected lung. Nat Immunol. 2015; 16(1): 57–63. PubMed Abstract | Publisher Full Text\n\nAchkar JM, Chan J, Casadevall A: B cells and antibodies in the defense against Mycobacterium tuberculosis infection. Immunol Rev. 2015; 264(1): 167–181. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJoosten SA, van Meijgaarden KE, Del Nonno F, et al.: Patients with Tuberculosis Have a Dysfunctional Circulating B-Cell Compartment, Which Normalizes following Successful Treatment. PLoS Pathog. 2016; 12(6): e1005687. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrados-Rosales R, Carreño L, Cheng T, et al.: Enhanced control of Mycobacterium tuberculosis extrapulmonary dissemination in mice by an arabinomannan-protein conjugate vaccine. PLoS Pathog. 2017; 13(3): e1006250. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKleinnijenhuis J, Quintin J, Preijers F, et al.: Bacille Calmette-Guerin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes. Proc Natl Acad Sci U S A. 2012; 109(43): 17537–17542. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcShane H, Williams A: A review of preclinical animal models utilised for TB vaccine evaluation in the context of recent human efficacy data. Tuberculosis (Edinb). 2014; 94(2): 105–110. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams A, Orme IM: Animal Models of Tuberculosis: An Overview. Microbiol Spectr. 2016; 4(4). PubMed Abstract | Publisher Full Text\n\nCardona PJ, Williams A: Experimental animal modelling for TB vaccine development. Int J Infect Dis. 2017; 56: 268–273. PubMed Abstract | Publisher Full Text\n\nKaufmann SH, Weiner J 3rd, Maertzdorf J: Accelerating tuberculosis vaccine trials with diagnostic and prognostic biomarkers. Exp Rev Vacc. (in press). 2017; 16(8): 845–853. PubMed Abstract | Publisher Full Text\n\nKaufmann SH, Evans TG, Hanekom WA: Tuberculosis vaccines: time for a global strategy. Sci Transl Med. 2015; 7(276): 276fs278. PubMed Abstract | Publisher Full Text\n\nMuller J, Matsumiya M, Snowden MA, et al.: Cytomegalovirus infection is a risk factor for TB disease in Infants. bioRxiv. 2017. Publisher Full Text\n\nSpertini F, Audran R, Chakour R, et al.: Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial. Lancet Respir Med. 2015; 3(12): 953–962. PubMed Abstract | Publisher Full Text\n\nSheehan S, Harris SA, Satti I, et al.: A Phase I, Open-Label Trial, Evaluating the Safety and Immunogenicity of Candidate Tuberculosis Vaccines AERAS-402 and MVA85A, Administered by Prime-Boost Regime in BCG-Vaccinated Healthy Adults. PloS One. 2015; 10(11): e0141687. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSatti I, Meyer J, Harris SA, et al.: Safety and immunogenicity of a candidate tuberculosis vaccine MVA85A delivered by aerosol in BCG-vaccinated healthy adults: a phase 1, double-blind, randomised controlled trial. Lancet Infect Dis. 2014; 14(10): 939–946. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSauerwein RW, Roestenberg M, Moorthy VS: Experimental human challenge infections can accelerate clinical malaria vaccine development. Nat Rev Immunol. 2011; 11(1): 57–64. PubMed Abstract | Publisher Full Text\n\nHarris SA, Meyer J, Satti I, et al.: Evaluation of a human BCG challenge model to assess antimycobacterial immunity induced by BCG and a candidate tuberculosis vaccine, MVA85A, alone and in combination. J Infect Dis. 2014; 209(8): 1259–1268. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartin CJ, Cadena AM, Leung VW, et al.: Digitally Barcoding Mycobacterium tuberculosis Reveals In Vivo Infection Dynamics in the Macaque Model of Tuberculosis. MBio. 2017; 8(3): Pii: e00312-17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHawn TR, Day TA, Scriba TJ, et al.: Tuberculosis vaccines and prevention of infection. Microbiol Mol Biol Rev. 2014; 78(4): 650–671. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEllis RD, Hatherill M, Tait D, et al.: Innovative clinical trial designs to rationalize TB vaccine development. Tuberculosis (Edinb). 2015; 95(3): 352–357. PubMed Abstract | Publisher Full Text\n\nTameris MD, Hatherill M, Landry BS, et al.: Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet. 2013; 381(9871): 1021–1028. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndrews JR, Hatherill M, Mahomed H, et al.: The dynamics of QuantiFERON-TB gold in-tube conversion and reversion in a cohort of South African adolescents. Am J Respir Crit Care Med. 2015; 191(5): 584–591. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMahomed H, Hawkridge T, Verver S, et al.: The tuberculin skin test versus QuantiFERON TB Gold® in predicting tuberculosis disease in an adolescent cohort study in South Africa. PLoS One. 2011; 6(3): e17984. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMahomed H, Hughes EJ, Hawkridge T, et al.: Comparison of mantoux skin test with three generations of a whole blood IFN-gamma assay for tuberculosis infection. Int J Tuberc Lung Dis. 2006; 10(3): 310–316. PubMed Abstract\n\nMangtani P, Abubakar I, Ariti C, et al.: Protection by BCG vaccine against tuberculosis: a systematic review of randomized controlled trials. Clin Infect Dis. 2014; 58(4): 470–480. PubMed Abstract | Publisher Full Text\n\nTameris M, McShane H, McClain JB, et al.: Lessons learnt from the first efficacy trial of a new infant tuberculosis vaccine since BCG. Tuberculosis (Edinb). 2013; 93(2): 143–149. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHatherill M, Verver S, Mahomed H, et al.: Consensus statement on diagnostic end points for infant tuberculosis vaccine trials. Clin Infect Dis. 2012; 54(4): 493–501. PubMed Abstract | Publisher Full Text\n\nBunyasi EW, Luabeya AK, Tameris M, et al.: Impact of isoniazid preventive therapy on the evaluation of long-term effectiveness of infant MVA85A vaccination. Int J Tuberc Lung Dis. 2017; 21(7): 778–783. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMulenga H, Tameris MD, Luabeya KK, et al.: The Role of Clinical Symptoms in the Diagnosis of Intrathoracic Tuberculosis in Young Children. Pediatr Infect Dis J. 2015; 34(11): 1157–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBunyasi EW, Tameris M, Geldenhuys H, et al.: Evaluation of Xpert® MTB/RIF Assay in Induced Sputum and Gastric Lavage Samples from Young Children with Suspected Tuberculosis from the MVA85A TB Vaccine Trial. PLoS One. 2015; 10(11): e0141623. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFletcher HA, Filali-Mouhim A, Nemes E, et al.: Human newborn bacille Calmette-Guérin vaccination and risk of tuberculosis disease: a case-control study. BMC Med. 2016; 14: 76. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "31832",
"date": "12 Mar 2018",
"name": "Rhea Coler",
"expertise": [
"Reviewer Expertise I have 20+ years of experience in studying the pathogenesis of a variety of infectious disease pathogens",
"including Mycobacterium tuberculosis",
"the pathogen that causes tuberculosis",
"as well as antigen discovery and adjuvant development in academic",
"biotech and non-profit environments",
"and have led vaccine development from the benchtop to human clinical 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\nI reviewed the manuscript “Progress and challenges in TB vaccine development” and it seems to me that the objective of this review is well defined and clearly stated.\nThe abstract is written clearly and objectively. Introduction is clear and presents a solid foundation for the review's objectives. The presentation of the various sub-topics is clear and the review of the bibliography is mostly complete and includes the main articles published in the area.\nThe section on animal models could be elaborated to discuss in greater details mouse and guinea pig TB models, in addition to the authors’ thoughtful perspective on the use of NHPs.\nSome discussion of the intricate network of events modulating inflammation in the context of TB and how a better understanding of this will aid in the development of more effective vaccines and host-directed therapies to curb TB is warranted.\nThe review should also be updated given the recent report of the first efficacy trial evaluating prevention of infection results for BCG revaccination or H4:IC31.\n\nThank you for the opportunity to review this excellent work.\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": "30908",
"date": "13 Mar 2018",
"name": "Ashley Birkett",
"expertise": [
"Reviewer Expertise Vaccine development"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, the authors rationally assert that compelling biological evidence for the induction of protective immunity associated with BCG vaccination, as well as by natural exposure, strongly support the feasibility for development of an improved TB vaccine. The manuscript highlights progress since the 2012 publication of the Blueprint for TB Vaccine Development, both in TB-specific and non-specific areas, that are critical to inform the design and testing of novel vaccines approaches. Progress is highlighted in areas such as the understanding of the basis for protective immunity, vaccine design (i.e. antigens, platforms and adjuvants), and improved sophistication of clinical trial design (i.e. enhanced inclusion/exclusion criteria associated with specific endpoints) to reduce sample size and trial duration, and therefore costs.\n\nDespite the documented advances, the main theme of the paper is one of frustration with the slow pace in advancing existing candidates and in the repopulation of the global vaccine portfolio with vaccines built on diverse approaches that leverage new scientific learnings. The authors argue that risk aversion on behalf of funding agencies is a critical factor in this regard. While I cannot speak to the current funding environment for TB vaccine development (convincing data on reduced investments in this area was not provided), the authors, quite rightly, suggest that a step-wise advancement (requiring investment risk), is more likely than the sudden transformational breakthrough likely to align with a risk averse investment approach. Appropriately, the authors acknowledge the need to identify approaches that induce clinical efficacy (including via nascent human challenge models) to ‘back-validate’ non-clinical models and thereby improve their predictive value of clinical outcomes going forward.\n\nThe manuscript would have benefited from a clearer communication on the Preferred Product Characteristics (PPCs) that underpin a (presumed) positive value proposition for the next generation target vaccines targeting the expressed indications, of which there were several.\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/7-199
|
https://f1000research.com/articles/7-198/v1
|
16 Feb 18
|
{
"type": "Case Report",
"title": "Case Report: Tibial and fibular osteochondroma as an unusual cause of popliteal artery entrapment syndrome",
"authors": [
"Jamil Victor de Oliveira Mariúba",
"Marcone Lima Sobreira",
"Winston Bonetti Yoshida",
"Eduardo Savio de Oliveira Mariúba",
"Hamilton de Almeida Rollo",
"Regina Moura",
"Matheus Bertanha",
"Rodrigo Gibin Jaldin",
"Rafael Elias Farres Pimenta",
"Paula Angeleli Bueno de Camargo",
"Mariana Thais Silva Secondo",
"Marcone Lima Sobreira",
"Winston Bonetti Yoshida",
"Eduardo Savio de Oliveira Mariúba",
"Hamilton de Almeida Rollo",
"Regina Moura",
"Matheus Bertanha",
"Rodrigo Gibin Jaldin",
"Rafael Elias Farres Pimenta",
"Paula Angeleli Bueno de Camargo",
"Mariana Thais Silva Secondo"
],
"abstract": "Background: Osteochondroma, or osteocartilaginous exostosis, is the most common benign neoplasm of bone, and accounts for 20-50% of all benign tumors. Vascular complications associated with osteochondromas are rare, and include pseudoaneurysm formation, vessel occlusion and vessel displacement. To date, only two cases of popliteal artery entrapment syndrome (PAES) caused by an isolated fibular osteochondroma have been reported. Case Report: This report describes a unique case of PAES. A 33-year-old woman had a history of multiple osteochondroma, including of the proximal tibia and fibula on the left, diagnosed at age two years and monitored clinically by an orthopedist. The patient presented at our facility with a one-year history of a progressive intermittent claudication, left-sided toe pain and pallor in cold weather. After a complete evaluation, we diagnosed an arterial occlusion of the left popliteal artery. We tried several attempts of revascularization, by different forms, without success. The case went to amputation surgery. Conclusion: We consider this an important case because, although the association of osteochondroma and PAES is rare, physicians should consider it early to avoid acute vascular complications. Moreover, to date, we believe this is the first description of a PAES related with multiple osteochondroma.",
"keywords": [
"popliteal entrapment syndrome",
"osteochondroma",
"tibia",
"fibula",
"popliteal artery"
],
"content": "Introduction\n\nPopliteal artery entrapment syndrome (PAES) is an infrequent condition, generally caused by embryonic abnormalities and characterized by deviation of the popliteal artery from its normal course and subsequent compression. During muscular exertion, blood flow to the leg muscles is reduced, leading to intermittent claudication, pallor, and coldness1–3. PAES may be classified into six types, with medial deviation of the popliteal artery around the medial head of the gastrocnemius being the most common form3.\n\nOsteochondromas are the most common benign tumors of bone (30%), and generally occur in the metaphyseal area of long bones4,5. The vast majority (85%) of osteochondromas present as single lesions and are not hereditary. Conversely, approximately 15% of cases present as multiple osteochondromas, which are characteristic of the autosomal dominant disease hereditary multiple osteochondromas (HMO), or hereditary multiple exostoses (HME); 62% of cases have a positive family history5,6.\n\nOsteochondromas occur in 3% of the overall population. The solitary form exhibits no gender predominance, whereas the hereditary form has a 1.5:1 male-to-female ratio; it is more common in the Caucasian population, but its overall prevalence is lower than that of solitary osteochondroma (0.9 to 2 cases per 100,000 population)5,6.\n\nOsteochondromas take the form of cartilage-covered masses that grow exophytically on the surface of bones. They develop in childhood and adolescence, usually as slow-growing, painless lesions. However, depending on the site of the tumor, significant symptoms may develop as a result of complications, including fractures, bone deformity, mechanical joint abnormalities, and neurovascular involvement. Malignant transformation may occur later in life, during adulthood (0.5–5% of cases), but metastases are rare. Proliferation of osteochondromas usually ceases after puberty5–7.\n\nThe treatment of choice is resection, provided the skeleton has matured5. Vascular compression, arterial and venous thrombosis, aneurysms, and pseudoaneurysms are common findings, occurring in 91% of cases; pseudoaneurysms are the most prevalent vascular complication5,8.\n\nOsteochondroma is an exceedingly rare cause of PAES. To date, only two case reports have been published in internationally indexed journals1,4. The present report describes a single case of popliteal artery entrapment secondary to multiple osteochondroma, treated by the present authors.\n\n\nCase report\n\nA 33-year-old woman had a history of multiple osteochondroma, including of the proximal tibia and fibula on the left, diagnosed at age two years and monitored clinically during childhood and adolescence by orthopedic specialists at an outside facility, with family history of osteochondroma (a sister, without complications). As the patient was pain-free and had no fractures or functional changes at the knee joint, resection was not indicated from an orthopedic standpoint.\n\nThe patient presented at our facility with a one-year history of left-sided toe pain and pallor in cold weather. Approximately one month before, she had experienced a sudden onset of intermittent claudication at 400 m; her maximum walking distance had decreased progressively to 100 m in the intervening time.\n\nPhysical examination revealed pallid discoloration of the hallux, second toe, and third toe of the left foot, with localized pain. The popliteal and distal pulses were absent in the left lower extremity, and a temperature gradient was felt on the affected foot.\n\nA preoperative arterial duplex scan revealed left popliteal artery occlusion. Arteriography also showed occlusion of the superficial femoral artery, consistent with a thrombus.\n\nA thromboembolectomy was performed using a Fogarty catheter inserted through the superficial femoral artery, with removal of fresh thrombi. The catheter could not be advanced past the juxta articular portion of the popliteal artery. Intraoperative angiography demonstrated a patent anterior tibial artery (Figure 1). The popliteal pulse returned at the end of the procedure, but distal pulses remained absent, although flow was present on Doppler analysis.\n\nA bony tumor is visible in the left fibular head.\n\nPostoperatively, the patient underwent CT angiography (Figure 2–Figure 4), which revealed a mass on the head of the fibula and tibial plateau, encircling the juxta articular portion of the popliteal artery. There was no contrast uptake in the arterial lumen distal to the site of the tumor.\n\nThe tumor is seen to completely envelop the left artery.\n\nA second Fogarty thromboembolectomy of the posterior tibial artery was then performed, with creation of a femoral-to-posterior tibial bypass greater saphenous vein graft placed in an inverted position, which was unsuccessful. A femoral-to-anterior tibial graft could not be fashioned due to anatomical limitations (the tumor obstructed the usual course of the bypass graft).\n\nThe patient was started on alprostadil (200 micrograms diluted into 200 milliliters of saline every 12 hours) which was discontinued after 24 days due to elevated liver enzymes. Toe pallor progressed to wet gangrene of the forefoot (Figure 5) and the patient developed intractable pain, fever, and confusion. We then performed a knee disarticulation amputation, which healed satisfactorily and produced clinical improvement (sensitivity and motor skills preserved without pain). The patient was discharged to outpatient follow-up (returned at 14 days to evaluate the healing, at 3 months to evaluate physical therapy and each year). She adapted successfully to a prosthesis and leads a virtually normal life.\n\n\nDiscussion\n\nIn younger patients, intermittent claudication, due to peripheral arterial disease, is a rare presentation. Its causes include early-onset atherosclerosis, trauma, tumors, arteritis, and fibrous dysplasias, but the most common etiology is PAES1–4. Entrapment may occur due to derangements in embryonic development of the popliteal artery or of the musculotendinous components of the popliteal fossa, leading to deviation of the artery from its normal course or to the development of anomalous structures that compress the artery. Consequently, distal blood flow is reduced during muscle contractions, producing intermittent claudication, pallor, and coldness of the extremity. These signs and symptoms usually remit at rest1–3.\n\nResection is usually indicated for asymptomatic, incidentally detected, solitary osteochondromas, so as to prevent future complications. Surgical resection is also recommended for painful or continuously growing lesions, joint involvement, vascular complications with intermittent claudication, or evidence of malignant transformation5.\n\nOsteochondromas are generally asymptomatic, and over 130 case reports of vascular complications have been published, with pseudoaneurysm and venous thrombosis being the most common such complications7. Eschelman et al. reported that among 56 cases of osteochondromas with vascular involvement, only six were tibial and only half of these caused arterial compression, as observed in our patient9.\n\nGuy et al4. reported a case of popliteal artery stenosis secondary to osteochondroma, presenting with left lower extremity pain and edema. The pain was initially exertional and resembled intermittent claudication, but eventually became continuous. Distal arterial pulses were palpable in both lower extremities, and a duplex scan of the popliteal arteries showed a normal triphasic waveform bilaterally. On the left side, blood flow velocity decreased during calf muscle contraction, and was followed by reactive hyperemia on relaxation, indicating popliteal artery entrapment. Signs of venous hypertension were also present (edema and varicose veins around the medial malleolus). Plain radiography and magnetic resonance imaging (MRI) revealed a fibular tumor compressing the artery4.\n\nHolzapfel et al.1 reported another case of popliteal artery entrapment in which Doppler ultrasonography and MRI revealed a partially compressed, but not occluded artery, with preserved distal pulses. Venography showed a completely compressed popliteal vein, mimicking deep venous thrombosis. The patient was treated for four weeks with low molecular weight heparin, to no effect, before being referred for correct diagnosis and management1.\n\nIn 2013, Henry et al.9 reported a case of intermittent claudication in a 23-year-old woman with a solitary tibial osteochondroma, which was treated by division of the soleus muscle and resection of the osteochondroma.\n\nA diagnosis of PAES should be considered in all young patients with intermittent claudication. Pain occurs in the foot and calf muscles, usually after strenuous exercise. Spastic claudication, in which patients are pain-free while running, but paradoxically experience pain while walking, may also occur; some patients report pain when standing on tiptoes3.\n\nDiagnostic confirmation usually begins with a duplex scan, which enables dynamic visualization of the popliteal artery and demonstrates its patency at rest and stenosis or occlusion in response to functional maneuvers3. MRI and CT angiography may aid in diagnosis by identifying the musculoskeletal or tendinous structures implicated in compression3.\n\nArteriography plays an important role in diagnosis and surgical treatment planning, and is indicated whenever arterial lesions, such as aneurysmatic degeneration or arterial thrombosis, are suspected. Osteochondroma (osteocartilaginous exostosis) is a cartilage-capped bony projection arising on the external surface of bone containing a marrow cavity, which is continuous with that of the underlying bone6. To establish the diagnosis of osteochondroma and to ensure good visualization of the bones, anteroposterior and lateral views of both lower extremities should be obtained, with the foot in passive dorsiflexion and active hyperextension.\n\nIn the case reported here, the patient presented with signs and symptoms of acute ischemia. This is an unusual manifestation of osteochondroma with arterial involvement, and this limited our approach to the case, because we were prepared for an embolus and what actually happened was a thrombosis. Even after two attempts at surgical revascularization for limb salvage, a major amputation of the affected extremity was required. Treatment failure in this case was attributed to the extent of local arterial involvement and by chronic distal occlusion.\n\nIn patients with osteochondromas in the vicinity of the popliteal artery, this vessel may be encircled by the tumor and chronically compressed, leading to endothelial injury, thrombus formation, and, eventually, critical ischemia. Although the association of osteochondroma and PAES is rare, it should be considered by physicians, particularly in the differential diagnosis of young patients with evidence of vascular involvement. Imaging should be performed early and periodically, so as to prevent diagnostic and therapeutic delay1.\n\n\nConsent\n\nWritten informed consent for the publication of the patient’s 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\nHolzapfel BM, Seppel G, Wagner R, et al.: Popliteal entrapment syndrome caused by fibular osteochondroma. Ann Vasc Surg. 2011; 25(7): 982.e5–10. PubMed Abstract | Publisher Full Text\n\nCairols MA, Blanes I, Gimenez A, et al.: An exceptional case of popliteal entrapment syndrome. Eur J Vasc Surg. 1994; 8(6): 754–6. PubMed Abstract | Publisher Full Text\n\nAlmeida MJ, Yoshida WB, Melo NR: [Popliteal artery entrapment syndrome]. J Vasc Bras. 2003; 2(1): 211–9. Reference Source\n\nGuy NJ, Shetty AA, Gibb PA: Popliteal artery entrapment syndrome: an unusual presentation of a fibular osteochondroma. Knee. 2004; 11(6): 497–9. PubMed Abstract | Publisher Full Text\n\nKitsoulis P, Galani V, Stefanaki K, et al.: Osteochondromas: review of the clinical, radiological and pathological features. In Vivo. 2008; 22(5): 633–46. PubMed Abstract\n\nBovée JV: Multiple osteochondromas. Orphanet J Rare Dis. 2008; 3: 3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreenway G, Resnick D, Bookstein JJ: Popliteal pseudoaneurysm as a complication of an adjacent osteochondroma: angiographic diagnosis. AJR Am J Roentgenol. 1979; 132(2): 294–6. PubMed Abstract | Publisher Full Text\n\nGomes FS, Lewin F, Mariotti GC, et al.: [Imaging of osteochondroma complications]. Rev Imagem. 2007; 29(2): 53–9. Reference Source\n\nHenry JC, Mouawad NJ, Phieffer L, et al.: Tibial osteochondroma inducing popliteal artery compression. J Vasc Surg. 2015; 61(6): 1595–8. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "34211",
"date": "04 Jun 2018",
"name": "Ali Khalifeh",
"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 report of a young female patient who had gradual growth of benign osteochondroma compromising blood supply to the lower extremity. This eventually lead to loss of limb despite multiple interventions to re-vascularize the lower extremity.\nThe authors should provide more diagnostic imaging such as Doppler imaging(distal flow/ABI), and complete angiography of left lower extremity(pelvis to foot). This will help readers understand the vascular anatomy that the patient presented with.\nWhat triggered the acute occlusion of the popliteal artery? It appears that the patient had a year history of claudication without evidence of collateral circulation on the presented angiogram.\nDid the authors discuss a possible combined case of tumor resection and revascularization? The patient had a largely patent AT which could have been sufficient for LE survival or atleast planning of lower amputation if needed.\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? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
},
{
"id": "34210",
"date": "02 Jul 2018",
"name": "Humberto Ferreira-Arquez",
"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\nThere are no negative comments to make on this manuscript.\nAn anatomical variable of very rare occurrence is approached in this article.\nOsteochondroma is the most common of the benign bone tumours, occurring in approximately one percent of the population. Although rare, periodic changes in blood flow can also occur. Vascular compression, arterial thrombosis, aneurysm, pseudoaneurysm formation and venous thrombosis are common complications and lead to claudication, pain, acute ischemia, and signs of phlebitis, while nerve compression occurs in about 20% of patients.\nThis case is of interest for the management of a patient with a family history of osteochondroma and/or circulatory disease. It is of great relevance to health professionals as well as specialists.\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
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https://f1000research.com/articles/7-198
|
https://f1000research.com/articles/7-196/v1
|
15 Feb 18
|
{
"type": "Systematic Review",
"title": "Zika virus infection as a cause of congenital brain abnormalities and Guillain-Barré syndrome: From systematic review to living systematic review",
"authors": [
"Michel Jacques Counotte",
"Dianne Egli-Gany",
"Maurane Riesen",
"Million Abraha",
"Teegwendé Valérie Porgo",
"Jingying Wang",
"Nicola Low",
"Dianne Egli-Gany",
"Maurane Riesen",
"Million Abraha",
"Teegwendé Valérie Porgo",
"Jingying Wang"
],
"abstract": "Background. The Zika virus (ZIKV) outbreak in the Americas has caused international concern due to neurological sequelae linked to the infection, such as microcephaly and Guillain-Barré syndrome (GBS). The World Health Organization stated that there is “sufficient evidence to conclude that Zika virus is a cause of congenital abnormalities and is a trigger of GBS”. This conclusion was based on a systematic review of the evidence published until 30.05.2016. Since then, the body of evidence has grown substantially, leading to this update of that systematic review with new evidence published from 30.05.2016 – 18.01.2017, update 1. Methods. We review evidence on the causal link between ZIKV infection and adverse congenital outcomes and the causal link between ZIKV infection and GBS or immune-mediated thrombocytopaenia purpura. We also describe the transition of the review into a living systematic review, a review that is continually updated. Results. Between 30.05.2016 and 18.01.2017, we identified 2413 publications, of which 101 publications were included. The evidence added in this update confirms the conclusion of a causal association between ZIKV and adverse congenital outcomes. New findings expand the evidence base in the dimensions of biological plausibility, strength of association, animal experiments and specificity. For GBS, the body of evidence has grown during the search period for update 1, but only for dimensions that were already populated in the previous version. There is still a limited understanding of the biological pathways that potentially cause the occurrence of autoimmune disease following ZIKV infection. Conclusions. This systematic review confirms previous conclusions that ZIKV is a cause of congenital abnormalities, including microcephaly, and is a trigger of GBS. The transition to living systematic review techniques and methodology provides a proof of concept for the use of these methods to synthesise evidence about an emerging pathogen such as ZIKV.",
"keywords": [
"Zika virus",
"causality",
"living systematic review",
"congenital abnormalities",
"Guillain-barre syndrome",
"microcephlay"
],
"content": "Introduction\n\nOutbreaks of Zika virus (ZIKV) infection in the Americas have caused international concern owing to the severity of neurological sequelae linked to the infection (WHO statement IHR 2005). During 2016, the number of countries affected by the ZIKV outbreak had grown from 33 countries (WHO situation report 05.02.2016) to 75 countries (WHO situation report 05.01.2017). By March 9, 2017, 31 countries had reported microcephaly or other congenital central nervous system (CNS) abnormalities potentially associated with ZIKV infection and 23 had reported an increase in the incidence of the immune-mediated condition Guillain-Barré syndrome (GBS) or laboratory confirmed ZIKV in persons with GBS (WHO situation report 10.03.2017). The causal association between ZIKV and adverse neurological outcomes has now been examined in many systematic and non-systematic reviews of research1,2. Case reports of other conditions in people with ZIKV infection, including immune-mediated idiopathic thrombocytopaenia purpura (ITP), have also been published3–6.\n\nThe World Health Organization (WHO) based its assessment, that there is “sufficient evidence to conclude that Zika virus is a cause of congenital abnormalities and is a trigger of GBS” (WHO Zika causality statement), on a review of systematically identified studies up to May 30, 2016 and nonsystematically identified studies up to July 29, 20167. The review addressed specific questions about 10 dimensions of causal associations, based on the work of Bradford Hill8 and organised as a causality framework (Supplementary Table 1) that covers: temporality (cause precedes effect); biological plausibility of proposed biological mechanisms; strength of association; exclusion of alternative explanations; cessation (reversal of an effect by experimental removal of, or observed decline in, the exposure); dose-response relationship; experimental evidence from animal studies; analogous cause-and-effect relationships found in other diseases; specificity of the effect; and the consistency of findings across different study types, populations and times. The review included 108 articles about congenital abnormalities or GBS but there was no, or insufficient evidence to answer questions in several dimensions of the causality framework7. The causality framework included questions about ITP, but the review authors judged the number of published articles to be too low to assess causality. Since the WHO statement and accompanying publication, about 200 scientific publications every month are added to the body of evidence about all aspects of research about ZIKV.\n\nA living systematic review would help to overcome some of the challenges of keeping up to date with the high volume of ZIKV research publications. A living systematic review is a systematic review that is “continually updated, incorporating relevant new evidence as it becomes available”9, which can help in fields where evidence is emerging rapidly and where new review outcomes might change policy or practice decision10. Technical solutions are available to facilitate the reviewing process, such as automated searching and deduplication and computer-assisted screening of article titles and abstracts, increase the efficiency and speed of a review team and transform the review into a living document.\n\nThis article aims to fulfil two separate objectives. First, we update our systematic review7 with new evidence published from May 30, 2016 to January 18, 2017 about all 10 dimensions of the causal associations between ZIKV and (a) congenital brain abnormalities, including microcephaly, in the foetuses and offspring of pregnant women and (b) GBS/ITP in any population. Second, we describe the transition of the review into a living systematic review.\n\n\nMethods\n\nWe performed the review according to the protocol registered in PROSPERO CRD42016036693 (PROSPERO protocol). The eligibility criteria, information sources and search strategy, study selection and data extraction are the same as reported in the protocol and in the previous publication7. In brief, the search covers PubMed, Embase and LILACS electronic databases; the Pan American Health Organization (PAHO), WHO, the Centers for Disease Control and Prevention (CDC) and the European Centre for Disease Prevention and Control (ECDC) websites; and several preprint databases (BioRxiv, PeerJ and ArXiv). Search terms included ‘Zika virus’ and ‘ZIKV’ and corresponding MESH terms. Two reviewers screen and select articles for inclusion and extract data independently. We included publications that held information on at least one of the ten dimensions of the causality framework, regardless of the study design7. We gathered publications systematically from May 30, 2016 to January 18, 2017 for this update. We refer to the previous version of the review as the baseline review7 and to this current update as update 1. Reporting of the results follows the Preferred Reporting Items of Systematic reviews and Meta-Analyses (PRISMA) statement (Supplementary File 1)11.\n\nTo keep up with the quantity of published research, we developed a living systematic review workflow (Supplementary File 2). We have identified three modules that could be automated (Figure 1). As of December 2017, module 1, searching and deduplication, and part of module 3, the output of the report have been automated. Reviewers can be notified daily with a list of new unique search results so that screening can be performed rapidly. Following manual data extraction and synthesis, the output can be updated semi-automatically. We use the online database Research Electronic Data Capture (REDCap)12 to maintain the references, perform screening and extract data into piloted extraction forms. We plan to update the review twice per year with formal peer reviewed updates (Figure 2), and continually through a web platform.\n\nBlue boxes and arrows represent the conceptual steps in a systematic review process. Automation is divided in three modules. Module 1 is the automation of the searching and deduplication of information from different data sources. Module 2 partly automates screening. Module 3 automates the production of tables and figures and outputs the data to a web platform (Data visualisation). Blue arrows represent automated information flows; red arrows represent manual input. The blue-red dashes arrow represents a blended form where reviewers verify automated decisions of the system. The white boxes show the practical implementation of the system and the data flow.\n\nThe baseline review (BR,7) and Update 1 (U1) this version classic, manual systematic review. During 2017 automation of the workflow was conducted resulting in a projected Update 2 (U2) and 3 (U3) with more rapid throughput. LSR, living systematic review.\n\nWe synthesised the findings as narrative summaries of the evidence according to causality dimension and outcome, as previously described7, and compare them with the the baseline review. We use the term ‘confirmation’ to summarise findings of new studies included in update 1 if they report the same findings as those in the baseline review. We use the term ‘expansion’ of evidence if studies included in update 1 provide new findings.\n\n\nResults\n\nBetween May 30, 2016 and January 18, 2017, we identified 2413 publications. After deduplication, we retained 1699 unique records. Based on screening of title and abstract, we discarded 1025 publications, retaining 674 items; after screening of the full text, 101 publications were included. Figure 3 shows the PRISMA flow diagram for this review11. Seventy-seven publications held information on one or more dimensions of the causality framework on adverse congenital outcomes and 25 on GBS or idiopathic thrombocytopaenia purpura. Table 1 compares the included publications, study types and the causality dimension(s) they address in the baseline review7 and update 1 of the review.\n\nOne publication can address multiple causality dimensions. Comparison between the current (U1) and the baseline review (BR, 7) stratified by outcome. GBS/ITP, adverse autoimmune outcomes (Guillain Barré syndrome/idiopathic thrombocytopaenia purpura). NA, not applicable; evidence about analogous conditions was not searched systematically; the dimension of consistency used information in items included for all other causality dimensions.\n\nA detailed overview of the new evidence is provided in Table 2 and Supplementary Table 2. In the search period for review update 1, an additional 548 cases of adverse congenital outcomes were described in 32 studies12–43. Adverse congenital outcomes described were: clinical microcephaly12–17,20–24,26–31,33,35,37,40–42, imaging confirmed brain abnormalities12,15,17,19–24,26–31,35,37,38,40,42, intrauterine growth restriction15,17,31,38,40,42, ocular disorders12,17,27–29,31,38,40 and auditory disorders12,18,29.\n\nEvidence is displayed for each dimension and for each question of the causality framework. Zika virus (ZIKV); Dengue virus (DENV); West Nile virus (WNV); Chikungunya virus (CHIKV); Toxoplasmosis, Other [Syphilis, Varicella-zoster, Parvovirus B19], Rubella, Cytomegalovirus, and Herpes infections (TORCH); Central Nervous System (CNS). NA, not applicable; evidence about analogous conditions was not searched systematically; the dimension of consistency used information in items included for all other causality dimensions. the baseline review (BR), update 1 (U1).\n\nTemporality. This update confirms the previous conclusion that ZIKV infection precedes the adverse congenital outcomes. We found an additional 21 publications in which ZIKV infection preceded the adverse congenital outcome at an individual level12,15–18,26-31,35-40,42,44,45 and at a population level45,46. Infections in the first and second trimester seemed to be related to the most adverse outcomes31,40. Cohort studies of pregnant women from French Guiana and Brazil found a higher proportion of congenital abnormalities in babies born from mothers infected in the first and the second trimester31,40.\n\nBiological plausibility. This update includes an additional 42 studies14,17,23,24,32–36,38,39,42,47–76, some of which expand the evidence base. Whereas in the baseline review, we found inconclusive evidence of whether ZIKV particles in infants were capable of replication, both in vivo and ex vivo studies now demonstrate that this is the case33,36,47,50,53-55. Furthermore, there was a strong expansion of the evidence clarifying how ZIKV causes adverse congenital outcomes. ZIKV uses receptors from the TAM family to enter cells47–52, where the virus induces cell death, primarily in developing neuronal cells60,61,64,65,67,69,70,75.\n\nStrength of association. We included five publications that confirm a strong association between ZIKV infection and adverse congenital outcomes21,22,31,40,41. The strength of association at an individual level was high but imprecise, owing to small sample sizes. Estimates from cohort studies31,40 appeared to be lower than those from case-control studies21,22,41. The definition of the outcomes and the outcomes assessed, varied between studies. The risk of any adverse congenital outcomes was higher and more variable than the risk of microcephaly. The risk ratio for microcephaly between ZIKV unexposed and exposed was 4.4 (95% CI: 0.2-80.8) in a cohort in Brazil31 and 6.6 (95% CI: 0.8-56.4) in a cohort in French Guiana40. In the Brazilian cohort31, the proportion of any adverse congenital outcomes among ZIKV infected women was high (41.9% [49/117]), compared with the uninfected group (5.2% [3/57]). In a prospective case- control study in Brazil, women with laboratory-confirmed ZIKV had 55.5 (95% CI: 8.6-infinity) times the odds of having a baby with microcephaly compared with women without evidence of ZIKV infection21. A retrospective case- control study in Hawaii found an odds ratio of 11.0 (95% CI: 0.8-147.9)41. In the latter, however, exposure was assessed retrospectively using serology.\n\nExclusion of alternatives. We included 23 new studies in this update12,14,17–19,21–28,30,31,34,36–38,40,42,45,77. Many studies included in this review that reported on adverse outcomes of congenital ZIKV excluded TORCH infections12,14,17–19,21–28,30,31,34,36–38,40,42,45,77; exposure to toxic chemicals12,14,18,23,28 or genetic conditions12,18,23,28,30,36,42. Maternal or foetal malnutrition, hypoxic-ischaemic lesions and underlying genetic conditions were not excluded. No single alternative explanation could be given to explain the relation between ZIKV and adverse congenital outcomes.\n\nCessation. We did not find any new publications for this causality dimension. Evidence is still lacking on the effect of intentional removal due to lack of vaccination or elimination of mosquitoes on a large scale.\n\nDose-response. There is still no direct evidence about the association between Zika viral load and probability of adverse congenital outcome in observational studies, or of an association between symptomatic status and outcome. In a study in the United States, Honein et al. found similar proportions of adverse congenital outcomes in symptomatic and asymptomatic ZIKV-infected mothers32.\n\nAnimal experiments. This update of the review includes an additional 11 studies63,71,78–86. These studies confirm a consistent relation between a range of contemporary ZIKV and adverse congenital outcomes, including from Brazil85, Puerto Rico79 and Mexico80,81. The body of evidence coming from animal studies has grown; both in mice and macaques, congenital anomalies such as intra-uterine growth restriction and signs of microcephaly were observed after ZIKV infection78,84,85.\n\nAnalogy. As for the baseline review, evidence for this dimension was not reviewed systematically because our search strategy did not include terms for other infections or conditions. Studies included in this version of the review confirm the analogy between congenital ZIKV and TORCH infections87. Vertical transmission of West Nile virus and dengue virus were summarised in the baseline review. In update 1, we included a case series from El Salvador that reported Chikungunya virus in 169 newborns of women with symptomatic infection; a minority had CNS infection, but microcephaly was not reported88. For most analogous pathogens, infections earlier in the pregnancy have a higher risk of adverse outcomes87.\n\nSpecificity. We included one study89, suggesting an expansion of evidence of a distinct congenital Zika syndrome (CZS)89. In a review of 34 published reports, the authors suggest five congenital abnormalities that, in conjunction, comprise a pattern that is unique to ZIKV: severe microcephaly with overlapping cranial structures, subcortical location of brain calcifications, macular scarring and retinal mottling, congenital contractures and early pyramidal and extrapyramidal symptoms89.\n\nConsistency. The studies included in this version of the review confirm the pattern of consistency observed in the baseline review. ZIKV infection in association with adverse congenital outcomes were reported in a range of study designs from different regions (WHO situation report 05.01.2017), although the proportion of affected infants varies over geographic region and time. ZIKV exposure resulted in adverse congenital outcome in people living in ZIKV endemic areas12–19,21–34,40–42,44,45,77,90,91 and in female travellers who returned to non-endemic countries34–39,92,93. Direct evidence from epidemiological studies comparing different lineages is lacking due to circulation of a single strain.\n\nConclusion. The evidence added in update 1 of the review confirms the conclusion of a causal association between ZIKV and adverse congenital outcomes. New findings expand the evidence base in the dimensions of biological plausibility, strength of association, animal experiments and specificity. In vitro and in vivo studies elucidate pathways on how these outcomes likely occur. Conclusive evidence on the strength of association is lacking. Studies provide crude overall measures of association, not taking into account potential co-factors.\n\nIn the search period for update 1 of the review, an additional 154 cases of ZIKV-related GBS95 and 11 ZIKV-related cases of ITP3–6 were described in 18 studies. Table 3 summarises the evidence for specific questions in each of 10 causality dimensions (detailed overview in Supplementary Table 3).\n\nEvidence is displayed for each dimension of the causality framework and for each question. Zika virus (ZIKV); Dengue virus (DENV); Guillain-Barré syndrome (GBS); immune-mediated idiopathic thrombocytopaenia purpura (ITP). NA, not applicable; evidence about analogous conditions was not searched systematically; the dimension of consistency used information in items included for all other causality dimensions. the baseline review (BR), Update 1 (U1).\n\nTemporality. We found an additional 17 publications that confirmed that ZIKV infection preceded the GBS or ITP at an individual level3,5,6,95–108 or at a population level103,109–111. ZIKV infections seems to be followed by GBS on average between 5 and 10 days. In one case series from Colombia103, the authors distinguished between rapid onset of GBS symptoms after ZIKV symptoms (para-infectious) and post-infectious onset, with an asymptomatic period after ZIKV symptoms before the start of GBS symptoms.\n\nBiological plausibility. We did not find any publications about the biological plausibility of ZIKV as a cause of GBS or ITP.\n\nStrength of association. We did not find any comparative observational studies during the search period for update 1. Several surveillance studies confirmed an increase in notified GBS cases during ZIKV outbreaks at the population level111. Rate ratios were significantly higher for Brazil, Colombia, the Dominican Republic, El Salvador, Honduras, Suriname and Venezuela when comparing pre-ZIKV GBS incidence and the incidence during the outbreak111; this ratio ranged from 2.0 (95% CI: 1.6-2.6) to 9.8 (95% CI: 7.6-12.5).\n\nExclusion of alternatives. We included 11 publications4–6,95,98,99,101,103,104,111,112 that expanded the list of alternative causes for autoimmune disease that were excluded, such as infections, vaccines, other system illnesses and medication, drugs or other chemicals. Many GBS cases in these publications had serological evidence of previous exposure to DENV, as seen in the baseline review. It remains unclear how large the potential role of co-factors such as antibody dependent enhancement are.\n\nCessation. We did not identify any publications with evidence about the effect of intentional removal/elimination/prevention of ZIKV on either GBS or ITP. An additional publication confirmed evidence that the natural removal of ZIKV resulted in a decrease in GBS cases in Brazil, Colombia, Dominican Republic, El Salvador, Honduras, Suriname and Venezuela104,111.\n\nDose-response. We did not identify any publications about this dimension for either GBS or ITP.\n\nAnimal experiments. No additional evidence from animal experiments was identified that support the association between ZIKV infection and GBS/ITP development.\n\nAnalogy. As for the baseline review, evidence for this dimension was not reviewed systematically because our search strategy did not include terms for other infections or conditions. We did not identify any new publications addressing this dimension for either GBS or ITP.\n\nSpecificity. We did not identify any new publications addressing this dimension for either GBS or ITP.\n\nConsistency. Studies included in update 1 confirmed the consistency of the evidence for 3 of 4 questions about the association between ZIKV and GBS. By geographical region, ZIKV transmission has been associated with the occurrence of GBS in 2 of 4 regions; increased GBS incidence has been reported in the WHO regions of the Americas and the Western Pacific region, but not in the African or Southeast Asian region, despite recent ZIKV circulation113. By study design, the association between ZIKV infection and GBS has been found at individual and population level and with different study designs. By population, ZIKV infection has been linked to GBS in ZIKV endemic regions4–6,95,96,98–101,103–105,109,111,114 and travellers from non-affected countries who were exposed in these endemic regions3,97,102,106,112. There was insufficient evidence to examine the consistency of evidence about ZIKV and ITP.\n\nConclusion. The body of evidence has grown during the search period for update 1 but only for dimensions that were already populated in the original publication for GBS. There is still a limited understanding of the biological pathways that potentially cause the occurrence of autoimmune disease following ZIKV infection. Additionally, prospective comparative epidemiological studies are still lacking. It remains unclear how co-factors such as age and previous exposure to flaviviruses influences the risk of developing GBS. The evidence supports a temporal association between ZIKV and ITP but there is an absence of evidence for other dimensions of causality.\n\nAutomated search and deduplication processes identified 2410 publications about any aspect of ZIKV infection. The next update of this review will address causality dimensions in the realm of epidemiological studies; strength of association, dose-response relationship, specificity and consistency.\n\n\nDiscussion\n\nStatement of principal findings. This systematic review confirms evidence of a causal association between ZIKV and adverse congenital outcomes and between ZIKV and GBS, although evidence about biological plausibility is still lacking. We assessed evidence about an association between ZIKV and ITP but found that this only addressed the dimension of temporality. The review is transitioning from classic systematic review methods to those of a living systematic review.\n\nStrengths and limitations of the study. The strengths of this study are the systematic approach to the identification, selection and extraction of data following a causality framework that provides a structure for the consideration of heterogeneous sources of evidence and a large set of review questions. Automation of the review output allows rapid updating of tables of results. We have also developed methods to automate search and deduplication of search results to make the transition to a living systematic review that will allow continual updating of results. The main limitation of the classic systematic review of such a complex topic is the high workload and time required to maintain it. Another limitation, resulting from the large number of review questions, is the time taken to resolve inter-reviewer differences in interpretation of eligibility criteria. This could have resulted in subjectivity over decisions about inclusion in the review. Although a second reviewer checked all extractions, changes in the review team could introduce inconsistency. As in the baseline review, we used case definitions as authors described them in individual publications. This potential source of information bias is likely to decrease over time as standardised case definitions and protocols are adopted115. As in the previous version, we did not systematically apply quality assessment tools to individual studies. Because much of the technical infrastructure was built as the evidence emerged, output was delayed. As much of the LSR methodology was novel, it took time to find a balance between speed and efficiency.\n\nStrengths and weaknesses in relation to other publications. Our systematic review differs from most standard reviews because of the number of questions within the dimensions of the causality framework and the number of outcomes. Other recent examples of living systematic reviews only distinguish between two study types (RCT and non-RCT)116 and are guided by only a small set of review questions117,118. Our review conclusion, confirming evidence for a causal association between ZIKV and GBS differs from that of a review119 of the findings of four case reports104,120–122 and one case-control study123. The authors found insufficient evidence to confirm the presence of an acute motor axonal neuropathy variant of GBS. They did not, however, suggest an alternative explanation for the increase in incidence of GBS in the countries that experienced ZIKV outbreaks. The two versions of our review included 64 publications about ZIKV and GBS across ten dimensions of causality.\n\nMeaning of the study: possible mechanisms and implications for basic researchers, clinicians or policymakers. The conclusions on the causal relation between ZIKV and adverse congenital outcomes and ZIKV and GBS did not change with this update. We found insufficient evidence about the association between ZIKV and ITP to state with certainty that there is a causal association. The total volume of evidence about the association between ZIKV and GBS is less than for the association with adverse congenital outcomes. There is, in particular a lack of published research to elucidate biological mechanisms for direct neuronal or autoimmune damage in GBS124. The descriptive data about the numbers and types of different studies over time illustrates how evidence about a new, or re-emerging, infection emerges over time. The evidence from many regions that were affected by the ZIKV outbreak remains limited to anecdotal evidence of adverse outcomes, in the form of case reports or case series. The slowing of ZIKV transmission in 2017 means that fewer people are being affected by ZIKV and its complications and fewer people are being enrolled into prospective studies. Further progress in epidemiological research will rely more heavily on research consortia who are contributing to joint analyses of data from existing studies.\n\nUnanswered questions and future research. As the volume and complexity of the evidence in different causality dimensions accumulates, the need for expert input and interpretation of the findings of this systematic review increases. The focus of research on ZIKV and causal associations with different types of adverse outcomes is also changing. For congenital abnormalities resulting from ZIKV vertical transmission, epidemiological research should examine CZS in comparative studies, quantify the strength of association with ZIKV, clarify associations with gestational age, symptomatology and viral load and further investigate potential co-factors such as previous dengue infection and flavivirus vaccination. WHO standardised study protocols provide suggestions for exclusion of alternative explanations and exploration of co-factors (Harmonization of ZIKV Research Protocols). For GBS, epidemiological studies are needed to quantify the association with ZIKV more precisely, but also to determine whether there are distinct phenotypes resulting from autoimmune mechanisms or direct neuronal involvement. For ITP, additional evidence across all causality dimensions is needed.\n\nPlanned updates of a living systematic review. Living systematic review methodology and techniques will continue to develop. Since a chain is only as strong as its weakest link, any processing step has the potential to slow down a living systematic review. Clearly defined protocols that define update frequencies and throughput speed of different actors in the publishing process are vital. The next update of the systematic reviews will use living systematic review methods to assess the evidence for 2017 and early 2018 (update 2, Figure 2). The review will, for the first time, separate evidence from epidemiological study designs from in vitro and in vivo laboratory studies. We will narrow down the inclusion criteria based on study type. Epidemiological evidence will address the causality dimensions ‘strength of association’, ‘dose-response’, ‘specificity’ and ‘consistency’. Several co-factors might play a role in the strength of association. Thus, we will continue to collect information on previous dengue virus infection, yellow fever vaccination status, socioeconomic status, gestational age and others factors that might play a role in the severity of the outcome. We will amend the protocol with a more focused search strategy and inclusion criteria (Supplementary File 3).\n\nSystematic reviews of questions addressed by laboratory studies are less frequent than those addressing epidemiological research questions. There is still need to update understanding of the causality dimensions ‘biological plausibility’ and ‘animal experiments’, particularly to increase our understanding of biological pathways for ZIKV effects on the peripheral nervous system and the immune system. We encourage and welcome collaboration from scientists with expertise in these fields to update systematic reviews for these causality dimensions.\n\nConclusion. This systematic review confirms previous conclusions that ZIKV is a cause of congenital abnormalities, including microcephaly and is a trigger of GBS. Evidence suggests an association with idiopathic thrombocytopaenia purpura but is not conclusive. The transition to living systematic review techniques and methodology provides a proof of concept for the use of these methods to synthesise evidence about an emerging pathogen such as ZIKV, ultimately leading to integration in the whole public health information cycle125. With the infrastructure for living systematic review methods and open source access to the software and outputs, we aim to enhance outbreak preparedness and the study of emerging and re-emerging pathogens.\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\nMJC received salary support from the Swiss National Science Foundation (project grants 320030_170069 and 320030_176233). DEG, YW and MR received salary support from the Swiss National Science Foundation (project grant 320030_170069).\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 thank the members of the WHO Zika Causality Working Group for their input on the conceptualisation of the causality framework and Anina Häfliger for her assistance on screening publications.\n\n\nSupplementary material\n\nSupplementary Table 1 – Bradford Hill’s “viewpoints” of causation.\n\nClick here to access the data.\n\nSupplementary Table 2 – Evidence table adverse congenital outcomes update 1.\n\nClick here to access the data.\n\nSupplementary Table 3 – Evidence table GBS/ITP update 1.\n\nClick here to access the data.\n\nSupplementary File 1 – PRISMA Checklist.\n\nClick here to access the data.\n\nSupplementary File 2 – LSR automation methodology.\n\nClick here to access the data.\n\nSupplementary File 3 – Search strategy update 2.\n\nClick here to access the data.\n\n\nReferences\n\nBroutet N, Krauer F, Riesen M, et al.: Zika Virus as a Cause of Neurologic Disorders. 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PubMed Abstract | Publisher Full Text\n\nAkl EA, Kahale LA, Hakoum MB, et al.: Parenteral anticoagulation in ambulatory patients with cancer. Cochrane Database Syst Rev. 2017; 9: Cd006652. PubMed Abstract | Publisher Full Text\n\nSpurling GK, Del Mar CB, Dooley L, et al.: Delayed antibiotic prescriptions for respiratory infections. Cochrane Database Syst Rev. 2017; 9: Cd004417. PubMed Abstract | Publisher Full Text\n\nLeis AA, Stokic DS: Zika Virus and Guillain-Barre Syndrome: Is There Sufficient Evidence for Causality? Front Neurol. 2016; 7(1): 170. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOehler E, Watrin L, Larre P, et al.: Zika virus infection complicated by Guillain-Barre syndrome--case report, French Polynesia, December 2013. Euro Surveill. 2014; 19(9): pii: 20720. PubMed Abstract | Publisher Full Text\n\nRozé B, Najioullah F, Fergé JL, et al.: Zika virus detection in urine from patients with Guillain-Barré syndrome on Martinique, January 2016. Euro Surveill. 2016; 21(9): 30154. PubMed Abstract | Publisher Full Text\n\nThomas DL, Sharp TM, Torres J, et al.: Local Transmission of Zika Virus--Puerto Rico, November 23, 2015-January 28, 2016. MMWR Morb Mortal Wkly Rep. 2016; 65(6): 154–8. PubMed Abstract | Publisher Full Text\n\nCao-Lormeau VM, Blake A, Mons S, et al.: Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet. 2016; 387(10027): 1531–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorrison TE, Diamond MS: Animal Models of Zika Virus Infection, Pathogenesis, and Immunity. J Virol. 2017; 91(8): pii: e00009-17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAkl EA, Meerpohl JJ, Elliott J, et al.: Living systematic reviews: 4. Living guideline recommendations. J Clin Epidemiol. 2017; 91: 47–53. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "33196",
"date": "04 May 2018",
"name": "Katrina J. Sullivan",
"expertise": [
"Reviewer Expertise Systematic reviews",
"living systematic reviews"
],
"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 systematic review assessed the causality of Zika virus in the development of congenital brain abnormalities and Guillain-Barre syndrome. To accomplish this, the 10 dimensions of casual associations were reviewed, and evidence obtained for each dimension were narratively reviewed. With the addition of 101 new studies, the authors were able to conclusively establish the causal association between Zika and adverse congenital outcomes. However, not all dimensions could be assessed concerning Guillain-Barre syndrome, even with the additional of new studies within this update. As a result, there is limited understanding within this field of research for Zika virus.\nThank you for allowing me to review this article. Overall it was well written, and did a good job addressing all aspects of it's complex objective. Please find below my comments:\nFor the methods I would have liked to see more information. This review should be able to stand on it's own without the reader having to find the baseline review to find important methodological information:\nI would have liked to see inclusion/exclusion criteria for the study (something that could easily be in a figure or table to save room). Please include your search strategy as a supplementary file (just Embase would be sufficient). It sounds like a very simple search the way you've described it in the methods. Also would have liked more information about who designed the search (e.g. information specialist, librarian, the researchers) and if it was peer reviewed using PRESS. Finally, in the \"analogy\" section of the results you indicate this wasn't reviewed in the baseline review as the search strategy didn't include terms for it. This indicates to me that you must have updated your search between the baseline review and the update, but you don't say this in your methods. There is no assessment for methodological quality, which you do address in your limitations. I think this is an important step when you're including different study designs (although can also be challenging for the same reason). Probably not appropriate to give the same weight to a case report as a cross-sectional study, or an animal experiment and an epidemiological study.\n\nGeneral:\nI find Table 1 a bit difficult to read, maybe because instinctively I'd expect the column for \"U1, N\" to be the total number of studies in the update, rather than just the new studies added with this update. This only becomes obvious when you look at rows like \"case series\" and you see the U1 has less studies than BR. If you keep this structure, it would be nice to add another column that has total N (as it was the total N that you're drawing conclusion from, the BR and U1 don't stand alone).\n\nLiving Systematic Review:\n\nDo the authors provide a clear rationale for the living systematic review? YES, it meets all criteria for doing a living systematic review (priority question for decision making, important level of uncertainty in the evidence, emerging evidence that will likely impact conclusions) Do the authors clearly state how this will be maintained as a living systematic review? Partly - While they detail the process of U1-U3, the time for search, between updates, etc. changes with each update (as they anticipate the technology getting better). Living systematic reviews require explicit decisions on how often searching, data extraction, analysis, etc. will be performed.\n\nI feel this review is premature in saying that it is transitioned to a living systematic review. The intention of the living systematic review is to provide the reader with up to date evidence so the lag between the search and publication is minimize. The authors even define a living systematic review as \"a systematic review that is continually updated, incorporating relevant new evidence as it becomes available\". The search for this study ended in January 2017, and technically is out of date even for a regular systematic review (which we try to publish within a year). The authors say that they plan to update the review twice per year with formal peer review updates, but if that was the case then another 2 reviews should have been done since this date. Additionally, they authors indicate that only module 1 and part of module 3 of their living systematic review automation were in place by December 2017, meaning this wasn't used for this update.\nFigure 2 already looks to be out of date - in it they propose that U2 would be published by Feb 2018. If they were able to accomplish this then U2 might have been more appropriate to call a living systematic review (where search looks to be from Jan to Dec 2017, and publication is Feb 2018).\n\nI think it would have been more appropriate to publish this as an update of the systematic review, publish a protocol for how they were going to transition to a living systematic review (where Figures 1 and 2 would be included), and then treat U2 as their 'baseline\" living systematic review, at which point the \"living\" aspect of the review is turned on and updates are given every 6 months (as they proposed).\n\nDo the authors provide a clear rationale for the Living Systematic Review? Yes\nDo the authors clearly state how this will be maintained as a Living Systematic Review? Partly\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? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
},
{
"id": "34944",
"date": "03 Jul 2018",
"name": "Hugh J. Willison",
"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 systematic review looking at the strength of evidence supporting the link between Zika virus (ZIKV) infection and neurological complications. The intention is to create a living review that is constantly updated as new publications arise, allowing researchers access to a data resource that keeps pace with the rapidly advancing evidence in the field. This is an extremely important initiative if it can provide truly up to date (i.e. as fast as a pubmed or similar search) and appropriately sifted information for those wishing to conduct spot checks on the current status of neuro-ZIKV.\nFrom an aspirational perspective I support it strongly, but does it work in practice? Setting aside many complex methodological considerations that this article raises, how useful is such a resource and who is it there to serve? We would all welcome tailor-made and up-to-date information at our fingertips to help navigate the mountain and minefield of information on ZIKV. I suspect that whilst this review will be widely consulted by those interested in neuro-ZIKV, from policy-makers to researchers, we will still refer back to primary publication for detailed up-to-date information in our specialist areas. At least it will provide a generic overview that facilitates more in depth access to and analysis of the primary data.\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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
},
{
"id": "31780",
"date": "10 Dec 2018",
"name": "Alfonso J. Rodriguez-Morales",
"expertise": [
"Reviewer Expertise Arboviral 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\nCertainly, a systematic review (SR) on a such highly relevant topic, as Zika association with congenital brain abnormalities and Guillain-Barré syndrome. However, I should suggest first, to include other Zika congenital associated abnormalities, as we have the congenital Zika syndrome (CZS) but also extra-Central Nervous System (CNS) abnormalities.\nThe idea to have a living SR is excellent, however very compromising. Right now, this first version, requires urgently to be updated. As the date of update was till January 2017, as a first update.\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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-196
|
https://f1000research.com/articles/6-1675/v1
|
11 Sep 17
|
{
"type": "Opinion Article",
"title": "Thermodynamic projection of the antibody interaction network: The fountain energy landscape of molecular interaction systems",
"authors": [
"József Prechl"
],
"abstract": "The adaptive humoral immune system of vertebrates functions by evolving a huge repertoire of binding proteins, which target potentially all molecules that come into contact with developing B cells. The key to endowing these binders with immunological activity is the adjustment of antibody structure and affinity against molecular targets. As a result, antibodies with a wide range of affinities and specificities evolve during the lifetime of an individual. I recently developed a quantitative model for the description of antibody homeostasis and suggested that a quantitative network can describe the dynamic antibody-antigen interaction space. Here, I project this molecular interaction space onto an energy landscape defined by conformational entropy and free energy of binding. I introduce the concept of binding fountain energy landscape, which allows the thermodynamic representation of binding events and paths of multiple interactions. I further show that the hypersurface of the binding fountain corresponds to the antibody-antigen interaction network. I propose that thymus independent and thymus dependent antibody responses show distinct patterns of changes in the energy landscape. Overall, the fountain energy landscape concept of molecular interactions allows a systems biological, thermodynamic perception and description of the functioning of the clonal humoral immune system.",
"keywords": [
"antibody",
"network",
"thermodynamics",
"binding",
"entropy",
"energy landscape"
],
"content": "\n\nBlood is a massive and critically important extracellular space of multicellular organisms. It is a fluid tissue with cellular, macro- and small molecular components that perfuses the whole multicellular organism, being in direct contact with vascular endothelial cells and blood cells. Its components are potentially derived from any cell of the organism via secretion and leakage (Anderson & Anderson, 2002). Such a hugely diverse molecular pool needs to be regulated with respect to the quality and quantity of its components. One of the mechanisms of regulation is the generation of antibodies by the humoral adaptive immune system (Prechl, 2017a; Prechl 2017b). Considering the diversity of antibodies and the diversity of molecular targets, the interaction landscape of the humoral immune system is presumably the most diverse in an organism. In this opinion article, I approach antibody homeostasis from the thermodynamic point of view, depicting antibody-antigen interactions in a novel energy landscape model. The currently used funnel energy landscape model is suitable for the description of folding and binding of one or a few molecules, but it would require landscapes of intractable sizes to depict a whole system, like adaptive immunity. I introduce the fountain energy landscape, a projection of the multidimensional binding landscape of antibodies to the dimensions of entropic penalty and energy of molecular interactions, to accommodate the vast range of interactions of antibodies.\n\n\nEnergy landscape and antibody binding\n\nMolecular interactions can be described by examining structural, kinetic and thermodynamic properties of the binding. Structural approaches aim to define the relative spatial positions of the constituting atoms of the interacting partners in the bound and unbound forms of a molecule. The advantage of the structural approach is the high resolution visual rendering of molecular structure that helps human perception. Systematic analysis of protein structures gives insight into the evolution of protein complexes and the dynamics of assembly and disassembly (Marsh & Teichmann, 2015). Structural information can reveal networks of protein interactions (Kiel et al., 2008). Kinetic studies follow temporal changes of association and dissociation of interacting partners. These observations are easily applicable to a simple system with a few components only, but it is difficult to describe complex systems and crowded molecular environments (Schreiber et al., 2009; Zheng & Wang, 2015). Thermodynamics examines the changes in free energy that accompany a binding event; providing statistical descriptions of enthalpic and entropic components of the interaction. Energy landscape theory resolves some shortcomings and integrates these approaches by assuming the presence of many different conformations that converge to thermodynamically stable forms -- the route taken to obtain this conformation dictating the kinetics of the events (Bryngelson et al., 1995). The intramolecular interactions of proteins lead to the emergence of the functional protein conformation, a process called folding. The energy landscape of folding is assumed to be funnel shaped, the stable form of the protein being at the bottom of the funnel with the lowest free energy state (Finkelstein et al., 2017; Wolynes, 2015).\n\nThe process of folding is obviously strongly dependent not only on general physical parameters, such as temperature and pressure, but on the quality and quantity of molecules present in the system. Water is the solvent of life and interactions with water molecules (Fogarty & Laage, 2014) are of key importance in all molecular interactions associated with life. The concentration of hydrogen ions (pH), cations and anions and small molecules modulate interactions. Macromolecules influence interactions not only by taking part in the interactions, but also by the excluded volume effect, restricting diffusional freedom (Zhou et al., 2008). A detailed definition of the binding environment is therefore indispensable for a realistic depiction of the binding energy landscape. Defining the antibody binding landscape in blood would therefore at least require a complete list of all constituents of blood, better involving abundance of each molecule.\n\nAntibodies are globular glycoproteins secreted into the blood and other biological fluids by plasma cells (Nutt et al., 2015). Antibodies are actually a family of oligomeric proteins, with distinct constant regions that qualify them into classes and subclasses, and with distinct variable domains that determine their binding specificity (Schroeder & Cavacini, 2010). While most of us think of antibodies as molecules with a well-defined specificity, in fact the majority of the circulating antibodies (especially of the IgM class) is not monospecific (specific to one target), but rather poly-specific and cross-reactive (Kaveri et al., 2012; Seigneurin et al., 1988). Any comprehensive systems approach to describe antibody function therefore must account for the presence of both highly specific and poly-specific antibodies. Our quantitative model of antibody homeostasis accordingly attempts to provide a unified framework for the complete humoral adaptive immune system (Prechl, 2017b). Antibodies are secreted by plasmablasts and plasmacells, descendants of B cells that had been stimulated by antigen. B cells are thus raised in an antigenic environment, the function of the immune system being the selection and propagation of B cells, which can respond to the antigenic environment. The essence of humoral immunity is therefore the definition and control of this antigenic environment by regulating molecular interactions. Thermodynamically this translates to the generation of a binding energy landscape suitable for maintaining molecular integrity of the host organism.\n\n\nThe binding fountain energy landscape\n\nThe funnel energy landscape is a theoretical approach used for the depiction of conformational entropy and free energy levels of one particular molecule (Bryngelson et al., 1995). Besides the description of intramolecular binding (folding) it can also be applied for the interpretation of homo- or hetero-specific binding, such as aggregation or ligand binding (Zheng & Wang, 2015). If we tried to describe antibody binding by the binding funnel energy landscape, we would face two interconnected problems, one deriving from antibody heterogeneity and the other from target heterogeneity. Antibody variable domains constitute the most diverse repertoire of all the proteins present in the organism, estimates being in the range of 109–1011 different primary structures at any particular time of sampling, the hard upper limit being the number of B cells in a human body, around 1012 cells (Bianconi et al., 2013). Even if the tertiary structures show orders of magnitude of lower diversity, we still face an immense variability. On the other side, poly-specific antibodies bind to a multitude of targets, with limits to the number of known targets being posited only by experimentation. A combination of these two factors implies that the binding funnel approach would not allow a clearly comprehensible yet thorough description of antibody-antigen binding. To resolve this issue, here I develop the concept of a binding fountain energy landscape model.\n\nFree energy decrease associated with binding can be resolved into components that act against or act favorably for binding. The loss of conformational entropy, a.k.a. entropic penalty, acts against binding while binding energies (enthalpic component), hydrophobic effect (conformational entropy of water molecules), contribute positively. The net difference between these events determines binding energy and protein stability (Figure 1). Conformational entropy loss of the antibody molecule thereby sets a minimum energy level that needs to be exceeded for any binding event to be stable. First, let us virtually collect all antibody binding events taking place in our system under examination, blood, and sort these events according to the entropic penalty of binding. For the sake of simplicity let us only consider entropic penalty of the variable domains of the antibodies. Second, let us plot free energy changes against conformational entropy. Since entropic penalty sets a minimum, all stable binding events should appear below the theoretical line, representing a gradually increasing entropic penalty (Figure 1). We can also set arbitrary limits for the free energy decrease, as the range of equilibrium constants for reversible antibody binding are known (Figure 1B) and we can obtain ΔG from Keq by the equation:\n\nΔG = -RT ln Keq\n\nwhere R is the universal gas constant and T is the thermodynamic temperature. The resulting plot will show the distribution of binding energies against conformational entropy loss. This latter entity is itself associated with the number of atoms at the binding interface and the buried surface area (Marillet et al., 2017). Experimental evidence suggests that reversible binding is characterized by a range of energies, limits observed both for maximal and minimal values, which are dependent on the magnitude of the interacting surface, whether characterized by the number of atoms or by buried surface area (Brooijmans et al., 2002; Smith et al., 2012).\n\n(A) By anchoring the axis of free energy change at zero entropic penalty we normalize binding events. Any antibody entering the binding landscape appears at the top can search for binding partners with favorable thermodynamic characteristics. (B) These binding events take place below the stability barrier, the line representing equality of entropic penalty and energies favoring binding. Theoretically we can collect and position all antibody interactions in this energy landscape.\n\nA binding funnel energy landscape focuses on the one (or few) native conformation(s) that can be reached via various conformational routes, as represented by a hypersurface of conformation, entropy and energy. The contribution of binding energies and conformational changes to free energy is described by the equation:\n\nΔG =ΔH -TΔS\n\nwhere ΔH is enthalpy change and ΔS is entropy change.\n\nIf instead of focusing on the one or few native conformations we would like to focus on the multitude of different conformational routes taken by several different antibodies while binding to different targets, we need a different kind of representation. To this end, we assume that all native unbound antibodies enter our landscape at the top of the energy landscape plot, where their starting conformational entropic penalty represents that associated with folding. In order to get a better resolution of the binding landscape let us spin our two-dimensional plot around the energy axis at the maximal entropy to obtain a conical hypersurface in three dimensions (Figure 2). Native unbound antibody molecules entering our landscape will move down along a path, while interacting with their targets with an increasing binding energy. This gradual increase in ΔG is accompanied by an increasing involvement of the binding site, called antibody paratope. All stable binding events take place under the theoretical conical surface generated from the stability barrier line. A binding path ends when the antibody finds its lowest state of energy, corresponding to binding to a target with the highest affinity. Where this point is located depends both on the antibody and the nature of its target (e.g. size, chemical characteristics). The hypersurface of conformations in the space of conformational entropy and free energy generated by this approach we shall call a binding fountain energy landscape.\n\n(A) By spinning the former representation in Figure 1 around the anchored axis we obtain a quasi-conical surface. This surface encloses all stable binding events of an antibody molecule and is suitable for displaying a binding path. (B) Thermodynamically defined subsets of binding events in the binding fountain can be obtained by looking at events in the isenergetic or isentropic rim.\n\nWhile the conical surface enclosing stable binding events is a theoretical surface, we can obtain descriptors of real binding events by looking at subsets of events of the interaction space. By cutting the binding fountain horizontally at a given ΔG value we obtain the isenergetic rim (Figure 2B). The isenergetic rim is the collection of binding events with identical ΔG and a range of corresponding ΔS. Thus, its ΔS distribution shows the range of entropic penalties that give rise to binding at the given ΔG in our system of study. By cutting the skirt of the cone at a given ΔS value, we obtain the isentropic rim (Figure 2B). The isentropic rim is the collection of binding events with identical ΔS and a range of corresponding ΔG values. Thus, its ΔG distribution shows the range of free energy changes and corresponding affinity values that give rise to binding at the given ΔS in our system of study. It shows how enthalpy and hydrophobic effects exceed entropic penalty. Please note that as these lines are derived from a hypersurface the lines are theoretical hyperlines themselves, comprising high-dimensional data that cannot be properly visualized in a simple 2D plot.\n\n\nProjections of the fountain energy landscape\n\nWe have so far worked out an energy landscape interpretation tool, which helps map all the binding events that occur in a molecularly complex environment, such as blood. We assumed that antibodies secreted into the blood gain their native unbound conformations then engage in binding events of various energies until they reach their specific target. The path leading to thermodynamic equilibrium can be rugged, caused by less specific contacts, or smooth, with few intermediate binding states (Figure 3A). It is important to note, however, that blood is the most heterogenous biological fluid, comprising potentially all molecules found in the organism (Anderson & Anderson, 2002). Besides a huge number of secreted molecules, any leakage from tissues, debris of cell death and foreign molecules may be present in blood. This vast molecular diversity generates a binding site diversity that we may assume to approach a randomized structural space, representing all potential variants of an antibody binding site covering up to 3000 Å2 (Marillet et al., 2017). Such a diverse binding space should approach a power law distribution of binding partners, with decay of partners as we increase binding energy or affinity (Figure 3B) (Zheng & Wang, 2015). A rugged start is therefore expected for all antibodies, with the path smoothing out depending on the paratope properties and the content of the binding landscape. As we approach higher energy and higher entropy, loss regions the epitope “sharpens”, as Irun Cohen termed (Cohen & Young, 1991) the gradually increasing affinity of antibodies (Figure 3C). This sharpening involves both a gradually increasing buried surface area and better fitting surfaces and various combinations of these components. It is also apparent that sectors of conformational entropy contain structurally related binding sites, since sharpening reveals more details of epitopes that appear identical at lower resolution (Figure 3D), later maturing into distinct conformational entities. This relationship also reflects the clonal relationship of antibodies going through affinity maturation, gaining sharper but constrained vision of targets by improving their fit (Kang et al., 2015).\n\n(A) Sequential binding of a given antibody appears as a path with more or less rugged track. (B) The frequency of interactions decreases by power law decay as we approach high energy binding with high entropic penalty. (C) The levels of contact accounting for the entropic penalty increase by improved fit with stronger binding forces and by increased buried interface area. (D) Conformations of binding surfaces share common origin with identical structural motifs closer to the “source” of the fountain, the region of low energy interactions.\n\n\nInterpretation of antibody function as a system of regulated binding landscape\n\nThe binding landscape is the set of all potential interactions in a given fluid with given constituents, each interaction being positioned according to the entropic penalty, conformation and free energy decrease. In the binding fountain representation we can trace the fate of a particular antibody in time as a binding path (Figure 4A) or display several different antibodies at an imaginary thermodynamic equilibrium (Figure 4B). Owing to the fact that blood is a highly heterogenous fluid with a vast diversity of potential binding sites, the frequency of low energy interactions is very high. At the tip of the fountain, antibodies are “surfing” along the ripples of low affinity interactions. Moving down the surface they encounter interaction partners with gradually improved fit, spending more and more time in an interaction, until the target with best fit, that is highest free energy decrease and largest entropic penalty, is found (Figure 4A).\n\n(A) An antibody entering the binding landscape engages in serial interactions with increasing energy, taking the molecule down a binding path. (B) At an imaginary equilibrium natural antibodies (blue beads) and affinity matured thymus dependent antibodies (red beads) fill the holes of binding, arranged according to their conformation, entropic penalty and free energy level. The distance between any two binding events can be expressed as ΔΔG, which represents the cross-reactivity of the two antibodies concerned. (C) We can further project these events into an interaction space where a network is formed based on distance and binding capacity.\n\nInteractions in the blood cannot reach thermodynamic equilibrium; molecules are continuously entering and leaving this compartment. On the other hand, due to the constant turbulent mixing, the distribution of molecules is constantly approaching homogeneity. Thus, we may display antibodies at an imaginary equilibrium where their position reflects their potential energy minimum in the system. This is where actually target antigen-bound antibody molecules are accumulating (Figure 4B). Registering the position of all the copies of a given antibody species should show a distribution of bound forms determined not only by ΔG, but also by the availability of the target molecules, which is antigen concentration [Ag]. The disappearance of the target ([Ag]≈ 0 M) will lead to the disappearance of the low energy position in the landscape. As a consequence, the antibody will accumulate in the interaction with the next available energy level, albeit the ratio of bound to free form will be lower as dictated by the higher KD value. Alternatively, the antibody can search the neighboring conformational space along the isenergetic rim for a binding site with similar ΔG. High concentrations of the target ([Ag]>>KD) will deplete antibody resulting in the potential overflow of related antibodies from the neighboring conformational space. The distance ΔΔG between any two interactions has three components: a free energy component, a conformational component and an entropic penalty component. These components are perceptible from the side view, top view and both views of the binding fountain, respectively (Figure 4B).\n\nAs suggested above, besides the presence of targets with a given ΔG, the actual concentrations of both Ab and Ag determine the frequency of their interactions and the development of the imaginary equilibrium. To appreciate these factors we can project the interactions of a binding fountain into a space where the distance of the interactions is defined by ΔΔG and the availability of antibody is expressed as the ratio of free antibody to the dissociation constant ([Ab]/KD). This latter value can be visualized as the radius of the circle representing the interaction (Figure 4C). Please note that this value corresponds to [AbAg]/[Ag], the ratio of bound and free antigen concentrations. This visualization corresponds to the network representation of antibody-antigen interactions as I recently described (Prechl, 2017b).\n\n\nThe immune response as a regulated binding landscape\n\nThe adaptive immune system responds to an antigenic stimulus by the production of antibodies reacting with the eliciting antigen. In our binding landscape an antigenic stimulus appears as an impression on the hypersurface representing antibody interactions, the position of the impression being determined by both the conformation of antigen and the conformation of fitting antibodies. The fact that an antigen can stimulate the humoral immune system implies that secreted antibodies that could efficiently bind to the antigen are not present. The antigen therefore binds to the membrane antibodies (B-cell receptors, BCR) of specific B cells (Figure 5). If BCR engagement reaches a threshold the affected B cells proliferate, differentiate and secrete antibodies (Prechl, 2017a). Depending on the nature of the antigen, the route of entry into the host, the presence of costimulatory signals, the ensuing response can proceed basically in two distinct ways. A thymus independent (TI) response will result in the generation of antibodies with binding properties identical to the parental B cell, since there is no affinity maturation. The structure of the binding site does not change, conformation, entropic penalty and ΔG of binding will be identical to the original interaction (Figure 5A). These interactions take place in regions with moderate conformational entropy loss and high interaction frequency, meaning that of the huge repertoire of BCRs several will respond. The response appears as a standing wave, the appearance of antigen showing as the development of the impression, the response of antibody secretion as the disappearance of the impression as free antigen is replaced by bound antigen and immune complexes are removed. This kind of response seems suited for keeping concentrations of target molecules stable. We can think of the response as a closely knit elastic net that regains its original shape after applying pressure to a point (Figure 5A). Thymus dependent (TD) responses will involve the affinity maturation of the antibody binding site, the sequential generation of antibodies with increasing affinity. As the binding site matures, the entropic penalty and ΔG increase. The interactions will take place at different positions of the binding landscape (Figure 5B). The response appears as a propagating wave sweeping down the slope of the binding fountain energy landscape. This wave is taking along the antigen, resulting in the efficient elimination of antigenic molecules.\n\n(A) Thymus-independent responses are characterized by antibodies of lower affinity. A closely knit network of antibody forming cells respond as an elastic net. (B) Thymus-dependent responses are characterized by the development of antibodies with increasing affinity. This corresponds to a wave of interactions sweeping down the slope of the fountain.\n\nIt is important to note the relative identity of binding partners in this landscape: an antibody can bind to antigens but can also be the target of another antibody. The unique binding site of an antibody, the paratope that determines idiotype (identity as a binder), is itself part of the binding landscape. This can be especially important for antibodies with high intrinsic specificity rate (Zheng & Wang, 2015) that are eager to bind and reach their conformation with lowest energy level. I suggest that in the absence of antigen these high affinity binders could be refrained from non-specific binding by engaging their binding sites in lower affinity interactions.\n\n\nSummary\n\nBlood carries potentially all the molecules expressed in the host, along with those originating from the environment. To ensure that all these molecules find their intended binding partners a regulated binding landscape evolved: the clonal immune system. The clonal humoral immune system generates a regulated binding landscape by constantly sampling the molecular environment via a huge repertoire of B-cell receptors and by the generation of antibodies with a wide range of specificities and affinities. To allow the thermodynamic representation of this multitude of interactions, I show here that this landscape can be visualized as a binding fountain, in an analogy with the folding funnel energy landscape. The binding fountain landscape is an anchored conformation landscape with the conformational entropic penalty of binding anchoring the axis of free energy. Binding sites appear as impressions of a hypersurface, which represents thermodynamically favorable binding events with negative ΔG values. This landscape can be further projected into a multidimensional space of the antibody-antigen interaction network. This systemic perception and interpretation of antibody function is expected to help reveal how the immune system actually functions as a whole, a thermodynamic network of interactions, taking us closer to the systems level understanding of adaptive humoral immunity.",
"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\nAnderson NL, Anderson NG: The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics. 2002; 1(11): 845–867. PubMed Abstract | Publisher Full Text\n\nBianconi E, Piovesan A, Facchin F, et al.: An estimation of the number of cells in the human body. Ann Hum Biol. 2013; 40(6): 463–471. PubMed Abstract | Publisher Full Text\n\nBrooijmans N, Sharp KA, Kuntz ID: Stability of macromolecular complexes. Proteins. 2002; 48(4): 645–653. PubMed Abstract | Publisher Full Text\n\nBryngelson JD, Onuchic JN, Socci ND, et al.: Funnels, pathways, and the energy landscape of protein folding: a synthesis. Proteins. 1995; 21(3): 167–195. PubMed Abstract | Publisher Full Text\n\nCohen IR, Young DB: Autoimmunity, microbial immunity and the immunological homunculus. Immunol Today. 1991; 12(4): 105–110. PubMed Abstract | Publisher Full Text\n\nFinkelstein AV, Badretdin AJ, Galzitskaya OV, et al.: There and back again: Two views on the protein folding puzzle. Phys Life Rev. 2017; 21: 56–71. PubMed Abstract | Publisher Full Text\n\nFogarty AC, Laage D: Water dynamics in protein hydration shells: the molecular origins of the dynamical perturbation. J Phys Chem B. 2014; 118(25): 7715–7729. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKang M, Eisen TJ, Eisen EA, et al.: Affinity Inequality among Serum Antibodies That Originate in Lymphoid Germinal Centers. PLoS One. 2015; 10(10): e0139222. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaveri SV, Silverman GJ, Bayry J: Natural IgM in immune equilibrium and harnessing their therapeutic potential. J Immunol. 2012; 188(3): 939–945. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKiel C, Beltrao P, Serrano L: Analyzing protein interaction networks using structural information. Annu Rev Biochem. 2008; 77: 415–441. PubMed Abstract | Publisher Full Text\n\nMarillet S, Lefranc MP, Boudinot P, et al.: Novel Structural Parameters of Ig-Ag Complexes Yield a Quantitative Description of Interaction Specificity and Binding Affinity. Front Immunol. 2017; 8: 34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarsh JA, Teichmann SA: Structure, dynamics, assembly, and evolution of protein complexes. Annu Rev Biochem. 2015; 84: 551–575. PubMed Abstract | Publisher Full Text\n\nNutt SL, Hodgkin PD, Tarlinton DM, et al.: The generation of antibody-secreting plasma cells. Nat Rev Immunol. 2015; 15(3): 160–171. PubMed Abstract | Publisher Full Text\n\nPrechl J: A generalized quantitative antibody homeostasis model: regulation of B-cell development by BCR saturation and novel insights into bone marrow function. Clin Transl Immunology. 2017a; 6(2): e130. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrechl J: A generalized quantitative antibody homeostasis model: antigen saturation, natural antibodies and a quantitative antibody network. Clin Transl Immunology. 2017b; 6(2): e131. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchreiber G, Haran G, Zhou HX: Fundamental aspects of protein-protein association kinetics. Chem Rev. 2009; 109(3): 839–860. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchroeder HW Jr, Cavacini L: Structure and function of immunoglobulins. J Allergy Clin Immunol. 2010; 125(2 Suppl 2): S41–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeigneurin JM, Guilbert B, Bourgeat MJ, et al.: Polyspecific natural antibodies and autoantibodies secreted by human lymphocytes immortalized with Epstein-Barr virus. Blood. 1988; 71(3): 581–585. PubMed Abstract\n\nSmith RD, Engdahl AL, Dunbar JB Jr, et al.: Biophysical limits of protein-ligand binding. J Chem Inf Model. 2012; 52(8): 2098–2106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWolynes PG: Evolution, energy landscapes and the paradoxes of protein folding. Biochimie. 2015; 119: 218–230. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZheng X, Wang J: The universal statistical distributions of the affinity, equilibrium constants, kinetics and specificity in biomolecular recognition. PLoS Comput Biol. 2015; 11(4): e1004212. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou HX, Rivas G, Minton AP: Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences. Annu Rev Biophys. 2008; 37: 375–397. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "25884",
"date": "09 Oct 2017",
"name": "Jordan D. Dimitrov",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Dr Prechl entitled “Thermodynamic projection of the antibody interaction network: The fountain energy landscape of molecular interaction systems” proposes theoretical framework for quantitative description of antibody-antigen interaction at repertoire level. The author presented in convincing manner a novel theoretical approach, referred to as “fountain energy landscape” for description of multidimensional space of antibody interactions. The text is written in an unambiguous language and the reader easily follows the logic of the author. This work is an important contribution to theoretical immunology and may be of interest of scientists from other fields.\nClarification of certain points in the manuscript would improve the work significantly.\nOn Page 2 the author correctly stated that intramolecular interactions are affected by solvent, pH and molecular crowding and that considering these parameters as state the author is “indispensable for a realistic depiction of the binding energy landscape”. However, this Reviewer did not find later in the text how these parameters are incorporated in the model.\nOn Page 2 (paragraph 4) for the sake of logic it would be better if the description of origin of antibodies is transferred before introduction of antibody polyspecificity and cross reactivity.\nIt would be nice if author comment how the model applies for antibodies with flexible binding sites as compared to antibodies with rigid antigen binding site. The studies of Manivel et al. (PMID: 12097393 and PMID: 11114374) demonstrated that interactions of antibodies with rigid binding sites can be driven by favourable (for free energy) entropy changes. Would energy hypersurface will have a similar topology for flexible (polyspecific) and rigid (monospecific) antibodies?\nOn Page 5, first paragraph, and Page 8, first paragraph, the author stated that the best fit of antibodies to their targets results in the highest entropic penalty and that maturation of the binding site increases the entropy penalty. These statements contradicts with previous studies where it was demonstrated that entropic penalty decreases with improvement of the fit (PMID: 12097393 and PMID: 11114374).\nHow this model would account in the changes in non-equilibrium (activation) thermodynamic parameters during antigen-antibody interactions?\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": "3410",
"date": "15 Feb 2018",
"name": "József Prechl",
"role": "Author Response",
"response": "I am thankful for the reviewer for highlighting the importance of different contributions of enthalpy and entropy to different stages of the immune response, and the role flexibility and rigidity may play there. These insights helped me further elaborate the theoretical backgrounds in the revised version of the paper. The present article only aims to establish a novel model and visualization method that allows the comparative representation of more than one interaction partners. As long as we assume that all interactions are taking place in the blood we can consider key parameters such as solvent, pH and molecular crowding identical and physiological. As a next step in the development of the model it will be exciting to incorporate the effects of such parameters, as suggested by the reviewer, when they change under pathological or therapeutical conditions. I also inserted a citation that allows readers to seek additional knowledge on this subject. Paragraph 4 on Page 2 was revised according to the suggestion of the reviewer. In accordance with both of the reviewers’ remarks I revised the interpretation of contribution of the entropic penalty component to the landscape. The revised version is also in accordance with the cited publications (PMID: 12097393, 11114374) and shows that topology is different for flexible binding of primary and rigid binding of secondary immune response antibodies. These papers proved very helpful in improving the present article. The revised version of the binding landscape is in agreement with the cited papers and with general knowledge about flexibility of antibodies at different stages of the immune response. Interestingly, the revised model also identifies natural antibodies as a unique subset with special thermodynamic properties. These properties should be related to the special biology of B1 cells that display such antibodies as B-cell receptors and secrete these antibodies. In my view the function of humoral immune system is to maintain a global antibody equilibrium by the antigen specific tuning of antibody production, affinity to target and effector quality. What we observe as immunological events are the fluctuations of the system: leaving from and returning to equilibrium. Non-equilibrium conditions arising as antigenic stimuli are now described in the last section of the article, as thymus independent and thymus dependent responses, with a distinction for primary and secondary responses in the second case."
}
]
},
{
"id": "26666",
"date": "24 Oct 2017",
"name": "Jonghoon Kang",
"expertise": [
"Reviewer Expertise Thermodynamics",
"immunology",
"kinetics"
],
"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\nVisualization of a scientific idea with diagrams is often time very useful. Examples of such visualization are phase diagrams in thermodynamics and reaction coordinate diagrams in kinetics. In the present paper, the author attempted to visualize complicated networks of antibody-antigen interactions in the immune system. The visual model that the author developed is analogous to the energy landscape diagram of protein folding, which has been very successful in describing protein folding. Even though the idea of the present paper is creative and ambitious, I found several mistakes and conceptual misunderstandings in the paper.\n\nHydrophobic effect does not originate from the conformational entropy of water molecules as stated in the paper. Its origin comes from the configurational freedom of water molecules. The structure (conformation) of water molecules is highly stable, so we don’t expect it to change in biologically relevant conditions.\n\nThe author was not clear about distinction between the free energy in standard state (ΔG°) and in any arbitrary states (ΔG) throughout the paper. The equation, ΔG = -RT ln K, in the paper should be presented as ΔG° = -RT ln K.\n\nIn the explanation of the equation, ΔG = ΔH - TΔS, ΔS was attributed to conformational changes. However, in most biochemical interactions, hydrophobic effects and dissociation and/or association of salts to binding molecules are the major contributors to ΔS.\n\nFigure 1 of the paper contains a theoretical line that the author claims represents a stability barrier. Thermodynamic stability of a complex is dependent only upon the ΔG for the association or dissociation. It is not clear why and how the stability line is a linear function of ΔS, as in the figure.\n\nFigure 2 presents a three dimensional version of Figure 1. The three dimensional version was obtained by spinning Figure 1. I am not sure what the mathematical or physical implications of the spinning of the graph are.\n\nThe author employed several physical concepts in describing Figure 5 such as hypersurface, elasticity, standing waves, propagating waves, and pressure. I am not sure how those concepts are related to the antibody-antigen bindings.\nDespite several creative and insightful points made in the paper, I found that the paper needs significant revision. Finally I am not sure how the complexity in the humoral immune reaction can be visualized graphically or presented algebraically. Suppose there are m different antibodies and n different antigens. The full description of the entire possible bindings of the system will require m × n binding equations. If m = 10^10 and n = 10^3, then it will produce a system of 10^13 parallel equations. Solving such systems will be challenging even with supercomputers.\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": "3409",
"date": "15 Feb 2018",
"name": "József Prechl",
"role": "Author Response",
"response": "I thank the reviewer for identifying the weaknesses of the article. I addressed these by correcting the mistakes and by further elaborating the thermodynamic backgrounds of the concept raised in the article. Please find my point-by-point responses below. It is indeed the organization and not the structure of water molecules that contributes to hydrophobic effects. I corrected this part of the article and further extended the description of entropic penalty. The paper now discriminates the standard state and non-equilibrium states, I made the requested correction. In the revised version I describe the components of entropy changes in more detail, paying more attention to properly define protein and solvent-related changes and contributions to entropic cost of binding. The theoretical line that I claimed represented a stability barrier is actually the line representing equilibrium states. In accordance with both of the reviewers’ remarks I revised the interpretation of contribution of the entropic penalty component to the landscape. Even though it is not stated explicitly, a simple funnel landscape is basically obtained by spreading potential energy states alongside the surface of a funnel. This is similar to spinning a free energy line plot around the energy axis, with the axis going through the bottom of the funnel. Potential routes leading to the bottom are spread out as a surface, conformational states with the highest free energy are placed farthest from the bottom of the funnel and creating a plane of the isenergetic substates of the unfolded protein. While visually appealing, a 3D surface diagram cannot properly display all the configurational states and their free energy levels. Rather it can give an impression of the key routes of configurational changes. In a similar manner, visualization of the fountain landscape as a 3D surface can only provide visual information on a subset of binding events. Nevertheless, in a mathematical sense the landscape can contain all the binding events of the high dimensions originating from the configurational variability. Thus, spinning the 2D energy diagram only serves the purpose of better visualization of energetically and conformationally related events. The physical concepts are now better explained in the revised text. The concept of hypersurface originates from the funnel energy landscape theory of folding. The two distinct forms of waves are used to explain the network properties of the landscape and are now shown in the figure. I agree that computer modeling of the complete system would be prohibitively challenging computationally; comparable to N-body simulations in cosmology. I see two potential areas of application. One is the general theoretical description of the humoral immune system, where only characteristic properties, ranges and averages of various descriptors – like distributions of events in the isentropic and isenergetic rim - are required for the schematic visualization of the system. The development of the immune system, responses to various challenges, dysfunctions such as autoimmunity could be approached and visualized this way. The other application is the exact experimental description of subsets of binding events. The behavior of one or a few cross-reactive antibody species could be analysed this way, potentially shedding light on immunological phenomena related to cross-reactivity in a quantitative manner."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1675
|
https://f1000research.com/articles/6-1039/v1
|
03 Jul 17
|
{
"type": "Software Tool Article",
"title": "GAC: Gene Associations with Clinical, a web based application",
"authors": [
"Xinyan Zhang",
"Manali Rupji",
"Jeanne Kowalski",
"Xinyan Zhang",
"Manali Rupji"
],
"abstract": "We present GAC, a shiny R based tool for interactive visualization of clinical associations based on high-dimensional data. The tool provides a web-based suite to perform supervised principal component analysis (SuperPC), an approach that uses both high-dimensional data, such as gene expression, combined with clinical data to infer clinical associations. We extended the approach to address binary outcomes, in addition to continuous and time-to-event data in our package, thereby increasing the use and flexibility of SuperPC. Additionally, the tool provides an interactive visualization for summarizing results based on a forest plot for both binary and time-to-event data. In summary, the GAC suite of tools provide a one stop shop for conducting statistical analysis to identify and visualize the association between a clinical outcome of interest and high-dimensional data types, such as genomic data. Our GAC package has been implemented in R and is available via http://shinygispa.winship.emory.edu/GAC/. The developmental repository is available at https://github.com/manalirupji/GAC.",
"keywords": [
"SuperPC",
"binary outcome",
"continuous",
"time-to-event",
"forest plot"
],
"content": "Introduction\n\nHeterogeneity in terms of tumor characteristics, prognosis, and survival among cancer patients has been a persistent problem for many decades. Currently, prognosis and outcome predictions are solely based on clinical factors. However, inaccurate prognosis and prediction may result from using only these. For example, based on clinical factors only, two tumor patients may have the same prognosis, but may not respond to the same treatment as the tumors may have a completely different molecular composition1–5.\n\nOne way to improve prognosis and clinical outcome predictions in cancer is to examine such associations using a tumor’s genomic profile. To achieve this goal, Bair and Tibshirani6 proposed a supervised principal component (SuperPC) method to identify subtypes of cancer. The method uses both gene expression and clinical data to diagnose future patients. This approach was applied to several publicly available datasets, showing that it was able to accurately predict the clinical outcome of interest based on a given gene expression profile. Since its inception, SuperPC has been introduced as a powerful tool for cancer diagnosis and treatment.\n\nCurrently, the SuperPC method has been developed as an R package, ‘superpc’ but as it stands, it is unable to address the following:\n\n1) clinical association based on a binary outcome (e.g. responders versus non-responders);\n\n2) ease of use for clinicians and researchers with limited programming skills; and\n\n3) a visual summary of results.\n\nTo address these limitations, we developed GAC: Gene Association with Clinical, an interactive, GUI-based web-based application for analysis of gene associations with various clinical outcomes of interest. We developed GAC based on the R packages ‘shiny’ and ‘superpc’. Our GAC tool enables the user to perform a SuperPC analysis for three types of outcomes: time-to-event, continuous, and binary, and provides a summary of results using forest plots that may be readily exported into a file.\n\n\nMethods\n\nSuperPC is a generalization of principal component analysis, which generates a linear combination of the features or variables of interest that capture the directions of largest variation in a dataset. Instead of using the whole dataset directly, SuperPC defines a list of genes based on their association with an outcome of interest. To select the list of genes, a univariate score for each gene is calculated and those features (a.k.a., genes) whose score exceeds a threshold are retained as input into a principal component analysis, based on the retained features. For details, refer to Bair and Tibshirani6.\n\nSuperPC for time-to-event was conducted using the ‘superpc’ package in R. Depending on the sample size of the original dataset; the researcher selects what proportion of the dataset to split into training and testing. The researcher can also specify how many numbers to test to check which the optimal threshold is. The number of folds for cross validation to determine the threshold also needs to be determined. There is also an option to run the analysis randomly, or upload fold IDs to replicate an analysis that was previously carried out. The association between the time-to-event outcome and the predicted principal component may be represented in a KM plot by dichotomizing the principal component using the median (Figure 1).\n\nThe interface shows an example SuperPC for time-to-event outcome. The left panel allows the user to select the various options such as data split, the optimal threshold, number of folds and a choice of generating new fold IDs or using a pre-existing set to replicate results. The right panel includes the results of the analysis. The KM plot displays the association of the outcome with the predicted principal component by the median. In addition, the univariate analysis regression scores and tables are also available for download.\n\nSuperPC for continuous outcomes is implemented using the ‘superpc’ package in R, with the same options as time-to-event analysis. The predicted principal component is presented visually as continuous values through a scatter plot along with Pearson’s correlation (Figure 2). The predicted principal component could also be presented as binary groups (cutoff at median) through a boxplot, with a t-test applied.\n\nThe interface shows an example SuperPC for continuous outcome. Similar to Figure 1, the user can choose the appropriate settings using the left panel and view the results of the analysis to the right. The association of the continuous outcome with the predicted principal component is summarized using a scatter plot (as seen). The user can alternatively choose to summarize these results through boxplots by dichotomizing the predicted principal component based on the median. As in Figure 1, the univariate regression scores and plots are available for download in the left panel.\n\nSuperPC for binary outcome follows the same analysis workflow as the other two outcomes. The predicted principal component can be visualized as either a continuous variable through a box plot, with a t-test to summarize the statistical association (Figure 3), or as binary groups (cutoff at median) using a bar plot, with a chi-square test to summarize the statistical association between the predicted and the observed outcome.\n\nThe interface shows an example SuperPC for binary outcome. The user can opt for similar options as in the previous figures. Also, as in Figure 2, the user has a choice to display the continuous predicted principal component as a scatter plot, or divide it into binary discrete groups (using median cut-off) to represent the association through a barplot. Similar download options are available.\n\nA forest plot is a graphical display of point estimates of association widely used in meta-analysis. It has become popular for displaying the associations between clinical and genomic data. With our GAC tool, users have the option to generate a forest plot to display results (Figure 4).\n\nThe interface shows an example forest plot. The left side comprises a user menu and the right includes the result plot. Users can upload their own summarized results with hazard ratio and confidence intervals for the survival outcome, or odds ratio and confidence intervals for the binary outcome. For graphical display, the researchers could choose to input different labels, font sizes and colors.\n\nThe GAC tool is written in R and tested using version 3.3.0. The interactive plots and data tables are made available using the shiny R package (www.rstudio.com/shiny).\n\nUsing a windows 7 Enterprise SP1 PC with a 32.0 GB RAM and a 3.30 GHz Intel® Xeon® Processor E5 Family, the time-to-event and regression analysis with 45 patients from the TCGA BRCA dataset was completed in 2.54 s and 1.80 s, respectively. The binary outcome analysis using the CoMMPass data of 135 patients was completed in 65.05 s. Source code is available at https://doi.org/10.5281/zenodo.803309.\n\n\nDiscussion\n\nGAC is a suite of tools that allows the user to conduct statistical analysis to identify and visualize the association between clinical outcomes of interest and genomic data using an interactive application in R.\n\n\nData and software availability\n\nFor SuperPC time-to-event and regression analysis, we used TCGA BRCA RNASeqV2 gene expression and clinical data, downloaded from the TCGA data portal (now accessed at https://portal.gdc.cancer.gov/)7. The data included 380 differentially expressed genes when favorable (patients who did not die with at least 7 years of follow up) and unfavorable (patients who died 30 months post-treatment) outcomes from 45 patients were compared.\n\nFor SuperPC binary outcome analysis, CoMMPass IA9 RNASeq expression and clinical data was downloaded from the publicly available Multiple Myeloma Research Foundation (MMRF) database (https://research.themmrf.org/rp/download). Patient cytogenetics for outcome dichotomization was obtained from the MMRF data portal’s ‘Analysis Tools’ section (https://research.themmrf.org/rp/explore/). 135 patients with clinical, gene expression and copy number data were classified as high risk based on cytogenetics and the remaining 343 as not high risk. Among these patients, the top 1450 most variable genes were used.\n\nThe example data used in this article is available in Zenodo (https://doi.org/10.5281/zenodo.803309).\n\nLicense: GAC is available under the GNU public license (GPL-3).",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nResearch reported in this publication was supported in part by the Biostatistics and Bioinformatics Shared Resource of Winship Cancer Institute of Emory University and NIH/NCI under award number P30CA138292. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.\n\n\nReferences\n\nAlizadeh AA, Eisen MB, Davis RE, et al.: Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000; 403(6769): 503–11. PubMed Abstract | Publisher Full Text\n\nSørlie T, Perou CM, Tibshirani R, et al.: Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001; 98(19): 10869–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan 't Veer LJ, Dai H, van de Vijver MJ, et al.: Gene expression profiling predicts clinical outcome of breast cancer. Nature. 2002; 415(6871): 530–6. PubMed Abstract | Publisher Full Text\n\nvan de Vijver MJ, He YD, van't Veer LJ, et al.: A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med. 2002; 347(25): 1999–2009. PubMed Abstract | Publisher Full Text\n\nLapointe J, Li C, Higgins JP, et al.: Gene expression profiling identifies clinically relevant subtypes of prostate cancer. Proc Natl Acad Sci U S A. 2004; 101(3): 811–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBair E, Tibshirani R: Semi-supervised methods to predict patient survival from gene expression data. PLoS Biol. 2004; 2(4): E108. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNCI and NHGRI: The Cancer Genome Atlas (TCGA) Data Portal. Accessed in December 2015. Reference Source"
}
|
[
{
"id": "26010",
"date": "18 Sep 2017",
"name": "Shengjie Yang",
"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 did a nice job to develop a web tool called GAC, which is used to explore the association between clinical and genomic data. Essentially, GAC is a GUI of R package ‘superpc’. They not only provided a handy web tool but also published the source code to allow researchers to do some further expansion according to their own specific study objects. The manuscript is well written, but I have some comments:\nSince the example data is from TCGA and this work aims to facilitate clinicians and researchers with limited programming skills, it is necessary to cite some literature to tell readers how to retrieve and process the TCGA Data, such as TCGA-Assembler (Nature methods, 2014), and it would be better if such tools can be embedded into GAC.\n\nThe authors stated that ‘superpc' is unable to address the limitation about clinical association based on a binary outcome in the Introduction. But in the Methods section, it said ‘SuperPC for binary outcome follows the same analysis workflow as the other two outcomes’. It confuses me and please explain the details.\n\nI find that the calculation will be automatically triggered when changes any one option in the sidebar. That is not an user-friendly experience, especially it happens in the ’Super PC Binary Outcome’ module which can not return the result immediately. Think about that if the users want to change two default options, they have to adjust the first one, wait a few seconds and then adjust the second option, which is not efficient. So adding a ‘submit’ button to trigger the calculation is a better way.\n\nBuild a user manual page to explain the details of each function and use an example to demonstrate the usage.\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? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "3044",
"date": "26 Sep 2017",
"name": "Manali Rupji",
"role": "Author Response",
"response": "We thank the reviewer for their thoughtful comments that have led to an improved paper. Below, we respond individually to each comment. Since the example data is from TCGA and this work aims to facilitate clinicians and researchers with limited programming skills, it is necessary to cite some literature to tell readers how to retrieve and process the TCGA Data, such as TCGA-Assembler (Nature methods, 2014), and it would be better if such tools can be embedded into GAC.Various tools are available for extracting TCGA data (now available through GDC data portal). Data can be downloaded directly from the GDC data portal (https://gdc-portal.nci.nih.gov/) or by use of R packages. Some of the more recent packages include TCGA Biolinks (Colaprico et. al, 2015) which is available through Bioconductor and the TCGA Assembler (Zhu et. al, 2014) available as an R package. Users may refer to the detailed tutorials of these tools for instructions on extracting TCGA data types. The authors stated that ‘superpc' is unable to address the limitation about clinical association based on a binary outcome in the Introduction. But in the Methods section, it said ‘SuperPC for binary outcome follows the same analysis workflow as the other two outcomes’. It confuses me and please explain the details.We have included a sentence in the methods section that clarifies how the superPC tool has been extended to include analysis of binary outcome data. I find that the calculation will be automatically triggered when changes any one option in the sidebar. That is not a user-friendly experience, especially it happens in the ’Super PC Binary Outcome’ module which cannot return the result immediately. Think about that if the users want to change two default options, they have to adjust the first one, wait a few seconds and then adjust the second option, which is not efficient. So adding a ‘submit’ button to trigger the calculation is a better way.A ‘Run Analysis’ submit button is now available under each analysis option that will update results based on user-specified options to multiple parameters, as opposed the previous version which triggered a calculation after each separate change. Build a user manual page to explain the details of each function and use an example to demonstrate the usage. We have included a tutorial as a separate tab within the tool to demonstrate the usage of each function with example data. References:Colaprico A, Silva TC, Olsen C, Garofano L, Cava C, Garolini D, Sabedot T, Malta TM, Pagnotta SM, Castiglioni I, Ceccarelli M, Bontempi G and Noushmehr H (2015). “TCGAbiolinks: An R/Bioconductor package for integrative analysis of TCGA data.” Nucleic Acids Research. doi: 10.1093/nar/gkv1507, http://doi.org/10.1093/nar/gkv1507.Genomic Data Commons (GDC) Data Portal: https://gdc-portal.nci.nih.gov/. NIH and NCI, accessed in August 2016.Y. Zhu, P. Qiu, Y. Ji. TCGA-Assembler: Open-Source Software for Retrieving and Processing TCGA Data. Nature Methods. Vol. 11, No. 6, pp:599-600, 2014. | doi:10.1038/nmeth.2956"
}
]
}
] | 1
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https://f1000research.com/articles/6-1039
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https://f1000research.com/articles/6-1606/v1
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30 Aug 17
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{
"type": "Research Article",
"title": "Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases",
"authors": [
"Ibai Goicoechea",
"Ricardo Rezola",
"María Arestin",
"María M. Caffarel",
"Ana Rosa Cortazar",
"Lorea Manterola",
"Marta Fernandez-Mercado",
"María Armesto",
"Carla Sole",
"Erika Larrea",
"Angela M. Araujo",
"Nerea Ancizar",
"Arrate Plazaola",
"Ander Urruticoechea",
"Arkaitz Carracedo",
"Irune Ruiz",
"Isabel Alvarez Lopez",
"Charles H. Lawrie",
"Ibai Goicoechea",
"Ricardo Rezola",
"María Arestin",
"María M. Caffarel",
"Ana Rosa Cortazar",
"Lorea Manterola",
"Marta Fernandez-Mercado",
"María Armesto",
"Carla Sole",
"Erika Larrea",
"Angela M. Araujo",
"Nerea Ancizar",
"Arrate Plazaola",
"Ander Urruticoechea",
"Arkaitz Carracedo",
"Irune Ruiz",
"Isabel Alvarez Lopez"
],
"abstract": "Background: The outcome for oestrogen receptor positive (ER+) breast cancer patients has improved greatly in recent years largely due to targeted therapy. However, the presence of involved multiple synchronous lymph nodes remains associated with a poor outcome. Consequently, these patients would benefit from the identification of new prognostic biomarkers and therapeutic targets. The expression of G-protein-coupled receptor kinase-interacting protein 1 (GIT1) has recently been shown to be an indicator of advanced stage breast cancer. Therefore, we investigated its expression and prognostic value of GIT1 in a cohort of 140 ER+ breast cancer with synchronous lymph node involvement. Methods: Immunohistochemistry was employed to assess GIT1 expression in a tissue microarray (TMA) containing duplicate non-adjacent cores with matched primary tumour and lymph node tissue (n=140). GIT1 expression in tumour cells was scored and statistical correlation analyses were carried out. Results: The results revealed a sub-group of patients that displayed discordant expression of GIT1 between the primary tumour and the lymph nodes (i.e. spatial intratumoural heterogeneity). We observed that loss of GIT1 expression in the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time. Moreover, multivariate analysis demonstrated that GIT1 expression was an independent prognostic indicator. Conclusions: GIT1 expression enabled the identification of a sub-class of ER+ patients with lymph node metastasis that have a particularly poor prognostic outcome. We propose that this biomarker could be used to further stratify ER+ breast cancer patients with synchronous lymph node involvement and therefore facilitate adjuvant therapy decision making.",
"keywords": [
"GIT1",
"breast cancer",
"immunohistochemistry",
"biomarker",
"lymph node"
],
"content": "Introduction\n\nBreast cancer is the most common type of cancer among Western women, and every year around 450,000 new cases are diagnosed in Europe alone1. Breast cancer is recognised as a very heterogeneous disease and several different subgroups have been identified on the basis of hormone receptor and Her2 status, or more recently by gene expression profiling2. Each subgroup shows different pathological, clinical and molecular characteristics, which in turn have different therapeutic options and prognostic outcome. The oestrogen-positive (ER+) breast cancer subtype is the most common of these subtypes representing around 70% of all cases. Although the majority of ER+ cases respond to anti-oestrogen treatments, some are resistant to endocrine therapies, in particular those patients with involved lymph nodes and distant metastases at time of presentation3,4. Indeed, the presence of synchronous lymph node metastasis is a strong indicator of poor prognostic outcome5. Therefore, there is a clear clinical need to identify new therapeutic targets and biomarkers for this group of patients including those that do not relapse.\n\nIt has been shown recently that advanced stage breast cancer and involved lymph nodes express high mRNA levels of G-protein-coupled receptor kinase-interacting protein 1 (GIT1)6, however, this study did not examine GIT1 protein expression, with the exception of nine tumours. As immunohistochemistry (IHC) exhibits greater potential for clinical application we decided to evaluate GIT1 immunoreactivity in an extensive cohort of ER+ breast cancer cases with synchronous lymph node (LN) involvement (n=140). We evaluated the association of GIT1 expression in the primary tumour and in the synchronous LN with clinical features and disease aggressiveness.\n\n\nMethods\n\nPrimary site and lymph node metastasis tissue samples were obtained from the Pathology Department of Donostia University Hospital from 140 breast patients who were diagnosed between 2000 and 2006, with follow-up data until 2014 with a median follow-up of 8 years and 8 months. Written consent was obtained from patients for the inclusion of their samples in this study and samples were collected in accordance with the Declaration of Helsinki, and approved by local ethics committees (Comite Etico de Investigación Clínica de Euskadi (CEIC-E)). Cases were selected and re-reviewed by two experienced breast pathologists independently. Clinical data was only available for 104 of these patients (Dataset 1). All patients were women with an age range between 27 and 86 years old (median age 58 years old). All primary site tumours were ER+ and 91% of them PR+. Eighty-six percent of patients presented with invasive carcinoma of no special type (NST) and 11% with invasive lobular carcinoma (ILC). Histological grades varied from grade I (21%), grade II (55%) to grade III (15%), according to the Elston-Ellis modification of Scarff-Bloom-Richardson grading system. All patients were surgically treated either by tumorectomy or by mastectomy with axillary lymphadenectomy. All patients underwent hormone therapy with the majority of them receiving radiotherapy (90%) and adjuvant chemotherapy (75%). Table 1 summarizes the clinical and histological characteristics of patients as we previously described7.\n\nAbbreviations: n (%) = number of patients (percentage within each feature), AI = Aromatase inhibitors, Tmx -> AI = Tamoxifen followed by Aromatase inhibitors. NST, invasive carcinoma of no special type; ILC, invasive lobular carcinoma. p values calculated with Chi-square contingency test\n\nRepresentative areas of high tumour load (>70%) were selected after H&E staining and two 1.5mm punch biopsies taken from both primary tumour and lymph node and arrayed non-adjacently to reduce staining bias using a Manual Tissue Arrayer MTA-1 (Beecher Instruments, USA).\n\nImmunohistochemical (IHC) staining was carried out on 5µm slices manually using the Immunohistochemistry Accessory Kit of Bethyl Laboratories (Montgomery, USA). Slides were deparaffinized in xylene and blocked in peroxidase for 30 min. Antigen retrieval was carried out in Epitope Retrieval Buffer (Bethyl Laboratories, Montgomery, USA). Slides were blocked in BSA for 30 min and then incubated for 1h at room temperature with anti-GIT1 rabbit polyclonal antibody (1:100) (IHC-00527, Bethyl Laboratories, Montgomery, USA). After 1h incubation with secondary anti-Rabbit IHC Antibody and slides counterstained with hematoxylin.\n\nGIT1 expression was scored in a blinded fashion by an experienced breast pathologist according to tumour cell staining intensity and categorical scores assigned as follows; 0= negative (0%); 1=1–10%; 2= 11–50%; 3= >50% (Dataset 2). Scores between non-adjacent cores were combined and categorised according to following criteria; GIT1 negative (combined score <1); moderate GIT1 expression (combined score 1–2); and high GIT1 expression (score >2). Examples of the different staining categories are shown in Figure 1 and Supplementary Figure S2. A selection of cases (n=8) were examined both as whole biopsy sections and TMAs, all scoring was concordant. Examples of whole section IHC staining with GIT1 are shown in Supplementary Figure S1 and Supplementary Figure S3.\n\nImages were enhanced from the original (Supplementary Figure S2). Representative intensity staining of GIT1 expression (primary tumour) depicting A. negative (score=0); B.weak (score=1); C. moderate (score=2); and D. strong (score=3). Magnification ×400.\n\nGIT1 gene expression levels were analyzed using publicly available databases. For this analyses we interrogated the TCGA dataset which included 525 mixed breast cancer tumours and 22 normal breast samples (Dataset 3)8, 2000 mixed breast tumours (Dataset 4)9, 570 metastatic breast tumours (Dataset 5)10, 252 lymph-node negative breast cancer patients (Dataset 6)11, 67 triple negative breast cancer patients (Dataset 7)12, and 19 primary breast cancer and 19 brain metastasis from HER2 positive breast cancer patients (Dataset 8)13.\n\nChi-square statistical test was used to determine association between GIT1 expression and lymph node metastasis (Table 3). Fisher’s exact test and Chi-square test were used to associate GIT1 expression with clinicopathological features (Table 1, Table 2 and Table 4). For survival analysis, Kaplan-Meier curves and univariate Log-rank (Mantel-Cox) analysis were performed (Figure 2 and Figure 3E). Statistical analyses were performed using GraphPad Prism v5.03 (GraphPad Software, La Jolla, CA, United States). Cox regression for multivariate analysis was performed with SPSS Statistics 20 (IBM, New York, USA) (Table 5).\n\nFor public dataset analysis, expression data were analyzed by t-test when comparing 2 groups or Anova when comparing more than 2 groups (Figure 3). Data was analyzed using GraphPad Prism v5.03 (CA, United States) and R (R Foundation for Statistical Computing, Vienna, Austria).\n\n\nResults\n\nWe observed both cytoplasmic and membrane expression of GIT1 in tumour cells of varying intensities, along with some perinuclear localisation and some weaker signal in stromal cells (Figure 1). This staining pattern is consistent with that previously observed6,14.\n\nIn order to ascertain the association of tumoural GIT1 expression with clinicopathological characteristics, we scored the expression according to intensity and % positive tumour cells in each case (based on two cores) as negative, moderate and high (Figure 1). Out of the 140 primary tumour specimens, we observed high expression of GIT1 in 47 cases (34%), moderate expression in 58 cases (42%) and no GIT1 expression in 35 cases (25%). There was no significant association between GIT1 expression and the 2012 WHO defined histological subtype (i.e. invasive carcinoma of no special type (NST; n=90 (86.5%)), invasive lobular carcinoma (ILC; n=12 (11.5%)) or other). Neither were there significant associations with hormone receptor status, tumour size, number of affected lymph nodes, histological grade or the presence of vascular lymphatic infiltrate (Table 2). We observed no significant difference in the overall survival (OS) (Figure 2A), or time to recurrence in patients according to the level of GIT1 expression in these primary tumours (data not shown).\n\nAbbreviations: n (%) = number of patients (percentage within each feature), AI = Aromatase inhibitors, Tmx -> AI = Tamoxifen followed by Aromatase inhibitors. p values calculated with Chi-square contingency test\n\nCurves were compared by univariate (log-rank) analysis. A. Cases sub-classified according to expression levels of GIT1 in the primary tumour (or LN) were not significantly different. Cases that were GIT1 +/- (n=31) had a shorter time to recurrence (B) and overall survival (C), and disease specific survival (D) than non GIT1+/- cases (n=73).\n\nA. GIT1 expression is significantly higher in breast cancer samples compared to normal breast cancer tissue. In silico meta-analysis of five databases representing 3452 cases. B. In two independent datasets GIT1 expression was lower in ER positive compared to ER negative tumours. C. GIT1 expression is highest in the basal breast cancer subtype which represents ER-cases (ANOVA analysis). D. GIT1 expression is lower in brain metastasis than primary breast tumour sites. E. Survival analysis of triple negative breast cancer patients show low GIT1 expression is associated with poorer clinical outcome. Patients were sub-classified on the basis of median GIT1 expression levels (univariate log rank test). Unless otherwise specified analysis was carried out by independent t-test.\n\nWhen we compared GIT1 expression in primary tumours with that of their counterpart lymph node metastases surprisingly we observed a significant decrease of GIT1 expression (p=0.0054, Table 3). Although both lymph node and primary biopsies showed a similar frequency of high GIT1 expression (29% vs 34% respectively), the percentage of lymph nodes with moderate GIT1 expression was lower than that of primary tumours (29% vs 41% respectively), and 43% of lymph node samples were negative for GIT1 compared with 25% of primary tumour samples (Table 3).\n\nAbbreviations: n (%) = number of patients (percentage with respect to all patients “140”). p value calculated with Chi-square contingency test\n\nTo explore this phenomenon further, we carried out a comparative analysis of GIT1 expression between the primary tumour site and matched synchronous lymph node metastasis. We found 64 cases (63%) with concordant GIT1 expression, either both positive for GIT1 expression (scores >1, n=45 (44%), or both negative for GIT1 expression (scores<1, n=19 (19%), and 38 cases (37%) with discordant GIT1 expression between the primary tumour and the lymph node.\n\nAs intratumoural heterogeneity has been suggested to be associated with resistance to therapy and prognostic outcome in breast cancer15, we investigated whether this spatial heterogeneity in GIT1 expression might be associated with clinical outcome (or clinicopathological characteristics) in our cohort. For this analysis we sub-classified cases into four groups: \"group 0\" cases were negative for GIT1 expression in both primary and lymph nodes (n=19); \"group ++\" cases were positive for GIT1 expression in both primary and lymph nodes (n=45); \"group +/-\" cases were positive for GIT1 expression in primary but not lymph nodes (i.e. loss of GIT1 expression; n=31); and \"group -/+\" cases were negative for GIT1 expression in primary but positive for GIT1 expression in lymph nodes (i.e. gain of GIT1 expression; n=7).\n\nWe did not detect any significant association between the aforementioned score and other clinicopathological features (Table 4). We analyzed the survival of these four groups and observed that group +/- showed a tendency towards shorter time to recurrence when compared to the rest of patients (p=0.05, hazard ratio = 2.902; Figure 2B), and that these patients had asignificantly poorer overall survival than the rest of the groups (p=0.03, hazard ratio = 2.996; Figure 2C). Furthermore, the disease specific survival of patients with +/- GIT1 expression was significantly worse than other patients (p<0.0001, hazard ratio = 7.423; Figure 2D) with a median survival time of only 67 months compared to 110 months, representing a reduction of ~40%. Comparing with other well defined prognostic indicators (histological grade, presence of distant metastasis) by multivariate analysis in our cohort, the loss of GIT1 expression in lymph nodes (+/- pattern) was an independent prognostic indicator (p=0.002; (Table 5)).\n\nAbbreviations: n (%) = number of patients (percentage within each feature), AI = Aromatase inhibitors, Tmx -> AI = Tamoxifen followed by Aromatase inhibitors. p values calculated with Chi-square contingency test\n\np values calculated with Cox regression for multivariate analysis test\n\nTo further examine GIT1 expression in breast cancer, we analyzed gene expression levels in several publicly available gene expression datasets. This revealed that GIT1 expression was significantly higher in breast cancer (n=144) compared to non-tumoural tissue (n=14) (P=4.87 x 10-4)8–10 (Figure 3A), and was particularly pronounced when comparing only NST cases (n=1556) with healthy breast tissue (n=144) (P=1.43 x 10-48). A comparison between the two main subtypes of breast cancer (i.e. NST and ILC) in the TCGA dataset (n=525) showed differences in expression levels with healthy breast tissue (n=64) (P=5.62 x 10-6) as did the dataset of Gluck et al (n=154 vs 4 (breast carcinoma vs healthy control) (P=0.002)) (Figure 3A)10. A comparison of GIT1 expression between ER+ and ER-tumours in two independent Datasets demonstrated a significantly lower level of expression in ER+ tumours compared to ER- tumours. These comparisons were carried out on the TCGA dataset (n=601 (ER+) vs. n=179 (ER-) and the Wang dataset (n=209 (ER+) vs. n=77 (ER-)8,11 (Figure 3B). Consistent with these findings, basal tumours, which are mostly ER negative, showed higher GIT1 expression than the rest of the molecular subgroups of breast cancer in the TCGA dataset (P=6.11 x 10-7; Figure 3C).\n\nWe also looked at GIT1 expression between the primary tumour and brain metastasis in a cohort of HER2-positive (mixed ER+ and ER- cases) breast cancers (n=19)13. We observed that GIT1 expression was significantly decreased in brain metastases (P=0.0279; Figure 3D). Furthermore, when we interrogated data from a publicly available cohort of triple negative breast cancer patients (n=67)12, we found that patients with GIT1 expression below the median had significantly poorer prognosis regarding event-free interval than those with GIT1 levels above the median (P=0.0411, hazard ratio = 1.625; Figure 3E).\n\n\nDiscussion\n\nGIT1 is a scaffold protein that forms part of the Arf and Rho family of GTPases16. It is involved in many cellular processes including cell adhesion, migration, lamellipodia formation, cell growth and angiogenesis17–20. In addition, GIT1 can activate many signalling pathways involved in carcinogenesis such as ERK1/2, Rho, AARF or P21-activated kinase (PAK)17,21. GIT1 has been demonstrated to be over-expressed in several cancers including hepatocellular carcinoma, colon cancer, lung cancer and melanoma17,22–25.\n\nIn the current study, we ascertained the clinical relevance of GIT1 expression by IHC in a cohort of ER positive breast cancer samples with involved synchronous lymph nodes. Although it has recently been reported that GIT1 is over-expressed in lymph nodes when compared to primary breast cancer, very few cases were examined by qRT-PCR (<30) and even fewer (<10) by IHC6, the most prevalent biomarker detection technique used in clinics. Furthermore, this study did not examine the potential prognostic value of GIT1 expression or its association with distant metastasis.\n\nWe observed a significant reduction in GIT1 expression in lymph node metastasis compared to matched primary breast tumours. Furthermore, we found that over a third of cases displayed a spatial intratumoural heterogeneous pattern of GIT1 expression between the primary tumour and the lymph node, with loss of GIT1 expression in lymph nodes being more common than its gain. Heterogeneity in protein expression is a well-established phenomenon in breast cancer, particularly regarding hormone receptor status, and has been associated with prognostic outcome26. However, these studies generally report lower levels of heterogeneity (<20%) between primary and synchrononus lymph node metastases7,27, suggesting that GIT1 could be a more sensitive indicator of heterogeneity in this cancer, and hence a more powerful prognostic indicator. It should be noted that those cases with heterogeneous expression of GIT1 were not the same cases as those with heterogeneous expression of hormone receptors in this cohort. Consistent with this idea, we found that loss of GIT1 in the lymph nodes (30% of patients in this study), was indicator of poor prognosis (time to recurrence and OS) by univariate analysis and an independent indicator of prognosis by multivariate analysis. It should be noted that further analysis in independent patient cohorts within a multi-centre setting is necessary to validate these findings further.\n\nWe extended these studies to other subtypes of breast cancer by looking at cohorts from publicly available databases (n= 3452) and found that GIT1 is over-expressed in breast cancer and its expression associates inversely with ER status. Furthermore, GIT1 levels were down-regulated between primary sites and distant metastases, and that was true not only in ER+ breast cancer but also other subtypes. Despite the clear evidence shown here that GIT1 is down-regulated in both lymph node and distant metastasis in breast cancer another study reported an increase in GIT1 expression between primary tumours and lymph node metastasis6. However, it should be borne in mind that the study of Chan et al. used a much smaller cohort of patients (n=26) and moreover the hormone receptor status of these patients was not provided. As our in silico analysis (Figure 3B) suggests that GIT1 expression varies significantly with ER status, it is plausible that the subtype analysed could influence the results. Our results support the notion that, at least in ER+ breast tumours, down-regulation of GIT1 in lymph node metastases is a sign of poor prognosis.\n\nIn summary, our study has shown that the expression of GIT1 in breast cancer could serve as a useful biomarker for the management of breast cancer patients in general and for ER+/LN+ patients in particular. The mechanistic reasons behind why the loss of GIT1 in these patients is indicative of poor prognosis remains to be determined, however it is tempting to suggest that further studies could lead to better management of these patients and ultimately improve their clinical outcome.\n\n\nData availability\n\nDataset 1: Clinical data of patient cohort. Table shows patients (numbered from 1 to 105) and their clinical features including histological subgroup, tumour size, number of affected lymph nodes, histological grade, vascular lymphatic infiltration, immunohistochemical initial status, treatment and patient follow up. 10.5256/f1000research.12393.d17543528\n\nDataset 2: GIT1 scoring. Table shows patients (numbered from 1 to 142) and associated primary tumour and lymph node GIT1 scoring. Categorical scores are assigned as follows according to tumour cell staining intensity; 0= negative (0%); 1=1–10%; 2= 11–50%; 3= >50%. 10.5256/f1000research.12393.d17544329\n\nDataset 3: Series mRNA expression matrix and clinical data information. GIT1 Expression Dataset consisting of 522 primary tumors, 3 metastatic tumors, and 22 tumor-adjacent normal samples. Data was median centered by genes. Platform: Affymetrix Human Genome U133A Array. Publicly available from https://tcga-data.nci.nih.gov/docs/publications/brca_2012/. 10.5256/f1000research.12393.d17544430\n\nDataset 4: Series mRNA expression matrix. Expression Dataset consisting of 2000 breast carcinoma. Platform: Affymetrix Human HT-12 V3 Array. Publicly available from http://www.cbioportal.org/study?id=brca_metabric#summary 10.5256/f1000research.12393.d17544531\n\nDataset 5: Series mRNA expression matrix. Expression Dataset consisting of one hundred fifty-four (154) invasive breast carcinoma samples and 4 normal breast samples. Platform: Agilent UNC Perou Lab Homo sapiens 1X44K Custom Array. Publicly available from Gluck Breast dataset (https://www.oncomine.org) 10.5256/f1000research.12393.d17544732\n\nDataset 6: Series mRNA expression matrix. Expression Dataset consisting of 252 lymph-node negative breast cancer samples. Platform: Affymetrix Human Genome U133A Array. Publicly available from https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse2034 10.5256/f1000research.12393.d17544933\n\nDataset 7: Series mRNA expression matrix. Expression Dataset consisting of 67 triple negative breast cancer samples. Platform: Affymetrix Human Genome U133A Array. Publicly available from https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31519 10.5256/f1000research.12393.d17545234\n\nDataset 8: Series mRNA expression matrix. Expression Dataset consisting of 19 HER2+ brain metastasis breast cancer samples and 19 HER2+ non-metastatic breast cancer samples. Platform: Affymetrix Human X3P Array. Publicly available from https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE43837 10.5256/f1000research.12393.d17545335\n\n\nConsent\n\nWritten informed consent for publication of the patients' details and their images was obtained from the patients.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nCHL and his research is supported by grants from the IKERBASQUE foundation for science, the Starmer-Smith memorial fund, Ministerio de Economía y Competitividad of Spanish central government and FEDER funds (PI12/00663, PIE13/00048, DTS14/00109, PI15/00275), Departamento de Desarrollo Económico y Competitividad y Departamento de Sanidad of Basque government, Asociación Española Contra el Cancer (AECC), and the Diputación Foral de Guipuzcoa (DFG). MFM acknowledges support from AECC and DFG. The work of AC is supported by FERO VIII call and the ERC starting grant (336343). IG also acknowledges support from AECC and Fundación Caja Navarra. MMC acknowledges support from IKERBASQUE foundation for science and Ministerio de Economía y Competitividad of Spanish central government. EL also acknowledges support from the Ministerio de Economía y Competitividad of Spanish central government.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary Figure S1: Examples of GIT1 expression in whole tumour sections. Images were enhanced from the original (Supplementary Figure S3). Patient A demonstrates a +/- (positive primary/negative lymph node) pattern of GIT1 expression in both TMA and whole section staining. Patient B shows a -/+ (weak positive primary/positive lymph node) pattern of GIT1 expression. Magnification x40.\n\nClick here to access the data.\n\nSupplementary Figure S2: Example of GIT1 expression patterns found in ER+ breast cancer. Original representative intensity staining photos of GIT1 expression (primary tumour) of Figure 1. A. negative (score=0); B. weak (score=1); C. moderate (score=2); and D. strong (score=3). Magnification x400.\n\nClick here to access the data.\n\nSupplementary Figure S3: Examples of GIT1 expression in whole tumour sections. Original photos of Supplementary Figure S1. Patient A demonstrates a +/- (positive primary/negative lymph node) pattern of GIT1 expression in both TMA and whole section staining. Patient B shows a -/+ (weak positive primary/positive lymph node) pattern of GIT1 expression. Magnification x40.\n\nClick here to access the data.\n\n\nReferences\n\nFerlay J, Steliarova-Foucher E, Lortet-Tieulent J, et al.: Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer. 2013; 49(6): 1374–1403. PubMed Abstract | Publisher Full Text\n\nDawson SJ, Rueda OM, Aparicio S, et al.: A new genome-driven integrated classification of breast cancer and its implications. EMBO J. 2013; 32(5): 617–628. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nIsabel Alvarez RR, Ruiz I, Plazaola A, et al.: Hormone receptors and HER2 expression in primary tumor and synchronous axillary lymph node metastasis in estrogen receptor positive breast cancer. J Clin Oncol. 2015; ASCO Annual Meeting 2015; 33: 15_suppl (May 20 Supplement). Reference Source\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\nCurtis C, Shah SP, Chin SF, et al.: The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature. 2012; 486(7403): 346–352. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlum JL, Kohles J, McKenna E, et al.: Association of age and overall survival in capecitabine-treated patients with metastatic breast cancer in clinical trials. Breast Cancer Res Treat. 2011; 125(2): 431–439. PubMed Abstract | Publisher Full Text\n\nWang Y, Klijn JG, Zhang Y, et al.: Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet. 2005; 365(9460): 671–679. PubMed Abstract\n\nRody A, Karn T, Liedtke C, et al.: A clinically relevant gene signature in triple negative and basal-like breast cancer. Breast Cancer Res. 2011; 13(5): R97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcMullin RP, Wittner BS, Yang C, et al.: A BRCA1 deficient-like signature is enriched in breast cancer brain metastases and predicts DNA damage-induced poly (ADP-ribose) polymerase inhibitor sensitivity. Breast Cancer Res. 2014; 16(2): R25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang N, Cai W, Yin G, et al.: GIT1 is a novel MEK1-ERK1/2 scaffold that localizes to focal adhesions. Cell Biol Int. 2010; 34(1): 41–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYates LR, Gerstung M, Knappskog S, et al.: Subclonal diversification of primary breast cancer revealed by multiregion sequencing. Nat Med. 2015; 21(7): 751–759. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchlenker O, Rittinger K: Structures of dimeric GIT1 and trimeric beta-PIX and implications for GIT-PIX complex assembly. J Mol Biol. 2009; 386(2): 280–289. PubMed Abstract | Publisher Full Text\n\nMajumder S, Sowden MP, Gerber SA, et al.: G-protein-coupled receptor-2-interacting protein-1 is required for endothelial cell directional migration and tumor angiogenesis via cortactin-dependent lamellipodia formation. Arterioscler Thromb Vasc Biol. 2014; 34(2): 419–426. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNola S, Sebbagh M, Marchetto S, et al.: Scrib regulates PAK activity during the cell migration process. Hum Mol Genet. 2008; 17(22): 3552–3565. PubMed Abstract | Publisher Full Text\n\nManabe R, Kovalenko M, Webb DJ, et al.: GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration. J Cell Sci. 2002; 115(Pt 7): 1497–1510. PubMed Abstract\n\nSchmalzigaug R, Garron ML, Roseman JT, et al.: GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin. Cell Signal. 2007; 19(8): 1733–1744. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Nieuw Amerongen GP, Natarajan K, Yin G, et al.: GIT1 mediates thrombin signaling in endothelial cells: role in turnover of RhoA-type focal adhesions. Circ Res. 2004; 94(8): 1041–1049. PubMed Abstract | Publisher Full Text\n\nPeng H, Dara L, Li TW, et al.: MAT2B-GIT1 interplay activates MEK1/ERK 1 and 2 to induce growth in human liver and colon cancer. Hepatology. 2013; 57(6): 2299–2313. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKao J, Salari K, Bocanegra M, et al.: Molecular profiling of breast cancer cell lines defines relevant tumor models and provides a resource for cancer gene discovery. PLoS One. 2009; 4(7): e6146. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang C, Sheng Y, Jia J, et al.: Identification of melanoma biomarkers based on network modules by integrating the human signaling network with microarrays. J Cancer Res Ther. 2014; 10(Suppl): C114–124. PubMed Abstract | Publisher Full Text\n\nChang JS, Su CY, Yu WH, et al.: GIT1 promotes lung cancer cell metastasis through modulating Rac1/Cdc42 activity and is associated with poor prognosis. Oncotarget. 2015; 6(34): 36278–36291. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYao ZX, Lu LJ, Wang RJ, et al.: Discordance and clinical significance of ER, PR, and HER2 status between primary breast cancer and synchronous axillary lymph node metastasis. Med Oncol. 2014; 31(1): 798. PubMed Abstract | Publisher Full Text\n\nYang YF, Liao YY, Yang M, et al.: Discordances in ER, PR and HER2 receptors between primary and recurrent/metastatic lesions and their impact on survival in breast cancer patients. Med Oncol. 2014; 31(10): 214. PubMed Abstract | Publisher Full Text\n\nGoicoechea I, Rezola R, Arestin M, et al.: Dataset 1 in: Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases. F1000Research. 2017. Data Source\n\nGoicoechea I, Rezola R, Arestin M, et al.: Dataset 2 in: Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases. F1000Research. 2017. Data Source\n\nGoicoechea I, Rezola R, Arestin M, et al.: Dataset 3 in: Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases. F1000Research. 2017. Data Source\n\nGoicoechea I, Rezola R, Arestin M, et al.: Dataset 4 in: Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases. F1000Research. 2017. Data Source\n\nGoicoechea I, Rezola R, Arestin M, et al.: Dataset 5 in: Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases. F1000Research. 2017. Data Source\n\nGoicoechea I, Rezola R, Arestin M, et al.: Dataset 6 in: Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases. F1000Research. 2017. Data Source\n\nGoicoechea I, Rezola R, Arestin M, et al.: Dataset 7 in: Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases. F1000Research. 2017. Data Source\n\nGoicoechea I, Rezola R, Arestin M, et al.: Dataset 8 in: Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases. F1000Research. 2017. Data Source"
}
|
[
{
"id": "25548",
"date": "05 Oct 2017",
"name": "Mona M. Mohamed",
"expertise": [
"Reviewer Expertise Cancer 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\nAbstract The authors mentioned that \"GIT1 expression in tumour cells was scored and statistical correlation analyses were carried out\". Authors should point with arrow to all tumour cells showing stain of GIT1 in tissue and differentiate between expression of GIT1 by cancer cells and stromal cells.\n\"We observed that loss fo GIT1 expression in the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time.\"\nAuthors should explain here that loss of \"GIT1 expression\" by which cells? Tumor metastatic cells in the lymph node or by cell populations of the lymph nodes (example: immune cells)?\nIn material and methods scoring of GIT1 is only in tumor cells so which cells are scored in lymph nodes. Please note that the cell populations of lymph nodes (LNs) is different from that of the tumor microenvironment and authors depends on scoring of GIT1 expression by the tumor cells (less in LNs) so this should be clarified by authors, since the IHC microscopic images provided is confusing and may show non-specific stain.\n\nMethods \"All primary site tumours were ER+ and 91% of them PR+.\" The authors should include ER- (ER negative) patients as a control to the studied group to provide their hypothesis that ’the expression of GIT1 in breast cancer could serve as a useful biomarker for the management of breast cancer patients in general and for ER+/LN+ patients in particular.\"\nAlthough the paper depends mainly on IHC the auhtors did not mention appropriate chromogen/substrate used to develop the color visualized using the microscope. This section should be written in details.\nI cannot read the Dataset files provided by the author, the columns and rows overlap.\nStatistical analysis \"Chi-square statistical test was used to determine association between GIT1 expression and lymph node metastasis (Table 3)\"\nWhat type of correlation test used here?\nDo the authors correlate between GIT1 expression in primary tumor and the \"number of metastatic lymph node\" this section should be clarified?\nResults The Supplementary Figures S1, S2 and S3 representing microscopic images for GIT1 show non-specific stains. The authors should present better microscopic images with high magnification showing the pattern of GIT1 by carcinoma cells and stromal cells.\n\"Spatial intratumoural heterogeneity of GIT1 expression in ER+ breast cancer: When we compared GIT1 expression in primary tumours with that of their counterpart lymph node metastases surprisingly we observed a significant decrease of GIT1 expression (p=0.0054, Table 3).\"\nThe authors did not take in consideration that cellular population of LN is different from that of carcinoma cells. A better IHC images should be presented showing which cell population express GIT1 in each of carcinoma tissues and lymph nodes.\n\"A comparison of GIT1 expression between ER+ and ER- tumours in two independent Datasets demonstrated a significantly lower level of expression in ER+ tumours compared to ER- tumours (8,11).\"\nThe references (8,11); assess GIT1 at mRNA level not at the protein level, in the present study authors should include ER- patients and their associated lymph nodes as control group to ER+.\nDiscussion Should be re-written presenting the mechanisms of GIT1 in carcinogenesis and scientific explanation for the authors findings \"loss of GIT1 expression in the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time\".\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?\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? Yes",
"responses": [
{
"c_id": "3354",
"date": "14 Feb 2018",
"name": "Ibai Goicoechea",
"role": "Author Response",
"response": "We thank the reviewer for their insightful comments and have made changes in the revised manuscript to address these concerns. Specifically: Abstract P1: The authors mentioned that \"GIT1 expression in tumour cells was scored and statistical correlation analyses were carried out\". Authors should point with arrow to all tumour cells showing stain of GIT1 in tissue and differentiate between expression of GIT1 by cancer cells and stromal cells. Only tumor cells were scored in this analysis not stromal or other microenvironment cell types \"We observed that loss fo GIT1 expression in the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time.\" Authors should explain here that loss of \"GIT1 expression\" by which cells? Tumor metastatic cells in the lymph node or by cell populations of the lymph nodes (example: immune cells)? Tumor cells (text changed in revised manuscript) In material and methods scoring of GIT1 is only in tumor cells so which cells are scored in lymph nodes. Please note that the cell populations of lymph nodes (LNs) is different from that of the tumor microenvironment and authors depends on scoring of GIT1 expression by the tumor cells (less in LNs) so this should be clarified by authors, since the IHC microscopic images provided is confusing and may show non-specific stain. Only tumor cells were scored in the involved lymph nodes, non-involved lymph nodes were used as negative controls in this instance. Methods \"All primary site tumours were ER+ and 91% of them PR+.\" The authors should include ER- (ER negative) patients as a control to the studied group to provide their hypothesis that ’the expression of GIT1 in breast cancer could serve as a useful biomarker for the management of breast cancer patients in general and for ER+/LN+ patients in particular.\" We agree that this would have been a good comparison to carry out. Unfortunately there were insufficient suitable ER- cases available for testing. We also decided to focus the study on ER+ cases as representing the majority of breast cancer cases Although the paper depends mainly on IHC the auhtors did not mention appropriate chromogen/substrate used to develop the color visualized using the microscope. This section should be written in details. The chromagen used was DAB. This information has been included in the revised manuscript I cannot read the Dataset files provided by the author, the columns and rows overlap. I have informed the editor of this problem Statistical analysis \"Chi-square statistical test was used to determine association between GIT1 expression and lymph node metastasis (Table 3)\" What type of correlation test used here? This was Chi-square analysis Do the authors correlate between GIT1 expression in primary tumor and the \"number of metastatic lymph node\" this section should be clarified? This is an interesting point and we have added this analysis to the revised manuscript. There was no correlation. Results The Supplementary Figures S1, S2 and S3 representing microscopic images for GIT1 show non-specific stains. The authors should present better microscopic images with high magnification showing the pattern of GIT1 by carcinoma cells and stromal cells. Whilst we agree with the reviewer that some GIT1 expression was non-specific this was very different staining from that seen in tumor tissue. This can be seen clearly from the figures S1 and a high magnification image is already given in Figure 1 whereby some light stromal staining is seen in the panel A compared to string tumour staining in panel D. \"Spatial intratumoural heterogeneity of GIT1 expression in ER+ breast cancer: When we compared GIT1 expression in primary tumours with that of their counterpart lymph node metastases surprisingly we observed a significant decrease of GIT1 expression (p=0.0054, Table 3).\" The authors did not take in consideration that cellular population of LN is different from that of carcinoma cells. A better IHC images should be presented showing which cell population express GIT1 in each of carcinoma tissues and lymph nodes. We disagree with the reviewer that we did not take into consideration the difference in non-tumoral staining between lymph node and primary material. The scoring was carried out by a breast pathologist with more than 25 years experience in the field. These differences are clearly shown in Supplementary Figure S1 which shows representative images of negative and positive lymph node staining, as well as primary tumor material. \"A comparison of GIT1 expression between ER+ and ER- tumours in two independent Datasets demonstrated a significantly lower level of expression in ER+ tumours compared to ER- tumours (8,11).\" The references (8,11); assess GIT1 at mRNA level not at the protein level, in the present study authors should include ER- patients and their associated lymph nodes as control group to ER+. As stated above we would have liked to included ER- cases as well. However, that was not possible Discussion Should be re-written presenting the mechanisms of GIT1 in carcinogenesis and scientific explanation for the authors findings \"loss of GIT1 expression in the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time\". The discussion has been rewritten to add possible explanations for this statement"
}
]
},
{
"id": "27272",
"date": "06 Nov 2017",
"name": "Richard T. Premont",
"expertise": [
"Reviewer Expertise Biochemical pharmacology of signal transduction pathways",
"discovered and characterized GIT1"
],
"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\nGoicoechea et al. have examined GIT1 expression in primary breast cancers and metastatic lymph nodes a cohort of Estrogen Receptor positive (ER+) patients. While high or low GIT1 overexpression in the primary tumor does not associate with patient survival, a discordance between high GIT1 in primary tumor and low GIT1 in metastatic lymph nodes is significantly associated with reduced time to metastatic recurrence and reduced patient survival. The authors suggest that this discordance is an independent prognostic indicator of poor outcome in ER+ patients. A further analysis of published datasets shows that GIT1 mRNA level is elevated in multiple breast cancer subtypes, but this overexpression is lowest in ER+ cancers, and that low GIT1 is associated with worse outcomes in triple negative breast cancer.\n\nThe strength of the paper is in the survival curves in Figure 2, showing that the GIT1-high primary tumor/GIT1-low lymph node metastatic (+/-) subgroup is significantly worse off than patients with either always high or always low GIT1 level. This could be a very significant finding.\nHowever, there are several major concerns:\n\nThe study relies on the specificity of a single GIT1 polyclonal antiserum from Bethyl. The authors should list the specific lots of antisera utilized, and describe the characterization they have performed to be assured that this antiserum is in fact detecting GIT1, and not also the related GIT2 protein or even other unrelated proteins. Is similar IHC data obtained using distinct anti-GIT1 antisera? The authors should also show and point out adjacent normal breast epithelial tissue (and lymph node tissue) for comparison. The authors should consider making all primary IHC image files available as supplemental material.\n\nRegarding the metastatic lymph nodes, what evidence is there that these cells are in fact metastatic? If other markers were used, they should be mentioned. The histology of the lymph nodes in Supp Fig 1, at the magnification shown, makes it impossible to distinguish normal lymphocytes from metastatic epithelial-like cells. Again, it would be helpful to include and mark adjacent non-tumor tissue.\n\nThe IHC figures should include scale bars in each micrograph for size. It is particularly worrying in Figure 1A that the cell nuclei are all substantially larger than those in the other panels of that figure. The cells themselves also appear larger.\n\nTable 5 appears confusing because the description is incomplete. Please clarify what was compared in each case (likely just explicitly referring to Table 4). That is, among the patient cohort, what number and percentage of patients had distant metastasis? Define what histology grades are compared here (I vs II vs III, pooling of subgroups I and II vs III, etc)? What are +/- patients compared to, all other patients or just a subgroup? The table as shown also does not include a description of how the p=0.002 value reported in the text was obtained. Surely the Hazard ratio of 300000+ in Table 5 is a typo?\n\nThe discussion of the functions and cancer-associations of GIT1 is inadequate and incorrect. GIT1 is not “part of the Arf and Rho families of GTPases” as it is not a GTPase itself, but instead is a GTPase regulator (GTPase activating protein - deactivator - for Arf). GIT1 functional interactions with the Rho pathway, and with PAK, require an additional GIT1 partner, PIX, which is a Rho guanine nucleotide exchange factor (activator). The authors should carefully read two recent reviews that discuss GIT1 and cancer to distill the current understanding of GIT1 function1,2, and then present a clearer and more accurate description of what GIT1 does and therefore why this discordance between primary and metastatic tumor might explain the differential patient survival rates they have discovered.\n\nMinor presentation/formatting issues:\n\nThe authors need to be consistent in their presentation of numerical values. Values are presented in the European style with a comma separating a number from fractional values in all of the data tables (“4,843”), but in the American style using a period in the text itself (“4.843”). This is confusing.\n\nThe datasets are provided as Excel spreadsheets, but with all values for each patient shown within a single cell, separated by semicolons. It would make this data more immediately accessible to provide either Excel spreadsheets with the distinct values for each patient in individual cells already, or to provide this data in a less platform-dependent manner, such as CSV tables with commas between values for each patient, which can be readily imported into any statistics program.\n\nIn Fig 2A and Fig 3E, GIT1 is labeled as “GIT-1”\n\nIt is beyond the scope of this study, but an interesting mechanistic question is, since GIT1 works hand-in-hand with PIX proteins and they regulate each others’ stability and expression, do PIX levels also change in GIT1 high vs low expressing tumors?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "3420",
"date": "14 Feb 2018",
"name": "Ibai Goicoechea",
"role": "Author Response",
"response": "Thank you very much for the useful comments made by this reviewer which have made further enriched the revised manuscript. I hope we are able to satisfy most of your concerns regarding the manuscript. In response to the specific points: The study relies on the specificity of a single GIT1 polyclonal antiserum from Bethyl. The authors should list the specific lots of antisera utilized, and describe the characterization they have performed to be assured that this antiserum is in fact detecting GIT1, and not also the related GIT2 protein or even other unrelated proteins. Is similar IHC data obtained using distinct anti-GIT1 antisera? The authors should also show and point out adjacent normal breast epithelial tissue (and lymph node tissue) for comparison. The authors should consider making all primary IHC image files available as supplemental material. We agree with the reviewer that many of the conclusions drawn in this study are dependent upon the specificity of the antibodies used. However, we would also like to point out that the immunohistochemistry results were also corroborated by our in silico analyses. As suggested by the reviewer, the specific lot of the antisera used has been included in the revised manuscript. Regarding the possibility of cross-specificity of the Ab with GIT2, even though the antisera is polyclonal, the immunogen used (i.e. between aa 375 and aa 425) is specific to GIT1 and this sequence is not present in GIT2. Furthermore, this antibody is the same that was used in the study of Chan et al (Oncogene 2014). That said we cannot rule out non-specific staining due to cross-reactivity against other proteins. This caveat has been made clear in the revised manuscript. Unfortunately, due to routine surgical protocols adjacent normal breast epithelial tissue was not available for these cases. Regarding the metastatic lymph nodes, what evidence is there that these cells are in fact metastatic? If other markers were used, they should be mentioned. The histology of the lymph nodes in Supp Fig 1, at the magnification shown, makes it impossible to distinguish normal lymphocytes from metastatic epithelial-like cells. Again, it would be helpful to include and mark adjacent non-tumor tissue. The identification of metastatic lymph nodes was carried out on the basis of H&E stains, by an expert breast cancer pathologist with more than 25 years’ experience. We agree it would be very useful to include adjacent non-tumor tissue in this study however this material was not available as immunohistochemical staining was carried out on TMAs rather than whole sections and cores were selected on the basis of high-tumour content so did not contain a significant non-tumoral component. 3. The IHC figures should include scale bars in each micrograph for size. It is particularly worrying in Figure 1A that the cell nuclei are all substantially larger than those in the other panels of that figure. The cells themselves also appear larger. The figures all represent magnified fields at 400x magnification and scale bars have been added to the figure as suggested. Table 5 appears confusing because the description is incomplete. Please clarify what was compared in each case (likely just explicitly referring to Table 4). That is, among the patient cohort, what number and percentage of patients had distant metastasis? Define what histology grades are compared here (I vs II vs III, pooling of subgroups I and II vs III, etc)? What are +/- patients compared to, all other patients or just a subgroup? The table as shown also does not include a description of how the p=0.002 value reported in the text was obtained. Surely the Hazard ratio of 300000+ in Table 5 is a typo? We agree that the description of Table 5 could be confusing and have therefore changed the description accordingly. The discussion of the functions and cancer-associations of GIT1 is inadequate and incorrect. GIT1 is not “part of the Arf and Rho families of GTPases” as it is not a GTPase itself, but instead is a GTPase regulator (GTPase activating protein - deactivator - for Arf). GIT1 functional interactions with the Rho pathway, and with PAK, require an additional GIT1 partner, PIX, which is a Rho guanine nucleotide exchange factor (activator). The authors should carefully read two recent reviews that discuss GIT1 and cancer to distill the current understanding of GIT1 function1,2, and then present a clearer and more accurate description of what GIT1 does and therefore why this discordance between primary and metastatic tumor might explain the differential patient survival rates they have discovered. We thank the reviewer for pointing out these references and the information contained within. The discussion has been re-written to reflect the comments of the reviewer. Minor presentation/formatting issues: The authors need to be consistent in their presentation of numerical values. Values are presented in the European style with a comma separating a number from fractional values in all of the data tables (“4,843”), but in the American style using a period in the text itself (“4.843”). This is confusing. This has been changed in the revised manuscript The datasets are provided as Excel spreadsheets, but with all values for each patient shown within a single cell, separated by semicolons. It would make this data more immediately accessible to provide either Excel spreadsheets with the distinct values for each patient in individual cells already, or to provide this data in a less platform-dependent manner, such as CSV tables with commas between values for each patient, which can be readily imported into any statistics program. This has been changed in the revised manuscript In Fig 2A and Fig 3E, GIT1 is labeled as “GIT-1” It is beyond the scope of this study, but an interesting mechanistic question is, since GIT1 works hand-in-hand with PIX proteins and they regulate each others’ stability and expression, do PIX levels also change in GIT1 high vs low expressing tumors?"
}
]
}
] | 1
|
https://f1000research.com/articles/6-1606
|
https://f1000research.com/articles/7-7/v1
|
03 Jan 18
|
{
"type": "Research Article",
"title": "Management of eight labor and delivery patients dependent on buprenorphine (Subutex™): A retrospective chart review",
"authors": [
"Solina Tith",
"Garinder Bining",
"Laurent A. Bollag",
"Solina Tith",
"Garinder Bining"
],
"abstract": "Background: Opioid use during pregnancy is a growing concern in the United States. Buprenorphine has been recommended by “The American College of Obstetrics and Gynecology” as an alternative to methadone to decrease risks associated with the use of illicit opioids during pregnancy. The partial μ-opioid agonists’ unique pharmacology, including its long half time and high affinity to the μ-opioid receptor, complicates patient management in a highly kinetic, and often urgent field like obstetric anesthesia. We reviewed our management and outcomes in this medically complex population. Methods: An Institutional Review Board (IRB) approved retrospective chart review was conducted of women admitted to the University of Washington Medical Center Labor and Delivery unit from July 2012 to November 2013 using buprenorphine. All deliveries, including intrauterine fetal demise, were included. Results: Eight women were admitted during this period to our L&D floor on buprenorphine. All required peri-partum anesthetic management either for labor and/or cesarean delivery management. Analgesic management included dilaudid or fentanyl PCA and/or continued epidural infusion, and in one instance ketamine infusion, while the pre-admission buprenorphine regimen was continued. Five babies were viable, two women experienced intrauterine fetal death at 22 and 36 weeks gestational age (GSA), respectively, and one neonate died shortly after delivery due to a congenital diaphragmatic hernia. Conclusions: This case series illuminates the medical complexity of parturients using buprenorphine. Different treatment modalities in the absence of evidence-based guidelines included additional opioid administration and continued epidural analgesia. The management of post-cesarean pain in patients on partial μ-opioid agonists remains complex and variable, and evidence-based guidelines could be useful for clinicians to direct care.",
"keywords": [
"Buprenorphine",
"Opioid tolerance",
"Pregnancy",
"Post Cesarean pain management",
"Regional anesthesia",
"Multimodal analgesia"
],
"content": "Introduction\n\nOpioid use during pregnancy is a growing concern in the United States. In a review of over 500,000 women, 76,742(15%) received at least one dose of an opioid during pregnancy and of these, 11,747 were dispensed opioids three or more times during pregnancy1. The U.S. Food and Drug Administration (FDA) highlighted the need for further investigation regarding the risks of pain medicine use during pregnancy in a recent Drug Safety Communication in order to inform clinical practice2. The FDA also emphasized that severe and persistent pain that is not effectively treated during pregnancy can result in maternal depression, anxiety, and high blood pressure2.\n\nThe American College of Obstetrics and Gynecology (ACOG) released their opinion regarding opioid abuse, dependence, and addiction in pregnancy. They recommended buprenorphine as an alternative to methadone to decrease risks associated with the use of illicit opioids during pregnancy3.\n\nBuprenorphine (SubutexTM) is a partial μ-opioid agonist and, at high doses, a weak κ-antagonist that is taken as a sublingual tablet4. Suggested advantages of buprenorphine over methadone in pregnancy include less severe withdrawal symptoms, a lower risk of opioid overdose, fewer drug interactions, better ability to be treated on an outpatient basis without daily visits to a treatment program, less severe neonatal abstinence syndrome (NAS), and possibly less analgesic pain medications postpartum3,5–7. On average, parturients taking buprenorphine did so for 131.6 (SD 98.7) days of their pregnancy1.\n\nAt our institution, it is not uncommon for parturients to present for delivery while currently taking buprenorphine. Managing such patients, who generally have a long history of opioid abuse and addiction, is challenging, particularly when addressing post-cesarean pain management. Perfect anticipation of labor and delivery timing is not always possible. Buprenorphine’s long duration of action conflicts with the desired goal of tapering to a pure μ-opioid agonist prior to delivery.\n\nThis case series illustrates a range of presentations and multimodal treatments for patients taking buprenorphine on the labor and delivery ward, and explores the role of alternative pain management options, including epidural catheters, in these challenging cases.\n\n\nMaterials and methods\n\nAfter receiving Institutional Review Board (IRB) approval from the University of Washington Human Subjects Division (IRB #51693, Committee D), we performed a retrospective chart review to find parturients using buprenorphine or neonates, who received postnatal morphine to determine if their mother had been taking buprenorphine during pregnancy. We included all deliveries, including intrauterine fetal demise, from July 2012 to November 2013, on the University of Washington Medical Center Labor and Delivery unit.\n\n\nResults\n\nThere were 2521 deliveries from 7/1/2012 through 11/30/2013, of which, 152 (6%) received neonatal morphine. A chart review of the biological mothers of each of these neonates found that eight had been taking buprenorphine during pregnancy. Table 1 to Table 4 show the demographic, labor analgesia, Obstetric/Maternal outcome and neonatal outcome data of the eight patients identified. Individual cases are presented below.\n\n• 24 hrs. Post Delivery Pain scores are presented as a range of lowest to highest reported pain score\n\n• Respiratory Depression assessed by continuous pulse oximetry\n\n37yo G3P1 at 39-1/7 weeks gestational age (GSA) who presented with vaginal bleeding and genital herpes. She had a history of polysubstance abuse and was started on buprenorphine (BUP) 8mg PO daily by an outside provider. On the day of admission (DOA), she had an urgent Cesarean section (CS) for possible abruption and fetal intolerance. Intraoperatively, a single shot spinal (SSS) with 100mcg of preservative-free (PF) morphine added to 12 mg of bupivacaine and 10mcg of fentanyl failed to provide adequate anesthesia. Subsequently, a combined spinal-epidural (CSE) using only 10mg of bupivacaine for the repeat spinal anesthesia was placed. Unfortunately, the patient complained of sharp incisional pain despite a negative Allis test to the T4 dermatome. She was then converted to a general anesthetic (GA).\n\nHer post CS pain management included BUP at her admission dose, PO OXY (15mg Q3H), APAP, and IBP. The patient additionally received three doses of 0.4mg IV HM to treat breakthrough pain. Her epidural was continued for 24 hours postoperatively with 0.0625% bupivacaine at 10ml/H. At this point, the patient had successfully transitioned to a PO pain regimen and the epidural was removed. She was discharged on POD 6 (reportedly with inadequate pain control) on her pre-operative BUP dose along with a 10-day supply of HM 2–4 mg PO Q4hrs (120 pills).\n\nTwo weeks after delivery, the patient was found pulseless and cold at a downtown hotel, and CPR was initiated for PEA arrest. The patient recovered to spontaneous circulation, and therapeutic hypothermia for 24 hours was initiated. Her MRI was consistent with anoxic brain injury demonstrating infarcts in the brainstem and cerebellum. A lumbar puncture was performed and creatine kinase bands (CK-BB) were exceptionally high. SSEPS noted an absence of cortical response. The patient was placed on comfort care and died 22 days after delivery. Donation of organs after cardiac death was declined. Opioid overdose was deemed the most likely etiology, based on the patient's history of heroin abuse and accounts from bystanders. OXY was identified in a post-arrest urine drug screen.\n\n27yo G1P0 at 39-1/7 weeks GSA with a history of opioid dependence. She successfully completed an inpatient addiction treatment and was on BUP 8mg daily for one year. She was admitted for IOL in the setting of premature rupture of membranes (PROM). A CSE was placed on the DOA for labor analgesia and later an urgent CS was called for fetal distress. The epidural in situ was dosed for anesthesia in the operating room, but the patient reported a positive Allis test and consequently required a GA.\n\nPost-operatively, the epidural was removed immediately since the epidural did not appear to provide operative anesthesia. She was not administered epidural morphine. Additional post-CS pain management included her pre-operative dose of BUP 8mg daily and a HM PCA; she was transitioned to PO OXY on POD 2. In addition, she did receive PO APAP and IBP throughout. On POD 1 she ambulated, met goals for symptom relief and was satisfied with her pain control. On POD 3, elevated blood pressures in the range of 120–150 mmHg systolic and 80–90 mmHg diastolic were measured and required treatment with furosemide and nifedipine.\n\nShe was discharged on POD 4 after a negative work-up of her hypertension. Discharge medications included a 7-day supply of OXY 5–15mg Q3H PRN (168 pills).\n\n34yo G5P2 at 22-5/7 weeks GSA with a history of bipolar disorder, morbid obesity, bicorneate uterus, and heroin abuse on buprenorphine-naloxone (SuboxoneTM) 8mg daily. Her obstetric history included two prior CS’s and an IUFD. She was admitted for IOL with an IUFD at 22 weeks GSA. A CSE was placed for labor analgesia on the DOA and provided adequate pain relief, but as her labor progressed, she required multiple top-up boluses.\n\nAfter an uneventful NSVD the patient required a dilation and curettage for retained products. Her epidural catheter in situ was successfully used for the surgery, and removed afterwards. Epidural morphine was not administered.\n\nPost-op pain management included PO OXY, APAP, and IBP and her home dose of Suboxone was re-initiated; the patient had discontinued it upon hospital admission.\n\nOn POD 1 the patient was diagnosed with a post-dural puncture headache and received an epidural blood patch with good effect. She required 10mg of PO OXY on 3 occasions during her hospital stay; however, at discharge on POD 1 she was not prescribed opioids.\n\n28yo G6P1 at 36 weeks GSA with cervical shortening, vaginal bleeding and pelvic pressure. She had a PMH significant for four years of BUP 8mg TID and alprazolam 1mg BID, opiate and benzo dependence, several 2nd trimester losses, and a CS at 40 weeks for 2nd stage arrest. She was diagnosed with an IUFD, and continued on her home dose of BUP and alprazolam while inpatient. The patient strongly desired GA for her CS.\n\nHer post CS pain management included a ketamine infusion that was started intra-operatively at 8mg/H and continued post-op for 24H, a fentanyl PCA, PO APAP and IBP, as well as PRN IV lorazepam for anxiety. The patient’s PCA use of fentanyl included 4500mcg (1st 24H), 2600mcg (next 24H) and 3–6 mg IV lorazepam per day. On POD 2 the PCA was discontinued and PO HM was started. BUP was continued throughout her stay. The patient met goals for symptom relief and was satisfied with her pain control. She was discharged on POD 2 with a 10-day supply of HM 4mg PO Q6H (120 pills).\n\n21yo G4P3 at 39 weeks GSA with a body mass index (BMI) of 44, a history of previous low transverse CS, followed by successful vaginal birth after CS (VBAC) twice before. She had a history of heroin abuse and was on BUP 4mg/day. In this pregnancy the fetus had been diagnosed with CDH (congenital diaphragmatic hernia). The patient desired a trial of labor after C-section (TOLAC) and received a CSE for labor analgesia. She remained on her pre-admission dose of BUP throughout her hospital stay. Due to the fetus’ likely poor prognosis, medical staff decided that expediting birth of the fetus would be the safest course of action. After the rupture of membranes and labor augmentation she delivered on the DOA. Unfortunately, the infant died within hours of birth due to complications from CDH.\n\nHer postpartum pain management included PO OXY, APAP and IBP, as well as her outpatient dose of BUP. Pain remained well controlled with this regimen. Her mood was somber and she was grieving appropriately. Postpartum complications included elevated blood pressures without features of pre-eclampsia on post-partum day 1 (PPD). With well controlled pain and appropriate functional status, she was discharged three days after delivery. By the end of the hospital stay, she only required scheduled PO APAP and IBP for pain; she was discharged with no additional short acting opioids.\n\n22yo G1PO at 30-6/7 weeks GSA who presented with preterm PROM. The pregnancy was complicated by heroin and methamphetamine abuse during the first trimester. After admission to the antepartum unit, her home dose of daily BUP 16mg for the remainder of her pregnancy was ordered. On the third day of the hospitalization, prolonged fetal decelerations prompted an urgent CS. A routine CSE was placed for CS anesthesia, and the surgery proceeded uneventfully. The spinal dose included bupivacaine 12.5mg, PF morphine 100mcg, and fentanyl 10mcg. Ketorolac 30mg IV was administered at the end of the case per routine protocol.\n\nHer post CS pain management included an epidural infusion of bupivacaine 0.0625% at 10cc/H, a HM PCA, PO APAP and IBP and her daily home dose BUP. The patient’s pain was well-controlled and she was fully satisfied with pain management. After successful transition to PO HM the epidural was removed. The patient remained satisfied with her pain relief and was discharged on POD 2 with 36 tabs of 2mg HM.\n\nThe neonate was newly diagnosed with congenital anomalies including imperforate anus and esophageal atresia, with a VATER association. The patient stayed with her baby at the local children’s hospital and was unable to visit her distant provider for BUP refills. It was noted that she subsequently relapsed into her previous abuse pattern, within three months postpartum.\n\n35yo G4P2 at 37-1/7 weeks GSA who presented for IOL in the setting of term IUFD in a previous pregnancy. She had a history of opioid dependence following an injury in the military requiring multiple reconstructive knee surgeries. She was placed on BUP 16mg daily for the remainder of her pregnancy and received this also throughout her hospital stay. During her IOL she received a CSE for labor analgesia, followed by an uncomplicated vaginal delivery 2 days after admission.\n\nHer postpartum pain management included PO APAP and IBP and her daily home dose BUP; epidural was removed after delivery. She did not require additional PO opioids during her hospital stay and was discharged without any additional short acting opioids on POD 2.\n\n34yo G1PO at 37-3/7 weeks GSA who presented with SROM. She had a history of opioid dependence following an MVA, in addition to current methamphetamine use. Her PMH was also significant for a congenital ventricular septal defect s/p surgery at age 1yo, with secondary pulmonary stenosis and a dilated right ventricle with mild dysfunction. In addition, the patient had a complex partial seizure disorder, tobacco use, and poor compliance with pregnancy care. She had been on BUP 2mg BID throughout the pregnancy, which was continued during her L&D stay. She received a CSE on the DOA for labor analgesia and required a CS for second stage arrest a day later; the epidural catheter in situ was successfully converted to provide anesthesia. She was not given epidural morphine.\n\nHer post CS pain management included a HM PCA, an epidural infusion (0.1% bupivacaine at 8cc/H), PO APAP, IBP and her home dose of BUP. On POD 2 the epidural infusion was discontinued. On POD 3 she was transitioned to PO OXY and the PCA stopped. She was counseled not to use amphetamines while breastfeeding. On POD 5 she was discharged to home with 30 tabs of 5mg OXY, with the plan of continuing BUP in the outpatient setting. In the following days, she returned to clinic requesting additional opioids due to breast pain; she was subsequently prescribed 20 tabs of 5mg OXY.\n\n\nDiscussion\n\nThis retrospective chart review shows the heterogeneity and complexity of peripartum pain management in patients on buprenorphine (SubutexTM) therapy. Neuraxial techniques, namely continued utilization of epidural catheters placed for labor and/or the cesarean delivery was the most common post-operative analgesic method used or offered to patients. While lumbar epidural analgesia provides effective analgesia8–10, the associated motor block hinders post-cesarean mobilization, often necessitating that epidural infusions be stopped on POD 2, in comparison to other surgical populations where epidural analgesia can be used longer11.\n\nIn addition to our standard post-CS multimodal analgesic regimen, which includes neuraxial opioids, PO APAP, NSAIDs, and OXY, IV ketamine is utilized mainly as a rescue medication for intractable pain. One patient with a non-viable fetus received a low dose (8mg/H) ketamine infusion post-operatively. NMDA receptor antagonist infusions are rarely used on our L&D floor, in part due to uncertainty of fetal central nervous system effects12,13. Similarly, gabapentinoids are reserved for cases where the pain management is complex, due to unclear fetal effects and reported maternal sedation14. None of our reported cases received this class of drug. Most patients received additional IV opioids after their CS’s. Fentanyl was used in one case, while HM was used in four cases. The most effective opioid in the setting of concurrent BUP remains unclear, some suggest using morphine15. The particular strong µ-opioid receptor affinity of BUP, however, complicates the titration of commonly used pure agonists for pain management. To allow for better titration, some suggest the use of shorter acting opioids, like fentanyl, which patient 5 received, in line with our acute pain service recommendations.\n\nIn many of the cases we described, transitioning patients from IV to PO opioid pain medication proved challenging and often required a prolonged hospital stay. OXY is our routine PO opioid and we found it to be effective in six cases; two women preferred PO HM. A retrospective study that matched patients treated with BUP to control patients found that patients maintained on BUP have similar intrapartum pain and analgesic needs during labor, yet experience more postpartum pain and use more opioid analgesia following cesarean delivery16. Theoretically, adding opioids to the local anesthetic epidural infusion for post-operative pain management, compared to an IV PCA system, may reduce maternal plasma levels and subsequent fetal opioid exposure. However we found this not feasible in our teaching institution setting.\n\nAll patients in our series were continued on their home dose of BUP throughout hospitalization. One key consideration is whether patients should be tapered off BUP prior to delivery when operative techniques may be necessary. One case report described a woman who tapered from 24mg of BUP starting at 14 weeks GSA17. The patient demonstrated increased withdrawal symptoms and her fetus showed signs of distress. The woman was re-initiated on BUP and delivered without complication. Further study is needed to investigate the appropriate tapering methods in this population, and each patient’s medical history and psychosocial background must be carefully evaluated. The potential risks of tapering, including autonomic effects and withdrawal symptoms, to both the mother and fetus may not be justified in many cases. The current evidence continues to support the relative safety of BUP; one study found that women who taper their BUP by more than 50% during pregnancy did not have significantly different neonatal outcomes compared to women who remained on the same dose.18. Also, fewer term NAS infants require drug treatment if exposed to BUP compared to methadone.19.\n\nThree women in our series relapsed into pre-pregnancy habits of opioid abuse. One woman unfortunately overdosed and a subsequent urine sample was positive for oxycodone and its metabolites. This emphasizes the importance of post-hospital care and follow-ups in this high-risk population. To this end, the University of Washington operates a perioperative pain clinic staffed with specialized physicians and pharmacists that follow-up with high risk patients. In this setting, opioid weaning can be professionally supported until the regimen is deemed manageable by the primary provider. Utilization of this service is patient dependent, and social disarray is a risk factor for poor compliance.\n\nThe management of post-cesarean pain in patients on partial μ-opioid agonists remains complex and variable, and evidence-based guidelines could be useful for clinicians to direct care. Pre-existing protocols, customized to provide flexibility, could be extremely valuable in a setting that is by its very nature, highly kinetic and often urgent. It is crucial that health care providers dealing with these complicated patients are aware of possible options that offer safe treatment.\n\n\nData availability\n\nAll gathered data was taken directly from patient files, de-identified and entered in the tables presented.\n\n\nEthics and consent\n\nApproval for the study was obtained from the Institutional Review Board (IRB) of the University of Washington Human Subjects Division (IRB #51693, Committee D). For this study a waiver of consent from patients was obtained from the IRB.",
"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\nBateman BT, Hernandez-Diaz S, Rathmell JP, et al.: Patterns of opioid utilization in pregnancy in a large cohort of commercial insurance beneficiaries in the United States. Anesthesiology. 2014; 120(5): 1216–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFDA Drug Safety Communication: FDA has reviewed possible risks of pain medicine use during pregnancy. Accessed March 30, 2017. Reference Source\n\nACOG Committee on Health Care for Underserved Women; American Society of Addiction Medicine: ACOG Committee Opinion No. 524: Opioid abuse, dependence, and addiction in pregnancy. Obstet Gynecol. 2012; 119(5): 1070–6. PubMed Abstract | Publisher Full Text\n\nLutfy K, Eitan S, Bryant CD, et al.: Buprenorphine-induced antinociception is mediated by mu-opioid receptors and compromised by concomitant activation of opioid receptor-like receptors. J Neurosci. 2003; 23(32): 10331–7. PubMed Abstract\n\nJohnson RE, Jones HE, Fischer G: Use of buprenorphine in pregnancy: patient management and effects on the neonate. Drug Alcohol Depend. 2003; 70(2 Suppl): S87–101. PubMed Abstract | Publisher Full Text\n\nGaalema DE, Scott TL, Heil SH, et al.: Differences in the profile of neonatal abstinence syndrome signs in methadone- versus buprenorphine-exposed neonates. Addiction. 2012; 107 Suppl 1: 53–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones HE, O’Grady K, Dahne J, et al.: Management of acute postpartum pain in patients maintained on methadone or buprenorphine during pregnancy. Am J Drug Alcohol Abuse. 2009; 35(3): 151–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRamin SM, Gambling DR, Lucas MJ, et al.: Randomized trial of epidural versus intravenous analgesia during labor. Obstet Gynecol. 1995; 86(5): 783–789. PubMed Abstract | Publisher Full Text\n\nDickinson JE, Paech MJ, McDonald SJ, et al.: Maternal satisfaction with childbirth and intrapartum analgesia in nulliparous labour. Aust N Z J Obstet Gynaecol. 2003; 43(6): 463–468. PubMed Abstract | Publisher Full Text\n\nSharma SK, McIntire DD, Wiley J, et al.: Labor analgesia and cesarean delivery: an individual patient meta-analysis of nulliparous women. Anesthesiology. 2004; 100(1): 142–148; discussion 6A. PubMed Abstract | Publisher Full Text\n\nComparative Obstetric Mobile Epidural Trial (COMET) Study Group UK: Effect of low-dose mobile versus traditional epidural techniques on mode of delivery: a randomised controlled trial. Lancet. 2001; 358(9275): 19–23. PubMed Abstract | Publisher Full Text\n\nZhao T, Li Y, Wei W, et al.: Ketamine administered to pregnant rats in the second trimester causes long-lasting behavioral disorders in offspring. Neurobiol Dis. 2014; 68: 145–55. PubMed Abstract | Publisher Full Text\n\nDong C, Rovnaghi CR, Anand KJ: Ketamine affects the neurogenesis of rat fetal neural stem progenitor cells via the PI3K/Akt-p27 signaling pathway. Birth Defects Res B Dev Reprod Toxicol. 2014; 101(5): 355–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMonks DT, Hoppe DW, Downey K, et al.: A Perioperative Course of Gabapentin Does Not Produce a Clinically Meaningful Improvement in Analgesia after Cesarean Delivery: A Randomized Controlled Trial. Anesthesiology. 2015; 123(2): 320–6. PubMed Abstract | Publisher Full Text\n\nSOAP 2013 Summer Newsletter: Education Committee: Post Cesarean Pain Management in the Buprenorphine (Subutex) Dependent Patient.\n\nMeyer M, Paranya G, Keefer Norris A, et al.: Intrapartum and postpartum analgesia for women maintained on buprenorphine during pregnancy. Eur J Pain. 2010; 14(9): 939–43. PubMed Abstract | Publisher Full Text\n\nWell-Strand GK, Kvamme O, Andreassen A, et al.: A woman’s experience of tapering from buprenorphine during pregnancy. BMJ Case Rep. 2014; 2014: pii: bcr2014207207. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWelle-Strand GK, Skurtveit S, Tanum L, et al.: Tapering from Methadone or Buprenorphine during Pregnancy: Maternal and Neonatal Outcomes in Norway 1996–2009. Eur Addict Res. 2015; 21(5): 253–261. PubMed Abstract | Publisher Full Text\n\nNanda S, Brant R, Regier M, et al.: Buprenorphine: a new player in neonatal withdrawal syndrome. W V Med J. 2015; 111(1): 16–21. PubMed Abstract"
}
|
[
{
"id": "29456",
"date": "01 Feb 2018",
"name": "Emily J. Baird",
"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\nTith et al. highlight the complexity of the analgesic management of labor and delivery in parturients receiving buprenorphine. The retrospective chart review details the peripartum course of 8 women on buprenorphine maintenance. Given the heterogeneity in patient demographics, buprenorphine dose, analgesic regimen, mode of delivery, and neonatal outcomes, it is difficult to extract meaningful conclusions. The vast disparateness of the peripartum management of parturients on buprenorphine vividly demonstrates the need for evidence-based practice guidelines.\nAlthough the details of the individual patient’s peripartum course are interesting, the comprehensiveness of each description is distracting. Since the focus of the review is the analgesic management of labor and delivery on patients receiving buprenorphine, consider omitting extraneous maternal and neonatal details. The patients’ descriptions should conclude with discharge. Details such as “two weeks after delivery, patient was found pulseless…,” “patient stayed with her baby at the local children’s hospital…,” and “in the following days, she returned to clinic requesting opioids due to breast pain” detract from the intention of the review. Similarly, the specifics of the neonate’s postdelivery course (i.e. diagnosis of imperforate anus) are irrelevant. Concise reconstruction of the results section will highlight the focus of this review.\nTables 1-4 are not referred to in the text. Without further explanation of the tables in the text, it is unclear what information the table is intended to convey.\nTable 2 (Labor Analgesia Data) is a bit misleading. For patient 1, the table indicates the patient had a CSE for labor that required no “top-ups” and resulted in a VAS score of zero. According to the results section, patient 1 had a failed single shot spinal, followed by a CSE, and ultimately needed a general anesthetic for cesarean delivery. This seems to suggest that the patient never received labor analgesia but rather the CSE was placed for surgical anesthesia.\nThe absences of a comprehensive legend for Table 3 makes it challenging to interpret. Twelve abbreviations are used in Table 3 which are not defined until the following page. Consider including a key to the abbreviations in the table legend. In addition, since respiratory depression did not occur in any parturient, consider removing it from the table.\nThe discussion section would be more meaningful if it offered some interpretation of the data rather than summarizing the results presented in the previous section. Specifically, why did 3 of the 5 women undergoing cesarean delivery have a general anesthetic? Based on the limited experience, what is the optimal labor analgesia regimen? Post vaginal delivery pain regimen? Post-operative regimen? Neonatal implications of intrauterine exposure to buprenorphine?\nTith et al.’s retrospective review of the periparturm course of parturients dependent on buprenorphine illustrates the heterogeneity of analgesic regimens for labor and delivery. The review highlights the need for research to help develop protocols and standards.\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 source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
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https://f1000research.com/articles/7-7
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https://f1000research.com/articles/7-179/v1
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12 Feb 18
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{
"type": "Brief Report",
"title": "A comparison of computationally predicted functional metagenomes and microarray analysis for microbial P cycle genes in a unique basalt-soil forest",
"authors": [
"Erick S. LeBrun",
"Sanghoon Kang",
"Erick S. LeBrun"
],
"abstract": "Here we compared microbial results for the same Phosphorus (P) biogeochemical cycle genes from a GeoChip microarray and PICRUSt functional predictions from 16S rRNA data for 20 samples in the four spatially separated Gotjawal forests on Jeju Island in South Korea. The high homogeneity of microbial communities detected at each site allows sites to act as environmental replicates for comparing the two different functional analysis methods. We found that while both methods capture the homogeneity of the system, both differed greatly in the total abundance of genes detected, as well as the diversity of taxa detected. Additionally, we introduce a more comprehensive functional assay that again captures the homogeneity of the system but also captures more extensive community gene and taxonomic information and depth. While both methods have their advantages and limitations, PICRUSt appears better suited to asking questions specifically related to microbial community P as we did here. This comparison of methods makes important distinctions between both the results and the capabilities of each method and can help select the best tool for answering different scientific questions.",
"keywords": [
"Metagenome",
"phosphorus",
"microbial communities",
"MiSeq",
"PICRUSt",
"GeoChip",
"nutrient cycling"
],
"content": "Introduction\n\nRelating the functionality of microbes to environmental factors is one of the primary goals in microbial ecology. With the advent of modern genomic technologies, such as next generation sequencing and microarray hybridization, there are more options than ever to test environmental community’s genomics and functional capabilities. Metagenome sequencing is one of the most thorough and comprehensive methods currently available for looking at microbial community gene compositions1–5, but can be costly and generate enormous data sets that require a large amount of work in processing, analysis, and storage. Two technologies currently in use for looking at community functional profiles that can be less expensive and more accessible than metagenome sequencing include computationally predicted functional metagenomes (PFMs)6 and microarray analyses7. These technologies both have known advantages and disadvantages8, but investigation into how they compare in the same system is still needed.\n\nHere we compare PFMs from Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt)6 to GeoChip9 microarray data. While both methods are distinct, they can each be applied to an environmental community gene pool to estimate the presence and abundance of genes within the community genomic landscape related to function. Resulting datasets from each technique are tables showing counts of genes or functions as determined by either probes (microarray) or reference data (PFMs), and therefore are directly comparable in the context of functional gene landscapes within the system. We utilize 20 sites in a unique basalt-soil Gotjawal forest on Jeju Island in Korea. Despite being both rocky, lava-formed basalt and having dense vegetation10, this forest is considered a wetland environment due to the homogenous, rocky soil and its capacity for absorbing water11. All 20 sites, though spatially separated by distance of 5 km to 65 km (Figure S1), showed strong homogeneity in bacterial/archaeal community assemblies in 16S rRNA gene taxonomic analysis (Figure S2) and so act as replicates in this system for the current study. This makes it ideal for comparing the technologies. We specifically look at how these technologies perform related to the same phosphorus (P) cycle genes as the unique basalt-soil environment has the potential to be a unique P environment12–14.\n\n\nMethods\n\nGeoChip 4.0 data for P cycle genes came from Kim et al.15. For sequencing data, we started with raw sequencing files also from the study by Kim et al.16. Paired-end reads were combined using the join-fastq algorithm from eautils17. Un-paired reads were discarded at this time. Additional sequence processing was performed using Quantitative Insights Into Microbial Ecology (QIIME) version 1.9.118. Sequences were then filtered with a maximum unacceptable Phred quality score of 20. Chimeric sequences were identified and removed using the UCHIME algorithm within USEARCH19. Operational taxonomic unit (OTU) picking was performed via open reference using uclust against the Greengenes 13_8 database with a 0.97 similarity cutoff20. Singleton sequences were removed during OTU picking and taxonomy was assigned with Greengenes 13_8 database as reference.\n\nOnly reads identified in closed reference picking were used for the PICRUSt analysis. Using PICRUSt6, predicted functional metagenomes (PFMs) were constructed from the resulting 16S rRNA sequences. PFMs were generated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database21,22 as a functional reference.\n\nThe GeoChip 4.0 data provided probe data for genes identified as “phytase”, “ppk”, and “ppx”. We identified these genes in the KEGG database to have the KEGG orthology (KO) numbers K01083 and K01093 for phytase, K00937 for ppk, and K01514 for ppx These KO numbers were the only PICRUSt results extracted for direct comparison. Additionally, we built another P assay in PICRUSt utilizing 417 KO numbers associated with P (Table S1).\n\nAll analyses were performed in the R software package v.3.2.323. The relationship between the PICRUSt and GeoChip data was tested using a Mantel test with the Pearson correlation method and 1,000 permutations through the vegan package24. Non-metric multidimensional scaling (NMDS) ordinations were constructed using Bray-Curtis dissimilarity through the vegan package. A PROcrustean randomization TEST of community environment concordance (PROTEST), a potentially more sensitive detection method than a Mantel test, was also used to compare the NMDS ordinations to each other25. Figures and plots were created using the ggplot2 package26.\n\n\nResults and discussion\n\nBoth PICRUSt and GeoChip appear to have captured the homogeneity of the system (Figure 1). PICRUSt captured much more diversity and depth in terms of taxa identified (Figure 1) and total counts (Figure 2) than GeoChip. PICRUSt identified organisms from 40 different phyla where GeoChip identified organisms from 15. Total counts at each site for the two methods were on a very different scale. When placed on a scale that shows the variation in each set of counts, it becomes apparent that the trends of total counts across sites do not match between methods (Figure S3). The Mantel test resulted in no significant statistic between the two data sets and Procrustes analysis confirmed this, showing no significant correlation either (Figure S4). The same analyses were performed with the data for each gene isolated and each of the three genes independently provided similar results of inconsistency between methods to the comparison of total gene datasets. There was no correlation between the datasets in Mantel or Procrustes analysis and gene counts and trends were markedly different.\n\nThe new PICRUSt assay with 417 P related genes captured the system homogeneity but with additional depth (Figure S5). The new assay identified organisms from 41 phyla similar to the smaller, comparative assay’s 40 but also provided data counts per site ranging from ~70,000 to ~110,000. The PICRUSt dataset from the new assay not only represents what is likely a better dataset for answering community functional questions within the P cycle than the previous, comparative PICRUSt or GeoChip datasets, but also illustrates an important difference between the two methods. While both methods could be considered “closed-format” technologies in that they are reliant on the available known references8, the process of adapting or updating the two methods contrasts. The method of using computational predictions is highly adaptable and allows for the easy inclusion or exclusion of additional genes6. Improving or expanding the reference database that computational prediction can be achieved through simply updating the curated reference database. The microarray method is more involved including the identification, creation, and inclusion of specific target probes into the manufacturing of a microarray7.\n\nIt is important to note that for our comparison we are specifically looking at functional genes within the P biogeochemical cycle. Both methods explored are designed for, and capable of looking a more comprehensive whole functional profile for communities. Computational functional prediction seems to be better suited to the task of viewing independent functional groupings as we did here. While microarrays have shown linear relationships to RNA and DNA levels in environmental systems16,27, they are limited in coverage and small sequence divergence can affect quantitative capability7. These quantitative limitations should be carefully considered in light of recent findings showing that the composition of P cycle genes in some microbial communities are more closely related to environmental P levels than absolute abundance1. Computational functional prediction again seems better equipped to handle questions related to functional gene composition due to the high specificity of probes to taxa and limited genes included in microarrays. It is also important to note that the data from both methods is representative of DNA present in microbial communities and not true expression levels or enzyme abundance.\n\n\nConclusions\n\nComputational functional prediction and microarray analysis of P cycle genes both captured system homogeneity. However, they did not agree in terms of capturing absolute abundance or taxonomic composition in P cycle genes. Computational functional prediction provided more count depth and taxonomic diversity than microarray analysis did. The ease with which computational functional prediction is adapted additionally allowed for the capture of additional genes and taxonomic diversity in P function along with increased depth by expanding the PICRUSt assay to include 417 KO numbers related to P function instead of the original 4 used in the microarray comparison. While we compared two methods for the exploration of functional P cycle genes within microbial communities to each other, an additional comparison to whole metagenome data in a system would further validate either method.\n\n\nData availability\n\nThe sequence data used in this study was deposited in the NCBI Sequence Read Archive (SRA) under the BioSample accession numbers SAMN06049757 to SAMN06049776. The GeoChip microarray data used in this study is available in OSF: http://doi.org/10.17605/OSF.IO/AT93H28.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors acknowledge Dr. Jong-Shik Kim (Geyongbuk Institute for Marine Bioindustry, Korea) for the data and the site map made available for this study.\n\n\nSupplemental material\n\nSupplementary File 1: File containing all supplementary figures referenced in main text (Figure S1–S5).\n\nClick here to access the data.\n\nSupplementary File 2: File containing the supplementary table referenced in main text (Table S1).\n\nClick here to access the data.\n\n\nReferences\n\nLeBrun ES, King RS, Back JA, et al.: A metagenome-based investigation of gene relationships for non-substrate associated microbial phosphorus cycling in the water column of streams and rivers. Rev. 2018.\n\nDaniel R: The soil metagenome--a rich resource for the discovery of novel natural products. Curr Opin Biotechnol. 2004; 15(3): 199–204. PubMed Abstract | Publisher Full Text\n\nUchiyama T, Abe T, Ikemura T, et al.: Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nat Biotechnol. 2005; 23(1): 88–93. PubMed Abstract | Publisher Full Text\n\nInskeep WP, Jay ZJ, Tringe SG, et al.: The YNP Metagenome Project: Environmental Parameters Responsible for Microbial Distribution in the Yellowstone Geothermal Ecosystem. Front Microbiol. 2013; 4: 67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQin J, Li Y, Cai Z, et al.: A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature. 2012; 490(7418): 55–60. PubMed Abstract | Publisher Full Text\n\nLangille MG, Zaneveld J, Caporaso JG, et al.: Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol. 2013; 31(9): 814–821. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHe Z, Deng Y, Zhou J: Development of functional gene microarrays for microbial community analysis. Curr Opin Biotechnol. 2012; 23(1): 49–55. PubMed Abstract | Publisher Full Text\n\nZhou J, He Z, Yang Y, et al.: High-throughput metagenomic technologies for complex microbial community analysis: open and closed formats. MBio. 2015; 6(1): pii: e02288-14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTu Q, Yu H, He Z, et al.: GeoChip 4: a functional gene-array-based high-throughput environmental technology for microbial community analysis. Mol Ecol Resour. 2014; 14(5): 914–28. PubMed Abstract | Publisher Full Text\n\nKim JS, Jung MY, Lee KC, et al.: The Archaea Community Associated with Lava-Formed Gotjawal Forest Soil in Jeju, Korea. J Agric Chem Environ. 2014; 03(03): 96. Publisher Full Text\n\nJang YC, Lee CW: Gotjawal Forest in Jeju Island as an Internationally Important Wetland. J Wetl Res. 2009; 11.\n\nToplis MJ, Libourel G, Carroll MR: The role of phosphorus in crystallisation processes of basalt: An experimental study. Geochim Cosmochim Acta. 1994; 58(2): 797–810. Publisher Full Text\n\nScheu S: Analysis of the microbial nutrient status in soil microcompartments: earthworm faeces from a basalt–limestone gradient. Geoderma. 1993; 56(1–4): 575–86. Publisher Full Text\n\nWells N, Saunders WMH: Soil studies using sweet vernal to assess element availability IV. Phosphorus. N Z J Agric Res. 1960; 3(2): 279–99. Publisher Full Text\n\nKim JS, Kim DS, Lee KC, et al.: Microbial Community Structure and Functional Potential of Lava-Formed Gotjawal Soils in Jeju, Korea. Rev. 2017.\n\nWu L, Thompson DK, Li G, et al.: Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment. Appl Environ Microbiol. 2001; 67(12): 5780–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAronesty E: Comparison of sequencing utility programs. Open Bioinforma J. 2013; 7(1): 1–8. Publisher Full Text\n\nCaporaso JG, Kuczynski J, Stombaugh J, et al.: QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010; 7(5): 335–336. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdgar RC: Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010; 26(19): 2460–2461. PubMed Abstract | Publisher Full Text\n\nDeSantis TZ, Hugenholtz P, Larsen N, et al.: Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 2006; 72(7): 5069–5072. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28(1): 27–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKanehisa M, Sato Y, Kawashima M, et al.: KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016; 44(D1): D457–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nR Core Team: R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. [Internet]. 2015. Reference Source\n\nOksanen J, Blanchet FG, Kindt R, et al.: vegan: Community Ecology Package. R package version 2.4-1. 2016.\n\nJackson DA: PROTEST: a PROcrustean randomization TEST of community environment concordance. Ecoscience. 1995; 2(3): 297–303. Publisher Full Text\n\nWickham H: ggplot: An Implementation of the Grammar of Graphics. R Package Version 210 [Internet]. 2006; [cited 2016 Oct 27]. Reference Source\n\nRhee SK, Liu X, Wu L, et al.: Detection of Genes Involved in Biodegradation and Biotransformation in Microbial Communities by Using 50-Mer Oligonucleotide Microarrays. Appl Environ Microbiol. 2004; 70(7): 4303–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeBrun ES, Kang S: Jeju Island Gotjawal GeoChip 4.0 Data for Phosphorus Genes. Open Science Framework. 2018. Publisher Full Text"
}
|
[
{
"id": "30837",
"date": "23 Mar 2018",
"name": "Ye Deng",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors reported a parallel comparison on two most popular high-throughput technologies on environmental microbiota studies, 16S sequencing and GeoChip. Other than taxa aspect, they focused on the comparison of functional gene involving in P. Overall, the methods and results are sound that 16S sequencing detected more taxa involving in P since 417 KO could be predicted by PtCRUST rather than 3 KO in GeoChip. It is unsurprising because functional gene microarray can only pick up limited number of genes in each bio-process as marked functional traits, while the high-throughput sequencing can be much more open to uncultured and novel taxa in environments. Zhou et al. already mentioned this opinion in their review paper1 as well. Thus, according to the policy of F1000, I think this paper can be indexed.",
"responses": []
},
{
"id": "32731",
"date": "17 Jul 2018",
"name": "Joy D Van Nostrand",
"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\nLeBrun and Kang compared two metagenomic functional gene datasets of the same microbial community, functional gene microarray and PICRUSt 16S rRNA sequencing data. PICRUSt detected a much higher gene diversity and abundance than the microarray. Some of that difference could be accounted for by group probes on the GeoChip microarray that cover two or more similar sequences, although PICRUSt offers a much larger database. The authors make a good point about the relative ease of expanding coverage by including additional genes for the PICRUSt method. Microarray require additional steps and manufacturing a new array compared to simply updating a database for PICRUSt. The methods used and the conclusions are sound. This paper can be indexed.\nMinor edit - The second Kim et al. reference in the methods section has an incorrect citation - I believe it should be 10 (or 15)",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-179
|
https://f1000research.com/articles/7-178/v1
|
12 Feb 18
|
{
"type": "Research Article",
"title": "Extended pharmacological miosis is superfluous after glaucoma angle surgery: A retrospective study",
"authors": [
"Hamed Esfandiari",
"Kiana Hassanpour",
"Mehdi Yaseri",
"Nils A. Loewen",
"Hamed Esfandiari",
"Kiana Hassanpour",
"Mehdi Yaseri"
],
"abstract": "Background: Pilocarpine is commonly used after angle surgery for glaucoma despite a host of side effects and risks. We hypothesized that a pharmacological miosis during the first two months does not improve short- and long-term results of trabectome-mediated ab interno trabeculectomy. Methods: In this retrospective comparative 1-year case series, we compared 187 trabectome surgery eyes with (P+) or without (P-) 1% pilocarpine for two months. Primary outcome measures were the surgical success defined as intraocular pressure (IOP) ≤ 21 mmHg and decreased ≥ 20%, and no secondary glaucoma surgery. Secondary outcome measures were the number of glaucoma medications, complications, and IOP. Results: We categorized 86 (46%) eyes as P- and 101 (54%) eyes as P+. The mean age was 69.8±10.1 in P- and 70.5±9.4 in P+ (P=0.617) with equal gender distribution (P=0.38). The cumulative probability of qualified success at 12 months was 78.1% in the P- and 81% in the P+ (P=0.35). The IOP was decreased significantly from 20.2±6.8 mmHg at baseline to 15.0±4.8 mmHg at 12 months follow-up in P- (P=0.001) and 18.8±5.3 and 14.7±4.0, respectively (P=0.001). The medications decreased significantly from 1.4±1.2 in P- and 1.4±1.2 in P+ at baseline to 1.0±1.2 and 0.7±1.0, respectively (P=0.183). P- and P+ did not differ in IOP or medications (all P>0.05). In Multivariate Cox Regression models, the baseline IOP and central corneal thickness were associated with failure. Conclusions: Use of postoperative pilocarpine does not improve the efficacy of trabectome surgery.",
"keywords": [
"Trabectome surgery",
"ab interno trabeculectomy",
"pilocarpine eye drop",
"miotics",
"peripheral anterior synechiae"
],
"content": "Introduction\n\nAb interno angle surgeries that disrupt or bypass the trabecular meshwork (TM) are bleb-less procedures without the need for office procedures and application of antifibrotics, the frequency of postoperative visits can be reduced1. Most surgeons administer pilocarpine hydrochloride 1% for two months to decrease the apposition between iris root and trabecular meshwork and minimize the risk of peripheral anterior synechiae (PAS) formation. Although this seems intuitive, miotics shallow the anterior chamber when the ciliary muscle contracts and can cause angle closure in phakic eyes2. There is no correlation between the degree of PAS formation and the amount of intraocular pressure (IOP) elevation after trabectome surgery3,4, a common and mature form of ab interno trabeculectomy5 appropriate for different types of glaucomas, including angle closure6, pigmentary7, exfoliative8, steroid-induced9, uveitic10, and advanced glaucomas11, as well as failed trabeculectomy and glaucoma shunt procedure12,13.\n\nPilocarpine is a nonselective muscarinic receptor agonist that has been used extensively in the medical management of glaucoma for almost 150 years14. It attaches to muscarinic receptors on ciliary smooth muscle and causes contraction of the longitudinal fibres. Following longitudinal muscle contraction, scleral spur is pulled back leading to expansion of juxtacanalicular portion of the TM and Schlemn’s canal (SC) and consequently enhance the aqueous outflow15,16. However, a TM-mediated IOP effect, for instance by adding cataract surgery, becomes negligible after trabectome surgery17,18, because the aqueous can bypass the remaining TM. Patients dislike miotics because of their side effects of headaches, nearsightedness, dim vision and more serious problems that include retinal detachment, worsening intraocular inflammation, paradoxical increase in IOP and cataract formation19–23.\n\nSince there is no evidence that pilocarpine decreases PAS formation after trabectome surgery24, along with the fact that PAS is not a risk factor for trabectome failure established without doubt25, we hypothesized that postoperative pilocarpine does not improve the outcome and stopped its use in our practice. The present study compares the outcome of operation with pilocarpine versus no pilocarpine to test out hypothesis.\n\n\nMethods\n\nInstitutional Review Board approval was obtained from the University of Pittsburgh Human Subjects Research Committee (approval number: PRO13120466). An informed consent from the patients was waived due to the low risk nature of this retrospective study. We followed the tenets of the Declaration of Helsinki and regulations of the Health Insurance Portability and Accountability Act.\n\nThis retrospective comparative study included all patients who underwent Trabectome surgery between July 2012 and October 2017 at the Eye and Ear Institute of the University of Pittsburgh, School of Medicine, in Pittsburgh, PA, United States. Patients were identified using current procedural terminology codes of Eye and Ear Institute, University of Pittsburgh.\n\nPatients were included regardless of same-session phacoemulsification, because the impact of same-session phacoemulsification on IOP is negligible in trabectome surgery17,18. Eyes were categorized as P- if no pilocarpine was used after the surgery, and P+ if pilocarpine was administered postoperatively.\n\nExclusion criteria were a history of incisional and angle surgeries, combined glaucoma surgeries, use of pilocarpine in the contralateral eye, any systemic medication with parasympathomimetic activities, and less than 3 months follow-up.\n\nInformation collected included demographic data, types of glaucoma, pre- and postoperative intraocular pressures (IOP), baseline ocular biometric characteristics including axial length (AL), central corneal thickness (CCT), anterior chamber depth (ACD), number of pre-and postoperative glaucoma medications, visual field mean deviation (MD), visual field index (VFI), type of surgery, use of pilocarpine eye drop after the surgery, and intra- and postoperative complications.\n\nThe primary outcome measure was success and defined as 5 < IOP ≤ 21 mmHg, ≥ 20% reduction of IOP from baseline at two consecutive visits, no need for further glaucoma surgery, and no loss of light perception. Qualified success was defined as achieving success with or without medications. Patients who achieved success without medication were labeled as complete success. A Kaplan Meier survival analysis was used to evaluate success rates. The secondary outcome measures were IOP, glaucoma medications, and complications.\n\nIn the case of combined phacoemulsification, trabectome (NeoMedix, Tustin, CA, USA) portion was performed first. Both the patient’s head and microscope were tilted 30° away from the surgeon. A temporal 1.6 mm incision was made 2 mm anterior to the limbus and parallel to the iris plane. The trabectome was inserted through the incision and positioned across the AC along the nasal angle. The tip of the trabectome was inserted into Schlemm’s canal and engaged with the TM. The aspiration and ablation was activated with the power set to 0.8 to 1W and the TM removed over approximately 160°. The tip was withdrawn from anterior chamber and the incision was hydrated to achieve watertightness. All glaucoma medications were discontinued after the surgery and were resumed if IOP was not within target range. In P+, 1% pilocarpine was used 4 times a day for one month followed by three times a day for a second month.\n\nAll analyses were performed using SPSS software (SPSS Statistics for Windows, Version 25, Armonk, NY, IBM Corporation). Frequency, percent, mean±SD, median, and range were used to describe the data. To evaluate the baseline differences between the two study groups, we used the T-test, Mann Whitney, and Chi-Square test. To compare the change in IOP and number of medications between the two groups or within a group, we used an interaction analysis within a linear mixed model. To compare the amount of IOP reduction between the groups adjusted for the baseline values, we used an Analysis of Covariance (ANCOVA). Kaplan-Meier survival plots were constructed to assess the long-term survival rates and compared by the log-rank test. A Cox proportional Hazard model was used to find risk factors for failure and to estimate the adjusted Hazard ratio of each factor. In the last step to obtain the most important factors, we used a backward variable elimination method based on the LR test. Statistical significance was set at p< 0.05. Success was defined as IOP <21 mmHg and a >20% reduction from baseline with no need for additional glaucoma surgery.\n\n\nResults\n\nA total of 187 eyes were included in this study. Eighty-six (46%) eyes in P- did not receive pilocarpine, while 101 (54%) in P+ did (Table 1). All patients were phakic at the time of surgery. Phacoemulsification was combined in 147 (79%) eyes (TP) while 40 eyes had trabectome surgery alone (T). The mean age of participants was 69.8±10.1 in P- and 70.5±9.4 in P+. Primary open-angle glaucoma was the most common diagnosis in both groups (73.3% and 63.2% in P- and P+, respectively, P=0.64). There were no significant differences between the study groups in terms of gender, preoperative intraocular pressure, central corneal thickness, axial length, visual field mean deviation, anterior chamber depth, baseline number of glaucoma medications, and type of glaucoma (Table 1).\n\n†Based on T-test. *Based on Chi-square. ‡ Based on Mann-Whitney test. AC: Anterior chamber, CACG: Chronic angle closure glaucoma, CCT: Central corneal thickness, HVF MD: Humphrey Visual Field Mean Deviation, IOP: intraocular pressure, POAG: primary open angle glaucoma, XFG: exfoliation glaucoma.\n\nKaplan-Meier survival curves (Figure 1) indicated a mean survival of 34.03±2.35 months in P- and 38.32±1.94 months in P+ with no statistically significant difference between two groups (log rank:0.87 P=0.35). The addition of phacoemulsification did not make a difference on survival (T: 33.9±3.2 months; TP: 32.3±1.7, log-rank:0.81 P=0.36). Similarly, pilocarpine did not have a significant effect on survival in T or TP: T in P- had a survival of 32.5±5.4 and T in P+ had a survival of 33.3±3.9 months (log-rank:0.06, P=0.8). TP in P- had a survival of 33.6±2.4 and TP in P+ had a survival of 36.2±2.2 months (log-rank:0.81, P=0.36). The multivariate Cox regression model was stratified by group, age, baseline IOP, number of medications, glaucoma type, CCT, AC depth, lens thickness, axial length, Humphrey visual field (HVF) MD and VFI (Table 2). The final model with backward elimination only included baseline IOP (mmHg) and CCT (micron) with HR of 4.1 for 22<IOP<28, 3.7 for IOP>28, and 1.03 for CCT. This means that in patients with 22<IOP<28 the risk of failure was 4.1 times higher compared to patients with IOP<18 (P=0.015). Every 10 micron increase in baseline CCT increased the risk of failure by 30% (P=0.005).\n\nSuccess rates were similar in both groups. Survival plots of P- and P+ for subgroup analysis separated by B) glaucoma surgery alone and C) same session phacoemulsification.\n\nResults from multivariate cox proportional hazard regression model. IOP, intraocular pressure; T, trabectome surgery; TP, trabectome surgery with phacoemulsification; CCT, central corneal thickness; AC, anterior chamber depth; HVF MD, Humphrey Visual Field Mean Deviation; HVF VFI, Humphrey Visual Field visual field index.\n\nIOP was decreased significantly from 20.2±6.8 mmHg at baseline to 15.0±4.8 mmHg at 12 months follow-up in P- (P=0.001, Figure 2). The corresponding numbers for P+ were 18.8±5.3 and 14.7±4.1, respectively (P=0.001). There was no significant difference in IOP at each follow-up visit between the two groups (Table 3). In subgroup analysis, considering the effect of phacoemulsification, there was no significant difference between IOPs of both groups in T and TP throughout the course of follow-up (Figure 2). Seven (8.1%) cases in P- and five (5%) in P+ experienced IOP spikes. There was no significant difference between the groups (P=0.153, based on the Chi-square test). The baseline number of glaucoma medications was 1.4±1.2 in P- and 1.4±1.2 in P+ (P=0.910). At month 12, the number of glaucoma medications was 1.0±1.2 drops in P- and 0.7±1.0 in P+ (P=0.082, Table 4).\n\nA) IOP in P- was similar to P+ through follow-up duration. IOP in both groups for subgroup analysis separated by B) glaucoma surgery alone. Patients who received pilocarpine showed lower IOPs during the first month but the difference was not statistically significant. C) Same session phacoemulsification. Error bars represent standard deviations.\n\nThe postoperative hyphema was observed in 49% of the eyes. The mean duration of hyphema was 10.68±9.96 days in P- and 12±4.30 days in P+ (P= 0.48).\n\n\nDiscussion\n\nMiotics are commonly used at the conclusion of cataract surgery to retract a floppy iris from the incision26 or to secure a sulcus lens implant after a complex cataract surgery27. It is, therefore, not surprising that ab interno glaucoma surgeons resort to the same medications despite reports of worsening of iritis28,29, retinal detachment and further angle narrowing, as the most serious complications19–23. Interestingly, a narrow angle is not a contraindication to trabectome surgery and yields similar results to wide angles6. In the current study, the survival, IOP reduction, and duration of postoperative hyphema were comparable regardless of pilocarpine use. Both groups experienced an IOP reduction like that observed in previous studies5,6,30.\n\nIt is unclear what may cause a late failure after trabectome surgery as the aqueous humor is not redirected into a conjunctival bleb maintained by antifibrotics or into tube shunts and microbypasses that cause a chronic foreign body reaction. Certainly, descemetization31 of the outer wall of Schlemm’s canal and PAS formation may be causes of surgical failure4,5,32, but it is not established whether miotics prevent this. Additionally, the incidence of PAS ranges from 20% to 40% which does not match the relatively high success rate observed with this procedure3–5. In one study by Minckler et al., the incidence of PAS was 38%33, while in another study by Wang et al. without pilocarpine, the incidence of PAS was 20%4.\n\nThe results of our study match established findings from other studies that higher baseline IOP is associated with larger IOP reduction5,25. It is consistent with the fact that after removing the site of the highest outflow resistance by trabectome, aqueous outflow would be limited only by downstream pathway. While post-trabectome IOPs tend to be similar in all patients, those with higher preoperative IOP experience larger IOP reduction11.\n\nOur study indicates a link between corneal biomechanics and risk of failure following trabectome surgery. Corneal thickness serves as an in vivo surrogate marker of corneal rigidity and is associated with biomechanical properties of the cornea34 and likely the sclera35–37. After trabecular ablation, outflow is only limited by the remaining outflow resistance in collector channels and episcleral veins. The collector channels and the intrascleral plexus have features that indicate they can regulate the outflow resistance like the remainder of the vascular system38,39. This novel finding invites further researches to clarify the role of corneal and scleral biomechanics in predicting the behavior of the downstream pathway in angle surgery.\n\nThis study was limited by its retrospective nature and number of patients. It was powered to detect a difference of 2 mmHg between the two groups (power 0.8, alpha 0.5), a clinically meaningful difference. It would have required close to 360 individuals in each group to detect a 1 mmHg difference. Patients were not randomized or matched and PAS was not assessed systematically.\n\nIn conclusion, our study indicates that administering pilocarpine eye drop after trabectome surgery does not improve the outcome of the procedure. Given the multitude of side effects, we recommend it be avoided. A thicker cornea was associated with a higher risk of failure.\n\n\nData availability\n\nDataset 1: Raw data collected from all study participants. Coding scheme for the data contained in Word document. DOI, http://dx.doi.org/10.5256/f1000research.13756.d19374940",
"appendix": "Competing interests\n\n\n\nHE, KH, and MY have no financial disclosure. NAL has received honoraria for trabectome wet labs and lectures from Neomedix Corp.\n\n\nGrant information\n\nWe acknowledge support from the Initiative to Cure Glaucoma of the Eye and Ear Foundation of Pittsburgh; NIH CORE Grant P30 EY08098 to the Department of Ophthalmology, and from an unrestricted grant from Research to Prevent Blindness, New York, NY.\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\nEsfandiari H, Pakravan M, Loewen NA, et al.: Predictive value of early postoperative IOP and bleb morphology in Mitomycin-C augmented trabeculectomy [version 2; referees: 2 approved]. F1000Res. 2017; 6: 1898. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDay AC, Nolan W, Malik AN, et al.: Pilocarpine induced acute angle closure. BMJ Case Rep. 2012; 2012: pii: bcr0120125694. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinckler D, Baerveldt G, Ramirez MA, et al.: Clinical results with the Trabectome, a novel surgical device for treatment of open-angle glaucoma. Trans Am Ophthalmol Soc. 2006; 104: 40–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Q, Harasymowycz P: Goniopuncture in the treatment of short-term post-Trabectome intraocular pressure elevation: a retrospective case series study. J Glaucoma. 2013; 22(8): e17–20. PubMed Abstract | Publisher Full Text\n\nKaplowitz K, Bussel II, Honkanen R, et al.: Review and meta-analysis of ab-interno trabeculectomy outcomes. Br J Ophthalmol. 2016; 100(5): 594–600. PubMed Abstract | Publisher Full Text\n\nBussel II, Kaplowitz K, Schuman JS, et al.: Outcomes of ab interno trabeculectomy with the trabectome by degree of angle opening. Br J Ophthalmol. 2015; 99(7): 914–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAkil H, Chopra V, Huang A, et al.: Clinical results of ab interno trabeculotomy using the Trabectome in patients with pigmentary glaucoma compared to primary open angle glaucoma. Clin Exp Ophthalmol. 2016; 44(7): 563–9. PubMed Abstract | Publisher Full Text\n\nTojo N, Abe S, Miyakoshi M, et al.: Comparison of intraocular pressure fluctuations before and after ab interno trabeculectomy in pseudoexfoliation glaucoma patients. Clin Ophthalmol. 2017; 11: 1667–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDang Y, Kaplowitz K, Parikh HA, et al.: Steroid-induced glaucoma treated with trabecular ablation in a matched comparison with primary open-angle glaucoma. Clin Exp Ophthalmol. 2016; 44(9): 783–8. PubMed Abstract | Publisher Full Text\n\nKaplowitz K, Loewen NA: Trabectome-Mediated Ab Interno Trabeculectomy for Secondary Glaucoma or as a Secondary Procedure. In: Advanced Glaucoma Surgery. Springer, Cham 2015; 15–29. Publisher Full Text\n\nLoewen RT, Roy P, Parikh HA, et al.: Impact of a Glaucoma Severity Index on Results of Trabectome Surgery: Larger Pressure Reduction in More Severe Glaucoma. PLoS One. 2016; 11(3): e0151926. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBussel II, Kaplowitz K, Schuman JS, et al.: Outcomes of ab interno trabeculectomy with the trabectome after failed trabeculectomy. Br J Ophthalmol. 2015; 99(2): 258–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMosaed S, Chak G, Haider A, et al.: Results of Trabectome Surgery Following Failed Glaucoma Tube Shunt Implantation: Cohort Study. Medicine (Baltimore). 2015; 94(30): e1045. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaqueur - Zbl Med Wiss L: Ueber eine neue therapeutische Verwendung des Physostigmin. 1876.\n\nLütjen-Drecoll E: Structural factors influencing outflow facility and its changeability under drugs. A study in Macaca arctoides. Invest Ophthalmol. 1973; 12(4): 280–94. PubMed Abstract\n\nSkaat A, Rosman MS, Chien JL, et al.: Effect of Pilocarpine Hydrochloride on the Schlemm Canal in Healthy Eyes and Eyes With Open-Angle Glaucoma. JAMA Ophthalmol. 2016; 134(9): 976–81. PubMed Abstract | Publisher Full Text\n\nParikh HA, Bussel II, Schuman JS, et al.: Coarsened Exact Matching of Phaco-Trabectome to Trabectome in Phakic Patients: Lack of Additional Pressure Reduction from Phacoemulsification. PLoS One. 2016; 11(2): e0149384. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeiweem AE, Bussel II, Schuman JS, et al.: Glaucoma Surgery Calculator: Limited Additive Effect of Phacoemulsification on Intraocular Pressure in Ab Interno Trabeculectomy. PLoS One. 2016; 11(4): e0153585. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZimmerman TJ, Wheeler TM: Miotics: side effects and ways to avoid them. Ophthalmology. 1982; 89(1): 76–80. PubMed Abstract | Publisher Full Text\n\nInoue K: Managing adverse effects of glaucoma medications. Clin Ophthalmol. 2014; 8: 903–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCamras CB, Toris CB, Tamesis RR: Efficacy and adverse effects of medications used in the treatment of glaucoma. Drugs Aging. 1999; 15(3): 377–88. PubMed Abstract | Publisher Full Text\n\nDetry-Morel:Side effects of glaucoma medications. Bull. Soc. belge Ophtalmol. 2006; 299: 27–40. Reference Source\n\nLama P: Systemic Side Effects of Glaucoma Medications. In: The Glaucoma Book. Springer, New York, NY 2010; 677–88. Publisher Full Text\n\nHwang JC, Khine KT, Rao NA, et al.: Assessment of the Anterior Chamber Angle after Trabectome Glaucoma Surgery by Optical Coherence Tomography, Histopathology, Ultrasound Biomicroscopy, and Scanning Electron Microscopy. Int J Ophthalmic Pathol. 2013; 2(4). Publisher Full Text\n\nOkeke CO, Miller-Ellis E, Rojas M, et al.: Trabectome success factors. Medicine (Baltimore). 2017; 96(24): e7061. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEnright JM, Karacal H, Tsai LM: Floppy iris syndrome and cataract surgery. Curr Opin Ophthalmol. 2017; 28(1): 29–34. PubMed Abstract | Publisher Full Text\n\nSpandau U, Scharioth G: Complications During and After Cataract Surgery: A Guide to Surgical Management. Springer, 2014. Publisher Full Text\n\nFreddo TF, Patz S, Arshanskiy Y: Pilocarpine’s effects on the blood-aqueous barrier of the human eye as assessed by high-resolution, contrast magnetic resonance imaging. Exp Eye Res. 2006; 82(3): 458–64. PubMed Abstract | Publisher Full Text\n\nBill A, WåLINDER PE: The Effects of Pilocarpine on the Dynamics of Aqueous Humor in a Primate (Macaca Irus). Invest Ophthalmol Vis Sci. 1966; 5: 170–5. Reference Source\n\nHu K, Gazzard G, Bunce C, et al.: Ab interno trabecular bypass surgery with Trabectome for open angle glaucoma. Cochrane Database Syst Rev. 2016; (8): CD011693. PubMed Abstract | Publisher Full Text\n\nStewart RM, Hiscott PS, Kaye SB: Endothelial migration and new descemet membrane after endothelial keratoplasty. Am J Ophthalmol. 2010; 149(4): 683; author reply 683–4. PubMed Abstract | Publisher Full Text\n\nFrancis BA, See RF, Rao NA, et al.: Ab interno trabeculectomy: development of a novel device (Trabectome) and surgery for open-angle glaucoma. J Glaucoma. 2006; 15(1): 68–73. PubMed Abstract | Publisher Full Text\n\nMinckler D, Mosaed S, Dustin L, et al.: Trabectome (trabeculectomy-internal approach): additional experience and extended follow-up. Trans Am Ophthalmol Soc. 2008; 106: 149–59; discussion 159–60. PubMed Abstract | Free Full Text\n\nKotecha A, Elsheikh A, Roberts CR, et al.: Corneal thickness- and age-related biomechanical properties of the cornea measured with the ocular response analyzer. Invest Ophthalmol Vis Sci. 2006; 47(12): 5337–47. PubMed Abstract | Publisher Full Text\n\nChang SW, Tsai IL, Hu FR, et al.: The cornea in young myopic adults. Br J Ophthalmol. 2001; 85(8): 916–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Alphen GW: Choroidal stress and emmetropization. Vision Res. 1986; 26(5): 723–34. PubMed Abstract | Publisher Full Text\n\nGoss DA, Van Veen HG, Rainey BB, et al.: Ocular components measured by keratometry, phakometry, and ultrasonography in emmetropic and myopic optometry students. Optom Vis Sci. 1997; 74(7): 489–95. PubMed Abstract | Publisher Full Text\n\nde Kater AW, Shahsafaei A, Epstein DL: Localization of smooth muscle and nonmuscle actin isoforms in the human aqueous outflow pathway. Invest Ophthalmol Vis Sci. 1992; 33(2): 424–9. PubMed Abstract\n\nGonzalez JM Jr, Ko MK, Hong YK, et al.: Deep tissue analysis of distal aqueous drainage structures and contractile features. Sci Rep. 2017; 7(1): 17071. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEsfandiari H, Hassanpour K, Yaseri M, et al.: Dataset 1 in: Extended pharmacological miosis is superfluous after glaucoma angle surgery: A retrospective study. F1000Research. 2018. Data Source"
}
|
[
{
"id": "32413",
"date": "28 Mar 2018",
"name": "Don S. Minckler",
"expertise": [
"Reviewer Expertise Optic nerve damage in glaucoma"
],
"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 Estandian et al. will be greatly appreciated by Trabectome and other glaucoma angle-surgeons. The logic of the discussion is outstanding and the references inclusive. I was an early enthusiast of Trabectome and well-remember that the rationale for utilizing pilocarpine post-operatively was based on non-proven concepts and the then fuzzier understanding of angle physiology. Not utilizing pilocarpine after these surgeries will greatly simplify the whole follow-up with great benefit to patients.\n\nI have no significant revisions to suggest.\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": "33024",
"date": "11 Apr 2018",
"name": "Sylvain Roy",
"expertise": [
"Reviewer Expertise Glaucoma surgery and glaucoma drainage devices"
],
"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 article about surgical technique and pharmacological requirements after glaucoma angle surgery. The details provided in this study will help better understanding this new technology and simplify postoperative management, while eliminating the burden of an old-fashioned miotic medication, i.e. the pilocarpine.\n\nI have few minor comments pertaining to this manuscript.\nPage 4 §1: For the sake of precision I would propose the following: Success was defined as IOP <21 mmHg and a >20% reduction from baseline with no need for additional glaucoma surgery and no loss of light perception. Page 4 Table 1: Please provide the units for Age (years); CCT (µm) Page 6 Figure 2 B and 2 C: For the sake of precision I would recommend also indicating the legend on the Y-axis, e.g. Mean IOP (mmHg) ± SD for Figure 2 B and Figure 2 C.\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/7-178
|
https://f1000research.com/articles/6-1765/v1
|
27 Sep 17
|
{
"type": "Research Article",
"title": "Two center experience of capsule endoscopy in Iran: Report on 101 cases",
"authors": [
"Fariborz Mansour-Ghanaei",
"Morteza Asasi",
"Farahnaz Joukar",
"Rahmatollah Rafiei",
"Alireza Mansour-Ghanaei",
"Ehsan Hajipour-Jafroudi",
"Morteza Asasi",
"Farahnaz Joukar",
"Rahmatollah Rafiei",
"Alireza Mansour-Ghanaei",
"Ehsan Hajipour-Jafroudi"
],
"abstract": "Background: Capsule endoscopy (CE) is a minimally invasive method for the visual examination of the small intestine, which may be for the evaluation and follow-up of patients with Crohn's disease. It can also be used to look at mucosal inflammation. Methods: This cross sectional study was used to determine the diagnostic efficacy of the CE system by performing a cross-sectional study of cases from 2011-2014. This study involved a total of 101 Iranian patients who were referred to the gastrointestinal and liver diseases outpatient clinics in Guilan (northern Iran) and in Isfahan (central Iran) for complaints of gastrointestinal problems. For all patients, definitive diagnosis had failed with the use of other diagnostic tools and CE was performed. Descriptive analysis was used. The patient population was represented by men and women equally, and the mean age of the patients was 42.3 ± 17.2 years (range: 16-89 years). Results: The final diagnoses were: non-specific enteritis (30.6%), Crohn's disease (20.7%), ulcers caused by aspirin or non-steroidal anti-inflammatory drugs (8.9%), mucosal erosion (5.9%) and angioectasia (4.9%); nearly 10% of the patients had normal findings. Analysis of the distribution of chief presenting complaints with patients stratified by the final diagnosis of Crohn's disease showed that the most frequently presented chief complaint was abdominal pain 42.9% and the least frequently presented chief complaint was diarrhea (4.8%). Conclusions: Small bowel evaluation by CE was well tolerated and capable of diagnosing Crohn's disease and gastrointestinal bleeding in patients who failed other diagnostic tests.",
"keywords": [
"Capsule Endoscopy",
"Diagnostic Imaging",
"Crohn disease",
"Gastrointestinal Diseases",
"Double-Balloon Enteroscopy"
],
"content": "Introduction\n\nCapsule endoscopy (CE) is a relatively new imaging technique used to visualize, evaluate and diagnose the gastrointestinal tract in a non-invasive manner. While it has proven to be feasible and well tolerated for examination of the small bowel, CE has not yet emerged as an efficacious alternative to traditional endoscopy for the esophagus, stomach, duodenum or colon1. Yet, its applications in small bowel continue to evolve and advance.\n\nThe recently developed double-balloon enteroscopy system is based upon the classical endoscopic approach of inserting a flexible endoscope, which is covered by a special tube that has two balloons at the end1,2. Inflation and deflation of the balloons allow for further advancement into the bowel and more extensive viewing of the mucosa. Although this device is able to facilitate biopsy taking and remedial actions, its application is very time consuming and it may not be feasible to examine the small bowel completely. CE is another recently developed technique and boasts the distinct advantage of being capable of providing endoscopic evaluation of the small bowel completely3,4.\n\nCompared to traditional endoscopy, CE has a higher sensitivity because it allows for examination of otherwise inaccessible areas of the small bowel and facilitates the operator’s ability to detect changes and diagnose disease5,6. Moreover, the latest advancement in the use of video capsule allows operators to visualize the complete small bowel7. During its 4-h to 6-h trek through the small bowel, a capsule will transfer captured images wirelessly to an external receiver that is worn by the patient. These images are of high quality and comparable to those taken by ordinary scopes8,9.\n\nCE has particularly high sensitivity and specificity for diagnosis of gastrointestinal lesions. As such, the most popular application of CE has emerged as determining the causes of gastrointestinal symptoms, such as gastrointestinal bleeding, malabsorption and unspecified abdominal pain; the main diagnoses are small bowel tumors, angiodysplasia and inflammatory diseases, such as Crohn's disease, infectious enteritis, celiac sprue and ulcers caused by use of non-steroidal anti-inflammatory drugs1,2,6. Several studies have also shown the utility of CE for diagnosis of celiac disease and its complications10–12, and for detection of polyps to screen for Peutz-Jeghers syndrome and familial adenomatous polyposis2,5,7. Hence, video CE would represent an alternative option for patients who are unable or unwilling to undergo esophagogastroduodenoscopy13. Contraindications to CE include known or suspected bowel obstruction, strictures or fistulas (which have been detected by other clinical imaging or tests prior), cardiac pacemakers, implanted electro-myocardial tools and swallowing disorders14.\n\nThe OMOM CE System, manufactured by Jinshan Science & Technology (Group) Co., Ltd (Chongqing, China), provides good quality images and is available at a reasonable price, making it a feasible option for smaller healthcare institutes and/or countries with developing economies15. In this study, we performed OMOM CE to diagnose gastrointestinal diseases in adult patients referred to two gastroenterology clinics in the Guilan and Isfahan provinces of Iran for evaluation of various gastrointestinal complaints between January 2011 and February 2014. The study was performed in order to distinguish the proficiency of OMOM CE and highlight its importance to other gastroenterologists.\n\n\nMethods\n\nThis cross-sectional study included patients that were referred to the gastrointestinal and liver disease outpatient clinic of Razi Hospital of the Guilan University of Medical Sciences (GUMS) in Rasht, north of Iran, and Dr Rafiei's gastroenterology clinic in Isfahan (private clinic), with a nonspecific age criteria and symptoms such as gastrointestinal bleeding of unknown origin; abdominal pain; chronic diarrhea; suspected inflammatory bowel disease; iron deficiency anemia; suspected tumors and/or polyps; malabsorption; unintentional weight loss without any diagnosis by other type of conventional evaluation. Patients were denied enrollment if any of the following were present: known or suspected bowel obstruction; strictures or fistulas detected by prior clinical imaging or tests; cardiac pacemakers; implanted electro-myocardial tools; swallowing disorders.\n\nAll the examinations were carried out under the following conditions. Iron supplementation was stopped 3 d before the examination. Consumption of antacids or bismuth components, which are known to coat the camera lens, was discontinued 1d before the examination. Starting at 8 am on the day before the test, the patient was permitted only clear liquids with a light breakfast. The patients consumed one dose (70 g) of polyethylene glycol laxative mixed in 250 mL water at 4 pm on the day before the test. All the patients were fully fasting starting at 8 pm on the day before the test, with the exception of any critical medications, which were given with sips of water. The procedure was performed at 8 am on the test day.\n\nThe following data was collected for each study participant: age; sex; clinical manifestation; chief complaint; preliminary tests performed, including hemoglobin and stool (ova & parasite, occult blood); final diagnosis with CE.\n\nThe acquired images were reviewed by the two coordinate gastroenterologists. For the descriptive analyses, quantitative variables are expressed as means with standard deviation values; data of analyses of qualitative variables are expressed as frequency and percentages.\n\nThe Medical Ethics Committee of GUMS (P/3/132) has approved the study design, protocol and informed consent procedure. All measurements were performed based on ethical guidelines of the 1975 Declaration of Helsinki. Informed written consent was obtained from each patient. All patients consented to the study.\n\n\nResults\n\nA total of 101 patients were enrolled in the study, based upon the inclusion and exclusion criteria. The study population was represented equally by the two sexes (48.5% male), and the mean age of the patients was 42.3 ± 17.2 years (range: 16–89 years). The most frequent chief complaints that led to evaluation by CE were abdominal pain and anemia, accounting for 40.6% and 21.8% of the cases, respectively (Table 1 and Table 2). In patients with Crohn’s disease, the most frequent chief complaint was abdominal pain 42.9% and the least frequent was diarrhea (4.8%).\n\nNSAIDs: non-steroidal anti-inflammatory drugs.\n\nWhen the patients were grouped by sex, Crohn’s disease was the most frequent diagnosis in both males (61.9%) and females (38.1%). When the patients were grouped by age, Crohn's disease was the most frequent diagnosis for both young adults (<30 years: 61.9%) and middle-aged adults (30–50 years: 28.6%), and angioectasia was the most frequent in patients > 50 years in age. Some of the observed results are shown in Figure 1.\n\n(A) Normal small bowel mucosa; (B) Angioectasia; (C) Ulcer; (D) Tumor; (E) Crohn’s disease; (F) Polyp.\n\n\nDiscussion\n\nCE was first described by its inventor Gavriel Iddan in 1981, and since has become a commonly applied clinical tool for evaluation of gastrointestinal disorders and diagnosis of gastrointestinal diseases. The current study, however, represents the first of its kind to be carried out in hospitals and patients in Iran. A similar study was conducted previously in New York in the United States16, but the patient population was slightly skewed towards the female sex (57% vs 48% in our study) and the mean age of patients was higher (61 years vs 43 years in our study). Moreover, the previous study focused on patients who had been referred for gastrointestinal bleeding, whereas our patients were referred for a variety of common gastrointestinal complaints.\n\nThe OMOM CE system is used throughout China and in many European and other Asian countries. The OMOM capsule is slightly larger than another popular small bowel capsules, such as the PillCam capsule, but our patients experienced no problems with its use. Similar to the PillCam capsule’s battery, the OMOM capsule’s battery can last 8 h. Two exclusive features of CE, as compared to traditional endoscopy, are its ability for real-time imaging and recording of information8. A new type of capsule, with a smaller size and a new shape, has just recently been developed and released to market by the OMOM manufacturer in China, and this will likely advance the use of CE even further.\n\nThe current study investigated patients treated with the older OMOM capsule, exclusively. The most frequent chief complaints that led to CE evaluation were abdominal pain, either in isolation or accompanied by diarrhea; this complaint distribution fits with the frequent diagnosis of Crohn's disease (28.6 % of patients that had abdominal pain and diarrhea, and 42.9% that had abdominal pain in isolation). These findings were different from those in the study by Ruuska et al.17, where abdominal pain in isolation was the most frequent chief complaint (42%), followed by diarrhea (17%) and weight loss (5%). Similarly, a study Mohan et al.18 examined the role of CE in evaluation of patients with suspected small bowel bleeding and identified the most frequent chief complaint as abdominal pain (43%) and a small portion of patients with the chief complaint of weight loss (6%).\n\nCE is commonly used to evaluate cases of occult gastrointestinal bleeding, such as that which underlies iron deficiency anemia, Crohn's disease and small intestinal tumors; its utility has also been proven for evaluating polyposis syndromes and refractory malabsorptive syndromes, such as celiac disease6. According to the literature, the most common uses of OMOM CE in China involve patients with occult gastrointestinal bleeding and who present with complaints of abdominal pain and chronic diarrhea6. CE findings of inflammation in the small bowel are indicative of inflammatory bowel disease, and help clinicians to diagnose Crohn's and other inflammation-related disorders19. A 2003 study of the technical performance and efficiency of CE reported by Mylonaki et al.20, which investigated 50 patients with gastrointestinal bleeding undiagnosed by colonoscopy and gastroscopy, found the diagnosis rate of occult gastrointestinal bleeding was ~43%.\n\nIncidence of Crohn's disease is increasing among the Chinese17 and Iran is facing a similar situation21. Zheng and colleagues22 warned that, while Crohn's disease incidence and prevalence rates in China are still lower than the rates reported from Western countries and even Asian industrialized countries, they are increasing rapidly. Previous studies, including one in 2007 by Fidder et al.23 have recommended the use of CE to diagnose Crohn's disease, based upon the reported evidence of its ability to detect the disease condition in a very small percentage of patients (0 to 4%).\n\nIn our study, two patients were diagnosed with small intestinal tumors. The 2010 study by Jung Wan Han et al.7 indicated that CE was capable of detecting particularly challenging or otherwise undetectable tumors in the small bowel. Without CE, many of these tumors may only become detectable by the other available technologies at the later or last stages of cancer, when therapies are less efficacious or feasible. Those authors also reported that CE has sufficiently higher diagnostic yield and sensitivity for definitive small bowel tumors.\n\nIn the present study, one patient was diagnosed with a tapeworm by CE. Several other studies have reported hookworm detection by CE, including a report of 26 Chinese patients with occult gastrointestinal bleeding published by Liao et al.17, in which 3.4% of the patients were diagnosed with hookworm. Another 6 case reports, 4 of which were from Asian countries, showed hookworm as the etiology of gastrointestinal bleeding, as diagnosed by CE19,24–28.\n\n\nConclusions\n\nMedical science is continually looking for ways to eliminate any aggressive (invasive and/or risky) methods of evaluating the body, and the CE method is an excellent way to detect small bowel diseases when traditional endoscopy cannot detect the problems. The OMOM CE system in particular, is a valuable device for small bowel evaluation because of the small size of its capsule, high-resolution images and low price, which supports its use in healthcare settings across the globe and in the diagnosis of common gastrointestinal complaints, especially Crohn's disease.\n\n\nData availability\n\nDataset 1: Data for all the variables collected for each study participant in SPSS and Excel format, i.e. age, sex, gender, clinical manifestation, preliminary tests performed (DX-primary), final diagnose with CE (DX-End). doi, 10.5256/f1000research.11288.d17863829",
"appendix": "Author contributions\n\n\n\nMansour-Ghanaei F and Asasi M designed the study; Joukar F supervised the surgical procedures; Mansour-Ghanaei F, Asasi M, Rafiei R, Mansour-Ghanaei A and Hajipour-Jafroudi E collected the data; Joukar F analyzed the data; Mansour-Ghanaei F and Joukar F wrote the manuscript; all authors approved the final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe would like to thank all members of Gastrointestinal & Liver Diseases Research Center (GLDRC). Also we would like to thank all the hospitals staff that assisted us in this study.\n\n\nReferences\n\nRao SS, Kuo B, McCallum RW, et al.: Investigation of colonic and whole-gut transit with wireless motility capsule and radiopaque markers in constipation. Clin Gastroenterol Hepatol. 2009; 7(5): 537–44. 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J Zhejiang Univ Sci B. 2008; 9(11): 857–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchnoll-Sussman F, Kulkarni K: Risks of capsule endoscopy. Tech Gastrointest Endosc. 2008; 10(1): 25–30. Publisher Full Text\n\nRuuska T, Vaajalahti P, Arajärvi P, et al.: Prospective evaluation of upper gastrointestinal mucosal lesions in children with ulcerative colitis and Crohn's disease. J Pediatr Gastroenterol Nutr. 1994; 19(2): 181–6. PubMed Abstract | Publisher Full Text\n\nKhadka M, Tao X, Chen DR, et al.: One year experience of OMOM Capsule Endoscopy for suspected intestine lesions. Journal of Nobel Medical College. 2011; 1(1): 27–39. Publisher Full Text\n\nLi ZS, Liao Z, Ye P, et al.: Dancing hookworm in the small bowel detected by capsule endoscopy: a synthesized video. Endoscopy. 2007; 39(Suppl 1): E97. PubMed Abstract | Publisher Full Text\n\nMylonaki M, Fritscher-Ravens A, Swain P: Wireless capsule endoscopy: a comparison with push enteroscopy in patients with gastroscopy and colonoscopy negative gastrointestinal bleeding. Gut. 2003; 52(8): 1122–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMansour-Ghanaei F, Haghkerdar M, Joukar F, et al.: Epidemiologic Features of Inflammatory Bowel Disease in Guilan Province, North of Iran, During 2002–2012. Middle East J Dig Dis. 2015; 7(2): 69–74. PubMed Abstract | Free Full Text\n\nZheng JJ, Zhu XS, Huangfu Z, et al.: Crohn's disease in mainland China: a systematic analysis of 50 years of research. Chin J Dig Dis. 2005; 6(4): 175–81. PubMed Abstract | Publisher Full Text\n\nFidder HH, Nadler M, Lahat A, et al.: The utility of capsule endoscopy in the diagnosis of Crohn's disease based on patient's symptoms. J Clin Gastroenterol. 2007; 41(4): 384–7. PubMed Abstract | Publisher Full Text\n\nChao CC, Ray ML: Education and imaging. Gastrointestinal: Hookworm diagnosed by capsule endoscopy. J Gastroenterol Hepatol. 2006; 21(11): 1754. PubMed Abstract | Publisher Full Text\n\nChen TH, Chen TY, Shyu LY, et al.: Hookworm infestation diagnosed by capsule endoscopy (with video). Gastrointest Endosc. 2006; 64(2): 277–8. PubMed Abstract | Publisher Full Text\n\nCroese J, Speare R: Intestinal allergy expels hookworms: seeing is believing. Trends Parasitol. 2006; 22(12): 547–50. PubMed Abstract | Publisher Full Text\n\nMorales CP, Ferrer G, Zuckerman MJ: Hookworm detected by capsule endoscopy. Gastrointest Endosc. 2005; 62(5): 782–3; discussion 783. PubMed Abstract | Publisher Full Text\n\nWu IC, Lu CY, Wu DC: Acute hookworm infection revealed by capsule endoscopy. Endoscopy. 2007; 39(Suppl 1): E306. PubMed Abstract | Publisher Full Text\n\nMansour-Ghanaei F, Asasi M, Joukar F, et al.: Dataset 1 in: Two center experience of capsule endoscopy in Iran: Report on 101 cases. F1000Research. 2017. Data Source"
}
|
[
{
"id": "28927",
"date": "12 Dec 2017",
"name": "Marco Pennazio",
"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 descriptive report on the use of capsule endoscopy for the investigation of a quite heterogenoeous population.\nI suggest to better specify:\nIn patients with Crohn's disease: was this diagnosis suspected before CE or Crohn's disease was already Known before CE\n\nspecify if, in patients with known Crohn's disease before CE, the subsequent endoscopic examination turned out to be negative\n\nif the diagnosis of Crohn's disease was only suspected before CE, please specify how the diagnosis was made on the basis of the capsule findings\n\ndid the authors observed any complication after CE (ie capsule retention)?\n\nin the discussion please underline that patients with known Crohn's disease are at increased risk of capsule retention and therefore every effort (patency capsule; MRE before CE) should be made in order to minimize this risk.\n\nwith reference to this last point please quote: Pennazio M, Spada C, Eliakim Small-bowel capsule endoscopy and device-assisted enteroscopy for diagnosis and treatment of small-bowel disorders: European Society of Gastrointestinal Endoscopy (ESGE) Clinical Guideline. Endoscopy 2015;47(4):352-76.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "3345",
"date": "12 Feb 2018",
"name": "Farahnaz Joukar",
"role": "Author Response",
"response": "Thank you very much for the insightful review comments. Here are the related point-by-point replies according to the comments. The diagnosis of questionable patients with Crohn’s disease before capsule endoscopy was based on existence of abdominal pain, anemia and the physician clinical suspicious The capsule endoscopy findings of Crohn’s disease among patients were after exclusion of NSAID consumption and diagnosis of parasitic infections. Furthermore, the patient’s treatment response was considered as the diagnosis confirmation. After capsule endoscopy no side effects were reported. It is added in the text as “Although capsule retention is one of the reported side effects of capsule endoscopy in patients with Crohn’s disease30, in our study this issue was not observed”. In the last part of the methods section we have added “Also, small bowel series were conducted for all patients”."
}
]
},
{
"id": "29793",
"date": "16 Jan 2018",
"name": "Eva Niv",
"expertise": [
"Reviewer Expertise Gastroenterology",
"small bowel diseases",
"Crohn's disease",
"15 years of experience in the field of capsule endoscopy"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is well-written and presents interesting data regarding the results of capsule endoscopy in Iran. It presents data about the prevalence of different small bowel pathologies in Iran population. I support the publication of this paper. For my opinion, there is no need in changes, besides minor language corrections.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1765
|
https://f1000research.com/articles/6-1958/v1
|
06 Nov 17
|
{
"type": "Research Article",
"title": "Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication",
"authors": [
"Ivan Nombela",
"Sara Puente-Marin",
"Veronica Chico",
"Alberto J. Villena",
"Begoña Carracedo",
"Sergio Ciordia",
"Maria Carmen Mena",
"Luis Mercado",
"Luis Perez",
"Julio Coll",
"Amparo Estepa",
"Maria del Mar Ortega-Villaizan",
"Ivan Nombela",
"Sara Puente-Marin",
"Veronica Chico",
"Alberto J. Villena",
"Begoña Carracedo",
"Sergio Ciordia",
"Maria Carmen Mena",
"Luis Mercado",
"Luis Perez",
"Julio Coll",
"Amparo Estepa"
],
"abstract": "Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of the type I interferon (ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs with TSS (stromal cell line from spleen) revealed the IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs Isobaric tag for relative and absolute quantification (iTRAQ) revealed that VHSV exposure can induce a global protein downregulation in trout RBCs, mainly related to RNA stability and proteasome pathways. The antioxidant/antiviral response is also suggested to be involved in the response of trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail. To our knowledge, this is the first report that implicates fish RBCs in the antiviral response against viruses not targeting RBCs.",
"keywords": [
"nucleated red blood cells",
"rainbow trout",
"VHSV",
"rhabdovirus",
"immune response",
"antiviral"
],
"content": "Introduction\n\nFish are the most primitive vertebrates possessing many of the immune system cells (lymphocytes, NK cells, macrophages, etc) and molecules (interleukins, chemokins, receptors, etc) found in higher vertebrates. In contrast to higher vertebrates, however, fish lack bone marrow, lymph nodes, IgG-switch, and have tetrameric rather than pentameric IgM, with a more limited binding repertoire than mammals1. Furthermore, fish poikilothermic nature results in a delayed antigen affinity maturation, memory and lymphocyte proliferation2. To compensate for those immune deficiencies, fish have unique phagocytic B lymphocytes3 and stronger innate immune responses, as shown in survivors of viral infection4. Furthermore, fish red blood cells (RBCs) have receptors capable of recognizing pathogen associated molecular patterns and respond to them with differentially expressed cytokine transcripts5,6 and cytokine-like factors7. Thus, fish RBCs generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them8–10, while their mammalian counterparts are unable to do this. In light of this evidence, an outstanding question is whether fish RBCs are able to respond to viral infections that are well known to replicate in other cells or tissues, and if they could further contribute with compensatory immune responses in order to physiologically combat viral infections that do not target RBCs.\n\nTo explore in vitro the above mentioned question, we used rainbow trout Oncorhynchus mykiss), an important aquacultured species, together with the Viral Haemorrhagic Septicemia virus (VHSV), a rhabdovirus also called the ‘fish ebola’, which causes important losses of high economic impact on world-wide salmonid aquaculture11. VHSV viruses are bullet-shaped enveloped virions with single-stranded negative-sense RNA with a genome of 11.2 kbp11–13. VHSV do not target specifically RBCs, and therefore represent a good model to investigate the immune response of RBCs to viruses targeting other cells or tissues.\n\nIn this study, we describe how in vitro cultures of trout RBCs upregulated the expression of some immune proteins as part of their antiviral immune response against VHSV, whose infection appeared to be halted in trout RBCs. Simultaneously, the interferon-inducible mx and pkr genes showed a downregulation tendency during VHSV early replication, after 6 hours postexposure (hpe). In addition, the protein levels corresponding to BD1 (β-defensin 1 – an anti-microbial peptide known to be involved in antiviral innate immunity14,15 and IL8 (Interleukin 8 – a neutrophil chemotactic factor), are shown, to our knowledge, for the first time, as characteristic of trout RBCs antiviral immune protein responses. Further, iTRAQ-based protein profiling of VHSV-exposed RBCs showed a global protein downregulation, mainly related to RNA stability and proteasome pathways. Related to this fact, the phosphorylation of the α-subunit of translational initiation factor 2 (eIF2α) and protein synthesis inhibition could be implicated in the inhibition of VHSV replication and RBCs proteome shut-off. Also, the antioxidant and related antiviral response is also suggested as involved in the response of trout RBCs to VHSV halted infection. In summary, we suggest a wide range of mechanisms implicated in the antiviral response of trout RBCs against VHSV halted infection\n\n\nMethods\n\nRainbow trout (Oncorhynchus mykiss) individuals (number of individuals used are indicated in each assay) of approximately 5–6 cm were obtained from a VHSV-free commercial farm (PISZOLLA S.L., CIMBALLA FISH FARM, Zaragoza, Spain), and maintained at University Miguel Hernandez (UMH) facilities at 14°C, with a re-circulating dechlorinated-water system, at a stocking density of 1fish/3L, and fed daily with a commercial diet (SKRETTING, Burgos, Spain). Prior to experiments, fish were acclimatized to laboratory conditions over 2 weeks.\n\nRabbit polyclonal antibodies against rainbow trout β-defensin (BD1) (RRID: AB_2716268) (unpublished) and rainbow trout Mx3 (RRID: AB_2716267)16,17 were produced at the laboratory of Dr. Amparo Estepa. Mouse polyclonal antibodies against rainbow trout IL1β (RRID: AB_2716269)18,19 , IL8 (RRID: AB_2716272)20, TNFα (RRID: AB_2716270)21, Hepcidin (RRID: AB_2716273)22, NKEF (RRID: AB_2716271)23, IFN1 (RRID: AB_2716274) (unpublished) and IFNγ (RRID: AB_2716275) (unpublished) were produced at the laboratory of Dr. Luis Mercado. Rabbit polyclonal antibody against human NF-κβ p65 antibody (Cat#ab7970, RRID: AB_306184) was purchased from AbCam (Cambridge, UK). This p65 antibody epitope corresponds to the C-terminal region of the p65 protein, similarly to other p65 antibodies used for teleost species24–26. To label VHSV, we used the mouse monoclonal 2C9 antibody (RRID: AB_2716276)27 against the N protein of VHSV (NVHSV) produced at Dr. Coll’s laboratory. Anti-Rabbit IgG (H+L) CF™ 488 antibody produced in goat and Anti-Mouse IgG (H+L) CF™ 488 antibody produced in goat were used as secondary antibodies (Sigma-Aldrich, Madrid, Spain). Rabbit polyclonal antibody against human eIF2α-P (Cat# E2152, RRID:AB_259283) and rabbit polyclonal antibody against human α-Actin (Cat#2066, RRID:AB_476693) were purchased from Sigma-Aldrich and used for western blotting.\n\nTrout RBCs were obtained from peripheral blood of fish sacrificed by overexposure to tricaine (tricaine methanesulfonate, Sigma-Aldrich; 0.2 g/l). Peripheral blood was sampled from the caudal vein using insulin syringes (NIPRO, Bridgewater, NJ). Blood samples were placed in a 2 ml eppendorf with RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fischer Scientific Inc., Carlsbad, CA) supplemented with 10% FBS (fetal bovine serum) gamma irradiated (Cultek, Madrid, Spain), 1 mM pyruvate (Gibco), 2 mM L-glutamine (Gibco), 50 µg/mL gentamicin (Gibco) and 2 µg/mL fungizone (Gibco), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (Sigma-Aldrich). Then, RBCs were purified by two consecutive density gradient centrifugations (7206g, Ficoll 1.007; Sigma-Aldrich). Purified RBCs were cultured in the above indicated medium at a density of 5·105 cells/ml in 24-well cell culture plates at 14°C and 5% CO2.\n\nThe fish cell lines TSS, RTG-2 and EPC, were also used in this work. TSS (Trout Stroma from Spleen)28 was donated by the laboratory of Dr. AJ Villena. TSS cells were maintained at 21°C in a 5% CO2 atmosphere in RPMI-1640 medium containing 20% FBS, 1 mM pyruvate, 2 mM L-glutamine, 50 µg/mL gentamicin and 2 µg/mL fungizone. RTG-2 (Rainbow Trout Gonad-2) cell line was purchased from the American Type Culture Collection (ATCC, 50643). RTG-2 cells were maintained at 21°C in a 5% CO2 atmosphere with MEM medium (Sigma-Aldrich) containing 10% FBS, 1 mM pyruvate, 2 mM L-glutamine, 50 µg/mL gentamicin and 2 µg/mL fungizone. EPC (Epithelioma Papulosum Cyprini)29 cell line was purchased from the ATCC (CRL-2872). Cells were maintained at 28°C, in a 5% CO2 atmosphere in RPMI-1640 10% FBS, 1 mM pyruvate, 2 mM L-glutamine, 50 µg/mL gentamicin and 2 µg/mL fungizone.\n\nViral haemorrhagic septicaemia virus (VHSV-07.71)30, isolated in France from rainbow trout, Oncorhynchus mykiss, was purchased from the American Type Culture Collection (ATCC, VR-1388) and propagated in EPC cells at 14°C, as previously reported31.\n\nRBCs and RTG-2 cells were infected with VHSV at different multiplicities of infection (MOI), at 14°C. After 3 hours of incubation for RBCs and 1.5 hours for RTG-2, cells were washed with cold RPMI, then RPMI 2% FBS was added and infection incubated at 14°C, at the different times indicated for each assay. In the case of the time-course assay, the virus was not removed.\n\nVirus titers present in VHSV-exposed RBCs supernatants were determined by plaque assays. Briefly, different dilutions of the supernatants (from 10-1 to 10-4) were added to EPC cell monolayers, grown in 24-well plates, at 14°C for 90 minutes. Then, the culture media were removed and infected cell monolayers covered with a solution of RPMI-1640 cell culture medium with 2% FBS and a 2% aqueous solution of methyl cellulose (Sigma-Aldrich). Cell plates were incubated at 14°C for 5 days and then the media with methyl cellulose was removed. Finally, EPC cell monolayers were stained with crystal violet-formalin to count plaques. Virus titers were expressed as plaque forming units (PFU) per ml.\n\nSeparately, NVHSV RT-qPCR was also used to quantify the viral RNA inside VHSV-exposed RBCs (see below).\n\nTo block endosomal low-pH, NH4Cl (Sigma-Aldrich) at 7 mM was added to RBCs during VHSV exposure, which was carried out as described in the previous section. No significant cell death was observed in RBCs treated with NH4Cl, since the concentration used is known as non-cytotoxic, but effective for reducing VHSV infectivity by 40%32. After the incubation period, the viral titer in the supernatant was calculated as described in the previous section.\n\nFicoll purified RBCs were pre-treated with 50 and 100 mU/ml of neuraminidase from Vibrio cholerae (Sigma-Aldrich), at 21°C for 30 minutes, before virus inoculation. After treatment, RBCs were washed once with PBS in order to completely remove the enzyme. After that, the pre-treated cells were inoculated with VHSV at MOI 1. RBCs inoculated with UV-inactivated VHSV were used as control. UV-inactivated VHSV was generated by exposure to UV-B at 1 J/cm2 using a Bio-Link Crosslinker BLX E312 (Vilber Lourmat, BLX-E312), as previously described33. The infection was monitored by RT-qPCR of the NVHSV gene 3 at 72 hpe.\n\nOne day prior to the co-culture, RBCs, extracted and seeded as indicated before, were stimulated using UV-inactivated VHSV over 24 hours. Subsequently, RBCs were washed once with cold RPMI and added to Corning® Transwell® polyester membrane cell culture inserts of 0.4 µm pore size (Corning, Sigma-Aldrich) on 24 well plates with previously cultured confluent TSS cells in RPMI 20% FBS. Co-culture was maintained for 24 hours at 14°C in RPMI 2% FBS. After that, cells were washed and stored in the indicated buffer and conditions for RNA extraction.\n\nE.Z.N.A. ® Total RNA Kit (Omega Bio-Tek, Inc., Norcross, GA) was used for total RNA extraction in accordance with the manufacturer’s instructions. Isolated RNAs were stored at −80 °C until used. DNAse treatment was done in order to eliminate residual genomic DNA using TURBO™ DNase (Ambion, Thermo Fischer Scientific Inc.), following the manufacturer’s instructions. RNA was quantified with a NanoDrop® Spectrophotometer (Nanodrop Technologies, Wilmington, DE). M-MLV reverse transcriptase (Invitrogen, Thermo Fischer Scientific Inc.) was used to obtain cDNA, as previously described34.\n\nReal-Time Quantitative PCR (RT-qPCR) was performed using the ABI PRISM 7300 System (Applied Biosystems, Thermo Fischer Scientific Inc.). Reactions were performed in a total volume of 20 μl comprising 12 ng of cDNA, 900 nM of each primer, 10 μl of TaqMan universal PCR master mix (Applied Biosystems, Thermo Fischer Scientific Inc.) with 300 nM of probe or 10 μl of SYBR green PCR master mix (Applied Biosystems, Thermo Fischer Scientific Inc.). The cycling conditions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Primers and probes used are listed in Table 1.\n\nGene expression was analyzed by the 2-ΔCt or 2−ΔΔCt method35 where 18S rRNA or ef1α gene (Applied Biosystems, Thermo Fischer Scientific Inc.) were used as endogenous control.\n\nRBCs were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) in RPMI 1640 medium for 20 minutes. Permeabilization of the RBCs was done in a 0.05% saponin (Sigma-Aldrich) buffer for 15 minutes. Primary antibodies were diluted in the permeabilization buffer at the recommended dilutions and incubated for 60 minutes at RT. Secondary antibodies were incubated for 30 minutes at RT. After every antibody incubation, RBCs were washed with the permeabilization buffer. Finally, RBCs were kept in PFA 1% in PBS. For nuclear staining, RBCs were stained with 1 µg/mL of 4′-6-Diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 minutes. RBCs were analyzed by flow cytometry (FC) in a BD FACSCanto™ (BD Biosciences) flow cytometer. Immunofluorescence (IF) images were performed in an IN Cell Analyzer 6000 Cell Imaging system (GE Healthcare, Little Chalfont, UK).\n\nTwo pools of eight samples (two control: C1 and C2, and two VHSV-exposed: V1 and V2), with 8·106 cells per sample, were used for iTRAQ 4plex protein profiling.\n\nThe pools, containing 6.4·107 cells, were pelletized by centrifugation (5 min, 700 × g). The supernatant was carefully removed and the RBC pellets (∼70–100 µL) were mixed with 250 µL of deionized water and frozen at – 80°C for 3 h. After thawing the lysate, it was centrifuged at 17000 × g for 20 min at 4°C to separate the cytosolic supernatant and the pelleted membrane fractions, as described in Nombela et al. (unpublished report, Nombela I, Ciordia S, Mena MC, Puente-Marin S, Chico V, Coll J, and Ortega-Villaizan M). Subsequently, a new proteomic analysis method was carried out that combines fractionation into cytosolic and membrane fractions, haemoglobin removal of the cytosolic fraction, protein digestion, pH reversed-phase peptide fractionation and finally LC ESI-MS/MS analysis of each of the fractions, as described in Nombela et al. (unpublished report, as before). Briefly, the haemoglobin of the cytosolic fraction was removed using a column of HemoVoidTM kit (Biotech Support Group, Monmouth Junction, NJ), following the manufacturer instructions36. For protein digestion of each fraction, 120 µg from haemoglobin-depleted cytosolic fraction were digested in the chaotropic buffer, and 40 µg of membrane fraction was precipitated by methanol/chloroform method and re-suspended in 20 µl of the chaotropic buffer. The digested samples (membrane and cytosol separately) were subsequently labelled using iTRAQ-4plex Isobaric Mass Tagging Kit (SCIEX), according to the manufacturer's instructions as follows: 114, C1 (Pool control 1); 115, V1 (Pool VHSV-exposed 1); 116, C2 (Pool control 2); 117, V2 (Pool VHSV-exposed 2). Then, offline high pH reversed-phase peptide fractionation of the peptides from the cytosolic RBC fraction was performed on a SmartLine (Knauer, Berlin, Germany) HPLC system using an XBridge C18 column (100 × 2.1 mm, 5 μm particle; Waters, Milford, MA). Thirty fractions were collected and then pooled alternatively into 5 fractions. After labelling, the samples were pooled, evaporated to dryness and stored at -20°C until LC−MS analysis.\n\nA 1 µg aliquot of labelled mixture was subjected to 1D-nano LC ESI-MSMS (Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric) analysis using a nano liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus, SCIEX,) coupled to high speed Triple TOF 5600 mass spectrometer (SCIEX) with a Nanospray III source. The analytical column used was a silica-based reversed phase Acquity UPLC® M-Class Peptide BEH C18 Column, 75 µm × 150 mm, 1.7 µm particle size and 130 Å pore size (Waters Corporation, Milford, MA). The trap column was a C18 Acclaim PepMapTM 100 (Thermo Fischer Scientific), 100 µm × 2 cm, 5 µm particle diameter, 100 Å pore size, switched on-line with the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 2 µl/min. The nano-pump provided a flow-rate of 250 nl/min and was operated under gradient elution conditions. Peptides were separated using a 250 minutes gradient ranging from 2% to 90% mobile phase B (mobile phase A: 2% acetonitrile, 0.1% formic acid; mobile phase B: 100% acetonitrile, 0.1% formic acid). Injection volume was 5 µl.\n\nData acquisition was performed with a TripleTOF 5600 System (SCIEX). Data was acquired using an ionspray voltage floating, 2300 V; curtain gas, 35; interface heater temperature, 150; ion source gas 1, 25; declustering potential, 150 V. All data was acquired using information-dependent acquisition (IDA) mode with Analyst TF 1.7 software (RRID: SCR_015785) (SCIEX). For IDA parameters, 0.25 s MS survey scan in the mass range of 350–1250 Da were followed by 30 MS/MS scans of 150ms in the mass range of 100–1800. Switching criteria were set to ions greater than mass to charge ratio (m/z) 350 and smaller than m/z 1250 with charge state of 2–5 and an abundance threshold of more than 90 counts (cps). Former target ions were excluded for 20 s. IDA rolling collision energy (CE) parameters script was used for automatically controlling the CE.\n\nMS/MS spectra were exported to MGF format using Peak View v1.2.0.3 (RRID: SCR_015786)(SCIEX) and searched using Mascot Server v2.5.1 (RRID:SCR_014322)(Matrix Science, London, UK), OMSSA v2.1.937, X!TANDEM 2013.02.01.138, and Myrimatch v2.2.14039 against a composite target/decoy database built from the Oncorhynchus mykiss sequences at Uniprot/Swissprot Knowledgebase (available here, last update: 2017/01/26, 50.125 sequences), together with commonly occurring contaminants. Search engines were configured to match potential peptide candidates with mass error tolerance of 25 ppm and fragment ion tolerance of 0.02D, allowing for up to two missed tryptic cleavage sites and a maximum isotope error (13C) of 1, considering fixed methyl methanethiosulfonate modification of cysteine and variable oxidation of methionine, pyroglutamic acid from glutamine or glutamic acid at the peptide N-terminus, acetylation of the protein N-terminus, and modification of lysine, tyrosine and peptide N-terminus with iTRAQ 4-plex reagents. Score distribution models were used to compute peptide-spectrum match P-values40, and spectra recovered by a FDR (False Discovery Rate) ≤ 0.01 (peptide-level) filter were selected for quantitative analysis. Approximately 1% of the signals with the lowest quality were removed prior to further analysis. Differential regulation was measured using linear models41, and statistical significance was measured using q-values (FDR). All analyses were conducted using Proteobotics software (Isobaric Tagging Analysis Workflow v.1.0, RRID:SCR_015787; Madrid, Spain). The cutoff for differentially regulated proteins was established at FDR q-value 5%.\n\nIn order to evaluate the functionally grouped Gene Ontology (GO) and pathway annotation networks of the differentially expressed proteins, pathway enrichment analysis was performed using ClueGO (RRID:SCR_005748)42 and CluePedia (RRID: SCR_015784)43 Cytoscape plugins (Cytoscape v3.4.0, RRID:SCR_003032,44). GO Biological process, GO Immunological process, KEGG (Kyoto Encyclopedia of Genes and Genomes), Wikipathways and Reactome functional pathway databases were used. A P-value ≤0.05 and Kappa score of 0.4 were considered as threshold values.\n\nControl and VHSV-exposed RBCs cell pellets were resuspended in 30 µl of PBS with a cocktail of protease inhibitors (Sigma-Aldrich). Cells were then frozen/thawed 3 times and protein concentration adjusted before loading. Samples were loaded in Tris–Glycine sodium dodecyl sulfate 17% polyacrylamide gels under reducing conditions. Electrophoresis was performed at 100 V for 90 min. For blotting, the proteins in the gel were transferred for 75 min at 100 V in transfer buffer (2.5 mM Tris, 9 mM glycine, 20% methanol) to nitrocellulose membranes (BioRad, Madrid, Spain). The membranes were then blocked with 8% dry milk, 1% Tween-20 in PBS and incubated with rabbit polyclonal antibody against human eIF2α-P (36.1 KDa) or rabbit polyclonal antibody against human α-Actin (42 KDa,) in PBS containing 0.5% dry milk, and 0.5% Tween-20 (PMT buffer), overnight at 4°C. Membranes were then washed 3 times with PMT buffer for 15 min before incubation with GAR-Po (Sigma-Aldrich) in PMT buffer for 45 min. Finally, the membrane was washed 3 times with PBS containing 0.5% Tween-20. Peroxidase activity was detected using ECL chemiluminescence reagents (Amersham Biosciences, Buckinghamshire, UK) and revealed by exposure to X-ray. Protein bands were analyzed by densitometry using the Scion Image 4.0.2 Software (RRID: SCR_008673) (www.scionorg.com).\n\nThe intracellular ROS level was assessed in VHSV-exposed RBCs using the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (DCFDA, Sigma-Aldrich). RBCs were exposed to VHSV at MOI 1, during 72 h, at 14°C. After that, RBCs were washed with I and incubated with 20 μM DCFDA in RPMI, for 30 min at RT. The fluorescence intensity of 2′,7′-dichlorofluorescin was measured using the POLARstar Omega microplate reader (BMG LABTECH, USA) at excitation 480 nm and emission 530 nm.\n\nGraphpad Prism 6 (RRID:SCR_002798, www.graphpad.com) was used for graphic representation and statistics calculation. The statistic tests and P-values associated with the graphics are indicated in each assay. Flow cytometry data was processed and analyzed using Flowing Software 2.5.1 (www.flowingsoftware.com/) (RRID: SCR_015781).\n\nAll experimental protocols and methods of the experimental animals were reviewed and approved by the Animal Welfare Body and the Research Ethics Committee at the University Miguel Hernandez (approval number 2014.205.E.OEP; 2016.221.E.OEP) and by the competent authority of the Regional Ministry of Presidency and Agriculture, Fisheries, Food and Water supply (approval number 2014/VSC/PEA/00205). All methods were carried out in accordance with the Spanish Royal Decree RD 53/2013 and EU Directive 2010/63/EU for the protection of animals used for research experimentation and other scientific purposes.\n\n\nResults\n\nFor this analysis we first purified RBCs (oval nucleated cells) to 99.9% (as evaluated by optical microscopy) and then exposed the purified RBCs to VHSV, for different times, to monitor the replication of VHSV in trout RBCs. For that, time course expression of the N gene of VHSV (NVHSV) was measured by RT-qPCR. Clearly, the expressions of NVHSV gene were significantly upregulated at 3 hours postexposure (hpe). However, they drastically decreased from 6 to 72 hpe, indicating that VHSV could replicate at early times postexposure, at the same levels as the VHSV susceptible trout cell line RTG-2. However, viral replication was halted in RBCs at later stages of infection, in contrast to RTG-2 (Figure 1A). On the other hand, after VHSV enters the cell, the first gene that starts to transcribe is the NVHSV gene, since it is the closest to the 3’ transcriptional start, and the more distal, excluding the polymerase, is the G glycoprotein gene (GVHSV) gene. Therefore, under a normal transcription scenario a high ratio between the NVHSV and GVHSV viral genes transcripts is to be expected, taking into account the attenuation phenomenon found in rhabdoviruses45. However, a ratio of 2 was observed in RBCs, compared to the ratio of 8 found in RTG-2 cells, at 1 and 3 hpe (Figure 1B).\n\n(A) Time course of VHSV gene replication in trout RBCs and RTG-2 cell line. N gene of VHSV (NVHSV) expression profile was quantified at time 0, 1, 3, 6, 24 and 72 hours postexposure (hpe) to VHSV, in RBCs (black bars) and RTG-2 (grey bars), with a multiplicity of infection (MOI) of 1 at 14°C. Gene expression was normalized against eukaryotic 18S rRNA and ef1α, respectively for RBCs and RTG-2 cells, and relativized to control cells (time 0) (fold of increase). Data represent the mean ± SD (n = 4 for RBCs and n=2 for RTG-2). (B) Ratio of NVHSV and GVHSV genes (NVHSV:GVHSV) viral genes at time 0, 1, and 3 hpe in RBCs (black bars) and RTG-2 (grey bars). Gene expression was normalized against ef1α. Data represent the mean ± SD (n = 3 for RBCs and n=2 for RTG-2). (C) Viral yield in VHSV-exposed trout RBCs. Viral titer (grey bars) (plaque forming units per millilitre, PFU/ml) and NVHSV gene expression (black bars) of VHSV-exposed RBCs, with MOI 1, 10 and 100, respectively corresponding to inoculum virus titers 2·106 (a), 2·107 (b) and 2·108 (c) PFU/ml, 72 hpe, at 14°C. Gene expression was normalized against ef1α. Data represent the mean ± SD (n = 3 for viral titer and n=4 for NVHSV gene expression). (D) VHSV internalization in trout RBCs is NH4Cl-sensitive. VHSV titers obtained in VHSV-exposed RBCs at MOI 1, at 3 and 6 days postexposure (dpe), at 14°C, in the absence (black bars) or in the presence (grey bars) of NH4Cl. Data represent the mean ± SD (n = 4). (E) Pre-treatment of RBCs with neuraminidase enhances early replication of VHSV. RBCs were inoculated with UV-inactivated or live VHSV, with a MOI of 1, at 14°C. Before infection, cells were pretreated with neuraminidase (NA) at 50 or 100 mU/ml during 30 minutes at 14°C. VHSV infectivity was quantified by NVHSV gene expression analysis at 3 hpe (grey bars) and 72 hpe (black bars). Gene expression was normalized against 18S rRNA gene and represented as arbitrary units (AU). Data represent the mean ± SD (n = 4). (F) Representative immunofluorescence of VHSV labelling in RBCs exposed to VHSV (MOI 100, 24 and 72 hpe, 14°C) stained from left to right with anti-NVHSV 2C9 (FITC), DAPI for nuclei stained and merged (IF representative of 32 images). (G) Representative flow cytometry overlay histograms showing untreated RBCs (grey filled histogram), VHSV-exposed RBCs with a MOI 100, at 14°C, 24 hpe (green filled histogram) and 72 hpe (black filled histogram). (H) Schematic representation of the VHSV infectivity in RBCs and RTG-2 cells, indicating the virus inoculation titer and recovered virus yield after 72 hpe in each cell line. Kruskal-Wallis Test with Dunn´s Multiple Comparison post-hoc test was performed for statistical analysis among all conditions. Values over the bars denote pairwise significant differences with the value-indicated time point or condition (P-value < 0.05).\n\nAlso, RBCs were exposed to different VHSV multiplicities of infection (MOI). The initial VHSV inoculum titer declined ~3-logs after 3 days of incubation at the indicated MOI assayed (1, 10 or 100, respectively corresponding to inoculum virus titers 2·106, 2·107, 2·108 PFU/ml) (Figure 1C), in contrast to the usual 1-log titer increment in RTG-2 cells infected in the same conditions (Figure 1H). Later on, RBCs showed only a minor ~1-fold increment of the VHSV titer as the time of infection increased from 3 to 6 days (Figure 1D). These low VHSV titers were due to true VHSV internalization and not to residual VHSV binding, since they were NH4Cl-sensitive, a characteristic of rhabdovirus infections (Figure 1D). NH4Cl acts as a lysosomotropic drug, blocking endosomal acidification and inhibiting rhabdoviral cytoplasmic entrance steps including those of VHSV46. NVHSV RT-qPCR also confirmed the presence of viral RNA in VHSV-exposed RBCs (Figure 1C).\n\nIn order to increase the amount of VHSV inside trout RBCs, RBCs were pre-treated with neuraminidase (NA) and then exposed to VHSV. NA has been shown to enhance rhabdovirus infection in NA pre-treated cells by favoring interaction with cellular membranes47. As a result, the VHSV RNA inside RBCs was increased about ten times at 3 hpe. However, seventy-two hpe the VHSV RNA drastically decreased to almost disappear, as indicated by NVHSV RT-qPCR (Figure 1E).\n\nBesides, NVHSV protein (2C9 antibody) was detected in RBCs exposed to VHSV MOI 100, at 24 hpe, but not at 72 hpe. IF images (Figure 1F) showed an intracellular stain along the cytoplasm and nucleus. FC histogram (Figure 1G) showed a slight increment of VHSV N protein in VHSV-exposed RBCs, at 24 hpe, but not at 72 hpe. VHSV could not be detected by IF or FC in RBCs exposed to lower MOIs. Strikingly, NVHSV protein stain was located mainly in the nuclear region of VHSV-exposed RBCs. Although it has not been described that NVHSV protein can be localized in the cell nucleus, another rhabdovirus proteins, such as rabies virus P3 protein48 and the IHNV NV protein49 have been localized in the nucleus of infected cells.\n\nWe next investigated whether trout RBCs exposed to VHSV could be capable of generating immune responses in vitro, by means of examining the differential expression profile of some genes characteristic of the fish antiviral response. First, a time course monitoring of the expression of the interferon-inducible mx and pkr genes was carried out at different time postexposure. The results showed that the mx and pkr genes exhibited the same increment peak at 3 hpe and the tendency to downregulation from 6 to 72 hpe, in parallel to NVHSV gene transcription levels tendency (Figure 2A and B, and Figure 1A). On the other hand, at 3 hpe, ifn1 gene expression already exhibited a statistically significant downregulation (Figure 2C), and a slight downregulation for tlr3 and irf7 genes.\n\nTime course of interferon-inducible antiviral genes mx (A) and pkr (B). RBCs were exposed to VHSV with a multiplicity of infection (MOI) of 1 at 14°C, and mx1-3 and pkr genes expression was quantified at time 0, 1, 3, 6, 12, 24, 72 hours postexposure (hpe). Data is displayed as mean ± SD (n = 3). Kruskal-Wallis Test with Dunn´s Multiple Comparison post-hoc test was performed among all conditions. (C) Interferon signaling at early time postexposure. RBCs were exposed to VHSV with a MOI of 1 at 14°C, and tlr3, irf7 and ifn1 gene expression profiles were quantified at time 0, and 3 hpe. Data is displayed mean ± SD (n = 3). Mann Whitney Test was performed for statistical analysis between the VHSV-exposed and control cells. Gene expression was normalized against eukaryotic 18S rRNA for mx, tlr3, irf7 and ifn1 genes and ef1α for pkr gene, and relativized to control cells (time 0, red line) (fold of increase). Asterisk denote statistically significant differences between the VHSV-exposed and the control cells (P-value < 0.05).\n\nThe changes in the RBCs immune protein response induced by VHSV exposure were assessed using specific antibodies. VHSV-exposed RBCs showed only an increment in individual protein levels of chemokine IL8 (Figure 3B and E, Figure S1) and antimicrobial peptide BD1 (Figure 3C and F, Figure S1), verified by means of FC and IF. Mx and IFN1 protein levels, according to the RT-qPCR results, did not change or downregulate, respectively (Figure 3A). Cytokines IL1β, IFNγ (Figure 3B), the antimicrobial peptide Hepcidin (Figure 3C) and the natural killer enhancing factor (NKEF) (Figure 3D) did not show regulation at 72 hpe.\n\nRelative immune protein expression levels, (A) interferon pathway related proteins (IFN1 and Mx), (B) antimicrobial peptides (BD1 and Hepcidin), (C) cytokines (IL8, IL1β and IFNγ) and (D) antioxidant protein NKEF, measured by flow cytometry and calculated by the formula MRFI (Mean Relative Fluorescence Intensity) = fluorescence in VHSV-exposed RBCs / fluorescence in non-exposed RBCs, at multiplicity of infection (MOI) 1, 72 hours postexposure (hpe), at 14°C, relative to control cells (red line). Data is displayed as mean ± SD (n=5). Mann Whitney Test was performed for statistical analysis between the VHSV-exposed cells and control cells. Representative immunofluorescences of control and VHSV-exposed RBCs stained with anti-IL8 (IF representative of 44 images) (E) and anti-BD1 (IF representative of 46 images) (F) (FITC) and DAPI for nuclei stain.\n\nIt is noteworthy to highlight the elevated inter-individual variability found in trout RBCs immune response, for most of the proteins and genes assayed, which could prevent to obtain statistical significance in most of the cases although regulations were apparent.\n\nThe rainbow trout spleen is an active hematopoietic organ50, and it is composed of various cell types, such as red blood cells, leukocytes and reticular or stromal cells51. It has been demonstrated that cytokines and soluble factors produced by the stromal cells are required for trout blood cells development in the spleen or head kidney52. In this regard, we wanted to evaluate the paracrine effects of the cytokines produced by VHSV stimulated RBCs over the stromal cell line from trout spleen, TSS28. For that, trout RBCs stimulated with VHSV UV-inactivated were co-culture with the TSS cell line, using a Transwell system to test whether a cross-stimulation mediated by soluble molecules was involved. The gene expression profiles for ifn1, and the interferon stimulated genes (ISGs) mx, viral inducible gene vig1, and interleukin il15 genes were examined for each cell line 24 hours post co-culture. Linear regression analysis of the RBCs ifn1 gene expression with their respective mx, vig1 and il15 genes showed a significant correlation between ifn1 and vig1 and il15, but not with mx gene (Figure 4A). ifn1 gene expression from RBCs and TSS cells also showed a significant correlation (Figure 4B). TSS cells showed significant correlation between ifn1 and mx, vig1 and il15 (Figure 4C). The results demonstrated an IFN crosstalk between the stimulated RBCs and TSS cells.\n\nControl and trout RBCs stimulated with VHSV UV-inactivated, multiplicity of infection (MOI) 1, were posteriorly co-culture with TSS cell line, at 14°C, and ifn1, mx, vig1 and il15 gene expression profiles were quantified at 24 hours postexposure (hpe) for RBCs and TSS. (A) Linear regression between ifn1 and the interferon stimulated genes (ISGs) mx, vig1 and il15 gene expression profiles in RBCs. (B) Linear regression between RBCs and TSS ifn1 gene expression profile. (C) Linear regression between ifn1 and the ISGs mx, vig1 and il15 gene expression profiles in TSS. Gene expression was normalized against eukaryotic 18S rRNA and relativized to control cells (red line) (fold of increase). Data is displayed as the linear regression line between the indicated cell lines and expressed genes (r2: coefficient of determination, asterisk denote statistical significance, P-value < 0.05) (n = 6). (D) Schematic representation of the RBCs and TSS co-culture assay and analysis.\n\nThe iTRAQ data showed a total of 9246 MS/MS Spectra, 2639 unique peptides with peptide-level FDR<0.01 and 872 inferred proteins common in all samples. Significant up/down regulations between samples were determined by a log2FoldChange)>1 with a q-value<0.05. In total, 64 proteins were significantly up or down-regulated during VHSV exposure (Figure 5). Specifically, 59 proteins were downregulated and only 5 proteins were upregulated during VHSV exposure. Cytoscape functional annotation was used to investigate the underlying biologically functional differences that may be related to VHSV exposure. The results showed four strongly represented networks of interest (mRNA stability, proteasome, viral process and cellular catabolic processes) (Figure 5 and Figure S2). Among the 59 down-regulated proteins (Figure 6, Table S1), the top-score network was the mRNA stability, being SNRPD3 (Small nuclear ribonucleoprotein D3 polypeptide) the most down-regulated protein with ̴ -3 log2FoldChange. This protein is a core component of the spliceosomal small nuclear ribonucleoproteins (snRNPs), the building blocks of the spliceosome, and therefore, it plays an important role in the splicing of cellular pre-mRNAs. Other proteins related to splicing processes were also highly downregulated (-2>log2FoldChange>-1), such as SRSF4 (Serine/arginine-rich splicing factor 4), which plays a role in alternative splice site selection during pre-mRNA splicing, RNPS1 (RNA binding protein S1, serine-rich domain), which is part of pre- and post-splicing multiprotein messenger ribonucleoprotein (mRNP) complexes. Apart from that, several heat shock chaperones were also downregulated (-2>log2FoldChange>-1), such as HSPA1L (Heat shock 70kDa protein 1-like) and HSPA5 (Heat shock 70kDa protein 5) both involved in the correct folding of proteins and degradation of misfolded proteins, and HSPA8 (Heat shock 70kDa protein 8), which may have a scaffolding role in the spliceosome assembly. Besides, another protein highly downregulated was NPEPL1 (Aminopeptidase-like 1), a novel protein which has been implicated in HIV replication53.\n\nRBCs were exposed to VHSV with a multiplicity of infection (MOI) of 1 at 14°C. Proteins were classified into five specific GO-Biological Process categories indicated in the x-axis. The y-axis indicates the number of proteins in each category, grey bars indicate upregulated proteins and black bars down-regulated proteins.\n\nBar plot of statistically significant differentially expressed proteins in VHSV-exposed RBCs compared to control cells (P-value< 0.05, FDR q-value < 0.05). Functional categories are labelled as follows: Blue = proteasome, pink = regulation of RNA stability, light green = cellular catabolic process, dark green= viral process, grey = proteins not associated to any function.\n\nOn the other hand, among the five upregulated proteins (Figure 6, Table S1), BANF1 (Barrier to Autointegration factor 1) has been directly implicated in viral processes and plays fundamental role in nuclear assembly, chromatin organization and gene expression. Besides, HNRNPR (Heterogeneous nuclear ribonucleoprotein R) plays an important role in processing precursor mRNA in the nucleus, and SRSF1 (Serine/arginine-rich splicing factor 1) is also implicated in mRNA splicing, via spliceosome.\n\nThe 59 downregulated proteins were analyzed using STRING v10.5 (RRID:SCR_005223, http://string.embl.de/)54 with a medium confidence score threshold of 0.4. An interactome network was built for these set of proteins to find out protein-protein interaction and predict functional associations. We found that proteins within spliceosome and proteasome networks interacted with each other as well as their partners. We also found that 17 proteins were involved in viral process category and that most of them interacted with each other as well as their partners (Figure 7).\n\nNodes represent DDPs and edges the interactions between two proteins. The colour of the edge indicates the interaction score (edge score). Red nodes highlight DDPs functionally annotated in viral processes.\n\nSince a global protein downregulation was observed in VHSV-exposed RBCs, we further investigated whether this phenomena could be due to the phosphorylation of the α-subunit of translational initiation factor 2 (eIF2α), a recognized key mechanism of global inhibition of translational initiation. For that, phosphorylation of eIF2α (eIF2α-P) was evaluated in VHSV-exposed RBCs compared to control cells by western blot (Figure 8A and B). The results showed a small upregulation of eIF2α-P in VHSV-exposed RBCs.\n\n(A) Representative western blot of eIF2α phosphorylation (eIF2α-P) in VHSV-exposed and control RBCs. (B) Bar plot of the eIF2α-P protein content of the stained bands estimated by densitometry, relative to α-Actin. Mann Whitney Test was performed for statistical analysis between the VHSV-exposed cells and control cells. Asterisk denote statistically significant differences (P-value < 0.05).\n\nFour eIF2α kinases have been identified to inhibit protein synthesis by phosphorylation of eIF2α: the double-stranded RNA-dependent eIF2α kinase (PKR), the mammalian orthologue of the yeast GCN2 protein kinase, the endoplasmic reticulum (ER) resident kinase (PERK) and heme-regulated eIF2α kinase (HRI)55. HRI, which was first discovered in reticulocytes under conditions of iron and heme deficiencies56,57, was later known to regulate the synthesis of both α- and β-globins in RBCs and erythroid cells by phosphorylation of eIF258, and therefore inhibiting protein synthesis. Besides, heme is also known to regulate the transcription of globin genes through its binding to the transcriptional factor Bach159. Taking this fact into account, we explored the RBCs β-globin gene expression during the course of VHSV exposure and the results showed that the β-globin gene was downregulated after 6 hpe (Figure 9), therefore suggesting an activation/phosphorylation of HRI and consequent phosphorylation of eIF2 and protein inhibition.\n\nRBCs were exposed to VHSV with a multiplicity of infection (MOI) of 1 at 14°C. Gene expression was quantified at time 0, 1, 3, 6, 12, 24, 72 hours postexposure (hpe). Gene expression was normalized against eukaryotic 18S rRNA and relativized to control cells (time 0, red line) (fold of increase). Data is displayed as mean ± SD (n = 3). Kruskal-Wallis Test with Dunn´s Multiple Comparison post-hoc test was performed among all conditions. Values denote pairwise significant differences with the value-indicated condition (P-value < 0.05).\n\nOxidative stress is known to be induced by viral infections, being one of the major pathogenic mechanisms by altering the balance of intracellular redox60. On the other hand, oxidative stress is known to activate HRI, which in turn phosphorylates eIF2α and inhibits protein translation. In order to evaluate the oxidative stress induced in VHSV-exposed RBCs as a possible causative mechanism for the proteome downregulation found in our study, we examined 72 hpe the ROS intracellular production by means of DCFDA (2′,7′-Dichlorofluorescin diacetate) fluorescence intensity. The results showed that VHSV-exposed RBCs significantly augmented DCFDA fluorescent intensity 72 hpe (Figure 10A), therefore VHSV halted infection in RBCs generated oxidative stress in trout RBCs. Besides, in order to evaluate the capability of RBCs to respond to the oxidative stress, the antioxidant response of VHSV-exposed RBCs was evaluated examining the transcript levels of the antioxidant genes fth (ferritin), gstp1 (glutathione S-transferase P), nkef (natural killer enhancement factor-like protein), sod1 (superoxide dismutase [Cu-Zn]) and trx (thioredoxin). The results depicted the increment in the transcript levels of fth, gstp1, nkef and trx (Figure 10B) as the time of exposure increased from 3 to 72 hours, demonstrating the capability of trout RBCs to counteract the oxidative stress.\n\nRBCs were exposed to VHSV with a multiplicity of infection (MOI) of 1 at 14°C, (A) DCFDA (2′,7′-Dichlorofluorescin diacetate) fluorescence intensity of VHSV-exposed RBCs relative to control cells, 72 hours postexposure (hpe). (B) Antioxidant genes (fth: ferritin, gstp1: glutathione S-transferase P, nkef: natural killer enhancement factor-like protein, sod1: superoxide dismutase [Cu-Zn], trx: thioredoxin) gene expression quantified 72 hpe. Gene expression was normalized against eukaryotic 18S rRNA and relativized to control cells (time 0, red line) (fold of increase). Data is displayed as mean ± SD (n = 3). Kruskal-Wallis Test with Dunn´s Multiple Comparison post-hoc test was performed among all conditions. Values denote pairwise significant differences (P-value < 0.05) with the value-indicated condition.\n\n\nDiscussion\n\nMost viral infections release their progeny to the outside of the cells (productive infections). However, viral infections can be also non-productive in non-permissive cells (also called abortive). Viral abortive infections occur when a virus enters a host-cell, then some or all viral components are synthesized but finally no infective viruses are released61. This situation may result from an infection with defective viruses or because the host cell is non-permissive and inhibits replication of a particular virus. Our results are consistent with VHSV binding and internalization, followed by viral genes transcription at early times of viral exposure and posterior quasi-inhibition inside trout RBCs. In this sense, trout RBCs could be classified as a non-permissive cell for VHSV replication, in contrast to other trout cells or tissues where VHSV is productive, such as RTG-2 cells62,63, fin cells64 or stroma65. Therefore, from our results, VHSV infection could be classified as halted in trout RBCs, since it enters the cell, but do not replicate at the levels comparable to the ~100-fold increase in titre of PRV and ISAV infections in salmon RBCs8,10. In fact, an apparent inhibition of the early viral genes transcription seemed to occur since NVHSV: GVHSV viral genes transcripts ratio was very low, and therefore did not follow the attenuation phenomenon found in rhabdoviruses45. However, strikingly, even though the recovered VHSV titer in the RBCs supernatant was very low 3 and 6 dpe, at 40 dpe almost the same virus titer could be recovered from the RBCs supernatant (data not shown), suggesting an ex vivo persistence of the halted VHSV inside RBCs.\n\nIn the literature, innate immune responses have been associated with viral abortive infections, including rhabdoviruses. Pham et al.66 speculated that the cause of the aborted VHSV infection in a trout macrophage cell line (RTS-11) could be the constitutive expression and/or upregulation of the mx genes. The abortive infection of the snakehead fish vesiculovirus (SHVV) in a zebrafish embryonic fibroblast cell line (ZF4) was associated with the activation of Retinoic acid-Inducible Gene I (RIG-I)-like receptors and interferon pathway by viral replicative intermediates67. Similarly, in mammals, Pfefferkorn et al.68 demonstrated that the abortive viral infection of astrocytes by rabies virus (RABV) and vesicular stomatitis virus (VSV) triggered a pattern recognition receptor signaling, which resulted in the secretion of IFN-β. On the other hand, it has been also described that alveolar macrophages are able to restrict the respiratory syncytial virus (RSV) replication even in the absence of type I IFNs (IFN1)69. In this sense, VHSV halted infection in trout RBCs did not seem to be related to IFN1 or IFN1-inducible genes, since inf1, mx and pkr genes as well as Mx and IFN1 proteins appeared poorly modulated or downregulated during VHSV exposure, in contrary to the 8-fold increase in ISAV productive infection in salmon RBCs10, the 50-fold increases in PRV productive infection in salmon RBCs8 or the 50-fold increases in IPNV non-productive infection in rainbow trout RBCs (unpublished study, Nombela I, Carrion A, Puente-Marin S, Chico V, Mercado L, Perez L, Coll J, and Ortega-Villaizan M). Alternatively, the high levels of constitutive Mx protein expression might have prevented its further increase in VHSV-exposed RBCs, like it is the case of the rainbow trout monocyte-macrophage RTS-11 cell line70. On the other hand, several cell mechanisms have been reported to suppress IFN1-mediated responses, which include downregulation of cell surface IFNα receptor (IFNAR) expression, induction of negative regulators (such as suppressor of cytokine signalling (SOCS) proteins and ubiquitin carboxy-terminal hydrolase 18 (USP18)), as part of a negative feedback loop to limit the extent and duration of IFN1 responses71. Separately, a putative antagonistic effect of the VHSV virus on the Mx induction has been previously reported72,73. Thus, it has been reported that VHSV NV protein interferes with the IFN signalling pathway, resulting in a poor induction of the Japanese flounder Mx promoter74. Furthermore, a lack of Mx upregulation has been speculated to be due to the immunosuppression caused by VHSV NV in trout injected with recombinant NV75. Recently, VHSV M protein has been also reported to suppress IFN1-induced gene expression76. From our results, in VHSV-exposed RBCs, the mx gene poor induction or slight downregulation could be probably supported by the existence of a VHSV antagonistic effect against the RBCs IFN response. To further clarify whether a viral antagonistic effect or a feedback loop of IFN1 and/or IFN1-inducible genes induction is related to or responsible for aborting or halting viral infections in trout RBCs remains to be studied, and are part of our ongoing research.\n\nSeparately, although the IFN levels were low, our results demonstrated the paracrine IFN crosstalk between RBCs, stimulated with UV-inactivated VHSV, and the spleen stromal the TSS cell line. The TSS cell line has been described to resemble the immune responses observed in cultures of head kidney macrophages77. Also, it has been demonstrated the ability of TSS to positively respond to conditioned supernatants from head kidney macrophage cultures exposed to poly I:C77. As well, after exposure to poly I:C, TSS produced a high upregulation of the Mx-1 gene78. Our results showed the correlated ifn1 regulation in both cell lines, as well as by the correlative regulation of the interferon-inducible mx gene in TSS, the regulation of il15, an interleukin that can activate antiviral responses via an interferon-dependent mechanism79, and the VHSV-inducible vig1, a gene induced by VHSV as well as by interferon80. Therefore, this crosstalk demonstrated the capacity of trout RBCs to exert a paracrine molecular antiviral communication with other cells with capacity to generate an immune response, as it is the case of the TSS cell line78. However, more extended research is need to identify further molecules involved in this crosstalk.\n\nOn the other hand, other immune proteins, such as BD1, IL1β and IL8, known to be involved in antiviral immunity, which were upregulated in VHSV- exposed RBCs, appeared to be part of the antiviral immune response of trout RBCs and could be implicated in the halted viral replication inside RBCs.\n\nTo further investigate the mechanisms implicated in the immune response of trout RBCs to VHSV, the comprehensive analysis of the differentially expressed proteins, obtained by means of iTRAQ proteome profiling, revealed the regulation of two typical mechanisms for viral subversive strategies: regulation of spliceosome, or splicing hijacking, and host-cell shut-off. However, even though these strategies usually lead to viral augmented replication and cell death, in the case of VHSV-exposed RBCs this is not observed. Therefore, how these strategies or another strategies contribute to halting viral replication yet remains elusive. Future research could be directed to investigate the role/implication of the small nuclear ribonucleoprotein SNRPD3, the aminopeptidase NPEPL1, the serine/arginine-rich splicing factor SRSF1 and the heterogeneous nuclear ribonucleoprotein HNRNPR, in the response of RBCs against VHSV replication, since these proteins were the more regulated ones and they have been shown to be implicated in HIV replication53,81–83).\n\nIt is noticeable that the iTRAQ-based protein profiling could not detect cytokines or other molecules related to the antiviral immune response, which could be detected by RT-qPCR, FC or IF. This fact could be due to the idiosyncratic limitations of the iTRAQ technique, such as its tendency to underestimate quantifications84, especially for low-represented proteins. This fact becomes especially critical in the case of RBC proteome analysis, since their protein production is lower compared to other cells. Further protein profiling by means of label-free protein quantification is ongoing.\n\nOn the other hand, the inhibition of both host and viral translation has been shown during infection with the prototype rhabdovirus vesicular stomatitis virus (VSV)85. During VSV infection, there is a rapid inhibition of host mRNA translation early after infection, followed by a later inhibition of viral mRNA translation, which has been associated to eIF2α phosphorylation86. Our results showed a slight increment in eIF2α phosphorylation in VHSV-exposed RBCs, indicating that this mechanism could be implicated in the inhibition of VHSV replication in trout RBCs. In this context, HRI, the heme-regulated eIF2α kinase, is one of the four kinases identified to inhibit protein synthesis by means of eIF2α phosphorylation. HRI is predominantly expressed in reticulocytes and erythroid precursors56,57, and it is known to regulate the synthesis of both α- and β-globins in RBCs and erythroid cells by phosphorylation of eIF258. Moreover, heme, the prosthetic group of hemoglobin, is known to inhibit eIF2α and therefore the transcription of globin genes through its binding to the transcriptional factor Bach1. From our results, we observed a decrease in the β-globin gene transcripts levels during the course of viral exposure, which accompanied with the observed phosphorylation of eIF2α could suggest a possible heme regulation mechanism of eIF2 pathway in response to VHSV exposure in trout RBCs. The mechanism by which heme is altered in trout RBCs during VHSV exposure remains to be investigated.\n\nAnother interesting mechanism found in trout RBCs in response to VHSV was the implication of the protective antioxidant enzymes genes fth, gstp1, nkef and trx in the defense of RBCs against the induction of ROS after VHSV exposure, since as the course of exposure increased, ROS slightly augmented in parallel to the transcript levels of these enzymes. These systems are known to contribute not only to repair the oxidative damage maintaining redox homeostasis, but also to the overall response of the cell to ROS by acting as oxidative sensors in signal transduction pathways87. Besides, regarding the implication of antioxidants activity against viral replication, it has been described that antioxidants can suppress virus-induced oxidative stress and reduce RNA virus production88. Separately, these antioxidant enzymes are known NF-κβ antioxidant targets in response to inflammation stimulus (reviewed in Morgan and Liu, 201187) and ROS can be sometimes produced in response to cytokines. Since NF-κβ appeared slightly activated in VHSV-exposed RBCs (Figure S3A and B), it is suggested that the cytokine response generated after VHSV exposure in trout RBCs would induce ROS production, and in turn this would modulate the NF-κβ response and NF-κβ target genes could attenuate ROS to promote RBCs survival. Apart from the observation of NF-κβ translocation to the nucleus in some of the RBCs, it is noteworthy that it is always accompanied by an increase in the protein levels of the p65 NF-κβ subunit in the cytoplasm. This phenomenon has been also observed in human foreskin fibroblasts during HCMV infection, where an increase in p65 mRNA levels correlated with the sustained increase in NF-kB activity during the course of infection89. On the other hand, the nuclear factor-erythroid 2 related factor 2 (Nrf2) and its downstream genes (i.e. Heme Oxygenase-1, HO1 and thrioredoxin) are also known as master genes of cellular defense against oxidative stress by scavenging ROS. Another fish rhabdovirus, the SVCV, has been reported to induce accumulation of ROS accompanied by the up-regulation of Nrf2 and its downstream genes. The overexpression of Nrf2 has been also reported to significantly suppress either entry or replication of several viruses (reviewed in 90), and Shao et al.90 also demonstrated that the activation of Nrf2 repressed the replication of SVCV. Therefore, future research could be directed to investigate the implication of the Nrf2 pathway in inhibiting VHSV replication in trout RBCs.\n\nIt is evident that the antiviral response of RBCs is low compared to other cells of the immune system. However, this fact could be explained by the inherent characteristic of the RBCs as the most abundant cell in the blood, where a unanimous high cytokine response by RBCs could lead to a septic shock. On the other hand, it is also noticeable the high inter-individual variability found for most of the genes and proteins assayed, which could be explained by the idiosyncratic presence of immune responders and non-responders in every assay.\n\nIn summary, this study unveils previously unobserved but important mechanisms for fish nucleated RBCs in the contribution to the defense against a viral aggression not involving RBCs as targets. To our knowledge, this is the first report that implicates fish RBCs as antiviral mediators against viruses targeting other tissues or cells. The recognition of body circulating viruses and the subsequent generation of immune defenses by RBCs may largely contribute to fish survival, given the large volume of RBCs and its rapid and wide distribution to the whole body. We are further investigating if similar mechanisms operate in vivo, the molecules that trigger such immune responses or the cellular factors implicated in the interaction with the virus.\n\n\nData availability\n\nF1000Research: Dataset 1. Excel file containing qPCR data. Each sheet contains the raw Ct values for the indicated figure numbers, organized by samples (rows) and genes (columns), 10.5256/f1000research.12985.d18283393\n\nF1000Research: Dataset 2. Excel file containing the virus titration data. Each sheet contains the virus titer (PFU/mL) results of the indicated figure number, 10.5256/f1000research.12985.d18283494\n\nF1000Research: Dataset 3. Flow cytometry data. Each folder contains the Flow Cytometry Standard (.fcs) format files. Source data files are organized by figure number, and then by antibody, sample number and condition, 10.5256/f1000research.12985.d18283595\n\nF1000Research: Dataset 4. Excel file containing the computed peptide spectrum match (PSM) raw data, and the spectra recovered in the iTRAQ 4-plex analysis., 10.5256/f1000research.12985.d18283696\n\nF1000Research: Dataset 5. Excel file containing the iTRAQ 4-plex quantitative analysis raw data., 10.5256/f1000research.12985.d18283797\n\nF1000Research: Dataset 6. Excel file containing the densitometry raw data of eIF2α-P and α-Actin western blots. Related uncropped blots are included., 10.5256/f1000research.12985.d18283898\n\nF1000Research: Dataset 7. Excel file containing DCFDA absorbance raw data., 10.5256/f1000research.12985.d18283999",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the European Research Council (ERC starting grant 2014 GA639249).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nSpecial acknowledgment is due to the co-author Dr. Amparo Estepa, passed away, who largely contributed to the initial ideas and enthusiasm behind this work. Thanks are due also to Beatriz Bonmati, Remedios Torres and Efren Lucas for their technical assistance. As well, thanks are due to Prof. Henny Gevers and Dr. Craig Plaisance for helping with English editing.\n\n\nSupplementary material\n\nFigure S1. Representative flow cytometry dotplots of immune protein responses of VHSV-exposed RBCs. RBCs were exposed to VHSV at MOI 1, at 14°C, and stained with anti-BD1 (A) and anti-IL8 (B), 72 hpe. Control and VHSV-exposed RBCs dotplots are shown. Y axis represents side scattering (SSC-A) and X axis FITC fluorescence intensity (FITC-A).\n\nClick here to access the data.\n\nFigure S2. Pathway network of significantly over-represented GO-terms in VHSV-exposed trout RBCs protein iTRAQ profiling.Big nodes represent significantly differentially expressed (down-regulated) proteins that have similar function; edges represent pairwise interactions; small nodes represent the proteins associated to each function. Functional groups are labelled as follows: Blue = proteasome, pink = regulation of RNA stability, light green = cellular catabolic process, dark green= viral process, grey = proteins not associated to any function. A list of all over-represented terms is provided in Table S1.\n\nClick here to access the data.\n\nFigure S3. NF-kß p65 protein labelling in VHSV-exposed RBCs. (A) Protein expression levels calculated by the formula MRFI (Mean Relative Fluorescence Intensity) = fluorescence in VHSV-exposed RBCs / fluorescence in non-exposed RBCs, at MOI 1, 10 and 100, 72hpe, at 14°C, relative to control cells (red line). Data represent mean ± SD (n=3). Mann Whitney Test was performed for statistical analysis between the VHSV-exposed cells and control cells. (B) Representative immunofluorescences of control and VHSV-exposed RBCs stained with anti-NF-kß (FITC) and DAPI for nuclei (IF representative of 20 images).\n\nClick here to access the data.\n\nTable S1. List of significantly over-represented GO-terms in VHSV-exposed trout RBCs protein iTRAQ profiling.\n\nClick here to access the data.\n\n\nReferences\n\nMagadan S, Sunyer OJ, Boudinot P: Unique Features of Fish Immune Repertoires: Particularities of Adaptive Immunity Within the Largest Group of Vertebrates. Results Probl Cell Differ. 2015; 57: 235–64. 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Vaccine. 2011; 29(4): 737–43. PubMed Abstract | Publisher Full Text\n\nNombela I, Puente-Marin S, Chico V, et al.: Dataset 1 in: Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication. F1000Research. 2017. Data Source\n\nNombela I, Puente-Marin S, Chico V, et al.: Dataset 2 in: Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication. F1000Research. 2017. Data Source\n\nNombela I, Puente-Marin S, Chico V, et al.: Dataset 3 in: Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication. F1000Research. 2017. Data Source\n\nNombela I, Puente-Marin S, Chico V, et al.: Dataset 4 in: Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication. F1000Research. 2017. Data Source\n\nNombela I, Puente-Marin S, Chico V, et al.: Dataset 5 in: Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication. F1000Research. 2017. Data Source\n\nNombela I, Puente-Marin S, Chico V, et al.: Dataset 6 in: Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication. F1000Research. 2017. Data Source\n\nNombela I, Puente-Marin S, Chico V, et al.: Dataset 7 in: Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication. F1000Research. 2017. Data Source"
}
|
[
{
"id": "27680",
"date": "27 Nov 2017",
"name": "Aleksei Krasnov",
"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 reports studies with red blood cells (RBC) of rainbow trout infected with Viral Haemorrhagic Septicaemia virus (VHSV). RBC most likely do not support propagation of VHSV and immune responses to the pathogen were small by magnitude. Still, experiments and analyses were well designed and implemented, applied diverse methods and therefore publication will be useful and interesting for experts in the area.\n\nMajor comment Suppression of innate antiviral immunity in infected RBC is included in the abstract as one of the key findings. However, of several genes analysed with qPCR only ifn1 showed down-regulation and only at one time-point. Other genes exhibited at best a slight tendency and differences from control were small. Reference to high variation is not convincing and does not overcome the lack of significance. Delete sentence “It is noteworthy to highlight the elevated inter-individual variability found in trout RBCs immune response, for most of the proteins and genes assayed, which could prevent to obtain statistical significance in most of the cases although regulations were apparent” (pages 9-10), this statement is trivial. M&M do not tell if each RBC culture was from an individual animal. If not, then variation was technical by character suggesting problems with methods. I also suggest to delete or at least shorten discussion of viral suppression of IFN-dependent responses in fish (page 16, 1st paragraph). I would emphasize strong induction of ROS scavengers as most impressive result of this study.\n\nSpecific comments\nFigure 2. Indicate method in the legend – qPCR? Change label of Y-axis: fold instead of fold of increase.\n\nFigure 4. The number of replicates (n = 6) is too small for regression and correlation analyses. I strongly suggest to plot empirical data, trend lines alone are not convincing. Judging from the figures, ifn1 levels were in the range from 0 to 15. Units should be explained. Furthermore, it is unclear how such differences was achieved taking into account minor responses of ifn1 to IHNV in trout RBC.\n\nFigure 10. Explain grey and black bars in the legend.\n\nPage 16. Delete paragraph “It is noticeable that the iTRAQ-based protein…” – no need to explain that proteomic analyses fail to detect low abundance proteins including cytokines.\n\nPage 17, 1st paragraph. NFkB can be mentioned but extensive discussion is not warranted since study did not produce any experimental data for this gene or protein.\n\n“It is evident that the antiviral response of RBCs is low compared to other cells of the immune system” – this statement is wrong. Virus infected fish RBC develop immune responses of huge magnitude. “Inter-individual variability” – if RBC cultures represented individuals, this must be explicitly stated in M&M (see comment above). Given small number of replicates, discussion of high and low responders is not supported with 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? 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 source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "3389",
"date": "09 Feb 2018",
"name": "Maria del Mar Ortega-Villaizan Romo",
"role": "Author Response",
"response": "Dear Dr. Aleksei Krasnov, We appreciate very much your thorough revision and positive and constructive comments on the manuscript. We have included your corrections and suggestions in the new version of the manuscript hoping that now the manuscript will be suitable for publication. Please find below the response to your comments: _____ The paper reports studies with red blood cells (RBC) of rainbow trout infected with Viral Haemorrhagic Septicaemia virus (VHSV). RBC most likely do not support propagation of VHSV and immune responses to the pathogen were small by magnitude. Still, experiments and analyses were well designed and implemented, applied diverse methods and therefore publication will be useful and interesting for experts in the area. Major comment Suppression of innate antiviral immunity in infected RBC is included in the abstract as one of the key findings. However, of several genes analysed with qPCR only ifn1 showed down-regulation and only at one time-point. Other genes exhibited at best a slight tendency and differences from control were small. Reference to high variation is not convincing and does not overcome the lack of significance. Delete sentence “It is noteworthy to highlight the elevated inter-individual variability found in trout RBCs immune response, for most of the proteins and genes assayed, which could prevent to obtain statistical significance in most of the cases although regulations were apparent” (pages 9-10), this statement is trivial. Response: We have deleted this sentence in order to avoid misunderstanding. M&M do not tell if each RBC culture was from an individual animal. If not, then variation was technical by character suggesting problems with methods. Response: We have indicated in Methods, Animals section, the following: The number of individuals used is indicated by an “n” in each experiment. I also suggest to delete or at least shorten discussion of viral suppression of IFN-dependent responses in fish (page 16, 1st paragraph). Response: As advised, we have shortened the discussion of viral suppression of IFN-dependent responses in fish (page 16, 1st paragraph). I would emphasize strong induction of ROS scavengers as most impressive result of this study. Response: As advised, we have emphasize the discussion related to ROS and antioxidant response in RBCs. Specific comments Figure 2. Indicate method in the legend – qPCR? Change label of Y-axis: fold instead of fold of increase. Response: We have indicated it as advised, in Figure 2 and in other figures as well. Figure 4. The number of replicates (n = 6) is too small for regression and correlation analyses. I strongly suggest to plot empirical data, trend lines alone are not convincing. Judging from the figures, ifn1 levels were in the range from 0 to 15. Units should be explained. Furthermore, it is unclear how such differences was achieved taking into account minor responses of ifn1 to IHNV in trout RBC. Response: As indicated we have included the individual values. The ifn1 response is normally downregulated in RBCs after VHSV-exposure. However, in a few individuals (outliers) we could find ifn1 upregulation, which correlated with the upregulation in TPS-2. Figure 10. Explain grey and black bars in the legend. Response: We have corrected it and explained it. Page 16. Delete paragraph “It is noticeable that the iTRAQ-based protein…” – no need to explain that proteomic analyses fail to detect low abundance proteins including cytokines. Response: We have deleted this sentence in order to avoid misunderstanding. Page 17, 1st paragraph. NFkB can be mentioned but extensive discussion is not warranted since study did not produce any experimental data for this gene or protein. Response: As advised, we have shortened the discussion related to NFkB. “It is evident that the antiviral response of RBCs is low compared to other cells of the immune system” – this statement is wrong. Virus infected fish RBC develop immune responses of huge magnitude. “Inter-individual variability” – if RBC cultures represented individuals, this must be explicitly stated in M&M (see comment above). Given small number of replicates, discussion of high and low responders is not supported with data. Response: As indicated before, we have indicated in Methods, Animals section, the following: The number of individuals used is indicated by an “n” in each experiment. Separately, in order to avoid misunderstanding we have eliminated the sentence “It is evident that the antiviral response of RBCs is low compared to other cells of the immune system”, “Inter-individual variability” and high and low responders discussion."
}
]
},
{
"id": "27745",
"date": "30 Nov 2017",
"name": "Johannes M. Dijkstra",
"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\nNombela et al. describe in what they claim to be defense mechanisms in trout red blood cells in response to halted replication of VHSV virus.\n\nMajor comments:\n\nThe study suffers from the effort to simultaneously answer a few unknowns. Can VHSV infect trout erythrocytes? Does infection by, or exposure to, VHSV virus modify the expression of genes with an anti-virus function? Do those altered gene expression levels have a measurable immune effect (which in this article is measured by the effect on cell line TSS)?\nIn my opinion, the authors show understanding of the complexity of those questions, but do not take sufficiently control of them. I am not satisfied with any of the story lines. My main concerns are that it is unclear for many of the presented data (i) whether the enhanced expression of immune molecules are due to RBC infection or due to other stimulations of the RBCs by the virus preparation, (ii) whether the changes in immune molecule expression are due to stimulation by virus preparation or due to time of culturing, and (iii) whether the expression levels of immune molecules reach meaningful levels or are just variation within what could be considered as “non-functional background levels”.\nAt the very positive side, the authors addressed an important question, and delivered an honest and elaborate piece of work. Therefore, I will not reject the paper, but I do request the addition of experimental data that in my opinion are necessary for better interpretation of the currently presented data in relation to actual VHS disease.\nThe authors should infect rainbow trout cells which they deem (sufficiently representative of) the natural host cells of VHSV, and use the UV-inactivated supernatant for stimulation of trout RBCs and compare the effect on RBC immune molecule expression with the effect after RBC incubation with VHSV. Alternatively, they can use inactivated serum of VHSV-infected trout. My guess is that the released cytokines have a much stronger effect on those erythrocytes than the viruses to which the erythrocytes are hardly receptive.\nThe authors should also use those supernatants of natural host cells, or sera from infected trout, for stimulation of TSS cells, and compare the effects quantitatively with those after stimulation with the supernatant of VHSV-exposed erythrocytes.\nThe above requested set of experiments (or modifications thereof, depending on the preferences of the authors) should help to quantitatively estimate the direct effect of VHSV on erythrocytes, and the effect of VHSV-stimulated erythrocytes on other cells, in comparison with other routes of immune stimulation during VHSV infection.\n\nDetailed comments:\n\nWhy did the authors use an MOI of 1? Even if such MOI is achieved, only half of the cells are expected to be infected. In this case the actual MOI for red blood cells probably was far below 1, because the MOI was calculated based on infection of the receptive EPC cells.\n\nHow were the viruses prepared? It seems that they were generated on EPC cells, but the details are important. Namely, other than the viruses, the infected cells also release cytokines which may have a cross-species effect.\n\nThe only presented data that I find convincing for that red blood cells were infected were the experiments shown in Fig. 1E and Fig. 1F, namely after pretreatment with neuraminidase (Fig. 1E) and infection with an MOI=100 (Fig. 1G; even in that case only 1/6 cells shows infection). However, none of the experiments on immune molecule expression was done under those conditions.\n\nIn the experiments, expression levels are compared with those of “control cells”. In some cases those control cells are not specified, while in other cases they are said to be the T=0 cells. However, this does not take into account that also the time of culture can have a significant effect on gene and protein expression levels. Most of the expression level effects reported in this article are quite small (e.g. from very low to only two-fold higher), and a possible “culture-time effect” should have been excluded.\n\nThe introduction should give detailed descriptions of what is known or unknown (i) about natural target cells and receptors used by VHSV for infection, and (ii) about fish erythrocytes and to what extent they have a normal metabolism. The introduction should also give an indication of the abundance of erythrocytes, which is relevant because many small amounts of cytokine could make a big amount, and also because any “intelligent” virus will do its best to avoid interaction with this abundant and for the virus non-productive cell type.\n\nIn the title, shouldn’t it be “in response to halted replication of VHS virus.”?\n\nIn the abstract and in the text: “after 6 hours postexposure” is double.\n\nIn the abstract, in the sentence “Co-culture assays of RBCs with TSS”, it should be made clear that those RBCs were stimulated with UV-inactivated VHSV.\n\nIn the introduction, a number of speculations are presented as facts:\nFish poikilothermic nature results in a delayed antigen affinity maturation, memory and lymphocyte proliferation. Fish have unique phagocytic B lymphocytes. (later than the reference, also mammalian B cells with phagocytic ability have been found) Fish have stronger innate immune responses. To compensate for those immune deficiencies, fish have unique phagocytic B lymphocytes and stronger innate immune responses.\n\nI don’t understand the “Thus” in the sentence “Thus, fish RBCs generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them”.\n\nIn Fig. 1A, how was the PCR value for N gene determined at T=0? Was that before or after addition of the viruses, and could the difference between T=0 and the other time points be explained by amplification from RNA in virions?\n\nFig. 1B seems to argue against the assumption that the RNAs amplified in Fig. 1A were derived from an infection (see also my previous point). In addition, although the relative comparison between the RTG2 and RBC results as presented in Fig. 1B should be OK, it is unclear to me from the materials and methods section how the absolute quantitative statement “However, a ratio of 2 was observed in RBCs, compared to the ratio of 8 found in RTG-2 cells, at 1 and 3 hpe (Figure 1B)” can be made.\n\nI am not convinced that Fig. 1D is evidence for replication between days 3 and 6, since the titer goes down >5000-fold from day 0 to day 3, and then stays very low. The authors should make clearer whether they feel that the small increase in virus titer between days 3 and 6 is only suggestive of virus replication, or that such replication is supported by proper statistics. As for the NH4Cl effect observed in Fig. 1D. Can a chemical effect of NH4CL on the integrity of virions stuck to the outside of RBCs be excluded from explaining the results? Furthermore, I would like the authors to elaborate, possibly in the introduction section, on endocytosis in regard to erythrocytes and VHSV infection. Could it be that only immature erythrocytes are expected to display efficient endocytosis, and might the Fig. 1F result be explained by differences in erythrocyte subpopulations?\n\nI don’t understand the sentence “As a result, the VHSV RNA inside RBCs was increased about ten times at 3 hpe”, because the increase seems to be from around 0.6 to 3.4, which is closer to a six-fold increase.\n\nFor discussion of the Fig. 1F result, the authors should explain the intracellular organization of RBCs (which are unusual cells), and where VHSV is expected. The sentence “along the cytoplasm and nucleus” can’t be understood, and gives the impression that the authors do not know where to expect (normal) cytoplasm in RBCs. Although they observe “along the nucleus”, which I think is the correct observation, the authors discuss the possibility of N protein being present in the nucleus. It is not wrong to present that as a possibility to partially explain their observations, but the authors should declare clearer that their observations do not necessitate that N protein is present in the nucleus. To superficial readers it now looks as if they claim detection of N protein in the RBC nucleus.\n\nIn Fig. 3 legend, the B and C order should be altered.\n\n(writing error) were co-culture with > were co-cultured with\n\nIn the Transwell system, the authors tried to get rid of RBC-attached virions with a single non-stringent wash. I doubt that such was sufficient for complete removal, and in the following 24 hours of co-incubation some virions or viral products may have diffused to the TSS cells.\n\nAs for Fig. 4. Is it OK to assume linear regression based on only two time points? Wouldn’t it be more proper to indicate all individual observations with dots? What is a 0-fold increase? In Figs. 4A and 4C, all three lines need explanation.\n\nI can’t trust the claims based on Fig. 8. In Fig. 8A, why were protein amounts loaded in the Control and VHSV lanes so different? I don’t believe that densiometry analysis technique for Western blot bands is sufficiently sensitive, especially not if comparing different ranges of band densities, to reliably claim an about 15% difference as done in Fig. 8B. In addition, for densiometry analysis, the Fig. 8A Actin blot is a horrible result because only half of the lane was properly exposed to the Western treatment. If the Fig. 8A result truly is a “representative” result, as claimed by the authors, the Fig. 8 based conclusions can’t be taken seriously.\n\nTrout erythrocytes are known to express MHC class I (Dijkstra et al. (2003)1; Sarder et al. (2003)2). Because MHC class I is a molecule upregulated during virus infection, it would be interesting to see the effect on its expression in RBCs by exposure to VHSV. Likewise, and especially because Nombela et al. discuss the proteasome, it would be interesting to see the regulation of the genes for the immuno-proteasome specific subunits.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "3390",
"date": "09 Feb 2018",
"name": "Maria del Mar Ortega-Villaizan Romo",
"role": "Author Response",
"response": "Dear Dr. Johannes M. Dijkstra, We appreciate very much your detailed revision and constructive comments and suggestions on the manuscript. We have included your corrections in the new version of the manuscript hoping that now the manuscript will be suitable for publication. Please find below the response to your comments: ______ Major comments: The study suffers from the effort to simultaneously answer a few unknowns. Can VHSV infect trout erythrocytes? Does infection by, or exposure to, VHSV virus modify the expression of genes with an anti-virus function? Do those altered gene expression levels have a measurable immune effect (which in this article is measured by the effect on cell line TSS)? In my opinion, the authors show understanding of the complexity of those questions, but do not take sufficiently control of them. I am not satisfied with any of the story lines. My main concerns are that it is unclear for many of the presented data (i) whether the enhanced expression of immune molecules are due to RBC infection or due to other stimulations of the RBCs by the virus preparation, (ii) whether the changes in immune molecule expression are due to stimulation by virus preparation or due to time of culturing, and (iii) whether the expression levels of immune molecules reach meaningful levels or are just variation within what could be considered as “non-functional background levels”. At the very positive side, the authors addressed an important question, and delivered an honest and elaborate piece of work. Therefore, I will not reject the paper, but I do request the addition of experimental data that in my opinion are necessary for better interpretation of the currently presented data in relation to actual VHS disease. The authors should infect rainbow trout cells which they deem (sufficiently representative of) the natural host cells of VHSV, and use the UV-inactivated supernatant for stimulation of trout RBCs and compare the effect on RBC immune molecule expression with the effect after RBC incubation with VHSV. Alternatively, they can use inactivated serum of VHSV-infected trout. My guess is that the released cytokines have a much stronger effect on those erythrocytes than the viruses to which the erythrocytes are hardly receptive. The authors should also use those supernatants of natural host cells, or sera from infected trout, for stimulation of TSS cells, and compare the effects quantitatively with those after stimulation with the supernatant of VHSV-exposed erythrocytes. The above requested set of experiments (or modifications thereof, depending on the preferences of the authors) should help to quantitatively estimate the direct effect of VHSV on erythrocytes, and the effect of VHSV-stimulated erythrocytes on other cells, in comparison with other routes of immune stimulation during VHSV infection. Response: This is an interesting suggestion. To answer this question we have carried out an experiment where RTG-2 cells were treated with UV-inactivated VHSV during 24h, after that UV-VHSV was removed and cells were culture 24h in RPMI fresh medium. This conditioned medium was used to stimulate rainbow trout RBCs, during 24h. RTG-2 is a known cell line susceptible to VHSV infection and with a high interferon response to VHSV, therefore we considered it as a proper cell line for this experiment. Please see the results in Figure 4D. In relation to the last sentence, we did not proceed with that assay since the results obtained with RTG-2 conditioned media on RBCs were conclusive. Detailed comments: Why did the authors use an MOI of 1? Even if such MOI is achieved, only half of the cells are expected to be infected. In this case the actual MOI for red blood cells probably was far below 1, because the MOI was calculated based on infection of the receptive EPC cells. Response: We have also infected RBCs with lower and higher MOIs, as can be observed in Figure 1C, in order to monitor course of infection. However, for immune response experiments we decided to use MOI 1 since over this value more defecting interfering virus will be present. Moreover, MOI1 is already a high titer in experiments of immune response to the infection. How were the viruses prepared? It seems that they were generated on EPC cells, but the details are important. Namely, other than the viruses, the infected cells also release cytokines which may have a cross-species effect. Response: VHSV was prepared in EPC cells as indicated in Methods. As suggested, we have expanded the details about it. As Dr. Dijkstra comments, we are aware that the clarified supernatant contains cytokines that may have a cross-species effect. However, the volume of supernatant/inoculum needed to infect may be less than 0.01 ul, since we usually have virus stock titter of 107 ffu/ml. Therefore, cytokines from EPC cells are diluted 1:100. The only presented data that I find convincing for that red blood cells were infected were the experiments shown in Fig. 1E and Fig. 1F, namely after pretreatment with neuraminidase (Fig. 1E) and infection with an MOI=100 (Fig. 1G; even in that case only 1/6 cells shows infection). However, none of the experiments on immune molecule expression was done under those conditions. Response: Yes, MOI values over 100 are needed to detect VHSV inside RBCs by immunofluorescence. However, MOI1 is already a high titer in experiments of immune response, since one or few viral particles per cell can induce detectable immune response. In the experiments, expression levels are compared with those of “control cells”. In some cases those control cells are not specified, while in other cases they are said to be the T=0 cells. However, this does not take into account that also the time of culture can have a significant effect on gene and protein expression levels. Most of the expression level effects reported in this article are quite small (e.g. from very low to only two-fold higher), and a possible “culture-time effect” should have been excluded. Response: For all the experiments, except for time course assays, control cells are uninfected cells culture in RPMI 2%FBS (viral infection medium), in the same plate as infected cells, and incubated the same time. To better explain it, we have indicated it in each figure legend. In the case of time course experiment (Figure 2), control cells refer to time=0h. For this assay, in order to evaluate the culture-time effect in the expression of mx and pkr genes, we have analysed gene expression in non-stimulated RBCs along the time course and observed that it does not change during the three days of culture ex vivo. We have added a Supplementary figure with these results (Figure S4), and commented it in the manuscript. The introduction should give detailed descriptions of what is known or unknown (i) about natural target cells and receptors used by VHSV for infection, and (ii) about fish erythrocytes and to what extent they have a normal metabolism. The introduction should also give an indication of the abundance of erythrocytes, which is relevant because many small amounts of cytokine could make a big amount, and also because any “intelligent” virus will do its best to avoid interaction with this abundant and for the virus non-productive cell type. Response: As advised, we have include information about VHSV targets and cell receptors in the introduction. About RBCs metabolism, we have talked about oxidative stress in the discussion. About the last item (“abundance of erythrocytes, which is relevant because many small amounts of cytokine could make a big amount”), we have talked about it the introduction and discussion. In the title, shouldn’t it be “in response to halted replication of VHS virus.”? Response: Yes, we have corrected it. In the abstract and in the text: “after 6 hours postexposure” is double. Response: We have corrected it. In the abstract, in the sentence “Co-culture assays of RBCs with TSS”, it should be made clear that those RBCs were stimulated with UV-inactivated VHSV. Response: Yes, we have added it. In the introduction, a number of speculations are presented as facts: Fish poikilothermic nature results in a delayed antigen affinity maturation, memory and lymphocyte proliferation. Response: We have deleted it to avoid misunderstanding. Fish have unique phagocytic B lymphocytes. (later than the reference, also mammalian B cells with phagocytic ability have been found) Response: We have deleted it to avoid misunderstanding. Fish have stronger innate immune responses. Response: We have deleted it to avoid misunderstanding. To compensate for those immune deficiencies, fish have unique phagocytic B lymphocytes and stronger innate immune responses. Response: We have deleted it to avoid misunderstanding. I don’t understand the “Thus” in the sentence “Thus, fish RBCs generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them”. Response: We have deleted it. In Fig. 1A, how was the PCR value for N gene determined at T=0? Was that before or after addition of the viruses, and could the difference between T=0 and the other time points be explained by amplification from RNA in virions? Response: Control cells are cells non-exposed to the virus, at t=0. We have better explained it in figure legend. Fig. 1B seems to argue against the assumption that the RNAs amplified in Fig. 1A were derived from an infection (see also my previous point). In addition, although the relative comparison between the RTG2 and RBC results as presented in Fig. 1B should be OK, it is unclear to me from the materials and methods section how the absolute quantitative statement “However, a ratio of 2 was observed in RBCs, compared to the ratio of 8 found in RTG-2 cells, at 1 and 3 hpe (Figure 1B)” can be made. Response: It is not clear to me why Fig. 1B argues that the RNAs amplified in Fig. 1A were derived from an infection. Please take into account that Fig. 1A is in log scale and the Nvhsv RNA level increment in RBCs at 3 hpe was low. About N:G genes ratio, which indicates RNA virus transcription, it was calculated as 2−ΔΔCt N VHSV: 2−ΔΔCt G VHSV. We have indicated it in the Figure 1B legend. I am not convinced that Fig. 1D is evidence for replication between days 3 and 6, since the titer goes down >5000-fold from day 0 to day 3, and then stays very low. The authors should make clearer whether they feel that the small increase in virus titer between days 3 and 6 is only suggestive of virus replication, or that such replication is supported by proper statistics. Response: This result is not statistically significant. VHSV titers are maintained in RBCs but do not increase. We have explained it in the manuscript. As for the NH4Cl effect observed in Fig. 1D. Can a chemical effect of NH4CL on the integrity of virions stuck to the outside of RBCs be excluded from explaining the results? Furthermore, I would like the authors to elaborate, possibly in the introduction section, on endocytosis in regard to erythrocytes and VHSV infection. Could it be that only immature erythrocytes are expected to display efficient endocytosis, and might the Fig. 1F result be explained by differences in erythrocyte subpopulations? Response: 7mM NH4Cl slightly increases cell pH and therefore inhibits endosome acidification. Thus, virus is kept into the endosome and not released into the cytoplasm. On the other hand, high alkaline pH (about 9) is needed to remove virus binded to cytoplasmic membrane. With 7mM NH4Cl, pH is only slightly increased to 7.5. In relation to RBCs endocytosis and virus endocytosis, as advised, we have talked about it in the introduction. However, in relation to the differences of endocytosis among RBCs subpopulations, it would be very interesting to evaluate but we do not have any evidence or reference about it. I don’t understand the sentence “As a result, the VHSV RNA inside RBCs was increased about ten times at 3 hpe”, because the increase seems to be from around 0.6 to 3.4, which is closer to a six-fold increase. Response: This comment refers to Figure 1E. Yes, we have now specified that 10 times increment at 3 hpe is in relation to VHSV-UV treated cells. For discussion of the Fig. 1F result, the authors should explain the intracellular organization of RBCs (which are unusual cells), and where VHSV is expected. The sentence “along the cytoplasm and nucleus” can’t be understood, and gives the impression that the authors do not know where to expect (normal) cytoplasm in RBCs. Although they observe “along the nucleus”, which I think is the correct observation, the authors discuss the possibility of N protein being present in the nucleus. It is not wrong to present that as a possibility to partially explain their observations, but the authors should declare clearer that their observations do not necessitate that N protein is present in the nucleus. To superficial readers it now looks as if they claim detection of N protein in the RBC nucleus. Response: Yes, thank you for your observation. After your observation, and in order to avoid misunderstandings, we prefer to eliminate the discussion about the localization of the N protein in the nucleus, and show it as an observation of intracellular localization. In Fig. 3 legend, the B and C order should be altered. Response: We have corrected it in figure 3 legend. (writing error) were co-culture with > were co-cultured with Response: We have corrected it. In the Transwell system, the authors tried to get rid of RBC-attached virions with a single non-stringent wash. I doubt that such was sufficient for complete removal, and in the following 24 hours of co-incubation some virions or viral products may have diffused to the TSS cells. Response: Yes, reviewer is right. However, more stringent washing conditions resulted to be cytotoxic for RBCs. As for Fig. 4. Is it OK to assume linear regression based on only two time points? Wouldn’t it be more proper to indicate all individual observations with dots? What is a 0-fold increase? In Figs. 4A and 4C, all three lines need explanation. Response: We have included individual observations and separated each line. In relation to your question about 0 fold-increase, for that point, TSS vig1 gene expression is 0.15 fold. I can’t trust the claims based on Fig. 8. In Fig. 8A, why were protein amounts loaded in the Control and VHSV lanes so different? I don’t believe that densiometry analysis technique for Western blot bands is sufficiently sensitive, especially not if comparing different ranges of band densities, to reliably claim an about 15% difference as done in Fig. 8B. In addition, for densiometry analysis, the Fig. 8A Actin blot is a horrible result because only half of the lane was properly exposed to the Western treatment. If the Fig. 8A result truly is a “representative” result, as claimed by the authors, the Fig. 8 based conclusions can’t be taken seriously. Response: We have replaced it by a better Western Blot. Trout erythrocytes are known to express MHC class I (Dijkstra et al. (2003)1; Sarder et al. (2003)2). Because MHC class I is a molecule upregulated during virus infection, it would be interesting to see the effect on its expression in RBCs by exposure to VHSV. Likewise, and especially because Nombela et al. discuss the proteasome, it would be interesting to see the regulation of the genes for the immuno-proteasome specific subunits. Response: Yes, it is an interesting observation. In fact we are working on it and the results wil be published in a different manuscript."
}
]
},
{
"id": "27746",
"date": "12 Dec 2017",
"name": "Uwe Fischer",
"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\nSummarized statement This is a comprehensive piece of work with a number of interesting experiments. However, I suggest to not accepting the paper in its present form.\n\nTo my opinion the authors have not convincingly shown an initial viral replication in RBCs, although this represents their primary experimental basis. All consecutive data are based on this initial assumption and this makes the whole story challengeable.\n\nIn contrast to the assumption of viral replication detection of viral RNA by RT-PCR could have been simply based on inoculated virus that could have been absorbed to the surface of RBCs or internalized by them. In order to prove viral replication de novo synthetized viral RNA or protein expression must be determined. In this system viral RNA replication can be determined on the mRNA level. For the RT step e.g. oligo(dT) primers could be used that only bind to the polyadenylated tail of mRNAs. The resulting cDNA must then be thoroughly treated with RNAse. This experiment is essential to make the experiments reliable.\n\nAlternatively, I suggest modifying the whole story and stressing the obvious responses of RBCs to the virus (avoiding the claim of virus replication). Thus the title could be changed into something like e.g. “Response of rainbow trout RBCs to VHSV”.\n\nSpecific comments Introduction: The authors claim: \"To compensate for those immune deficiencies, fish have unique phagocytic B lymphocytes\". This is not fully true since also B lymphocytes of some mammalians can execute phagocytosis.\n\nThey further claim: \"To compensate for those immune deficiencies, … and stronger innate immune responses, as shown in survivors of viral infection\". This is highly speculative and can’t be concluded from a single paper.\n\nMaterial and methods: The anti-β-defensin (BD1) anti -IFN1 and anti -IFNγ antibodies have not been described or characterized elsewhere. Their suitability can be questioned. The anti-Mx3 antiserum has been produced against a predicted Mx peptide. However, I did not find information on the characterization of this antiserum. The anti-IL1β and the NKEF antibodies have only been tested against the immunizing peptide and there is no further information if the antibodies react with the respective native proteins. The rabbit polyclonal antibody against human NF-κβ p65 has not been shown to be suitable in any fish species. Abcam only declares that it reacts with mouse, rat, chicken, human and Indian muntjac (Heterocephalus glaber). Those species are phylogenetically not closely related to teleosts.\n\nThe authors describe: \"Separately, NVHSV RT-qPCR was also used to quantify the viral RNA inside VHSV-exposed RBCs ….\" This is not a proof that virus was inside erythrocytes. The virus could have been simply attached/absorbed to RBCs. To show viral replication RT-PCR with mRNA is required.\n\n\"RBCs were fixed with 4% paraformaldehyde (PFA)…\" Unfixed RBCs should have been used in addition to fixed RBCs in order to distinguish between intracellular and membrane bound virus.\n\nResults: The authors conclude: “…to monitor the replication of VHSV in trout RBCs. … Clearly, the expressions of NVHSV gene were significantly upregulated at 3 hours postexposure.”. Again, I can’t support this statement since replication can’t be shown by RT-PCR for an RNA virus. Here simply increasing attachment to RBCs might have occurred which explains lower Cq values (higher amounts of viral RNA, respectively). This needs to be verified on the mRNA level.\n\nThe authors write: “On the other hand, after VHSV enters the cell, the first gene that starts to transcribe is the NVHSV gene,…”. This observation has been made in cell cultures that are permissive for VHSV. However, it has not convincingly been shown in this paper that N gene transcription took place in RBCs (see my comments above).\n\n“NVHSV RT-qPCR also confirmed the presence of viral RNA in VHSV-infected RBCs (Figure 2B).” Similarly to the above mentioned, this just says that VHSV was associated with RBCs, but not if the virus was inside RBCs. Attached virus could have resulted in positive RT-PCR results as well.\n\nTo further illustrate my concerns regarding viral replication in RBCs I have a few comments regarding Figure 1: The authors write that “The initial VHSV inoculum titer declined ~3-logs after 3 days\". In Figure 1H, however, I see a 4 log (from 10-to-the-6 to 10-to-the-2) reduction after 3 days (72 hours).\n\nAs for Figure 1H a negative control is missing where the same inoculum should have been added to the corresponding amount of cell-free medium or, even better, to inactivated (irradiated) erythrocytes. At 14°C, viral titers would probably also drop in the absence of cells (RBCs).\n\nIn Figure 1H the viral titers with RBCs drop within 72 hours by log4. However, in Figure 1D I can see an increase (although minor) in viral titers between 72 hours and 6 days in untreated cells. Is this statistically approved?\n\nIn Figure 1D the authors claim that “VHSV internalization in trout RBCs is NH4Cl-sensitive.” I can’t follow this conclusion. If internalization was NH4Cl-sensitive why it did not fully block virus internalization? The difference between untreated and treated RBCs is about only 2 fold. Is this statistically approved? Why the decrease in internalization wasn’t checked 3 hours after exposure when the N gene expression was claimed to be highest in RBCs, and why was it recorded 3 days post exposure when viral titers have been dropped by 4 logs anyway?\n\nThe NH4Cl blocking experiment should have been done with RTG-2 cells as a positive control?\n\nEthical statement The anti-IL1β antibody and the NKEF antibodies have been produced by ascitic tumor induction. This method is not in line with European standards of animal welfare. Since there are already two reports on this manuscript by two other referees, which I agree on, I will not repeat their objections and suggestions.\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "3391",
"date": "09 Feb 2018",
"name": "Maria del Mar Ortega-Villaizan Romo",
"role": "Author Response",
"response": "Dear Dr. Uwe Fischer,Thank you for your revision and comments on the manuscript. We have included your corrections in the new version of the manuscript hoping that now the manuscript will be suitable for publication.Please find below the response to your comments:_____This is a comprehensive piece of work with a number of interesting experiments.However, I suggest to not accepting the paper in its present form. To my opinion the authors have not convincingly shown an initial viral replication in RBCs, although this represents their primary experimental basis. All consecutive data are based on this initial assumption and this makes the whole story challengeable. In contrast to the assumption of viral replication detection of viral RNA by RT-PCR could have been simply based on inoculated virus that could have been absorbed to the surface of RBCs or internalized by them. In order to prove viral replication de novo synthetized viral RNA or protein expression must be determined.In this system viral RNA replication can be determined on the mRNA level. For the RT step e.g. oligo(dT) primers could be used that only bind to the polyadenylated tail of mRNAs. The resulting cDNA must then be thoroughly treated with RNAse. This experiment is essential to make the experiments reliable.Response: De novo synthetized viral RNA has been determined by Oligo(dT) RT-qPCR. We have included this result as supplementary Figure (Figure S1). As it can be observed, the kinetics of VHSV mRNA expression closely matched total virus RNA synthesis. Alternatively, I suggest modifying the whole story and stressing the obvious responses of RBCs to the virus (avoiding the claim of virus replication). Thus the title could be changed into something like e.g. “Response of rainbow trout RBCs to VHSV”.Response: We appreciate the suggestion, however, in view of other reviewers’ comments, we would like to maintain the title as it is. Specific commentsIntroduction:The authors claim: \"To compensate for those immune deficiencies, fish have unique phagocytic B lymphocytes\".This is not fully true since also B lymphocytes of some mammalians can execute phagocytosis.Response: We have removed this sentence.They further claim: \"To compensate for those immune deficiencies, … and stronger innate immune responses, as shown in survivors of viral infection\".This is highly speculative and can’t be concluded from a single paper.Response: We have removed this sentence.Material and methods:The anti-β-defensin (BD1) anti -IFN1 and anti -IFNγ antibodies have not been described or characterized elsewhere. Their suitability can be questioned.Response: The antiserum against β-defensin has been validated by our laboratory. Figure S2 depicts western blotting validation for anti-BD1 antibody. anti -IFN1 and anti -IFNγ antibodies have been validated by Dr. Luis Mercado laboratory. Figure S3 depicts western blotting validation for anti -IFN1 and anti –IFNγ.The anti-Mx3 antiserum has been produced against a predicted Mx peptide. However, I did not find information on the characterization of this antiserum.Response: anti-Mx3 antiserum production was described in Chico et al. 2010, Journal of Virology. In Martinez-Lopez et al. 2014 and Ortega-Villaizan et al. 2011 they showed the Mx protein expression in stimulated RTG-2 cells, using anti-Mx3 antibody. The anti-IL1β and the NKEF antibodies have only been tested against the immunizing peptide and there is no further information if the antibodies react with the respective native proteins.Response: For anti-IL1β, in Schmitt et al. 2015 and Rojas et al. 2015, we can find theexpression of IL1β in RTS11 and RTgutGC cells, respectively, by immunofluorescence and western blot. For anti-NKEF, Bethke et al. 2012, show western blotting of NKEF protein in serum of Atlantic salmon. Also Tafalla et al. 2011 show the expression of NKEF in RTS11 as evaluated by western blotting and by flow cytometry.The rabbit polyclonal antibody against human NF-κβ p65 has not been shown to be suitable in any fish species. Abcam only declares that it reacts with mouse, rat, chicken, human and Indian muntjac (Heterocephalus glaber). Those species are phylogenetically not closely related to teleosts.Response: We indicated references where this antibody has been used for teleost species:Encinas P, Garcia-Valtanen P, Chinchilla B, et al.: Identification of multipath genes differentially expressed in pathway-targeted microarrays in zebrafish infected and surviving spring viremia carp virus (SVCV) suggest preventive drug candidates. PLoS One. 2013;8(9):e73553. 24069208 10.1371/journal.pone.0073553 3772095García-Valtanen P, Martinez-Lopez A, Ortega-Villaizan M, et al.: In addition to its antiviral and immunomodulatory properties, the zebrafish β-defensin 2 (zfBD2) is a potent viral DNA vaccine molecular adjuvant. Antiviral Res. 2014;101:136–47. 24286781 10.1016/j.antiviral.2013.11.009Abós B, Castro R, González Granja A, et al.: Early activation of teleost B cells in response to rhabdovirus infection. J Virol. 2015;89(3):1768–80. 25410870 10.1128/JVI.03080-14 4300759The authors describe: \"Separately, NVHSV RT-qPCR was also used to quantify the viral RNA inside VHSV-exposed RBCs ….\"This is not a proof that virus was inside erythrocytes. The virus could have been simply attached/absorbed to RBCs.To show viral replication RT-PCR with mRNA is required.Response: As previously indicated, we have included that result as supplementary Figure (Figure S1). As it can be observed, the kinetics of VHSV mRNA expression closely matched total virus RNA synthesis.\"RBCs were fixed with 4% paraformaldehyde (PFA)…\"Unfixed RBCs should have been used in addition to fixed RBCs in order to distinguish between intracellular and membrane bound virus.Response: Yes, it is right. However, we have tried to do immune staining of unfixed RBCs and it is very difficult since RBCs are lysed during the antibody incubation. Results:The authors conclude: “…to monitor the replication of VHSV in trout RBCs. … Clearly, the expressions of NVHSV gene were significantly upregulated at 3 hours postexposure.”.Again, I can’t support this statement since replication can’t be shown by RT-PCR for an RNA virus. Here simply increasing attachment to RBCs might have occurred which explains lower Cq values (higher amounts of viral RNA, respectively). This needs to be verified on the mRNA level.Response: As previously indicated, we have included that result as supplementary Figure (Figure S1). As it can be observed, the kinetics of VHSV mRNA expression closely matched total virus RNA synthesis. The authors write: “On the other hand, after VHSV enters the cell, the first gene that starts to transcribe is the NVHSV gene,…”.This observation has been made in cell cultures that are permissive for VHSV. However, it has not convincingly been shown in this paper that N gene transcription took place in RBCs (see my comments above).“NVHSV RT-qPCR also confirmed the presence of viral RNA in VHSV-infected RBCs (Figure 2B).”Similarly to the above mentioned, this just says that VHSV was associated with RBCs, but not if the virus was inside RBCs. Attached virus could have resulted in positive RT-PCR results as well.Response: The reviewer is correct, RT-qPCR data do not tell if the virus is inside or attached to the cell. But we would like to highlight that Figure 1E shows that in RBCs exposed to UV-inactivated VHSV, Nvhsv levels did not increase in comparison with live VHSV exposed RBCs. This experiment demonstrates that live VHSV replicated early post-exposure, in comparison with UV-inactivated VHSV. In order to emphasize this result we have re-written its explanation. To further illustrate my concerns regarding viral replication in RBCs I have a few comments regarding Figure 1:The authors write that “The initial VHSV inoculum titer declined ~3-logs after 3 days\".In Figure 1H, however, I see a 4 log (from 10-to-the-6 to 10-to-the-2) reduction after 3 days (72 hours). Response: It was a mistyping error, we have corrected it.As for Figure 1H a negative control is missing where the same inoculum should have been added to the corresponding amount of cell-free medium or, even better, to inactivated (irradiated) erythrocytes. At 14°C, viral titers would probably also drop in the absence of cells (RBCs).Response: VHSV titer in cell-free medium 3 d at 14ºC does not drop more than one log. In Figure 1H the viral titers with RBCs drop within 72 hours by log4. However, in Figure 1D I can see an increase (although minor) in viral titers between 72 hours and 6 days in untreated cells. Is this statistically approved?Response: Yes, we observed a minor increase at 6 days post exposure (from ̴ 200 to ̴ 300 PFU/ml), and not statistically significant. In Figure 1D the authors claim that “VHSV internalization in trout RBCs is NH4Cl-sensitive.” I can’t follow this conclusion. If internalization was NH4Cl-sensitive why it did not fully block virus internalization? The difference between untreated and treated RBCs is about only 2 fold. Is this statistically approved?Why the decrease in internalization wasn’t checked 3 hours after exposure when the N gene expression was claimed to be highest in RBCs, and why was it recorded 3 days post exposure when viral titers have been dropped by 4 logs anyway?Response: You are right, the sentence is quite confusing, we have re-written it. We would like to clarify that NH4Cl blocks fusion of viruses to cell membrane, not internalization. On the other hand, the concentration used for NH4Cl (7 mM) was selected since it is non-cytotoxic for RBCs and other cells, such as EPC and RTG-2, but effective for reducing VHSV infectivity by 40% in EPC cells (Mas et al. 2002, Journal of General Virology), similar to the reduction that we observe in RBCs. The NH4Cl blocking experiment should have been done with RTG-2 cells as a positive control?Response: We have previously carried out this assay in RTG-2 cells and observed that, at 10 mM NH4Cl, VHSV infection is reduced by 90% in RTG-2 cells, quantified by Nvhsv RT-qPCR. However, a reduction of 50% was observed by Mas et al. 2002 at the same NH4Cl concentration, in EPC cells, although in this case it was quantified by FFU/ml.Ethical statementThe anti-IL1β antibody and the NKEF antibodies have been produced by ascitic tumor induction. This method is not in line with European standards of animal welfare.Response: Only anti-NKEF, produced at the laboratory of Dr. Luis Mercado (Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile), was developed in ascities fluids, because this antibody was obtained in 2011, when this technique was allowed in Chile. Nowadays, anti-NKEF is no longer produced by ascitic tumor induction, following the Chilean standards for animal welfare."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1958
|
https://f1000research.com/articles/6-1751/v1
|
26 Sep 17
|
{
"type": "Research Article",
"title": "Dermal fibroblasts from patients with Parkinson’s disease have normal GCase activity and autophagy compared to patients with PD and GBA mutations",
"authors": [
"Lucy M Collins",
"Janelle Drouin-Ouellet",
"Wei-Li Kuan",
"Timothy Cox",
"Roger A Barker",
"Janelle Drouin-Ouellet",
"Wei-Li Kuan",
"Timothy Cox",
"Roger A Barker"
],
"abstract": "Background: Recently, the development of Parkinson’s disease (PD) has been linked to a number of genetic risk factors, of which the most common is glucocerebrosidase (GBA) mutations. Methods: We investigated PD and Gaucher Disease (GD) patient derived skin fibroblasts using biochemistry assays. Results: PD patient derived skin fibroblasts have normal glucocerebrosidase (GCase) activity, whilst patients with PD and GBA mutations have a selective deficit in GCase enzyme activity and impaired autophagic flux. Conclusions: This data suggests that only PD patients with a GBA mutation have altered GCase activity and autophagy, which may explain their more rapid clinical progression.",
"keywords": [
"GBA mutations",
"Parkinson’s disease",
"Gaucher disease",
"fibroblasts",
"lysosome",
"autophagy"
],
"content": "Introduction\n\nGaucher disease (GD) is an autosomal recessive condition that is caused by defective glucocerebrosidase (GCase) enzyme. GCase (GBA gene) normally functions to breakdown lipids in the lysosomes and mutations in GCase cause accumulation of its substrate, glucoceramide (GlcCer)1. These mutations result in characteristic engorged macrophages, known as Gaucher macrophages (GMs) or ‘Gaucher cells’2. The GlcCer buildup in macrophages manifests as a multi-systems disorder, with signs such as hepatosplenomegaly, anaemia, thrombocytopenia, and bony impairments, reviewed by3. GD is common in Ashkenazi Jewish populations, although the disorder is pan-ethnic4. GD has been historically divided into three types, based on the severity of clinical features and neurological involvement3,5. Type I GD is classically defined as non-neuropathic, although neurological deficits have been described in some of these patients6 including Parkinsonism7–10. Type II disease usually begins in infancy, with severe neurological involvement. Type III GD is an extended form of type II, also with neuropathic problems but patients live into adolescence and adulthood largely due to the development of enzyme replacement therapy (ERT)3,5.\n\nIt is now apparent that there is a strong genetic connection between Gaucher and Parkinson’s disease (PD). Studies have shown that in large multi-center patient cohorts, patients with PD have an increased incidence of carrying GBA mutations11–14, including 3% of sporadic PD patients and up to 15% in Ashkenazi Jewish populations with PD15. Thus, we now know that heterozygote GBA mutations are the single commonest genetic risk factor for familial16 and sporadic PD; leading to a more rapid progression of PD with an early onset dementia12–14.\n\nThe cellular pathology in lysosomal storage disorders (LSD) is centered around the misfolding of GCase and lysosomal dysfunction. Lysosomes have also been implicated in PD and several other neurodegenerative disorders including Alzheimer’s disease and Huntington’s disease17,18, suggesting that similar underlying defects in autophagy and lysosomal dysfunction may link the pathophysiology of PD to GD19. The accumulation of substances in the lysosome impacts on their function and other intracellular pathways, resulting in secondary changes such as an impairment of autophagy20. Lysosomal and autophagy impairments are apparent in cell line models, mouse models and in post-mortem tissue from PD patients. In PD mice models, components of lysosomal function are affected, causing a reduction in lysosomal number and accumulation of autophagosomes21. Genetic mutations causing PD have also implicated the lysosome; these include ATPase Type 13A2 (ATP13A), which encodes a lysosomal ATPase, maintains lysosomal pH, and inhibits α-syn misfolding22. α-synuclein aggregation in the lysosome, as seen in PD and LSD, may in turn accelerate its own aggregation.\n\nIn this study, we sought to investigate autophagic function and GCase enzyme function using cells derived from patients. We tested patients with PD with and without GBA mutations as well as individuals with GD. We sought to define the extent of reduced GCase enzyme activity in all cases, and how this relates to autophagic flux. The activity of three other housekeeping lysosomal enzymes was also looked at, to see the extent to which the enzyme defect was specific.\n\n\nMethods\n\nFibroblasts were derived from dermal skin biopsies. In Table 1 the characteristics of the 17 patient derived fibroblast cell lines are summarised. Written informed consent was taken from each participant, and the ethics for this study was approved by the Cambridge Central Research Ethics Committee (REC09/H0311/88). HFL1 (ATCC-CCL-153) cells were obtained from the American Type Culture Collection (ATCC), expanded in standard fibroblast medium and used as controls. Skin biopsies were taken from patients with:\n\n(i) Both GD and PD (homozygous, PD/GD), n=3\n\n(ii) PD with one GBA mutation (heterozygous, PD GBA), n=3\n\n(iii) PD with no GBA mutations, idiopathic PD (iPD), n=3\n\n(iv) Healthy controls, n=4\n\n(v) GD only (homozygous), with no PD (GD), n=4\n\nWT = Wildtype, (?) = Unknown genotype.\n\nTo detect protein expression, samples obtained from the lysed cells were loaded on to a 4–12% SDS-PAGE gel (Invitrogen). Cells were harvested using either Radioimmunoprecipitation assay buffer (RIPA), containing 1.0% sodium dodecyl sulfate (SDS), 0.5% Sodium Deoxycholate, 0.1% Triton X 100, 150 mM NaCl, 50 mM Tris-HCl pH 8.0 and protease inhibitors, or NP40 (Nonidet P40) buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 8.0, NP-40 1.0% and protease inhibitors. The samples were sonicated at 1 second intervals for 10 secs, centrifuged at 20,000 g for 20 minutes at 4°C and the supernatant was snap frozen and stored at -80°C. The total protein concentration was quantified by a BCA assay (Thermo Scientific). The gel was run in 3-(N-morpholino) propanesulfonic acid (MOPS) buffer at 100 V, 40mA for 1 hour. The proteins on the SDS gel were transferred to a polyvinylidene fluoride (PVDF) membrane at 30 V, 100 mA for 2 hours. Then the membrane was incubated with Ponceau red to detect the proteins before being blocked (room temperature (RT), 1 hr) with 5 % w/v semi skimmed milk, 1% bovine serum albumin (BSA) PBS with tween (PBS-T 0.3 % v/v) and incubated with primary antibodies GBA(GCase) rabbit polyclonal, sigma (G4046) 1:1000, LC3 rabbit polyclonal New England Biolabs (NEB) (2775S) 1:1000, and actin rabbit polyclonal Abcam ab8229 1:1000 overnight at 4°C. Following washes in PBS-T (3 X 5 min), horseradish peroxidase labeled secondary anti rabbit antibodies (Bio-Rad 1662408EDU) were applied at 1:10,000 dilution in 5 % milk, 1% BSA (room temperature, 1 hr). Membranes were washed again with PBS-T and protein bands visualized by chemiluminescence (Thermo Pierce) estimating band molecular weights relative to standard protein markers in the range of 10–120 kDa (Novex® Sharp Pre-stained Protein Standard, Life Technologies). Protein bands were quantified by densitometry using Image J (Fiji) software.\n\nThe cellular localisation of the GBA protein in dermal fibroblasts was investigated using antibodies against GBA (Sigma, G4046, rabbit polyclonal 1:50) and Lamp1 (Abcam, ab25630, mouse monoclonal 1:50). Fibroblasts were grown to 50,000 cell/well confluency on 0.1% gelatin coated covers-slips in 24-well plates overnight in DMEM containing 10% FBS, and 1% penicillin/streptomycin (pen/strep) and fixed in 4% PFA for 30 minutes. Cells were washed twice with PBS, blocked in PBS, 10% FBS and 0.2% Triton and incubated for 1 hour at room temperature. After this the cells were then incubated overnight at 4°C with the relevant primary antibody (GBA (Sigma, G4046, rabbit polyclonal 1:50) and Lamp1 (Abcam, ab25630, mouse monoclonal 1:50)) in PBS, 0.2% Triton and 2% FBS. The cells were washed three times with PBS the following morning and incubated with the secondary antibodies (goat anti rabbit (green) and goat anti mouse (red) antibodies (Bio-Rad) were applied at 1:10,000 dilution in PBS, 0.2% Triton and 2% FBS in the dark for 2 hours at room temperature and washed with PBS three times for 5 minutes each and stained with Hoescht 1mg/ml in PBS for 15 minutes at room temperature in the dark. The cells were then washed with PBS 3 times for 5 minutes and imaged on a confocal microscope. In each well 10 fields of view were captured and this was repeated in triplicate.\n\nThe dermal fibroblasts were seeded and grown at a density of 72,000 cells per well in 24-well plates overnight in DMEM with 10% FBS and 1% pen/strep. Bafilomycin A1 from Sigma-Aldrich (B1793) was used at a concentration of 0.1 mM and added to the cells for 2 hours, this concentration was derived from titrations of Bafilomycin A1 and 0.1 mM and found to be the concentration best tolerated by the cells. The cells were either treated with 1) Bafilomycin A1 alone, 2) Bafilomycin A1 and starvation medium (Hanks’ Balanced Salt Solution (HBBS) with 5% Sodium Bicarbonate) 3) starvation alone and 4) untreated cells as a control. The cells were incubated for 2 hours at 37°C and then washed twice with ice cold PBS. To harvest, the cells were incubated with prechilled NP-40 lysis buffer for 30 minutes with shaking at 4°C. The cells were then collected and spun at 20,000 g for 15 minutes, the supernatant was snap frozen and stored at -80°C until processed for western blotting analysis.\n\nCell preparation. The enzyme activity assays were performed on fibroblasts harvested from 24-well plates. Cells were seeded at a density of 100,000 cells per well in DMEM containing 10% FBS and 1% pen/strep. After 24 hours the cells were harvested in ice-cold lysis buffer specific to each reaction described below. The lysate was spun for 20 minutes at 20,000 g at 4°C. The cell pellet was then resuspended in lysis buffer and sonicated for 1 minute, while keeping the sample on ice. A BCA assay (Thermo Fisher) was performed to quantify protein concentration.\n\nFor the GCase activity assay, cells were harvested in lysis buffer containing 30 mM Citrate Phosphate buffer pH 5.5, 0.65% Sodium Taurochlorate and 0.65% triton-X 100. Enzyme activity was measured using 4-Methylumbelliferyl (4MU) β-D-glucopyranoside, Sigma (M3633), as the substrate in running buffer containing 30 mM Citrate Phosphate buffer and 0.6% Sodium Taurochlorate pH 4.4 (to inhibit GCase 2 and 3). Each well in a 96 well black microplate, contained 20 μg (for each sample ~20 μl of protein and 30 μl of buffer) of sample protein, 75 μl running buffer and 25 μl of 15 mM substrate (which was made up immediately before the assay at 59.15 mg/ml in Dimethyl sulfoxide (DMSO) and diluted 1/10 in running buffer and kept in the dark). Each assay was repeated in triplicate for each cell sample. The sample alone was added in triplicate as a blank and also, the buffer with the substrate alone was added as a blank. The samples were incubated for 1 hour at 37°C and the reaction was stopped using 100 μl of 0.2 M Glycine pH 10.5. The fluorescence was read on the FLUOstar Omega plate reader at excitation 355 nm and emission 460 nm.\n\nTotal hexosaminidase activity was measured using fibroblasts harvested with lysis buffer containing 0.01 M Citrate phosphate buffer, pH 4.4, and 0.2 M Na2HPO4. A solution of 4 μg cell lysate (for each sample of ~10 μl of protein), 95 μl of 0.01 M Citrate Phosphate buffer and 20 μl of 2.5 mM 4-MU N-acetyl-b-D-glucosaminide, Sigma (M2133), in 0.01 M Citrate phosphate buffer was added to each well, the assay was performed in triplicate and blanks were included in lysate alone and a separate blank (buffer and substrate alone). The reaction ran for 20 minutes at 37°C and was stopped by adding 100 μl of 0.17 M Glycine pH 9.8 (made up from 2.5 g glycine and 3.6 g Na2CO3). The activity was measured on the FLUOstar Omega plate reader at excitation 360 nm and emission at 415 nm.\n\nTo measure α-galactosidase activity, 15 μg of cell lysate (each sample contained ~20 μl of protein and 30 μl of buffer), was incubated with 75 μl 0.2 M Citrate Phosphate buffer pH 4.4 and 20 μl of 10 mM 4-MUl α-D-galactopyranosidase, Sigma (M7633). This cell lysate solution was added to each well, the assay was performed in triplicate and blanks included lysate alone and a separate blank (buffer and substrate alone). The reaction was incubated for 30 minutes at 37°C and stopped by adding 100 μl of 0.17 M Glycine pH 9.8. The activity was measured on the FLUOstar Omega plate reader at excitation 360 nm and emission at 415 nm.\n\nTo measure mannosidase activity, 15 μg of cell lysate (for each sample ~20 μl of protein and 40 μl of buffer), was incubated with 50 μl of 0.2M Citrate Phosphate buffer pH 4.4 and 15 μl of 5 mM 4-MU α-D-mannopyranoside, Sigma (M3657). This cell lysate solution was added to each well, the assay was performed in triplicate and blanks included lysate alone and a separate blank (buffer and substrate alone). The reaction was incubated for 20 minutes at 37°C and stopped by adding 100 μl of 0.17M Glycine pH 9.8. The activity was measured on the FLUOstar Omega plate reader at excitation 360 nm and emission at 415 nm.\n\nThe fibroblasts were plated at a density of 50,000 cells/well on 0.1% gelatin coated coverslips and placed in a 24-well plate. The cells were grown over night in DMEM with 10% FBS and 1% pen/strep. The following day Gold nanoparticles were added to the cells (AuNPS). The cells were incubated with the AuNPS for 24 hours and then washed with cell culture medium to remove the AuNPS. After 16 hours the cells were fixed using 4% formaldehyde and imaged on a brightfield microscope.\n\nAll analysis was performed using SPSS version 20.1 (IBM). Normality for all the variables was tested using a one-sample Kolmogorov–Smirnov tests. Variables which did not follow a normal distribution, were log transformed and retested for normality. Non-parametric variables were compared with a Mann-Whitney U-test or Kruskal Wallis test. Parametric variables were compared with a t-test or ANOVA. Bonferroni post analysis was applied. Each experiment was repeated a minimum of three times.\n\n\nResults\n\nWe started by assessing enzyme activity as an indication of overall lysosomal health and the extent to which this is restricted to GCase activity. A panel of lysosomal enzymes were screened for activity levels and included GCase, α-galactosidase, hexosaminidase and mannosidase. The enzyme activity was assessed in all the patient fibroblast lines (n=17), and then grouped in to their respective disease phenotypes, based on GBA genotype for the analysis. GCase activity was found not surprisingly to be significantly lower in the PD GBA, GD and PD/GD compared to controls. In contrast, the iPD group was not significantly different compared to any other groups (Figure 1A and Dataset 1). There was no significant difference seen in the other lysosomal enzymes that were looked at (Figure 1 B–D) and Dataset 1).\n\n(A) GCase activity was significantly lower in the PD GBA (83.76 ± 8.16) (p=0.023), GD (55.33 ± 11.88) (p=0.006) and PD/GD (45.56 ± 2.99) (p=0.013) compared to controls (229.67 ± 79.96). The iPD group was not significantly different compared to any other groups (134.20 ± 33.94) (p>0.05) (B) α-galactosidase activity for PD (212.42 ± 22.30), PD GBA (189.81 ± 12.51), GD (188.10 ± 10.02), control (257.63 ± 17.13) and PD/GD (179.00 ± 16.25) (p>0.05). (C) Hexosaminidase activity for PD GBA (540.10 ± 178.66), iPD (539.61 ± 203.33), GD (485.08 ± 153.99), control (931.33 ± 120.93) and PD/GD (350.65 ± 111.99) (p>0.05). (D) Mannosidase activity for iPD (245.33 ± 28.76), PD GBA (222.67 ± 24.81), GD (234.83 ± 26.06), control (304.00 ± 14.00) and PD/GD (210.44 ± 19.92) (p>0.05). Enzyme activity measured by the FLUOstar Omega plate reader (Ex max = 360nm, Em max.= 415nm. (E) Western blot of GCase protein levels. No reduction in GCase expression in GD (0.74 ± 0.08), PD GBA (1.41 ± 0.60) and the HLF (1.96 ± 0.48) (all p>0.05). PD/GD (0.33 ± 0.16) was significantly lower compared to control (1.95 ± 0.15) (p=0.042) and compared to the iPD (2.05 ± 0.55) (p=0.044). Ratio of GCase protein over total actin expression as a loading control. (F) Fibroblasts were stained for GCase (green), Lamp1 (red) and DAPI (blue). Representative images of Control and PD/GD lines. Density was measured in Image J. Western blot analysis was repeated in three independent experiments for each cell line. (A.U arbitrary units).*p<0.05. Data was found to be parametric and analysed by ANOVA and Bonferroni post hoc test. Analysis was repeated in three independent experiments for each cell line. Data are presented as mean ± s.e.m. (G) Brightfield images of cell lines with AuNP uptake. No significant difference was found when measuring gold nanoparticle density in control (1.91 × 106 ± 5.89 × 105), PD/GD (1.31 × 106 ± 1.79 × 105), GD (1.24 × 106 ± 3.41 × 105), PD GBA (1.19 × 106 ± 9.04 × 104) and iPD (1.17 × 106 ± 2.11 × 105) (all p>0.05) (A.U) arbitrary units. Density was measured in Image J. For each cell line 30 fields of view were analysed from three separate repeats. The AuNP uptake data was parametric and analysed by ANOVA and Bonferroni post hoc test. Analysis was repeated in three independent experiments for each cell line. Data are presented as mean ± s.e.m. Scale bar = 100μm.\n\nEndogenous GCase protein expression was assessed by western blot to check if there were differences in the protein concentration between the lines (n=17). The PD/GD skin fibroblasts had significantly lower GCase protein compared to control, and iPD. There was no significant difference seen between GD, PD GBA and the HFL1 (Figure 1E and Dataset 1). Fluorescence immunostaining revealed that the endogenous GCase and Lamp1 protein co-localised in the control lines but this was not the case in the PD/GD lines (Figure 1F and Dataset 1).\n\nWe then measured lysosomal function by looking at the uptake of gold nanoparticles (AuNP), and did not find any significant difference when comparing all cell lines (Figure 1G Dataset 1). Taken together, our result show a decrease in GCase levels and activity in GD and PD patients carrying GBA mutations, compared to iPD and healthy donors. However, the lysosomal basal function and content are not affected.\n\nAutophagy has a role in quality control in the cell, and to examine this we looked at autophagosome maturation as a measure of autophagic flux in the patient fibroblasts (n=17). We first investigated the autophagosome content by measuring the ratio of LC3IIb over total actin, in cells with or without Bafilomycin A1 treatment followed by starvation. Bafilomycin A1 is a specific inhibitor of vacuolar (H+)-ATPases, and a blocker of autophagosome-lysosome fusion. If the cell has a well-regulated autophagic flux, Bafilomycin A1 should increase LC3IIb without any starvation. If the cell has normal basal flux, starvation should increase LC3IIb, due to activation of autophagy. Using a combined starvation and Bafilomycin A1 treatment should produce the maximum amount of autophagosomes possible under these starvation conditions.\n\nIn light of this, autophagic flux was found to be elevated following treatment with Bafilomycin A1 and starvation in control, iPD and PD/GD fibroblasts (Figure 2 A–C and Dataset 2). In the GD and PD GBA lines, Bafilomycin A1 treatment had no effect on LC3IIb expression levels, indicating that autophagic flux was impaired in these cells (Figure 2 and Dataset 2).\n\n(A, B) Western blot of LC3IIb and GBA for all patient lines. Ratio of LC3 IIb/actin for control (NT) (0.41 ± .14) compared to (B+S) (1.06 ± 0.20) (p=0.053), iPD (NT) (0.17 ± .07) compared to (B+S) (1.13 ± 0.32) (p=0.032). This was also true for PD/GD (NT) (0.03 ± 0.01) compared to (B+S) (0.74 ± 0.09) (p=0.048), PD GBA (NT) (0.50 ± 0.07) compared to (B+S) (0.95 ± 0.21).GD (NT) (0.86 ± 0.23) compared to (B+S) (0.54 ± 0.49) (p>0.05). Optical density of western blot bands was measured in Image J. (C) Data was analysed by ANOVA and Bonferroni post hoc test. Analysis was repeated in three independent experiments for each cell line. Data are presented as mean ± s.e.m.\n\n\nDiscussion\n\nOur results show, for the first time in a large variety of PD/GD patient fibroblasts that the only lysosomal enzyme affected by GBA mutations is GCase. This finding is consistent with other’s work, who have shown GCase to be impaired in fibroblasts from GD and PD patients although in smaller patient numbers23–26. However we have now extended these findings to show that GCase is also abnormal in fibroblasts from patients with PD/GD and normal in iPD fibroblasts. We also found that the impairments are not due to low protein amounts in line with other studies24,25. This is in contrast with findings in brain post mortem tissue, where GCase amounts have been reported to be decreased in both PD GBA and iPD cases27. In contrast to other studies, we did not find that the iPD line had significantly lower GCase activity compared to controls, although we were looking at fibroblasts rather than specific neuronal cells.\n\nThe screening of such a large number of GBA carriers with PD and GD, using a panel of lysosomal enzymes has not been previously undertaken. During this screening we found that there were no impairments in any other lysosomal enzymes measured besides GCase. These findings contradict published work which have reported that Hexosaminidase is elevated in PD GBA, GD and healthy GBA carriers fibroblasts24. However, in this paper the enzyme activity was not directly measured, and the amount was determined by western blot, thus the protein amount may be elevated, but the specific activity may not be impaired.\n\nIn terms of looking at autophagic function, we used both Bafilomycin A1 and starvation treatments and observed a normal LC3IIb level in the PD/GD, iPD and control lines. However, in the case of the PD GBA and GD lines there was no increase in LC3IIb flux upon treatment. Most of the GD lines had N370S mutations and the PD GBA lines were carriers of N370S and E326K. Our results are in agreement with recent publications that found impaired autophagic flux was apparent in fibroblasts from a GD patient with homozygous L444P mutations28 and in cases with compound heterozygous (N370S/L444P) mutations29. However, other studies have found no impairment in autophagy, but these GD patients had Saposin C, not GBA, mutations30.\n\nAll of the PD/GD lines in our study were compound heterozygous and two out of the three cases included L444P. These cases had normal autophagic flux, and this was the first study to assess autophagy in patients with both diseases. It may be that having both diseases together also impacts on autophagy, but autophagic function differs depending on the GBA mutation or cell type studied. There are mixed findings in relation to autophagy in PD depending on the model being tested. Autophagy has been found to be increased in fibroblasts from patients with LRRK2 mutations31, but in cell lines it is decreased when α-synuclein is overexpressed32. Autophagy as also been found to be impaired in iPS derived DA neurons from a PD patient with GBA mutations33,34. In our study, autophagic flux was normal in iPD patients but abnormal in the PD GBA lines. This could be further assessed by correcting the GBA mutation using CRISPR/Cas9 gene editing to see if the impairment in autophagy is dependent on GBA gene status. In addition it may be that GCase activity and autophagy are affected by mitochondrial function, which has been implicated in PD GBA previously35, but as of yet, not extensively studied. Another factor to consider is that the affected GCase in GBA related PD, may be in the wrong location such as trapped in the ER, which has previously been observed to occur24.\n\nIn summary, the GBA mutated patient fibroblast lines had impairments of 20–60% GCase activity compared to controls. These diseased cells had normal lysosomal function, with independent lysosomal uptake. However, PD GBA and GD lines display abnormal autophagy. Our results from these genetically relevant patient cell models provide evidence that GBA mutations could lead to impaired GCase and autophagic function, which may translate to CNS neurons and thus clinical expression and progression. If so, this cell model could be used in novel drug development.\n\n\nData availability\n\nDataset 1: Raw data underlying the results presented in Figure 1.\n\nFigure 1A. GCase assay raw data and statistics.\n\nFigure 1B. α-galactosidase assay raw data and statistics.\n\nFigure 1C. Hexominidase assay raw data and statistics.\n\nFigure 1D. Mannosidase assay raw data and statistics.\n\nFigure 1E. Western blot GCase raw data, blots and statistics.\n\nFigure 1F. Immunocytochemistry fibroblasts were stained for GCase (green), Lamp1 (red) and DAPI (blue). Representative images of control and PD/GD lines.\n\nFigure 1G. Gold nanoparticles, images, raw data and statistics.\n\nDOI, 10.5256/f1000research.12090.d17824436\n\nDataset 2: Raw data underlying the results presented in Figure 2.\n\nFigure 2A. Western blots for autophagy assay with iPD, PD GBA, PD/GD and control.\n\nFigure 2B. Western blots for autophagy assay with GD.\n\nFigure 2C. Autophagy assay statistics.\n\nDOI, 10.5256/f1000research.12090.d17824637",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWe are grateful to an NIHR award of a Biomedical Research Centre to Addenbrookes Hospital and the University of Cambridge. We are also grateful to the Rosetrees Trust, the WT-MRC Stem Cell Institute and the Canadian Institutes of Health Research (CIHR) fellowship (358492) for the funding for this work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe wish to thank Dr. Anna Huefner for the gold nanoparticles, Dr. Aviva Tolkovsky for her input to the study and Dr. Elena Pavlova and Dr. Begona Cachon Gonzalez for their input to the enzyme assays.\n\n\nReferences\n\nBrady RO, Kanfer JN, Shapiro D: Metabolism of Glucocerebrosides. II. Evidence of an Enzymatic Deficiency in Gaucher's Disease. Biochem Biophys Res Commun. 1965; 18(2): 221–5. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nPlatt FM, Boland B, van der Spoel AC: The cell biology of disease: lysosomal storage disorders: the cellular impact of lysosomal dysfunction. J Cell Biol. 2012; 199(5): 723–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDehay B, Bové J, Rodríguez-Muela N, et al.: Pathogenic lysosomal depletion in Parkinson's disease. J Neurosci. 2010; 30(37): 12535–44. PubMed Abstract | Publisher Full Text\n\nGitler AD, Chesi A, Geddie ML, et al.: Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity. Nat Genet. 2009; 41(3): 308–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBendikov-Bar I, Maor G, Filocamo M, et al.: Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase. Blood Cells Mol Dis. 2013; 50(2): 141–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcNeill A, Magalhaes J, Shen C, et al.: Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells. Brain. 2014; 137(Pt 5): 1481–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRon I, Dagan A, Gatt S, et al.: Use of fluorescent substrates for characterization of Gaucher disease mutations. Blood Cells Mol Dis. 2005; 35(1): 57–65. PubMed Abstract | Publisher Full Text\n\nSun Y, Liou B, Xu YH, et al.: Ex vivo and in vivo effects of isofagomine on acid β-glucosidase variants and substrate levels in Gaucher disease. J Biol Chem. 2012; 287(6): 4275–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGegg ME, Burke D, Heales SJ, et al.: Glucocerebrosidase deficiency in substantia nigra of parkinson disease brains. Ann Neurol. 2012; 72(3): 455–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde la Mata M, Cotán D, Oropesa-Ávila M, et al.: Pharmacological Chaperones and Coenzyme Q10 Treatment Improves Mutant β-Glucocerebrosidase Activity and Mitochondrial Function in Neuronopathic Forms of Gaucher Disease. Sci Rep. 2015; 5: 10903. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLay I, Tkachyova I, Tropak M, et al.: Macroautophagy-lysosomal system (mals) in gaucher patients carrying L444P and N370S mutations. FEBS J. 2012; 279(Supplement: 1): 248. Reference Source\n\nVaccaro AM, Motta M, Tatti M, et al.: Saposin C mutations in Gaucher disease patients resulting in lysosomal lipid accumulation, saposin C deficiency, but normal prosaposin processing and sorting. Hum Mol Genet. 2010; 19(15): 2987–97. PubMed Abstract | Publisher Full Text\n\nBravo-San Pedro JM, Niso-Santano M, Gómez-Sánchez R, et al.: The LRRK2 G2019S mutant exacerbates basal autophagy through activation of the MEK/ERK pathway. Cell Mol Life Sci. 2013; 70(1): 121–36. PubMed Abstract | Publisher Full Text\n\nWinslow AR, Chen CW, Corrochano S, et al.: α-Synuclein impairs macroautophagy: implications for Parkinson's disease. J Cell Biol. 2010; 190(6): 1023–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFernandes HJ, Hartfield EM, Christian HC, et al.: ER Stress and Autophagic Perturbations Lead to Elevated Extracellular α-Synuclein in GBA-N370S Parkinson's iPSC-Derived Dopamine Neurons. Stem Cell Reports. 2016; 6(3): 342–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchöndorf DC, Aureli M, McAllister FE, et al.: iPSC-derived neurons from GBA1-associated Parkinson’ s disease patients show autophagic defects and impaired calcium homeostasis. Nat Commun. Nature Publishing Group; 2014; 5: 4028. PubMed Abstract | Publisher Full Text\n\nCleeter MW, Chau KY, Gluck C, et al.: Glucocerebrosidase inhibition causes mitochondrial dysfunction and free radical damage. Neurochem Int. 2013; 62(1): 1–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCollins L, Drouin-Ouellet J, Kuan WL, et al.: Dataset 1 in: Dermal Fibroblasts from patients with Parkinson’s Disease have normal GCase activity and autophagy compared to patients with PD and GBA mutations. F1000Research. 2017. Data Source\n\nCollins L, Drouin-Ouellet J, Kuan WL, et al.: Dataset 2 in: Dermal Fibroblasts from patients with Parkinson’s Disease have normal GCase activity and autophagy compared to patients with PD and GBA mutations. F1000Research. 2017. Data Source"
}
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[
{
"id": "26387",
"date": "13 Oct 2017",
"name": "Dario Besusso",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript by Collins L.M. et al., the authors sought to investigate glucocerebrosidase (GCase) activity and autophagic flux in fibroblast derived from Parkinson’s (PD) and Gaucher disease patients (GD). GD is specifically caused by mutations in the GCase gene that drive defective enzymatic activity. Since GCase mutation is the most single commonest genetic risk factor in PD and is associated with poorer prognosis, the authors set to compare PD, GD and PD-GD patient-derived fibroblast for enzymatic activity and autophagic flux.\n\nThe major point of concern is the physiological relevance of the cell models studied. Stating the obvious, since both PD and GD have neurological components, it would have been more appropriated to reprogram the fibroblasts in iPSCs and then convert these in the relevant cell type to assess GCase activity and autophagy. Unfortunately, this requires a good year of work.\n\nInterestingly, the authors show that mutations to the GCase gene affect both enzymatic activity (Figure 1A) and amount of protein in PD/GD (Figure 1E - Maybe also in other GBA context, but the statistical power is too low to discern this aspect). Since the first can be a consequence of the latter, as a mere suggestion, it would be of interest to investigate this aspect in more detail maybe looking at protein stability, although also this goes beyond the aim of the present manuscript.\nTo this point, the authors state in the discussion about the GCase activity in PD/GD “We also found that the impairments are not due to low protein amounts in line with other studies”. As far as this reviewer understand, there is a significant difference between PD/GD and controls as indicated in Figure 1E. Authors may want to clarify this point.\n\nMajor comments:\nThe most obscure point to this reviewer is the fact that PD/GD fibroblasts carrying homozygous mutation in DBA show reduced enzymatic activity but normal autophagic flux when PD GBA and GD (carrying similar mutations) have abnormal flux. Could the authors elaborate better their considerations to this point in the discussion? The patient-derived cell lines are actually 13 and not 17 since 4 are derived from healthy donor. Please, amend the entire text on this regard. In Figure 1A, multiple bands are visible in the WB. It is not clear which band is accounted for quantification. Please, highlight the quantified band in the figure, if possible. Figure 1F will benefit from a colocalisation plot and quantification. It is not clear how many cells have been recorder for this phenotype.\n\nMinor comments:\nThe second column of Table 1 should specify “GBA genotype” in the title, for clarity. Acronyms are first presented in the method session. Since many readers do not take the time to go through the methods, I kindly suggest repeating the acronyms in the main text. Figure 2C is missing legend for the color-coding. Although, is the same as previous figure, a reminder would improve reading. Legend of Figure 1F it may contain text that actually belong to the panel 1E. In Figure 2A, please specify the subtype of LC3 in the legend. The antibody used for LC3 WB usually recognizes both the LC3II and LC3IIb form of the protein. I wonder whether the authors have an explanation for not detecting the LC3II.\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": "3330",
"date": "09 Feb 2018",
"name": "Lucy Collins",
"role": "Author Response",
"response": "Reviewer’s comments: The major point of concern is the physiological relevance of the cell models studied. Stating the obvious, since both PD and GD have neurological components, it would have been more appropriated to reprogram the fibroblasts in iPSCs and then convert these in the relevant cell type to assess GCase activity and autophagy. Unfortunately, this requires a good year of work. Thank you kindly for your comments. We really appreciate your review of our manuscript. As you point out it would have been more valuable as a module of neurodegeneration to convert these cells to iPS and neurons relevant to the disease. Janelle is now pursuing this through iN models with success in different starting from different cell types. Interestingly, the authors show that mutations to the GCase gene affect both enzymatic activity (Figure 1A) and amount of protein in PD/GD (Figure 1E - Maybe also in other GBA context, but the statistical power is too low to discern this aspect). Since the first can be a consequence of the latter, as a mere suggestion, it would be of interest to investigate this aspect in more detail maybe looking at protein stability, although also this goes beyond the aim of the present manuscript. Thank you for the suggestion. Indeed it would have been really useful to assess the protein stability and we will consider this for future work, in the lab. To this point, the authors state in the discussion about the GCase activity in PD/GD “We also found that the impairments are not due to low protein amounts in line with other studies”. As far as this reviewer understand, there is a significant difference between PD/GD and controls as indicated in Figure 1E. Authors may want to clarify this point. To clarify this point in the case of PD/GD and GD the GCase protein and activity was reduced which was expected, however, in the PD GBA the protein amount was not reduced but the activity was reduced in this case, this leads us to believe in the case of PD GBA there is an additional factor, outside the GCase protein, causing pathology in these cells which could lead to dysfunction. We have clarified this point in the text. Major comments: The most obscure point to this reviewer is the fact that PD/GD fibroblasts carrying homozygous mutation in DBA show reduced enzymatic activity but normal autophagic flux when PD GBA and GD (carrying similar mutations) have abnormal flux. Could the authors elaborate better their considerations to this point in the discussion? In the PD GBA and GD lines there was no increase in LC3II flux upon treatment, and this impairment could be due to the GBA mutations in these cases. Most of the GD lines had N370S mutations and the PD GBA lines were carriers of N370S and E326K. Autophagy may vary between different patients and indeed different GBA mutations/ combinations of GBA mutations. However, our results from the GD line are in agreement with a recent publication where it was found that impaired autophagic flux was apparent in fibroblasts from a GD patient with homozygous L444P mutations (de la Mata et al., 2015). Also in agreement with our study, it was shown that three GD fibroblasts lines with compound heterozygous N370S/L444P mutations had decreased autophagic flux (Lay et al., 2012). Other studies have 144 Lysosomal Function in Fibroblasts From Gaucher and Parkinson’s Disease found no impairment in autophagy (Pacheco et al., 2007; Tatti et al., 2012), but this was seen in GD patients with Saposin C mutations (Vaccaro et al., 2010). All of the PD/GD lines in our study were compound heterozygous and two out of the three cases included L444P. These cases had normal autophagic flux, and this was the first study to assess autophagy in patients with both diseases. It may be that having both diseases together also impacts on autophagy, but autophagic function differs depending on the GBA mutation or cell type studied. It is very rare to have both PD and GD so it would also be interesting to assess autophagic function in iN cells with a-syn in these cases. Indeed recently it has been shown that loss of GCase caused impairment in autophagy that was associated with PP2A (protein phosphatase 2A), further leading to a-syn accumulation, albeit in a neuroblastoma cell line (Du et al., 2015). It would be interesting to test the function of PP2A in future experiments in the patient fibroblasts. The patient-derived cell lines are actually 13 and not 17 since 4 are derived from healthy donor. Please, amend the entire text on this regard. Text has been amended: Fibroblasts were derived from dermal skin biopsies. In Table 1 the characteristics of the 13 patient derived and 4 healthy control fibroblast cell lines are summarised. Autophagy has a role in quality control in the cell, and to examine this we looked at autophagosome maturation as a measure of autophagic flux in the patient and healthy control fibroblasts (n=17). The enzyme activity was assessed in all the patient and healthy control fibroblast lines (n=17), and then grouped In Figure 1A, multiple bands are visible in the WB. It is not clear which band is accounted for quantification. Please, highlight the quantified band in the figure, if possible. The whole band was taken for the quantification as there are multiple forms of GBA depending on cellular location and trafficking. Similar western blots of GBA have been seen by others (McNeill et al., 2014). Figure 1F will benefit from a colocalisation plot and quantification. It is not clear how many cells have been recorder for this phenotype. This is a good suggestion. However, the images were captured during my PhD study and I no longer have access to a microscope or image analysis to complete this. For this study the images were included as qualitative data to display the distribution of GCase. Others prior to this study have already shown that GCase is indeed sequestered in the ER. (McNeill et al., 2014). For this qualitative study 10 fields of view were taken for each fibroblast line in three separate wells. Minor comments: The second column of Table 1 should specify “GBA genotype” in the title, for clarity. This has been updated on page 23 Acronyms are first presented in the method session. Since many readers do not take the time to go through the methods, I kindly suggest repeating the acronyms in the main text. I have added main acronyms to the main text. Figure 2C is missing legend for the color-coding. Although, is the same as previous figure, a reminder would improve reading. This has been updated and included. Legend of Figure 1F it may contain text that actually belong to the panel 1E. The text has been updated to correct this. In Figure 2A, please specify the subtype of LC3 in the legend. The subtype used was LC3IIb, this has been added to the legend The antibody used for LC3 WB usually recognizes both the LC3II and LC3IIb form of the protein. I wonder whether the authors have an explanation for not detecting the LC3II. The LC3II band was quite faint in these experiments. This could be due to the pathological affect of GBA in the disease states on the lysosome, as in the control lines the bands were more detectable. This could also be due to experimental technique as part of the bafilomycin assay requires washing and harvesting the cells and LC3 quite a small protein. It is possible that LC3 levels in the cell lines are very low, on the limit of detection. Also in these patient and healthy control lines there could be a rapid turnover of LC3."
}
]
},
{
"id": "26388",
"date": "14 Nov 2017",
"name": "Mia Horowitz",
"expertise": [
"Reviewer Expertise Lysosomal storage diseases",
"Gaucher disease"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present manuscript, the authors used dermal fibroblasts to assess possible changes in acid- b-glucocerebrosidase (GCase) levels and changes in autophagy markers between three Gaucher disease (GD) patients with Parkinson disease, three carriers of GD mutations with PD, three idiopathic PD patients, four healthy controls and three GD patients with no known PD signs. They also had one human fetal lung fibroblast line (HFL1).\nThe authors concluded that only PD patients with a GBA mutation have altered GCase activity and autophagy, which may explain their most rapid clinical progression. The authors mentioned that there was no decrease in GCase activity in iPD patients. However, the results in Figure 1A indicate reduced GCase activity in iPD patients. It may not be significant due to the small sample used in the study, but it is obvious and cannot be overlooked. Likewise, there is a notable decrease in the level of hexosaminidase and mannosidase activity.\n\nThe authors state that: \"the impairments in (GCase) activity are not due to low protein amounts, in line with other studies\". However, several reports have already documented decreased GCase amounts in GD derived cells. McNeill et al. (Reference 24 in the present manuscript) showed that: “Glucosylceramidase protein levels, assessed by western blot, were significantly reduced in fibroblasts from Gaucher disease (median glucosylceramidase levels 42% of control, P < 0.001) and heterozygous mutation carriers with (median 59% of control, P < 0.001) and without (median 68% of control, P < 0.001) Parkinson's disease\". Other studies described a decrease in the amount of mutant GCase in cells that derived from GD patients and carriers of GD mutations. (Jonsson et al.1; Ron et al.2; Lu, et al.3; Bendikov-Bar et al.4).\nThe authors mention in their discussion that: “Another factor to consider is that the affected GCase in GBA related PD may be in the wrong localization such as trapped in the ER”. It has already been shown that mutant GCase is recognized as misfolded in the ER, undergoes ER retention and, depending on the ability of the ER chaperones to refold it, it will either undergo ERAD or traffic to the lysosomes (Ron et al.2).\n\nTypographical errors:\nThe authors mention HFL1 or HLF cells and I guess they are the same.\n\nGBA (or better GBA1, since there are three GBA genes: GBA1, GBA2 and GBA3) is the gene encoding acid-b-glucocerebrosidase (GCase). It is the protein (GCase) that localizes in the cell and not the GBA protein. It could also be referred to as the GBA1 encoded protein.\n\nIn Materials and Methods: bafilomycin assay: ….”this concentration was derived from titrations of Bafilomysin A1 and 0.1mM and found …” The second and should be replaced by was. In the same paragraph the authors mention that after NP-40 treatment “the cells were collected”. NP40 is a detergent that disrupts the plasma membranes so following its treatment there are lysates, not cells.\n\nIn the galactosidase assay the authors mention: “4-MUI a-D-galactopyranosidase”, should be: 4-MU a-D-galactopyranoside.\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": [
{
"c_id": "3331",
"date": "09 Feb 2018",
"name": "Lucy Collins",
"role": "Author Response",
"response": "I have made these changes, thank you"
}
]
}
] | 1
|
https://f1000research.com/articles/6-1751
|
https://f1000research.com/articles/6-296/v1
|
20 Mar 17
|
{
"type": "Data Note",
"title": "A collection of annotated and harmonized human breast cancer transcriptome datasets, including immunologic classification",
"authors": [
"Jessica Roelands",
"Julie Decock",
"Sabri Boughorbel",
"Darawan Rinchai",
"Cristina Maccalli",
"Michele Ceccarelli",
"Michael Black",
"Cris Print",
"Jeff Chou",
"Scott Presnell",
"Charlie Quinn",
"Puthen Jithesh",
"Najeeb Syed",
"Salha B.J. Al Bader",
"Shahinaz Bedri",
"Ena Wang",
"Francesco M. Marincola",
"Damien Chaussabel",
"Peter Kuppen",
"Lance D. Miller",
"Davide Bedognetti",
"Wouter Hendrickx",
"Jessica Roelands",
"Julie Decock",
"Sabri Boughorbel",
"Darawan Rinchai",
"Cristina Maccalli",
"Michele Ceccarelli",
"Michael Black",
"Cris Print",
"Jeff Chou",
"Scott Presnell",
"Charlie Quinn",
"Puthen Jithesh",
"Najeeb Syed",
"Salha B.J. Al Bader",
"Shahinaz Bedri",
"Ena Wang",
"Francesco M. Marincola",
"Damien Chaussabel",
"Peter Kuppen"
],
"abstract": "The increased application of high-throughput approaches in translational research has expanded the number of publicly available data repositories. Gathering additional valuable information contained in the datasets represents a crucial opportunity in the biomedical field. To facilitate and stimulate utilization of these datasets, we have recently developed an interactive data browsing and visualization web application, the Gene Expression Browser (GXB). In this note, we describe a curated compendium of 13 public datasets on human breast cancer, representing a total of 2142 transcriptome profiles. We classified the samples according to different immune based classification systems and integrated this information into the datasets. Annotated and harmonized datasets were uploaded to GXB. Study samples were categorized in different groups based on their immunologic tumor response profiles, intrinsic molecular subtypes and multiple clinical parameters. Ranked gene lists were generated based on relevant group comparisons. In this data note, we demonstrate the utility of GXB to evaluate the expression of a gene of interest, find differential gene expression between groups and investigate potential associations between variables with a specific focus on immunologic classification in breast cancer. This interactive resource is publicly available online at: http://breastcancer.gxbsidra.org/dm3/geneBrowser/list.",
"keywords": [
"Breast Cancer",
"Immune Subtypes",
"Cancer Immune Phenotype",
"Gene Expression Browser",
"Immunologic Constant of Rejection"
],
"content": "Introduction\n\nTechnological progress in the field of biomedical research has resulted in an increased utilization of platforms generating information on a system-scale, e.g. genome, transcriptome and proteome. As researchers are typically willing and often required to share their data collections, the availability of ‘big data’ is expanding rapidly. At this moment, the NCBI Gene Expression Omnibus (GEO), a public repository of transcriptome profiles, holds over 2 million individual transcriptome profiles from more than 76,000 studies (“Home - GEO - NCBI”, 2016). This large amount of available transcriptomic data provides major opportunities as well as challenges to researchers. Identification of differential gene expression in healthy versus diseased individuals, for example, has the potential to increase our understanding of the disease process, can lead to the identification of novel disease biomarkers or to the recognition of potential therapeutic targets. However, utilization of the available system-scale information can be challenging, since data repositories often lack the analytical and visualization tools needed for data assessment and interpretation. For this reason, proper analysis relies on elevated bioinformatics skills.\n\nTo overcome the challenges faced when analyzing transcriptomic data, we previously developed a web application called gene expression browser (GXB), which makes datasets more accessible and interactive (Speake et al., 2015). The application graphically visualizes gene expression data in bar chart or box plot representation and is capable of dynamically changing its interface views upon user input. GXB allows users to upload microarray data, add data annotations, which enables overlay of clinical data, explore gene rank lists based on their differential expression patterns between groups, view the data on a gene-by-gene basis and compare different datasets and diseases. These capabilities stimulate the acquisition of new knowledge from public datasets, as demonstrated by the first paper that employed GXB to identify a previously unknown role of a specific transcript during immune-mediated processes (Rinchai et al., 2015).\n\nIn recent years, a large number of transcriptional studies have been conducted with the aim to characterize breast cancer on a genetic basis. GEO holds about 1297 datasets relating to breast cancer. One of the main impacts gene expression profiling has had on our understanding of breast cancer has been through the classification of breast cancer into intrinsic molecular subtypes (IMS). Three main methods have been described to achieve this, which have the same subtypes, but actually use different gene sets to stratify the patients (Hu et al., 2006; Parker et al., 2009; Sorlie et al., 2003). Four major IMS of breast cancer have been identified: Luminal A, Luminal B, HER2-enriched and Basal-like. A less common molecular subtype called Claudin-low has been characterized at a later time point (Prat et al., 2010). Stratified IMS groups present critical differences in incidence, survival and response to treatment, and most importantly add prognostic information that is not provided by classical stratifications, like estrogen receptor status, histologic grade, tumor size, and node status (Parker et al., 2009).\n\nRecent breakthroughs in the field of cancer immunotherapy and especially the application of checkpoint blockade inhibitors has ignited a fierce drive to understand the genetic basis for the huge differences observed between patients with different immune phenotypes. Several papers have shown that expression profiles are able to distinguish between those patients that have an active immune environment and those that do not (Galon et al., 2013; Herbst et al., 2014; Ji et al., 2012; Ribas et al., 2015; Wang et al., 2013). A clear correlation can be seen both regarding prognosis (survival) and prediction of therapeutic effectiveness of immune regulatory therapies. The expression of genes observed in association with tissue-specific destruction in a broader context, defined as the immunological constant of rejection (ICR), can distinguish between breast cancer patients with different prognosis. This immunological classification is based on the consensus clustering of ICR genes (Galon et al., 2013), e.g. genes underlying Th1 polarization, related chemokines, adhesion molecules and cytotoxic factors, in combination with immune regulatory genes IDO1 and FOXP3, PDCD1, CTLA4 and CD274/PD-L1 (Figure 1A) (Bedognetti et al., 2015). In Miller et al. (2016), a novel survival-based immune classification system was devised for breast cancer based on the relative expression of immune gene signatures that reflect different effector immune cell subpopulations, namely antibody-producing plasma B cells (the B/P metagene), cytotoxic T and/or NK cells (the T/NK metagene), and antigen-presenting myeloid/dendritic cells (the M/D metagene). The system defines a tumor’s immune subclass based on its survival-associated immunogenic disposition status (IDS), which discriminates between poor immunogenic disposition (PID), weak immunogenic disposition (WID) and favorable immunogenic disposition (FID). The ability of IDS to distinguish patients with differential prognosis is dependent on the tumor’s immune benefit status (IBS), which is defined by IMS and the expression of cell proliferation markers. The IBS classification segregates immune benefit-enabled (IBE) and immune benefit-disabled (IBD) tumors. In IBE tumors, but not IBD tumors, FID status confers a protective survival benefit compared to WID and PID status (Figure 1B) (Miller et al., 2016; Nagalla et al., 2013). In this data note, we demonstrate the use of GXB to evaluate cancer gene expression across immunologic classifications of breast cancer.\n\n(A) Consensus clustering based on ICR genes segregates breast cancer patient in four different groups: ICR1, 2, 3 and 4. Patients with tumors categorized as ICR4 have the highest expression of the ICR gene signature and have a better prognosis compared with other ICR groups. (B) Immune metagene model based on the relative expression of immune metagenes (B/P, T/NK and M/D) distinguishes PID, WID and FID tumors (horizontal axis: genes, vertical axis: individual cases). This classification has prognostic value in IBE tumors, and not in IBD tumors. Diagrams are based on Hendrickx et al., 2017 (A) and Miller et al., 2016 (B). ICR, Immunologic Constant of Rejection; IBE/D, Immune Benefit Enabled OR Disabled; F/P/WID, Favorable OR Poor OR Weak Immune Disposition.\n\nSince the amount of possible datasets to be included in GXB is enormous, we chose to start with the GEO datasets underlying the immunologically classified breast cancer datasets by (Miller et al., 2016). In Hendrickx et al. (2017), these same datasets were classified according to ICR. This will allow us to share our immune related classifications in a comprehensible way and allow others to reuse them. A harmonization effort of the other available clinical data had been undertaken and should help the downstream analysis of the expression data. Therefore, gathering these datasets with their detailed study and sample information will facilitate the identification of clinically-relevant genetic signatures for biomarker and/or therapeutic purposes.\n\nIn this data note, using GXB, we have made available a curated compendium of 13 public datasets relevant to human breast cancer, representing a total of 2142 cases.\n\n\nMethods\n\nThe starting point of our selection of breast cancer datasets are the patient cohorts included in the multi-study breast cancer database described by Nagalla et al. (2013). These 13 NCBI GEO datasets (GEO accession numbers: GSE45255, GSE2034, GSE5327, GSE12093, GSE9195, GSE11121, GSE1456, GSE2603, GSE6532, GSE7390, GSE7378 and GSE4922) resulted in 2142 cases initially uploaded in GXB. 22 of these cases reflect data from breast cancer cell lines and were therefore excluded from our data collection. A total of 1839 cases represent primary invasive breast tumors sampled at the time of surgical resection without prior neoadjuvant treatment and were therefore annotated with survival data, IMS, IBS, IDS and ICR status (Hendrickx et al., 2017; Miller et al., 2016). 281 of the cases did not fulfill these criteria and were therefore not annotated. Of note, 115 cases of original meta-cohort used Nagalla’s study (n=1954) were not shared within GEO, but shared within other platforms (caArray and ArrayExpress). For this reason, these samples were not included in our GXB collection (Figure 2).\n\nBreast cancer cases included in 13 NCBI GEO datasets were uploaded in GXB (n=2142). 22 cases described data from breast cell lines and were excluded from our data collection. We annotated 1839 cases with survival data, IMS, IBS, IDS and ICR status. 281 cases were either neoadjuvant treated, did not represent a primary invasive tumor, were not sampled at the time of surgery or without available survival data and were therefore not annotated. The total collection includes 1839 cases from the original cohort described in Nagalla et al. (2013) (n=1954). Of note, 115 cases of this cohort are not included in our collection as these were not shared via GEO. *251/1839 cases have been classified for IMS “Normal-like”. IDS is not applicable for normal-like breast cancer tissue; therefore, IDS is non-classified for these samples. DMFS, Distant Metastasis Free Survival; GXB, Gene Expression Browser; IMS, intrinsic molecular subtype; IBS, immune benefit status; IDS, immune disposition status; ICR, immunologic constant of rejection.\n\nThe datasets that comprise our collection are listed in Table 1 and can be searched interactively in GXB. All GEO datasets consist of unique cases with the exception for 36 cases from NUH Singapore, which are both present in the Bordet Radcliff NUH (GSE45255) dataset and the Uppsala and Singapore (GSE4922) dataset.\n\nData of the 1839 GEO-cases annotated with survival data that were previously combined and used in the Nagalla study, have been uploaded to GXB in the dataset “Nagalla 2013 reconstituted public dataset”.\n\nAll datasets were downloaded from NCBI GEO in SOFT file format and were uploaded into GXB with the exception of the Guy's hospital dataset (GUYT2; GSE9195). Expression data in the SOFT file of this dataset was expressed as fold change. Therefore, we had to revert to reprocessing of the CEL files found attached to the GSE on GEO. In this case, the cell files were read into R (v3.2.2) using the ‘affy’ package (v1.50.0). Data was normalized using the RMA (Robust multichip averaging) and gene annotation data was added using the hgu133plus2.db package (v3.2.3).\n\nGSE records containing data generated with different or multiple platforms have been split by platform using the import process of GXB. GSEs containing data from both clinical as in vitro origin (GSE2603) have been split manually using the GXB Graphical interface.\n\nMetadata of the different studies was added to GXB both from the descriptive information found on GEO or from the method sections of the publications linked to these datasets. Short links to PMID (Pubmed) and GEO records were added.\n\nThe constitution of the complete cohort has previously been described by (Nagalla et al., 2013). The dataset “Nagalla 2013 reconstituted public dataset” available in GXB contains only the samples that were publicly available via GEO. Briefly, raw data (CEL files) were extracted from GEO. The array platforms employed for these 13 datasets were Affymetrix U133A, U133A2, and U133 PLUS 2.0 gene chips; the 22,268 probe sets present in each of these platforms were included in the gene expression file. Data were MAS5.0 normalized using the justMAS function in the simpleaffy library from Bioconductor (Gentleman et al., 2004) using a trimmed mean target intensity of 600 without background correction. COMBAT empirical Bayes method was used to correct for batch effects (Johnson et al., 2007).\n\nGene expression data is accompanied with clinical data in CSV file format. Gene expression data and clinical data are coupled to the sample via variable “Sample ID”. We annotated a total of 1839 cases with 10-year survival (time and event), IBS (IBE, IBD), IDS (PID, WID and FID) (Miller et al., 2016) and ICR (ICR1, ICR2, ICR3 and ICR4) immune classifications (Hendrickx et al., 2017) (Figure 2). IMS (i.e., Basal-like, HER2-enriched, Luminal A and Luminal B) were defined using the Single Sample Predictor (SSP) algorithm by Hu (Hu et al., 2006) utilized by (Fan et al., 2006). Claudin-low tumors were identified using the method of (Prat et al., 2010). Of the 1839 samples, 251 samples were “Normal-like” in IMS classification. Therefore, these samples are not classified according to IDS. For the separate dataset containing samples of in vitro origin (GSE2603), survival annotations and immune classifications are not applicable. A final 281 cases were not annotated and non-classified, since for these cases either samples were not taken at the time of surgical resection, were neoadjuvant-treated or cases were not annotated with distant metastasis free survival (DMFS) time and event.\n\nTo enable comparisons between datasets and to facilitate efficient data analysis, the clinical data was harmonized to reflect a nomenclature similar to that of The Cancer Genome Atlas (TCGA). Clinical variable names and availability in datasets are listed in Table 2. In general, variable values have been replaced by descriptive values (e.g. “1” and “0” are replaced by “ER+” and “ER-”, respectively). For disease free survival, variable values have been adapted to “DiseaseFree” or “Recurred/Progressed”, and for distant metastasis survival to “DistantMetastasisFree” and “DistantMetastasis”. Numeric values of variable “tumor size” have been converted to units in cm for all datasets. This variable was used to generate the additional variable pathology T stage according to the 7th edition of the AJCC staging system for breast cancer (Edge & Compton, 2010). For tumors with a diameter larger than 5 cm, pathology T stage could be either T3 or T4, therefore value “T3/T4” has been assigned to these cases.\n\nStandardized clinical datasets can be found in the ‘downloads’ tab in GXB under the heading “additional files”. All datasets start with the following 21 clinical variables in fixed order: \"sample.ID\", \"array sample id\", \"sample title\", \"series\", \"IMS\", \"IBS\", \"IDS\", \"ICR\", \"DMFS_10Y_EVENT\", \"DMFS_10Y_TIME\", \"disease free survival event\", \"disease free survival years\", \"distant met free survival event\", \"distant met free survival\", \"age at initial pathologic diagnosis\", \"lymph node status\", \"ER status\", \"PR status\", \"histology differentiation grade\", \"tumor size cm\", and \"pathology T stage\". In case one of these variables is not available in a specific dataset, values in this column are all NA.\n\nGroup sets for IBS/IDS, ICR cluster, Lymph Node (LN) Status, IMS, Histological grade, stage and Estrogen Receptor (ER) status were defined with matching differential gene expression rank lists. Rank lists are based on differential gene expression between two relevant groups for each group set: IBD-FID vs IBE-FID (IBS/IDS); ICR1 vs ICR4 (ICR1/ICR4); LN+ vs LN- (LN status); G1 vs G3 (histological grade); ER+ vs ER- (ER status). For IMS, no rank list was generated, as this variable is not ordered. For tumor stage, no rank list was generated because the spread of samples between categories was small.\n\n\nDataset demonstration\n\nThe GXB software has been described in detail in a recent publication (Speake et al., 2015). This custom software interface provides users with a means to easily navigate and filter the dataset collection available at http://breastcancer.gxbsidra.org/dm3/landing.gsp. A web tutorial is also available online: http://breastcancer.gxbsidra.org/dm3/tutorials.gsp#gxbtut.\n\nIn GXB, users can search interactively for a specific gene of interest. Differential expression across different group sets can be observed in the graphical interface, either in bar or box plots. For illustrative purposes, we choose to evaluate the abundance of the HLA-G transcripts across ICR groups.\n\nHLA-G is a non-classical class I gene of human Major Histocompatility Complex that is primarily expressed on fetal derived placental cells (Ellis et al., 1990). In contrast to its classical counterparts, HLA-G does not initiate immune responses, but instead has immunosuppressive effects (Naji et al., 2014; Rouas-Freiss et al., 1997). Expression of HLA-G has been reported in a variety of cancers, including breast cancer, and has been assigned a role in tumor immune escape (Naji et al., 2014; Rouas-Freiss et al., 1997; Swets et al., 2016; Zeestraten et al., 2014).\n\nConcerning its role in tumor immunity, it may be of interest to investigate whether HLA-G expression is elevated in breast tumors of specific immune phenotypes. The ICR gene signature segregates breast tumors into four immune phenotype groups based on the expression of genes underlying immune-mediated tissue-specific destruction, with ICR1 having the lowest and ICR4 the highest expression of this signature (Bedognetti D et al., in press).\n\nTo compare HLA-G expression across ICR groups using the breast cancer datasets uploaded to GXB, we start by selecting one of the datasets. After opening this dataset: 1) the gene of interest, HLA-G, can be identified using the search box in the upper left corner of the user interface. Upon selection of “HLA-G” in the left panel, the central panel displays the expression values of this gene for all samples as a bar chart. 2) Sample grouping is default as “All sample”, it is changed by selecting “Immunologic Constant of Rejection” and 3) plot type is set to “Box Plot” in drop down menus in the central panel. The central panel now presents a graphical display of the observed abundance of HLA-G transcripts in breast cancer samples across the different ICR groups, each sample is represented by a single point in a boxplot (Figure 3A). A tendency of increased HLA-G expression in groups with the highest expression of ICR genes can be observed. 4) To verify whether this trend can also be observed in other breast cancer datasets, GXB’s “Cross Project View” is used. By selecting “Cross Project View” in the “Tools” drop-down menu located in the top right corner of the user interface, a list of available datasets/projects appears in the left pane. By consecutive selection of single datasets, box plots with HLA-G transcripts across ICR groups are displayed for each individual dataset.\n\n(A) Cross Project View in GXB showing HLA-G expression across ICR groups. ICR represents the immune gene signatures observed in association with tissue-specific destruction. In this view of GXB, expression of HLA-G can be visualized across projects listed on the left. (B) Boxplots of HLA-G expression across ICR groups of three additional representative datasets selected from the dataset collection and the complete dataset including all annotated cases (right bottom plot). Plots indicate an increased HLA-G expression in breast tumors with a high expression of ICR genes. ICR, Immunologic Constant of Rejection.\n\nEach of the boxplots corresponding to the 13 datasets show a similar pattern, indicating an increased HLA-G expression in breast tumors with a high expression of ICR genes (representative plots are shown in Figure 3B). In the combined dataset containing the total of 1839 annotated cases from these datasets, this trend is also observed (Figure 3B). From a biological perspective, increased expression of an immunosuppressant in an immunologically active tumor would be in line with our current view of the tumor microenvironment. Pro-inflammatory tumor environments, as observed in ICR4 tumors, also show counter regulatory mechanisms to suppress the immune system (Bedognetti et al., 2015; Galon et al., 2013).\n\nThis observation made by exploring transcriptome data in GXB provides an interesting starting point for further analysis. Statistical analysis of this potential association is required and, of course, the clinical relevance of the observed difference in abundance of transcripts should be determined. Most importantly, the functional relevance of HLA-G expression depends on its interaction with inhibitory receptors including ILT2, ILT4 and KIR2DL4 (LeMaoult et al., 2005). Therefore, combined analysis of both HLA-G and these inhibitory receptors is suggested in future analyses.\n\nThis example illustrates the convenience of exploring gene expression data in GXB. The browser facilitates intuitive navigation and visualization of gene expression across different group sets.\n\nThe breast cancer datasets uploaded in GXB are provided with a rich context of immune classifications and clinical parameters. As opposed to start a search with a specific gene of interest, as presented in the HLA-G example case, differential gene expression between groups of interest can be explored in GXB by evaluation of gene rank lists. Here, we demonstrate the use of GXB to explore differential gene expression across IBS/IDS groups.\n\nThe IDS group set is based on an immune metagene model segregating breast tumors in groups of different immunogenic dispositions: PID, WID and FID (Nagalla et al., 2013). The prognostic value of this classification is dependent on the molecular subtype and the proliferative capacity of the tumor, hereby segregating tumors in IBE and IBD groups, with and without prognostic value of the IDS, respectively. Since the hypothesis is that IBE-FID tumors confer metastasis-protective potential and IBD-FID tumors do not, transcriptional differences between these specific subgroups are of particular interest and have systematically been analyzed by Miller et al. (2016).\n\nThe Nagalla 2013 reconstituted dataset containing all annotated cases of this GXB breast cancer instance (n=1839) is used to explore differential gene expression between IBE-FID and IBD-FID tumors in GXB. Group set “Immune Benefit Status” is selected and corresponding gene rank list “IBD-FID vs IBE-FID” will load in the left panel by default. Filtering for specific immune gene categories, e.g. cytokine and chemokine ligands, cytokine and chemokine receptors, B and T cell signaling, and antigen presenting cell processing, is possible by selecting gene list category in the rank list menu. Exploring the expression of genes with known roles in tumor immunology reveals two important observations: 1) markers of immune cell infiltration, including CD8, CD3, CD19 and CD2, show similar expression in IBD-FID and IBE-FID subgroups (Figure 4A); while (2) markers of immune functional orientation, including CXCL10 (tissue rejection chemokine), GZMB (cytotoxic effector molecule), INFG and STAT1 (Th1 polarization), show differential expression across IBD-FID and IBE-FID groups (Figure 4B). A comprehensive statistical analysis of expression of these and other immune-related genes confirmed these observations, suggesting that while the composition of the immune infiltrate is similar in these tumors, the functional molecular orientation determines the metastasis-protective phenotype (Miller et al., 2016).\n\n(A) Expression values of CD8 and CD19, indicators of immune cell infiltration, are similar in IBD-FID and IBE-FID groups, indicating equal immune cell infiltration in these subgroups. (B) Expression values of CXCL10, GNZB, IFNG and STAT1, markers of immune functional orientation, are increased in the IBE-FID group compared with IBD-FID, indicating a differential functional orientation of the immune infiltrate between IBD-FID and IBE-FID tumors. IBE/D, Immune Benefit Enabled OR Disabled; F/P/WID, Favorable OR Poor OR Weak Immune Disposition.\n\nThis demonstration indicates that GXB allows for easy and efficient visualization of differential gene expression between subgroups. Subsequently, elaborate statistical analysis is required to confirm the differences in gene expression observed in GXB.\n\nSince this GXB data collection is provided with multiple immune classifications of breast cancer, it is interesting to visualize the relationship between these classifications in GXB. The overlay feature in GXB can be used to visualize the assignment of different classifications to individual samples simultaneously.\n\nTo illustrate this overlay option, we choose to select the Erasmus Medical Center dataset 2 (EMC2) with CXCL9 expression, as this is one of the chemokines included in the ICR gene signature. Graphical representation in GXB is set to bar plot and group set ICR is selected. As anticipated, the CXCL9 expression gradually increases from ICR1-ICR4. The drop down menu “Overlays” is used to add multiple layers of additional variables, “IBS”, “IDS” and “IMS”. Boxes underneath the individual bars (each bar represents a single case) display the assigned classifications (Figure 5A). When comparing IBS classifications across ICR groups, it is evident that IBE tumors are frequently assigned to the higher ICR clusters, ICR3 and ICR4, while IBD tumors tend to concentrate to the clusters with a low expression of the ICR signature (ICR1, ICR2) (Figure 5A). This result is consistent with our previous observations: pathways that distinguish IBE and IBD are associated with the immune functional orientation of the tumor, and genes in these same pathways are crucial components of the ICR signature (Bedognetti et al., 2015; Miller et al., 2016).\n\n(A) Bar graph showing CXCL9 expression in individual samples from Erasmus Medical Center (EMC) dataset 2 split by ICR (single bar represents single case). Overlay of additional variables IBS, IDS and IMS is shown (http://breastcancer.gxbsidra.org/dm3/miniURL/view/Lv). (B) Frequency plot showing number of breast cancer cases across IBS/IDS subgroups split by ICR cluster. ICR, Immunologic Constant of Rejection; IBE/D, Immune Benefit Enabled OR Disabled; F/P/WID, Favorable OR Poor OR Weak Immune Disposition.\n\nIDS relates to the ICR classification in a similar manner. FID tumors are mostly assigned to ICR4, while WID tumors are frequently classified to intermediate clusters (ICR2 and ICR3), and PID tumors prevail in the ICR1 cluster (Figure 5A). This observation is also in line with our expectations, the IDS classification is based on an immune metagene model that relies on immune gene subclusters that reflect the relative abundance of tumor-infiltrating immune cells (Nagalla et al., 2013). As markers of immune cell infiltration are also included in the ICR signature, IDS is closely associated with ICR.\n\nFor a more comprehensive overview of the relationship between different immune classifications in breast cancer, the overlay of immune classifications was evaluated in the Nagalla 2013 reconstituted public dataset (n=1839). The observations made in the EMC2 dataset (n=58; Figure 5A) are also apparent in the dataset containing all annotated cases of this GXB breast cancer instance (Figure 5B). Moreover, in this dataset it is clearly visible that IBS/IDS subgroups with an improved prognosis are more prevalent in the ICR4 cluster. For example, IBE-FID tumors are relatively more frequently assigned to ICR4 compared with IBD-FID. Vice versa, IBD-PID tumors are proportionally more frequently observed in the ICR1 cluster compared with IBE-PID tumors, which are in comparison more frequently assigned to ICR2 ICR3.\n\nThe overlay of the different immune classifications demonstrates a coherency between the IBS/IDS classification and the ICR clusters. Bearing in mind that the ICR signature is associated with a broader phenomenon of immune-mediated, tissue-specific destruction, this coherency strengthens the hypothesis of a common final pathway of tissue destruction.\n\n\nConclusions\n\nIn this data note, we highlighted the opportunities provided by the availability of public datasets. We uploaded 13 public datasets on human breast cancer, including a combined dataset, with harmonized clinical data annotation and immune classification to GXB to facilitate the reuse of gene expression data. The use of GXB to explore gene expression and the different possible approaches were illustrated by the following: (1) an example case of a specific gene of interest, HLA-G; (2) comparison of gene expression between specific subgroups, IBD-FID vs IBE-FID; and (3) the evaluation of the relationship between different categorical variables, IBS/IDS and ICR immune classifications. To conclude, GXB provides a convenient environment to explore gene expression profiles in the context of breast cancer.\n\n\nData availability\n\nAll datasets included in our curated collection are available publicly via the NCBI GEO website: http://www.ncbi.nlm.nih.gov/geo/, and are referenced throughout the manuscript by their GEO accession numbers (e.g. GSE7390). Signal files and sample description files can also be downloaded from the GXB tool under the “downloads” tab.",
"appendix": "Author contributions\n\n\n\nJR: curated, uploaded and annotated datasets, interpreted the data and drafted the manuscript and accepted final version. JD: critically reviewed the datasets annotation, drafted the manuscript and accepted final version. SBo: installed the software, uploaded datasets, programmed portions of the web application, tested the software, and assisted in drafting the manuscript. DR: critically reviewed gene expression data, sample annotation, drafted the manuscript and accepted final version. CM: reviewed manuscript and accepted final version. MC: technical support annotating datasets and accepted final version of manuscript. MB: assembled and curated datasets and accepted final version of manuscript. CP: assembled and curated datasets and accepted final version of manuscript. JC: assembled and curated datasets and accepted final version of manuscript. SP: participated in the design of the software, programmed portions of the original web application, installed the software, tested the software, assisted in drafting the manuscript and accepted final version. CQ: participated in design and programmed portions of the original web application, tested the software, assisted in drafting the manuscript and accepted final version. PJ: critically reviewed sample annotation and gene expression data and accepted final version of manuscript. NS: critically reviewed quality of gene expression data and accepted final version of manuscript. SBa: reviewed manuscript and accepted final version. SBe: reviewed manuscript and accepted final version. DC: participated in software and study design, tested the software, assisted in drafting the manuscript and accepted final version. EW, FM: critically reviewed the manuscript. PK: Interpreted the data, critically reviewed manuscript and accepted final version. LM: assembled and curated datasets, provided immunological attributes, interpreted the data, drafted the manuscript and accepted final version. DB: designed the study, provided immunological attributes, supervised the project, interpreted the data, drafted the manuscript and accepted final version. WH: coordinated the uploading and annotation of datasets, contributed to the study design, provided immunological attributes, interpreted the data, drafted the manuscript and accepted final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nJD, SB, DR, DC, DB, WH received support from the Qatar Foundation. JR received support from Qatar National Research Fund (grant number: JSREP07-010-3-005).\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 acknowledge all the investigators who decided to make their datasets publically available by sharing them in NCBI GEO.\n\n\nReferences\n\nBedognetti D, Tomei S, Hendrickx W, et al.: Toward the Identification of Genetic Determinants of Responsiveness to Cancer Immunotherapy. In: Developments in T Cell Based Cancer Immunotherapies. Cancer Drug Discovery and Development 2196–9906. Springer, 2015. in press; 99–127. Publisher Full Text\n\nBedognetti D, Tomei S, Hendrickx W, et al.: Toward the Identification of Genetic Determinants of Responsiveness to Cancer Immunotherapy. In: Developments in T Cell Based Cancer Immunotherapies, edited by Paolo A. Ascierto, David F. Stroncek, and Ena Wang, Cancer Drug Discovery and DevelopmentCancer Drug Discovery and Development. Springer International Publishing. 2015; 99–127. Publisher Full Text\n\nDesmedt C, Piette F, Loi S, et al.: Strong time dependence of the 76-gene prognostic signature for node-negative breast cancer patients in the TRANSBIG multicenter independent validation series. Clin Cancer Res. 2007; 13(11): 3207–3214. PubMed Abstract | Publisher Full Text\n\nEdge SB, Carolyn CC: The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol. 2010; 17(6): 1471–74. PubMed Abstract | Publisher Full Text\n\nEllis SA, Palmer MS, McMichael AJ: Human trophoblast and the choriocarcinoma cell line BeWo express a truncated HLA Class I molecule. J Immunol. 1990; 144(2): 731–35. PubMed Abstract\n\nFan C, Oh DS, Wessels L, et al.: Concordance among gene-expression-based predictors for breast cancer. N Engl J Med. 2006; 355(6): 560–69. PubMed Abstract | Publisher Full Text\n\nGalon J, Angell HK, Bedognetti D, et al.: The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures. Immunity. 2013; 39(1): 11–26. PubMed Abstract | Publisher Full Text\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\nHendrickx W, Simeone I, Anjum S, et al.: Identification of Genetic Determinants of Breast Cancer Immune Phenotypes by Integrative Genome-Scale Analysis. OncoImmunology. 2017; 6(2): e1253654. Publisher Full Text\n\nHerbst RS, Soria JC, Kowanetz M: Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature. 2014; 515(7528): 563–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHome - GEO - NCBI. Accessed November 28. 2016. 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FASEB J. 2005; 19(6): 662–64. PubMed Abstract | Publisher Full Text\n\nLoi S, Haibe-Kains B, Desmedt C, et al.: Definition of clinically distinct molecular subtypes in estrogen receptor-positive breast carcinomas through genomic grade. J Clin Oncol. 2007; 25(10): 1239–1246. PubMed Abstract | Publisher Full Text\n\nLoi S, Haibe-Kains B, Desmedt C, et al.: Predicting prognosis using molecular profiling in estrogen receptor-positive breast cancer treated with tamoxifen. BMC Genomics. 2008; 9: 239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiller LD, Chou JA, Black MA, et al.: Immunogenic Subtypes of Breast Cancer Delineated by Gene Classifiers of Immune Responsiveness. Cancer Immunol Res. 2016; 4(7): 600–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinn AJ, Gupta GP, Padua D, et al.: Lung metastasis genes couple breast tumor size and metastatic spread. Proc Natl Acad Sci U S A. 2007; 104(16): 6740–6745. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinn AJ, Gupta GP, Siegel PM, et al.: Genes that mediate breast cancer metastasis to lung. Nature. 2005; 436(7050): 518–524. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNagalla S, Chou JW, Willingham MC, et al.: Interactions between immunity, proliferation and molecular subtype in breast cancer prognosis. Genome Biol. 2013; 14(4): R34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaji A, Menier C, Morandi F, et al.: Binding of HLA-G to ITIM-Bearing Ig-like Transcript 2 Receptor Suppresses B Cell Responses. J Immunol. 2014; 192(4): 1536–46. PubMed Abstract | Publisher Full Text\n\nParker JS, Mullins M, Cheang MC, et al.: Supervised Risk Predictor of Breast Cancer Based on Intrinsic Subtypes. J Clin Oncol. 2009; 27(8): 1160–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPawitan Y, Bjöhle J, Amler L, et al.: Gene expression profiling spares early breast cancer patients from adjuvant therapy: derived and validated in two population-based cohorts. Breast Cancer Res. 2005; 7(6): R953–964. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrat A, Parker JS, Karginova O, et al.: Phenotypic and Molecular Characterization of the Claudin-Low Intrinsic Subtype of Breast Cancer. Breast Cancer Res. 2010; 12(5): R68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRibas A, Robert C, Hodi FS, et al.: Association of Response to Programmed Death Receptor 1 (PD-1) Blockade with Pembrolizumab (MK-3475) with an Interferon-Inflammatory Immune Gene Signature. J Clin Oncol Res. suppl; abstr 3001. Oral Abstract Session, Developmental Therapeutics—Immunotherapy. 2015. Reference Source\n\nRinchai D, Kewcharoenwong C, Kessler B, et al.: Abundance of ADAM9 transcripts increases in the blood in response to tissue damage [version 1; referees: 3 approved with reservations]. F1000Res. 2015; 4: 89. Publisher Full Text\n\nRouas-Freiss N, Gonçalves RM, Menier C, et al.: Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis. Proc Natl Acad Sci U S A. 1997; 94(21): 11520–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmidt M, Böhm D, von Törne C, et al.: The Humoral Immune System Has a Key Prognostic Impact in Node-Negative Breast Cancer. Cancer Res. 2008; 68(13): 5405–5413. PubMed Abstract | Publisher Full Text\n\nSorlie T, Tibshirani R, Parker J, et al.: Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A. 2003; 100(14): 8418–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpeake C, Presnell S, Domico K, et al.: An interactive web application for the dissemination of human systems immunology data. J Transl Med. 2015; 13: 196. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwets M, König MH, Zaalberg A, et al.: HLA-G and classical HLA class I expression in primary colorectal cancer and associated liver metastases. Hum Immunol. 2016; 77(9): 773–79. PubMed Abstract | Publisher Full Text\n\nWang E, Bedognetti D, Marincola FM: Prediction of response to anticancer immunotherapy using gene signatures. J Clin Oncol. 2013; 31(19): 2369–71. PubMed Abstract | Publisher Full Text\n\nWang Y, Klijn JG, Zhang Y, et al.: Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet. 2005; 365(9460): 671–679. PubMed Abstract\n\nZeestraten EC, Reimers MS, Saadatmand S, et al.: Combined analysis of HLA class I, HLA-E and HLA-G predicts prognosis in colon cancer patients. Br J Cancer. 2014; 110(2): 459–68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang Y, Sieuwerts AM, McGreevy M, et al.: The 76-gene signature defines high-risk patients that benefit from adjuvant tamoxifen therapy. Breast Cancer Res Treat. 2009; 116(2): 303–309. PubMed Abstract | Publisher Full Text\n\nZhou Y, Yau C, Gray JW, et al.: Enhanced NF kappa B and AP-1 transcriptional activity associated with antiestrogen resistant breast cancer. BMC Cancer. 2007; 7: 59. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "24330",
"date": "24 Jul 2017",
"name": "Benjamin Haibe-Kains",
"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, Roelands et al. describe the implementation of the gene expression browser (GXB), a database and web-application integrating 13 breast cancer datasets containing gene expression and clinical data for a total of almost 2000 patients. The authors focused their case studies on the investigation of immune-related genes and subtypes and made their curated data publicly available. I think GXB is a welcome contribution to the breast cancer research community and its public availability will help other scientists to leverage this valuable collection of datasets. However, I have several concerns that need to be addressed before indexing, as listed below.\nThe manuscript is well written and easy to understand.\n\nMajor comments\nIn Table 1 and on the front page of the web-application, Princess Margaret Cancer Centre is listed in the sample set name even though these data have not been generated in this institution, which is misleading.\nWhen selecting \"Breast Cancer\", which I assumed contained the whole database, many datasets got filtered out, which is confusing.\nI tried AURKA and selected GSE9195 as dataset. Then I played a bit with the barplot to overlay different kinds of information. I added DMFS 10Y (categorical) and sorted the patients based on this but there were some patients with very low DMFS at the beginning and the end of the plot, which is confusing.\nSince the authors are dealing with survival data, I was expecting survival statistics and/or survival curves but I did not see any in GXB or the manuscript. Figure 1 is somehow misleading as these plots do not seem to be part of the web-application.\nThe authors decided to limit their database to Affymetrix data. In that case, I suggest the authors reprocess all the CEL files consistently using (f)RMA and up-to-date chip description files (CDF), such as BrainArray CDFs. This would increase the consistency across all the datasets.\nGiven the authors' large collection of datasets, mining each dataset separately is cumbersome. Implementing meta-analysis pipelines would help draw an overall conclusion before digging into dataset-specific results (e.g., Figure 3). This is what the authors seem to have done with the \"Nagalla\" dataset that is a compendium of all the datasets reprocessed using MAS5 and further corrected with ComBat. It is not clear why the authors used MAS% to normalize the data in this case vs (f)RMA for some other datasets. Please clarify. Why not recommending users to first explore Nagallia before going after each dataset separately to detect trends that may be (in)consistent with the majority of the datasets?\nThere have been many molecular subtyping schemes published, including the Integrative Subtypes (IntClust; Curtis et al, Nature 2012), the Subtype Classification Models (SCMGENE, SCMOD1, SCMOD2; Haibe-Kains et al, JNCI 2012) and the Absolute Assignment of Breast Cancer Intrinsic Molecular Subtype (AIMS; Paquet et al, JNCI 2015). The authors should discuss the rationale for their choice of relying solely on PAM50.\n\nMinor comments\nTable 2 would be better represented as a barplot or a similar figure.\nGXB would benefit from the inclusion of normal samples (from healthy patients or adjacent normal samples). METABRIC, TCGA and GTEx are relevant data sources for such gene expression profiles.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "3041",
"date": "18 Sep 2017",
"name": "Wouter Hendrickx",
"role": "Author Response",
"response": "Answers are in Italic and bold Major comments In Table 1 and on the front page of the web-application, Princess Margaret Cancer Centre is listed in the sample set name even though these data have not been generated in this institution, which is misleading. Names of datasets have been changed from “Princess Margaret Cancer Centre dataset - GSE6532.GPL96” and “Princess Margaret Cancer Centre dataset - GSE6532.GPL97”, to “John Radcliff Hospital (OXFU, OXFT) dataset- GSE6532.GPL96” and “John Radcliff Hospital (OXFU, OXFT) dataset- GSE6532.GPL97” respectively. “Princess Margaret Cancer dataset- GSE6532.GPL570” was changed to “Guys hospital (GUYT) dataset- GSE6532.GPL570”. Tables and figures throughout the manuscript have been revised accordingly. When selecting \"Breast Cancer\", which I assumed contained the whole database, many datasets got filtered out, which is confusing. Thank you for noticing this. Only a single disease type can be assigned per dataset. We can change ER+ and LN- datasets to disease type: Breast Cancer , effectively disabling the user to filter datasets based on ER+ and LN status. We preferred changing the name of Breast cancer (general) to \"mixed breast cancer types\" for clarity. I tried AURKA and selected GSE9195 as dataset. Then I played a bit with the barplot to overlay different kinds of information. I added DMFS 10Y (categorical) and sorted the patients based on this but there were some patients with very low DMFS at the beginning and the end of the plot, which is confusing. We were able to reproduce the barplot you described. By sorting by the categorical variable DMFS 10Y EVENT, you only sort based on this variable (DistantMetastasisFree or DistantMetastasis). The continuous counterpart of this variable (DMFS 10Y TIME) is not taken into account when you perform the sorting, explaining your observation. An alternative is to sort based on the continuous variable DMFS 10Y TIME, though this sorting results in a plot that mixes DistantMetastasisFree and DistantMetastasis categories. Unfortunately, subsequent sorting based on 2 variables is not possible using GXB. We have suggested this improvement to the GXB developing team. Since the authors are dealing with survival data, I was expecting survival statistics and/or survival curves but I did not see any in GXB or the manuscript. Figure 1 is somehow misleading as these plots do not seem to be part of the web-application. Survival statistics are not yet part of the GXB web-application. To prevent any misconception, we have added an extra sentence in the legend of figure 1: “This figure is for explanatory purposes only and does not serve as a demonstration of the GXB web application.” The authors decided to limit their database to Affymetrix data. In that case, I suggest the authors reprocess all the CEL files consistently using (f)RMA and up-to-date chip description files (CDF), such as BrainArray CDFs. This would increase the consistency across all the datasets. The purpose of this data note is to share existing data from GEO in a more comprehensible format and does not involve any data processing. We had to make an exception for dataset GSE9195.GPL570, since only raw format was available on GEO. Given the authors' large collection of datasets, mining each dataset separately is cumbersome. Implementing meta-analysis pipelines would help draw an overall conclusion before digging into dataset-specific results (e.g., Figure 3). This is what the authors seem to have done with the \"Nagalla\" dataset that is a compendium of all the datasets reprocessed using MAS5 and further corrected with ComBat. It is not clear why the authors used MAS5 to normalize the data in this case vs (f)RMA for some other datasets. Please clarify. Normalization of the complete Nagalla cohort was previously performed as described in Nagalla et al. 2013 and Miller et al. 2016 To make this data available as published in this article, we used the same data file to upload to GXB. Normalization is different between datasets because the data was transferred straight from GEO and different contributors use different methods depending on the times it was performed and the need of their specific work. Why not recommending users to first explore Nagallia before going after each dataset separately to detect trends that may be (in)consistent with the majority of the datasets? In this dataset collection, it is indeed possible to first explore the Nagalla dataset, which is a dataset that includes all samples from the collection. This advantage is specific for this breast cancer compendium of datasets, so your suggestion is a very good strategy to explore these datasets. We have added this recommendation in the text of the Dataset Demonstration section of the data note. There have been many molecular subtyping schemes published, including the Integrative Subtypes (IntClust; Curtis et al, Nature 2012), the Subtype Classification Models (SCMGENE, SCMOD1, SCMOD2; Haibe-Kains et al, JNCI 2012) and the Absolute Assignment of Breast Cancer Intrinsic Molecular Subtype (AIMS; Paquet et al, JNCI 2015). The authors should discuss the rationale for their choice of relying solely on PAM50. Categorization using the PAM50 molecular subtyping was performed as part of the research performed by Nagalla et al. 2013 and Miller et al. 2016. These annotated datasets were uploaded in GXB as described in this data note. The choice to rely on this 50-gene classifier was dependent on its high prognostic and predictive value of this subtyping method in combination with the high clinical applicability of PAM50 testing. Minor comments Table 2 would be better represented as a barplot or a similar figure. For the purpose of listing which clinical variables are available in how many datasets from the collection, we found a table the more appropriate option. GXB would benefit from the inclusion of normal samples (from healthy patients or adjacent normal samples). METABRIC, TCGA and GTEx are relevant data sources for such gene expression profiles. Comparing healthy control tissue with cancerous tissue on gene expression level indeed represents an interesting aspect that can be explored using the GXB browser. At this moment, different types of dataset collections have already been uploaded to GXB, including case versus control comparisons. For example, a dataset with gene expression of whole blood samples from both lung cancer patients and healthy controls and a dataset with head and neck cancer and cervical cancer tissue samples, matched with site-matched normal epithelial samples are currently available. The TCGA, METABRIC dataset and datasets in the GTEx portal are valuable resources that can be used to upload new datasets into GXB. We will definitely take this suggestion into consideration for our new dataset collection to upload into GXB. Discussed changes will be applied in version 2 of the manuscript."
}
]
},
{
"id": "24824",
"date": "26 Sep 2017",
"name": "Christos Hatzis",
"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\nRoelands et al present a web application that provides real-time analysis and visualization of a large compendium of microarray datasets generated from primary breast cancer biopsies. The uniqueness and distinct utility of this dataset is the focus on immune-related signatures. Before discussing potential shortcomings according to this reviewer's opinion, the authors should be commended for investing the energy and resources to develop and implement a focused public repository that would facilitate further analyses.\nAlthough the overall objective behind this website and tools is laudable, I believe that the approach followed, as presented in the manuscript, has several potential shortcomings that would be worth addressing.\nRegarding the protocols followed, I would echo the comments by the first reviewer and question the use of MAS5 and COMBAT, given that more robust algorithms are now available. This may be a key issue given the quantitative nature of many of the indices used. Also, it is not clear that this approach removes bias between the older Affymetrix platforms and the newer ones (PLUS2.0). On page 5 it is mentioned that \"the 22,268 probe sets present in each of these platforms were included\", but the PLUS 2.0 chips include twice as many. They probably meant to say \"present in all platforms\".\n\nIn terms of whether sufficient details were provided to allow replication, it would be great if the authors provided the complete set of scripts used to pre-process and normalize the data included in the analysis. This will allow transparent assessment of the methods and replication of the dataset if needed.\n\nRelated to the type of data included, I believe that the resource would be more valuable if additional clinically-relevant information was provided with each sample. For example, disease progression clearly depends on treatment and treatment information is missing entirely - we do not know whether patients even received any treatment at all. It would be useful to curate treatment information available in the original datasets. Also, it may be worth providing annotations for additional immune signatures (e.g. Rody et al, 20111) and also of additional breast cancer subtypes (e.g. Lehmann et al, 20162). This will allow a more thorough assessment of the overlap between the various signature-defined subtypes. Finally, although there are not as many profiles from breast cancer metastasis available in GEO, it may be worth considering curating those and including them in the platform. It would be extremely interesting to know how the immune signatures differ between primary and metastatic samples, and whether the trends suggest immune evasion.\n\nFinally, from a usability standpoint, I have a few suggestions outlined below:\n- Although the functionality may be available, I found it very difficult to filter samples based on a particular feature. For example, I was trying to do the HLA-G by ICR group analysis only for the ER-negative or basal cases, but could not find an easy way to do that.\n- It would be useful to provide tools for correlational analysis of two or more genes (e.g. co-expression patterns) and heat maps. The one gene at a time visualization is very restrictive.\n\nMinor points:\nWorth considering having another table after Table 1 that summarizes the immune subtype signatures and annotations with references. Intrinsic subtype signatures used can also be summarized in that table. That will also eliminate the need to explain the acronyms (e.g. Table 2 is marred by too many acronyms IMS, IBC, IDS, ICR that are not explained).\n\nWhen running the HLA-G example as outlined in the manuscript, I was presented with 4 different HLA-G transcripts with no explanation as to what was the difference between them. Are these different probe sets? If so, that should probably be explained. Also, in that case, is there a recommendation as to which of these probe sets is the most representative (e.g. based on specificity, range, variance etc).\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly",
"responses": [
{
"c_id": "3366",
"date": "09 Feb 2018",
"name": "Wouter Hendrickx",
"role": "Author Response",
"response": "Major points: Regarding the protocols followed, I would echo the comments by the first reviewer and question the use of MAS5 and COMBAT, given that more robust algorithms are now available. This may be a key issue given the quantitative nature of many of the indices used. Also, it is not clear that this approach removes bias between the older Affymetrix platforms and the newer ones (PLUS2.0). On page 5 it is mentioned that \"the 22,268 probe sets present in each of these platforms were included\", but the PLUS 2.0 chips include twice as many. They probably meant to say \"present in all platforms\". The primary goal of this data note was to share the transcriptomic and annotation data associated with this previous publication (in GXB defined as “the Nagalla 2013 reconstituted public dataset”) and the breast cancer transcriptomic datasets from GEO that it constitutes of in a more interactive, comprehensible format to facilitate usage of the data and did not involve data processing. The selection of 22,268 probe sets, combination and normalization of the 13 datasets was also previously performed (Nagalla et al). For your second point, we indeed meant the probe sets that are present in all platforms, for this reasons probe sets that are exclusively present in Affymetrix U133 PLUS2.0 are not included in these 22,268 probe sets. We have revised this sentence in the manuscript accordingly. In terms of whether sufficient details were provided to allow replication, it would be great if the authors provided the complete set of scripts used to pre-process and normalize the data included in the analysis. This will allow transparent assessment of the methods and replication of the dataset if needed. The uploads from individual GEO datasets into GXB did not involve any data processing. For the Nagalla et al this has been performed as part of the work described in Nagalla et al. This data note did involve clinical data harmonization performed in R, scripts have been made available on Github. Related to the type of data included, I believe that the resource would be more valuable if additional clinically-relevant information was provided with each sample. For example, disease progression clearly depends on treatment and treatment information is missing entirely - we do not know whether patients even received any treatment at all. It would be useful to curate treatment information available in the original datasets. Also, it may be worth providing annotations for additional immune signatures (e.g. Rody et al, 20111) and also of additional breast cancer subtypes (e.g. Lehmann et al, 20162). This will allow a more thorough assessment of the overlap between the various signature-defined subtypes. Finally, although there are not as many profiles from breast cancer metastasis available in GEO, it may be worth considering curating those and including them in the platform. It would be extremely interesting to know how the immune signatures differ between primary and metastatic samples, and whether the trends suggest immune evasion. When uploading these GEO datasets into GXB, all available clinical data on GEO was also transferred to GXB. For some of the datasets (e.g. GSE4922), a treatment parameter is available which can be selected as a parameter using the overlay function in the bar plot generated in GXB. We completely agree that information on adjuvant treatment is essential for interpretation of the supplied survival data (Distant Metastasis Free Survival and Disease Free Survival). For proper survival analysis, we recommend users to download the csv file supplied which can be found under the “Downloads”-tab of each dataset and use available treatment information for stratification.Addition of more gene signatures can indeed be valuable to compare immune based classifications as demonstrated in this data note for Immunological Constant of Rejection (ICR) classification and Immune Benefit Status (IBS)/ Immune Disposition Status (IDS). Similarly, uploading additional GEO datasets that contain matched primary and metastatic samples would be very interesting and allow for comparison of both immune microenvironments. Although not in the scope of the current data note, we will definitely take these suggestions into consideration for further development of the GXB breast cancer instance. Finally, from a usability standpoint, I have a few suggestions outlined below: -Although the functionality may be available, I found it very difficult to filter samples based on a particular feature. For example, I was trying to do the HLA-G by ICR group analysis only for the ER-negative or basal cases, but could not find an easy way to do that.It is indeed possible to visualize the expression of a gene of interest, for example HLA-G, in a specific subgroup of samples like all ER-negative or basal cases. The most convenient way to do this is to (1) select barplot, (2) set group by Molecular Subtype and (3) overlay the barplots with ICR group annotation. To make the figure easier to analyze, you can subsequently sort by ICR. - It would be useful to provide tools for correlational analysis of two or more genes (e.g. co-expression patterns) and heat maps. The one gene at a time visualization is very restrictive.We realize that gene per gene visualization has its limitations and analysis of groups coordinately expressed genes provides more biological significance. For this reason, the GXB development team has already created a module analysis tool (MAT) that analyses expression data to find pre-defined groupings of co-expressed genes (modules) to obtain a molecular fingerprint of gene expression for each individual sample in your dataset. MAT has already been applied to many datasets with samples of various immune related diseases and we are considering to also use this platform for cancer transcriptomic datasets. As the desired number of group comparisons in breast cancer (i.e., Molecular Subtypes, Immunologic classifications, Pathology T Stage) is higher compared to the existing group comparisons, some work is required before we can implement these dataset in this tool.Minor points: Worth considering having another table after Table 1 that summarizes the immune subtype signatures and annotations with references. Intrinsic subtype signatures used can also be summarized in that table. That will also eliminate the need to explain the acronyms (e.g. Table 2 is marred by too many acronyms IMS, IBC, IDS, ICR that are not explained). We have added the suggested table to the manuscript. This is both useful when reading the manuscript as well as for the use of this breast cancer GXB application, thank you for this suggestion. When running the HLA-G example as outlined in the manuscript, I was presented with 4 different HLA-G transcripts with no explanation as to what was the difference between them. Are these different probe sets? If so, that should probably be explained. Also, in that case, is there a recommendation as to which of these probe sets is the most representative (e.g. based on specificity, range, variance etc). These different HLA-G transcripts are indeed different probe sets. If you are interested to find and select a specific probe ID, you can select “Show Probe ID” under “Tools”. This GXB data portal does not give an explanation on the difference between these probes. For this information we would like to refer to the Affymetrix website. For the HLA-G example, we just took a single probe at random: 211528_X_AT. We added this information in version 2 of the manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/6-296
|
https://f1000research.com/articles/6-1845/v1
|
16 Oct 17
|
{
"type": "Method Article",
"title": "Measuring evolutionary rates of proteins in a structural context",
"authors": [
"Dariya K. Sydykova",
"Benjamin R. Jack",
"Stephanie J. Spielman",
"Claus O. Wilke",
"Dariya K. Sydykova",
"Benjamin R. Jack",
"Stephanie J. Spielman"
],
"abstract": "We describe how to measure site-specific rates of evolution in protein-coding genes and how to correlate these rates with structural features of the expressed protein, such as relative solvent accessibility, secondary structure, or weighted contact number. We present two alternative approaches to rate calculations, one based on relative amino-acid rates and the other based on site-specific codon rates measured as dN/dS. In addition to describing the specific analysis protocols we recommend, we also provide a code repository containing scripts to facilitate these kinds of analyses.",
"keywords": [
"Protein evolution",
"protein structure",
"evolutionary rate",
"relative solvent accessibility",
"weighted contact number",
"multiple sequence alignment"
],
"content": "Introduction\n\nDifferent sites within a protein-coding gene evolve at different rates1,2. This evolutionary rate heterogeneity across protein sites results from a complex interplay of both functional and structural constraints3. For example, residues that are critical to a given protein’s function, such as residues involved in enzymatic activity, in protein–protein interactions, or in protein–ligand interactions, tend to evolve more slowly than other residues in the protein4–10. In addition, protein structure has been found to play a major role in shaping protein evolutionary rates across the entire protein sequence, because the imperative for a protein to stably fold produces an overarching evolutionary constraint. Structurally-important protein residues (namely residues in the protein core) tend to be highly conserved and evolve very slowly, but residues with a minor influence on structure (namely surface residues) evolve more rapidly4,9,11–19.\n\nTo study evolutionary conservation in a structural context, we need methods to (i) measure evolutionary rates at individual sites in a protein alignment, (ii) map those rates onto the protein structure, and (iii) quantify site-level structural properties. Here, we describe in detail how to perform these three steps, considering a few commonly used alternatives at both steps (i) and (iii). In addition, we provide extensive notes highlighting specific technical issues and/or describing alternative analysis approaches. At step (i), we demonstrate how evolutionary rates can be measured using either amino-acid or codon data. For amino-acid data, we consider relative amino-acid rates, i.e., rates of evolutionary variation normalized by the mean of the rate in the entire protein10,20. For codon data, we consider site-specific dN/dS values. These are site-specific rates of nonsynonymous variation normalized by (whole-gene) rates of synonymous variation21,22. At step (iii), we discuss two related but somewhat distinct structural measures. First, we consider the solvent accessibility, which measures the extend to which a site is exposed to the solvent environment. Specifically, we consider the relative solvent accessibility (RSA)23, which is the solvent accessibility of a residue in a structure normalized by the maximally possible solvent accessibility of that residue in a Gly-X-Gly tripeptide. Second, we consider the packing density, which measures the proximity and number of neighboring residues. Specifically, we consider the side-chain weighted contact number (WCN)19, which is calculated relative to the geometric center of the residue side-chain atoms and employs an inverse-square weighting term.\n\n\nMaterials\n\nBelow we list the software packages needed to perform the analysis. Please download the most recent version of each software, unless a specific version is specified in the text. The links provided contain instructions for installing and testing the software.\n\n1. HyPhy (see Note 1)\n\nHyPhy is a general-purpose software platform for inference in a phylogenetic framework24. To install, clone the HyPhy git repository to your desired directory. The HyPhy repository can be found at https://github.com/veg/hyphy.git. Instructions for installation are available from http://hyphy.org/installation.\n\n2. MAFFT\n\nMAFFT is a program for generating multiple sequence alignments25. Download MAFFT from http://mafft.cbrc.jp/alignment/software/.\n\n3. RAxML\n\nRAxML is a tool for phylogenetic inference using maximum likelihood26. Clone the RAxML repository to a local directory. The RAxML git repository can be found at https://github.com/stamatak/standard-RAxML. Analyses presented here utilize the raxmlHPC-SSE3 executable, which can be compiled with Makefile.SSE3.gcc or Makefile.SSE3.mac. Note that this executable does not allow threading. (See Note 2 for information on how to enable threading.)\n\n4. mkDSSP\n\nmkDSSP is a tool that calculates solvent accessibilities and parses secondary structure assignments from a PDB input file into a standardized format27. This format follows that of the entries in the DSSP database28. Download the mkDSSP software from https://slackbuilds.org/repository/14.2/academic/mkDSSP/.\n\n5. Python\n\nDownload Python from https://www.python.org/downloads/.\n\n6. Biopython\n\nBiopython is a python library for computational molecular biology29. Download Biopython from http://biopython.org/wiki/Download.\n\nBiopython has several dependencies that also need to be installed. You can find the information about installing the dependencies in the link provided.\n\n7. argparse\n\nargparse is a python module providing user-friendly command-line interfaces. We use argparse in most of our custom python scripts. Install argparse using the link https://pypi.python.org/pypi/argparse.\n\n8. pandas\n\npandas is a python module for data manipulation and analysis. You can download pandas from https://pandas.pydata.org/getpandas.html.\n\n9. R\n\nDownload R from https://www.r-project.org/. We recommend to use RStudio to execute and edit R scripts. RStudio can be installed from https://www.rstudio.com/. We will use R for data visualization. Our scripts require the packages dplyr, readr, cowplot, and their dependencies. You can install an R package by typing the command install.packages(\"dplyr\") (for installing dplyr) in the R shell. By default, this command will also install any dependencies needed for the package to work.\n\n10. Custom scripts (see Note 3)\n\nAll our custom python, R, and HyPhy scripts can be found at: https://github.com/clauswilke/proteinER/tree/master/src.\n\n\nProtocols\n\nIn the following, we provide four separate protocols to (i) measure relative amino acid rates, (ii) measure site-specific codon evolutionary rates (expressed via the metric dN/dS), (iii) measure structural quantities such as RSA and WCN, and (iv) combine the measured quantities into a combined analysis. To provide an example, we demonstrate all four protocols on an empirical dataset consisting of mammalian orthologs of histamine receptor 1 (ENST00000438284) and an accompanying PDB structure. This dataset was originally analyzed by Spielman and Wilke30. Throughout, we assume that we are working on a UNIX-like command line interface. For your convenience, we have provided a git repository at https://github.com/clauswilke/proteinER/ that contains the input and output files used in each step. Our overarching strategy throughout this work is to first infer a given measurement (e.g., dN/dS or RSA) for each site in the multiple sequence alignment or protein structure. To compare the different measurements, we then map them all to columns in the multiple sequence alignment.\n\nThe input and output files used in this section can be found at: https://github.com/clauswilke/proteinER/tree/master/measuring_aa_rates.\n\n1. Align sequences with MAFFT\n\nStore all of the sequences you wish to align into one file. The file must be in the FASTA format. The FASTA format contains two pieces of information for each sequence: the sequence ID preceded by a \">\" sign and followed by a new line, and then the sequence itself. We will use the FASTA file HRH1_unaligned.fasta that contains homologous sequences that are not aligned. We align them with the command:\n\n\n\nArguments above correspond to the following:\n\n• --auto, Select the optimal alignment algorithm for the given data.\n\n• --inputorder, Output sequences in the same order in which they were provided. Without this option, the order of the sequences in the alignment is arbitrary.\n\nThe output file HRH1_aligned.fasta will contain the aligned sequences.\n\n2. Infer tree with RAxML (see Notes 2, 4)\n\nUsing the file with the alignment HRH1_aligned.fasta, run RAxML with the following command:\n\n\n\nArguments above correspond to the following:\n\n• -s, The multiple sequence alignment file.\n\n• -n, The extension for the outputted tree files. Here, the outputted files will contain HRH1_tree in their names.\n\n• -m, The model of sequence evolution, in this case the LG amino-acid model31 with RAxML’s “CAT\" model32 of sequence heterogeneity.\n\n• -p, The random number seed initializing this phylogenetic inference. To reproduce the exact phylogeny we have, specify this random seed.\n\nThe desired tree file is RAxML_bestTree.HRH1_tree.\n\n3. Infer site-wise rates with HyPhy (see Note 5)\n\nTo run HyPhy, the file runRelativeProtRates.bf must be edited to specify the directories and file names that will be used in the analysis. Edit these two lines of runRelativeProtRates.bf\n\n\n\nHere, \"0\" specifies the full path to the alignment file HRH1_aligned.fasta, and \"1\" specifies the full path to the tree file RAxML_bestTree.HRH1_tree.\n\nRun HyPhy with the command\n\nHYPHYMP runRelativeProtRates.bf\n\nAn output file HRH1_aligned.fasta.site-rates.json is written to the folder that contains the alignment.\n\n4. Parse HyPhy output (see Note 3)\n\nFor further downstream processing, the HyPhy output file in JSON format needs to be converted to CSV format. The custom python script parse_prot_rates.py will extract the site’s position, rate, and other site information outputted by HyPhy. Parse the JSON file with the command\n\n\n\nArguments above correspond to the following:\n\n• -j, JSON file outputted by HyPhy.\n\n• -r, The output CSV file. If not specified, the output file is site_rates.csv.\n\n5. Calculate relative site-wise rates\n\nAs discussed by Jack et al.10, we recommend calculating relative evolutionary rates by normalizing inferred site-specific rates by their average. In other words, to compute the relative amino-acid rates, calculate the mean rate of the entire sequence and divide each site’s rate by this mean rate. Once normalized, a rate below 1 will indicate a site that evolves more slowly than average. For example, a rate of 0.5 implies that the corresponding site evolves half as fast as the average. Similarly, a rate above 1 will indicate a site that evolve more quickly than average. For example, a rate of 2 implies that the corresponding site evolves twice as fast as the average.\n\nThe input and output files used in this section can be found at: https://github.com/clauswilke/proteinER/tree/master/measuring_dNdS.\n\n1. Translate codon sequences (see Note 3)\n\nIn this section, both codon and amino-acid sequences are required to perform site-wise rate calculations. Store all of the desired nucleotide sequences into one FASTA file. Use our custom script to convert a codon FASTA file to an amino acid FASTA files. We use the FASTA file HRH1_unaligned_codon.fasta that contains homologous nucleotide sequences we wish to translate. Translate with the command:\n\n\n\nArguments above correspond to the following:\n\n• -n, The input file with codon sequences. Both aligned and unaligned sequences are accepted.\n\n• -o, The output file with amino acid sequences. If not specified, the script outputs aa_aln.fasta. If the input file contains aligned sequences, the output file will also contain aligned sequences.\n\n2. Align amino acid sequences with MAFFT\n\nAlign amino acid sequences using step 1 in Protocol 1.\n\n3. Back-translate the amino acid alignment into a codon alignment (see Note 3)\n\nThis step requires the original codon sequences and the amino acid alignment. Note that the amino acid alignment is retained, and the script simply inserts corresponding codons in place of amino acids at each column of the alignment. The command to back-translate the sequences is:\n\n\n\nArguments above correspond to the following:\n\n• -a, The inputted amino-acid alignment.\n\n• -n, The file of codon sequences. The script accepts either aligned or unaligned sequences.\n\n• -o, The output file to contain the codon alignment. This argument is optional, and, if it is missing, the script outputs a file codon_aln.fasta.\n\n4. Infer tree with RAxML\n\nThe following step is the same as step 2 in Protocol 1. Use the amino-acid alignment file HRH1_aligned_aa.fasta to infer the tree.\n\n5. Infer site-wise rates with HyPhy (see Note 6)\n\nTo run HyPhy, the file runFEL.bf must be edited to specify the directories and file names that will be used in the analysis. Edit the following two lines of the runFEL.bf script:\n\n\n\nHere, \"1\" specifies the full path to the alignment file HRH1_aligned_codon.fasta, and \"2\" specifies the full path to the tree file RAxML_bestTree.HRH1_tree.\n\nRun HyPhy with the following command:\n\nHYPHYMP runFEL.bf\n\nAn output file\n\nHRH1_aligned_codon.fasta.FEL.json is written to the folder that contains the alignment file.\n\n6. Parse HyPhy output (see Note 3)\n\nFor further downstream processing, the HyPhy output file in JSON format needs to be converted to CSV format. The custom python script parse_FEL.py will extract the site’s position, dN/dS, and other site information outputted by HyPhy:\n\n\n\nArguments above correspond to the following:\n\n• -j, JSON file from the FEL analysis.\n\n• -r, The output CSV file. If not specified, the output file is site_rates.csv.\n\n7. Change dN/dS values for conserved sites (see Note 3)\n\nThe FEL method as implemented in HyPhy assigns dN/dS = 1 to sites without any synonymous and any non-synonymous substitutions. We recommend to express rate at entirely conserved sites with dN/dS = 0. We provide a custom script that will assign dN/dS = 0 to completely conserved sites. This script will not change extracted_HRH1_dNdS.csv’s original format.\n\n\n\nArguments above correspond to the following:\n\n• -a, The amino acid alignment file.\n\n• -r, The CSV file with parsed FEL rates.\n\n• -o, The output CSV file. If not specified, the script outputs processed_dNdS.csv.\n\nAll structural features in this section are calculated from an example PDB file, 3rze.pdb. This PDB file defines the crystal structure of a transmembrane protein fused to an unrelated lysozyme protein. The lysozyme is required for crystallization, but is not biologically relevant. We have pre-processed the PDB file to exclude residues from the lysozyme protein (residue numbers 1000 and above). The input and output files used in this section can be found at: https://github.com/clauswilke/proteinER/tree/master/measuring_structural_features.\n\n1. Calculate relative solvent accessibility (RSA) from the PDB file (see Note 3)\n\nWe provide a custom script calc_rsa.py that will run mkdssp27,28, extract absolute solvent accessibilities, and calculate relative solvent accessibilities23. The first argument is the PDB file, and the second optional argument (-o 3rze) is the prefix used for the output files.\n\npython calc_rsa.py 3rze.pdb -o 3rze\n\nThis command will generate two output files: 3rze.asa.txt containing the raw mkdssp output, and 3rze.rsa.csv containing RSA values and secondary structure classifications.\n\n2. Calculate weighted contact numbers (WCN) from the PDB file (see Note 3)\n\nWCN measures amino acid packing density, and may be calculated with respect to either the α-carbon or the geometric center of the side-chain33,19. We provide a custom script that will calculate both types of WCN values. The command line arguments follow the same format as the calc_rsa.py script.\n\npython calc_wcn.py 3rze.pdb -o 3rze\n\nThe above command will produce an output file 3rze.wcn.csv that contains both side-chain WCN and α-carbon WCN values for each position in the input PDB file.\n\nThe input and output files used in this section can be found at: https://github.com/clauswilke/proteinER/tree/master/map_structural_features.\n\n1. Generate sequence alignment map (see Note 3)\n\nTo map site specific evolutionary rates to residues in a PDB structure, we first align the sequence of amino acids extracted from the PDB structure to the multiple sequence alignment used for rate inference. We provide a script that calls mafft to align a PDB sequence to a multiple sequence alignment and reformat the output.\n\n\n\nRunning the above command produces a CSV file with four columns. The first column contains, for each residue, the numbered position of that residue in the alignment used for rate inference. The second column similarly contains the numbered position of each residue in the PDB structure. Numbered positions are extracted directly from the PDB input file and may include PDB insertion codes (see Note 8). The third and fourth columns contain the single-letter amino acid present in the PDB structure and the PDB chain, respectively. If an amino acid is in the alignment but not in the PDB structure, the PDB position is assigned a value of NA. Likewise, if an amino acid is in the PDB structure but not the alignment, the alignment position is assigned NA.\n\n2. Map rates to structural features (see Note 3)\n\nAfter mapping the alignment used for rate inference to the sequence of the PDB structure, we align rates with structural features. We provide a script that uses the map generated above to combine rates and structural features into a single CSV.\n\n\n\nArguments above correspond to the following:\n\n• The input file containing a map between the alignment residue positions and the structure residue positions.\n\n• -r, The rates files.\n\n• -f, The structural feature files.\n\n• -o, The CSV output file. If not specified, the script outputs a file <pdb_id>.rates_features.csv. Here, <pdb_id> is the name of the PDB ID used to make the map file.\n\nThe output from this command provides all the data needed to compute correlations between rates and structural features and corresponding visualizations. See Figure 1 as an example.\n\n(a–d) Each point represents a residue in the structure of the HRH1 protein (PDB: 3rze). The Pearson correlation coefficients r between structural features (RSA or WCN) and rates (dN/dS or amino acid) are as follows for each panel: (a) –0.39, (b) 0.43, (c) –0.39, (d) 0.42.\n\n\nConclusions\n\nWe have provided four separate protocols that jointly enable the analysis of protein evolutionary rates in a structural context. The first two protocols are used to measure site-specific evolutionary rates from multiple-sequence alignments, either at the amino-acid or the codon level. Any actual study will generally employ only one of these protocols. The third protocol is used to quantify local characteristic of a protein structure, such as relative solvent accessibility or weighted contact number, and the fourth protocol maps the structural quantities to the evolutionary rates and vice versa. We hope that these protocols will be useful for further research into disentangling structural and functional constraints on protein evolution.\n\n\nNotes\n\n1. The minimum required HyPhy version for FEL dN/dS inference is 2.3.3. The minimum required version for relative amino-acid rate inference is 2.3.4.\n\n2. To thread RAxML, compile the raxmlHPC-PTHREADS-SSE3 executable with Makefile.SSE3.PTHREADS.gcc or Makefile.SSE3.PTHREADS.mac. The options to call RAxML stay the same. Add the option -T to thread, and run RAxML with\n\n\n\n3. All of our custom python scripts provide documentation when called with the options -h or --help. For example, to view the documentation for the script calc_rsa.py, run the command\n\npython calc_rsa.py -h\n\nThe script’s use and required input files will be described in the documentation. Additionally, where applicable, the documentation also provides a description of the information stored in the output files.\n\n4. RAxML can also infer trees from nucleotide sequence data in addition to amino-acid data. Importantly, if the analyzed sequences are highly diverged, trees inferred from nucleotide sequences may yield better rate predictions. To infer a tree from nucleotide data with RAxML, issue the following command (specifically, -m PROTCATLG has been changed to a GTR nucleotide model with CAT heterogeneity, -m GTRCAT):\n\n\n\nFurthermore, if the dataset of interest contains fewer than 50 taxa, it is not recommended to adopt the CAT model of heterogeneity32. Instead, a discrete Gamma distribution should be used. To specify this model, simply replace the phrase CAT with GAMMA: For amino-acids, use the model specification -m PROTGAMMALG, and for nucleotides use the model specification -m GTRGAMMA.\n\n5. As an alternative method to infer site-wise amino acid rates one can use Rate4Site. Rate4Site is a tool for inferring site-wise evolutionary rates in amino acid sequences20. Download Rate4Site from https://www.tau.ac.il/~itaymay/cp/rate4site.html. Analyses presented here use Rate4Site downloaded as rate4site.3.2.source.zip and compiled with the Makefile_slow file.\n\nThe options to run Rate4Site may be different for different Rate4Site installation files. We recommend using the rate4site -h command to find the proper options for your version, as opposed to using the software’s website.\n\nRun the following command to infer site-wise rates:\n\n\n\nArguments above correspond to the following:\n\n• -Mw, Specify the WAG model of amino-acid evolution (see Note 7).\n\n• -s, The multiple sequence alignment file.\n\n• -t, The input phylogeny.\n\n• -o, The output file of normalized amino-acid rates.\n\n• -y, The output file of raw amino-acid rates.\n\nRate4Site normalizes rates by converting them into z-scores. The z-scores are written to HRH1_norm_rates.txt. Rate4site also outputs the raw (unnormalized) scores in HRH1_orig_rates.txt. We advise you to use raw scores and to normalize them by the average score in the sequence, as discussed in protocol 1 step 5. Note that Rate4Site also outputs a new tree file TheTree.txt and an empty rates file r4s.res. These files are not needed for further analysis.\n\nFor further downstream processing, the Rate4Site output file needs to be converted to a CSV file. The following command will extract the site’s position, amino acid, and Rate4Site score (see Note 3).\n\n\n\nArguments above correspond to the following:\n\n• The Rate4Site output file.\n\n• -o, The output CSV file name.\n\nBy default, the Rate4Site software will output rates for the sites in the first sequence of the alignment file. That is, a gap in the first sequence will not be assigned a rate, even though a rate is inferred for every site in the alignment. To circumvent losing information outputted from Rate4Site, we suggest finding the sequence in the alignment with least amount of gaps and using it as the reference sequence for the output. The reference sequence for Rate4Site can be specified with the option -a sequence_ID, where sequence_ID is the name of the sequence in a FASTA file provided for rate inference.\n\n6. The file runFEL.bf implements fixed-effect likelihood (FEL) inference without synonymous rate variation, which is sometimes referred to as a one-rate FEL model. The model infers one dN value per site and one dS value per the entire sequence21. The one-rate FEL model has been found to infer more accurate dN/dS values than other HyPhy methods22.\n\n7. For reasons that are beyond the scope of this paper, it turns out that the specific matrix choice has little effect on the final rates, as long as rates are normalized relative to their means as we do here. The underlying reason for this insensitivity to matrix choice is that the available matrices were all derived by pooling data from many sites in many proteins (see e.g.31), and this pooling yields matrices that are close to uninformative34,35.\n\n8. The residue numbers in PDB files are not strictly sequential or numeric. If multiple residues share the same numeric value, they will be distinguished by a single letter insertion code (e.g. 53A or 53B)36. These insertion codes appear when there are several homologous proteins with crystal structures. Generally, each new structure retains the numbering of the earliest crystalized structure to preserve the alignment among structures of homologous proteins. If the new structure contains deletions relative to the original structure, the PDB file will skip residue numbers. If the new structure contains insertions, the PDB file will have residue numbers with insertion codes.\n\nAll information required to reproduce the analysis is provided at https://github.com/clauswilke/proteinER. Version 1.0 of this code is archived at https://doi.org/10.5281/zenodo.100594237.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by National Science Foundation Cooperative (agreement no. DBI-0939454; BEACON Center), National Institutes of Health (grant R01 GM088344), and Army Research Office (grant W911NF-12-1-0390).\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\nKimura M, Ohta T: On some principles governing molecular evolution. Proc Natl Acad Sci U S A. 1974; 71(7): 2848–2852. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPerutz MF, Kendrew JC, Watson HC: Structure and function of haemoglobin: II. Some relations between polypeptide chain configuration and amino acid sequence. J Mol Biol. 1965; 13(3): 669–678. Publisher Full Text\n\nEchave J, Spielman SJ, Wilke CO: Causes of evolutionary rate variation among protein sites. Nat Rev Genet. 2016; 17(2): 109–121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDean AM, Neuhauser C, Grenier E, et al.: The pattern of amino acid replacements in alpha/beta-barrels. Mol Biol Evol. 2002; 19(11): 1846–1864. PubMed Abstract | Publisher Full Text\n\nKimura M, Ohta T: Mutation and evolution at the molecular level. Genetics. 1973; 73(Suppl 73): 19–35. PubMed Abstract\n\nHuang YW, Chang CM, Lee CW, et al.: The conservation profile of a protein bears the imprint of the molecule that is evolutionarily coupled to the protein. Proteins. 2015; 83(8): 1407–1413. PubMed Abstract | Publisher Full Text\n\nMintseris J, Weng Z: Structure, function, and evolution of transient and obligate protein-protein interactions. Proc Natl Acad Sci U S A. 2005; 102(31): 10930–10935. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim PM, Lu LJ, Xia Y, et al.: Relating three-dimensional structures to protein networks provides evolutionary insights. Science. 2006; 314(5807): 1938–1941. PubMed Abstract | Publisher Full Text\n\nFranzosa EA, Xia Y: Structural determinants of protein evolution are context-sensitive at the residue level. Mol Biol Evol. 2009; 26(10): 2387–2395. PubMed Abstract | Publisher Full Text\n\nJack BR, Meyer AG, Echave J, et al.: Functional sites induce long-range evolutionary constraints in enzymes. PLoS Biol. 2016; 14(5): e1002452. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMirny LA, Shakhnovich EI: Universally conserved positions in protein folds: reading evolutionary signals about stability, folding kinetics and function. J Mol Biol. 1999; 291(1): 177–196. PubMed Abstract | Publisher Full Text\n\nZhou T, Drummond DA, Wilke CO: Contact density affects protein evolutionary rate from bacteria to animals. J Mol Evol. 2008; 66(4): 395–404. PubMed Abstract | Publisher Full Text\n\nRamsey DC, Scherrer MP, Zhou T, et al.: The relationship between relative solvent accessibility and evolutionary rate in protein evolution. Genetics. 2011; 188(2): 479–488. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScherrer MP, Meyer AG, Wilke CO: Modeling coding-sequence evolution within the context of residue solvent accessibility. BMC Evol Biol. 2012; 12: 179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShahmoradi A, Sydykova DK, Spielman SJ, et al.: Predicting evolutionary site variability from structure in viral proteins: buriedness, packing, flexibility, and design. J Mol Evol. 2014; 79(3–4): 130–142. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYeh SW, Liu JW, Yu SH, et al.: Site-specific structural constraints on protein sequence evolutionary divergence: local packing density versus solvent exposure. Mol Biol Evol. 2014; 31(1): 135–139. PubMed Abstract | Publisher Full Text\n\nYeh SW, Huang TT, Liu JW, et al.: Local packing density is the main structural determinant of the rate of protein sequence evolution at site level. BioMed Res Int. 2014; 2014: 572409. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang TT, del Valle Marcos ML, Hwang JK, et al.: A mechanistic stress model of protein evolution accounts for site-specific evolutionary rates and their relationship with packing density and flexibility. BMC Evol Biol. 2014; 14: 78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarcos ML, Echave J: Too packed to change: side-chain packing and site-specific substitution rates in protein evolution. PeerJ. 2015; 3: e911. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPupko T, Bell RE, Mayrose I, et al.: Rate4Site: an algorithmic tool for the identification of functional regions in proteins by surface mapping of evolutionary determinants within their homologues. Bioinformatics. 2002; 18(Suppl 1): S71–S77. PubMed Abstract | Publisher Full Text\n\nKosakovsky Pond SL, Frost SD: Not so different after all: a comparison of methods for detecting amino acid sites under selection. Mol Biol Evol. 2005; 22(5): 1208–1222. PubMed Abstract | Publisher Full Text\n\nSpielman SJ, Wan S, Wilke CO: A comparison of one-rate and two-rate inference frameworks for site-specific dN/dS estimation. Genetics. 2016; 204(2): 499–511. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTien MZ, Meyer AG, Sydykova DK, et al.: Maximum allowed solvent accessibilites of residues in proteins. PLoS One. 2013; 8(11): e80635. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPond SL, Frost SD, Muse SV: HyPhy: hypothesis testing using phylogenies. Bioinformatics. 2005; 21(5): 676–679. PubMed Abstract | Publisher Full Text\n\nKatoh K, Standley DM: MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30(4): 772–780. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStamatakis A: RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9): 1312–1313. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKabsch W, Sander C: Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983; 22(12): 2577–2637. PubMed Abstract | Publisher Full Text\n\nJoosten RP, te Beek TA, Krieger E, et al.: A series of PDB related databases for everyday needs. Nucleic Acids Res. 2011; 39(Database issue): D411–D419. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCock PJ, Antao T, Chang JT, et al.: Biopython: freely available python tools for computational molecular biology and bioinformatics. Bioinformatics. 2009; 25(11): 1422–1423. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpielman SJ, Wilke CO: Membrane environment imposes unique selection pressures on transmembrane domains of G protein-coupled receptors. J Mol Evol. 2013; 76(3): 172–182. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLe SQ, Gascuel O: An improved general amino acid replacement matrix. Mol Biol Evol. 2008; 25(7): 1307–1320. PubMed Abstract | Publisher Full Text\n\nStamatakis A: Phylogenetic models of rate heterogeneity: a high performance computing perspective. In Proc. of IPDPS2006. 2006. Publisher Full Text\n\nYeh SW, Liu JW, Yu SH, et al.: Site-specific structural constraints on protein sequence evolutionary divergence: local packing density versus solvent exposure. Mol Biol Evol. 2014; 31(1): 135–139. PubMed Abstract | Publisher Full Text\n\nGoldstein RA, Pollock DD: The tangled bank of amino acids. Protein Sci. 2016; 25(7): 1354–1362. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEchave J, Wilke CO: Biophysical Models of Protein Evolution: Understanding the Patterns of Evolutionary Sequence Divergence. Ann Rev Biophys. 2017; 46: 85–103. PubMed Abstract | Publisher Full Text\n\nProtein Data Bank Contents Guide: Atomic Coordinate Entry Format Description. wwPDB, 2012; Version 3.30. Reference Source\n\nSydykova DK, Jack BR, Spielman SJ, et al.: github.com/clauswilke/proteinER: v1.0, first complete release. Zenodo. 2017. Data Source"
}
|
[
{
"id": "27048",
"date": "02 Nov 2017",
"name": "Ugo Bastolla",
"expertise": [
"Reviewer Expertise Computational biology of proteins: Folding stability",
"evolution",
"protein dynamics with elastic network models. Theoretical ecology."
],
"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 structural properties of a protein site in the native state of the protein are known to strongly influence its evolutionary rate, according to research of which the corresponding author has been a prominent exponent. This finding gives useful insights on how natural selection targets the structural properties of proteins.\nThis paper presents computational protocols for measuring site-specific substitution rates in protein families, either at the amino-acid or at the codon level, and mapping them to the site-specific structural descriptors that have been found to influence the rates most strongly, such as relative solvent accessibility and weighted contact number. All the necessary software is clearly illustrated and all the relevant input and output files are made available to researchers willing to repeat the exercise, to whom this report will be for sure useful.\nHowever, I think that the paper falls a bit short in motivating why it is interesting to perform this computation for a protein family of interest, besides recovering the known correlations between evolutionary rates and structural descriptors. Secondly, different methods and software exist to compute substitution rates. The authors show that, for the protein family that they choose, rates at the amino-acid and at the nucleotide level obtained with the same software yield almost identical results. How do results compare using different software (Rate4site versus HyPhy)? How do the authors interpret the sites with estimated dN/dS>1, which is considered an indication of positive selection, in the context of the protein structure?\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "3378",
"date": "09 Feb 2018",
"name": "Claus Wilke",
"role": "Author Response",
"response": "Dear Dr. Ugo Bastolla,We thank you for your helpful comments and suggestions. We now cite the new LEISR paper that demonstrates that Rate4Site and HyPhy’s method LEISR produce similar rates. We reference this paper in the note about Rate4Site."
}
]
},
{
"id": "27042",
"date": "06 Nov 2017",
"name": "Yu Xia",
"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 provides a complete and detailed recipe for calculating evolutionary rates and structural features of residues in proteins, and for correlating site-specific evolutionary rates with structural features. Such analysis is important for better understanding of protein structure-evolution relationships at the residue level. This paper describes all the computational steps in detail including the required computer programs and their installation, provides scripts to run the different programs and to process the results, and introduces a repository with all the scripts. This paper very clearly explains the steps needed to fully perform such analyses. The computational pipeline provided is expected to become a useful resource to the scientific community. This paper could potentially benefit from addressing a few minor issues described below.\n1. While the paper aims to describe a complete analysis, the initial step of how to properly collect protein sequences for subsequent evolutionary analyses is not described. For the sake of completeness and to help interested readers to perform such analyses from scratch, it will be beneficial to at least mention this step (and preferably provide a more detailed description).\n2. A full and automated procedure for analyzing relative amino acid rates already exists with the ConSurf server. At the same time, the second protocol described in this paper can be especially useful for site-specific codon rate calculations, which is not included in ConSurf. It will be beneficial to provide more context regarding other available procedures and how the procedure described in this paper complements these other available procedures.\n3. It will be beneficial to include the scripts for normalizing the inferred relative rates as well as for finding the sequence in the MSA with smallest number of gaps. These steps are described in the paper but it appears that the corresponding scripts are not included.\n4. The file name of the output for Step 1 of Protocol 4 (pdb_id.map.csv) is not described in the text.\n5. It is unclear how the order of the lines in the output pdb_id.rates_features.csv is decided. Because this is the final output of the entire analysis, it will be useful to organize this output as clearly as possible.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "3377",
"date": "09 Feb 2018",
"name": "Claus Wilke",
"role": "Author Response",
"response": "Dear Drs. Yu Xia and Avital Sharir-Ivry,We thank you for your helpful comments and suggestions. We have added an additional step about collecting homologous sequences to both protocols 1 and 2. We also added a paragraph in the Conclusions about other methods to infer site-wise evolutionary rates. We also now discuss the differences between the different methods. See also our response to Dr. Mario dos Reis’s comments. Further, we now provide the name of the output file for step 1 of protocol 4, and we have added a note to step 6 of protocol 1 to state that our script to plot the figure contains code to normalize inferred rates. When we worked on version 1 of our publication, HyPhy’s LEISR method was not yet published. Both Rate4Site and LEISR infer similar rates. We encourage users to use LEISR over Rate4Site to circumvent little issues Rate4Site has, such as not outputting site-wise rates for all site in the alignment.We have refrained from organizing the order of rows in the output file pdb_id.rates_features.csv. There are two columns by which we could order: the site position of the protein structure or the site position of the alignment. Because the sequence of the PDB structure is typically different from the ones in the alignment, the two will not align perfectly to each other. This introduces gaps in either of the two or both sequences. It is, therefore, unclear what the order of lines with gaps should be."
}
]
},
{
"id": "27041",
"date": "07 Nov 2017",
"name": "David D Pollock",
"expertise": [
"Reviewer Expertise Molecular evolution",
"evolutionary genomics",
"protein structure function and evolution",
"mathematical 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 other two reviewers did a good job, such that I am going to be more philosophical about my review by focusing on two questions: Is this the best format for this kind of information, and am I (or the other two reviewers) the right person to review it? Other than a brief introduction and conclusion, the manuscript is mostly a detailed recipe of a specific pathway to assess correlations between evolutionary rates and some crystal structural features (when such information is available). This might be useful to a range of users such as undergraduates and beginning graduate students for whom it would serve as a gateway to thinking about these kinds of questions. The described pipeline is not a substantive modification to existing methods or an innovative application to to new scientific questions, but rather is a tool designed to facilitate performance of experiments in a particular way. I would hope that more advanced students would quickly want to modify the experiments and tools that make up this pipeline, asking questions about the assumptions that the included tools make, and asking questions about alternative methods of analysis. Should one use only a maximum likelihood “best tree”, or should one consider a bootstrap or Bayesian posterior distribution? How does model choice interact with inference of site-specific rates? What assumptions were made in the alignment, and do those assumptions and errors in the alignment interact with the phylogenetic and rates inferences? In many ways, rather than a fixed and published document, the author’s pipeline might be better implemented in the context of an interactive and perhaps continuously modified lesson plan to teach students about the underlying issues, with more direction and encouragement to substitute in different methods and models. This leads to the question of whether I, or other professors, are the right people to review this. I trust Wilke and his laboratory members to have tested and made this pipeline function, and I don’t have time in the context of reviewing a paper to install and implement and test their scripts. But what they and I and other professors or advanced students are going to be bad at anyway is figuring out how hard it is and how it can go wrong in the hands of beginning students. So my suggestion is that it is beginning students who should be reviewing this pipeline, trying to implement it and trying to break it (or breaking it without trying). That would seem to me to be a more meaningful review.\n\nAs an aside, publishing answers to simplistic bubble questions seems inane to me, especially without the choice to opt out, and that is the context in which they were filled.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\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? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "3376",
"date": "09 Feb 2018",
"name": "Claus Wilke",
"role": "Author Response",
"response": "Dear Dr. David Pollock,We thank you for your comments and ideas. For specific feedback, you referred us to the other reviewers, and we have addressed their comments and modified our paper where appropriate."
}
]
},
{
"id": "27040",
"date": "17 Nov 2017",
"name": "Mario dos Reis",
"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 Sydykova et al. is written in tutorial style, with the aim of demonstrating how to use a series of software tools to calculate site-specific rates of evolution in proteins and protein-coding sequences. In brief, the user starts with a molecular sequence alignment as input data, and then proceeds to estimate a phylogenetic tree, site-specific evolutionary rates, and other statistics related to protein structure and evolution.\nThe things I look for in a good tutorial are clarity and brevity. This manuscript achieves both. Brevity is important as, in my opinion, a tutorial user should not be bogged down with lengthy discussions of theory, caveats, comparisons to other methods, etc. However, what I do expect from a good tutorial is a set of key sentences pointing out to the user where to find the appropriate theory papers, simulation studies, and work by others that may have developed similar methods.\nI have the following recommendations:\nPerhaps at the beginning of the protocol section, the authors could clearly state to the user what level of knowledge is expected from them. Does the user need to know how to use the command line? R? Must they have some basic experience of using phylogenetic software? This is important, specially in this case, where the user base may come from a very mixed background (physical sciences, computer science, chemistry or biology).\n\nThe authors may consider adding a section at the end of the paper with general recommendation for this type of analysis, and where they point the user towards additional literature methods. By putting this section at the end, the user is not bogged down with this information in the middle of the tutorial. For example, Yu Xia recommends mentioning the ConSurf server. This appears a reasonable request. I think the authors should also consider mentioning here other related methods, as this will be useful information to the user. For example, the works by Rodrigue et al.1-2 and Tamuri et al.3 could be mentioned in reference to population genetic ways to measure site-specific rates and dN/dS. It may also be desirable to cite some of the more modern theoretical works on this, for example Halpern and Bruno4, Jones et al.5, etc. These are just suggestions. Similarly, Ugo Bastolla suggests adding some mention of what the results would like under other software, for example, in comparison with Rate4site. Although an extensive analysis of this topic may seen out of the scope of the tutorial, I think a set of references from the literature would be in order.\n\nFinally, I think the tutorial would benefit with the inclusion of a few additional figures. For example, the authors may want to consider adding a histogram of dN/dS vs. protein site, and perhaps also add screenshots of key program outputs. A well-illustrated tutorial is more appealing to students. Screenshots of program output are essential as it allows the user to check whether they are indeed obtaining the correct results. Perhaps also include tables summarising result values for key parameters.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "3375",
"date": "09 Feb 2018",
"name": "Claus Wilke",
"role": "Author Response",
"response": "Dear Dr. Mario dos Reis,We thank you for your helpful comments and suggestions. We have added additional notes, where appropriate, to indicate other theory papers, methods, and simulations a user might find useful. In the note about Rate4Site, we have added a brief paragraph that discusses the differences between Rate4Site and HyPhy’s LEISR method. We have also added a paragraph in the Conclusions describing other relevant works. In that paragraph, we have cited the papers you suggested.In the protocols section, we have added some information about the level of skill needed to run our analysis. We have replaced “Throughout, we assume that we are working on a UNIX-like command line interface” with the following: “Throughout, we assume that we are working on UNIX-like command line interface. We recommend that a user is comfortable with command execution and syntax, which includes flags, arguments, and directories. No prior knowledge of any of the listed software is essential. For python and R scripts, we provide detailed description for each script’s function. As such, it is not strictly necessary that a user knows python or R to execute our pipeline. However, if more detailed understanding of the custom scripts is desired, a user should be familiar with python and R.”We also wanted to address your comment to include screenshots with program outputs. We have provided the git repository for this purpose. The git repository contains all input and output files the pipeline has generated. We feel that this is sufficient for the user to check their execution of our pipeline. A user can compare the output files we have provided to the output files he or she generated. We also have provided detailed descriptions of the output files for each step. We believe those should be enough to assess if the software has run and whether it has run correctly."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1845
|
https://f1000research.com/articles/7-171/v1
|
09 Feb 18
|
{
"type": "Opinion Article",
"title": "Matchmaking in Bioinformatics",
"authors": [
"Ewy Mathé",
"Ben Busby",
"Helen Piontkivska",
"Team of Developers",
"Ben Busby",
"Helen Piontkivska"
],
"abstract": "Ever return from a meeting feeling elated by all those exciting talks, yet unsure how all those presented glamorous and/or exciting tools can be useful in your research? Or do you have a great piece of software you want to share, yet only a handful of people visited your poster? We have all been there, and that is why we organized the Matchmaking for Computational and Experimental Biologists Session at the latest ISCB/GLBIO’2017 meeting in Chicago (May 15-17, 2017). The session exemplifies a novel approach, mimicking “matchmaking”, to encouraging communication, making connections and fostering collaborations between computational and non-computational biologists. More specifically, the session facilitates face-to-face communication between researchers with similar or differing research interests, which we feel are critical for promoting productive discussions and collaborations. To accomplish this, three short scheduled talks were delivered, focusing on RNA-seq, integration of clinical and genomic data, and chromatin accessibility analyses. Next, small-table developer-led discussions, modeled after speed-dating, enabled each developer (including the speakers) to introduce a specific tool and to engage potential users or other developers around the table. Notably, we asked the audience whether any other tool developers would want to showcase their tool and we thus added four developers as moderators of these small-table discussions. Given the positive feedback from the tool developers, we feel that this type of session is an effective approach for promoting valuable scientific discussion, and is particularly helpful in the context of conferences where the number of participants and activities could hamper such interactions.",
"keywords": [
"computational biology",
"bioinformatics",
"biology",
"speed dating",
"collaboration",
"matchmaking"
],
"content": "Introduction\n\nInformal, face-to-face communication between participants is a vital piece of a scientific conference, just as important, if not more important, as formal activities such as keynote addresses and formal talk sessions (Saunders et al., 2009). However, as the number of attendees grows, coupled with multiple research plenary sessions that often run concurrently (a regular feature of conferences in bioinformatics and other fields), the time available for individual contact with conference participants drops dramatically. Further, for new attendees, it can be difficult to navigate abstracts, posters, and talks to figure out the key people to engage with. While social media interactions via Twitter and other similar social media platforms (Biospace, 2009; Saunders et al., 2009; Tachibana, 2014), or dedicated online communities (Budd et al., 2015) have their own role in facilitating conversations, face-to-face conversations remain invaluable (Budd et al., 2015; Fuller et al., 2013).\n\nEven for those of us who conduct most of our interactions online, face-to-face interactions can solidify relationships, spur novel ideas and research directions, further promote collaborations, and speed up project implementations. Moreover, it is critical for tool developers to carefully assess the utility (e.g., is their tool addressing an unmet need?) and usability (e.g., how streamlined and simple to use is the tool?) of their software. In the open source community especially, these aspects often tend to be overlooked or there are not enough resources to implement them (Al-Ageel et al., 2015). To assess utility and usability, developers need to establish a network of potential users, and need to get direct input from those users, including whether the software is sufficiently user-friendly to enable the user to focus on hypothesis- generation and testing in lieu of tool tweaking (Kumar & Dudley, 2007). These interactions can be key in addressing specific needs and/or offering a vision and/or a wish-list for further development (e.g., addition of new features).\n\nFor users, another source for finding tools of interest is via formal publication (peer-reviewed). However, this avenue is relatively slow, and is occasionally inefficient and/or insufficient in reaching a broader audience. Pre-peer-reviewed venues, e.g. bioRxiv, Figshare (Huang & Lapp, 2013), Zenodo, are trying to address this gap. Nonetheless, often the user’s needs are not well articulated (or even formalized), and that’s where face-to-face discussions can be much more helpful.\n\n\nDeveloping novel tools that are usable to the wider community\n\nWhile many tools are being developed, a relatively fewer number are routinely used by the larger biological and medical community. In fact, the average lifespan of an open-source Bioinformatics software is often relatively short, frequently limited by the transient nature of work contracts of developers, many of whom are post-docs or graduate students (Ahmed et al., 2014). Through literature mining, a recent study reported that many database and software resources are mentioned only in the Bioinformatics literature, while only a fraction of the tools are mentioned in the biological and medical literature (Duck et al., 2016). Specifically, only 5% of the resources account for 47% of total usage and over 70% of the resources are only mentioned once in the literature (Duck et al., 2016). This striking bias suggests that while the Bioinformatics community promotes development of novel software, the biological and medical communities only access a fraction of what is available. It is quite reasonable to think that these latter communities only access software that are intuitive and usable, and that perhaps usability could trump accuracy of analyses performed (Huang & Lapp, 2013; Pavelin et al., 2012).\n\nOf note, two broad approaches could be undertaken when developing Bioinformatics software. First, developers can develop a tool that solves a known issue in the field (e.g. RNA-seq analysis, omics integration), and then can seek users and data to test their approach and software. With this approach, it may be difficult for their tool to have visibility outside the Bioinformatics community, since 1) it is less likely that non-computational users are aware of your tool, and/or 2) your tool may not be user-friendly to non-computational users, and/or 3) your tool may not be readily adaptable to answer specific biological questions, or to accommodate a specific dataset format. With the surge in volume and variety of data types in high-dimensional biological data, adaptability is becoming more and more of a challenge. For example, a novel tool that integrates high-throughput omics data that is collected in the same samples may not be readily adaptable to data that is collected in different samples. Second, developers can develop Bioinformatics solutions that try to answer a specific biological or biomedical question, and can then broaden the utility of the tool by developing an associated software. Because the emphasis is on the biology, the resources and time available to generalize the software to other datasets are oftentimes lacking. This often results in a gap between a goal of developing a user-friendly software and ‘on the ground’ availability of low-level computational infrastructure (which is frequently scripting based) (Kumar & Dudley, 2007). We believe that this gap could be narrowed by further communication between biologists, computational biologists, clinicians, and users.\n\nImportantly, it is worth noting that developers of widely adopted tools have often formally assessed utility and usability, enabling them to broadly disseminate their software. Guidelines for adopting a user-centered design when developing software have been formally assessed (Ahmed et al., 2014; Pavelin et al., 2012), and if applied, could yield highly usable software and could facilitate novel scientific discoveries. These formal assessments typically require face-to-face meetings between developers and users, and require developers to understand what problems need to be addressed, and how users will interact with the software. While taking these aspects into consideration prior to developing software can be lengthy, the resulting software will surely be useful and used by a wider community. Creating useful software can also provide a lot of job satisfaction to developers.\n\n\nReproducibility and software in biomedical research\n\nCreating sustainable computational solutions can have a strong, positive impact on reproducibility of analysis results. With the recent rising concerns in reproducibility of scientific research (Clark, 2017; Editorial, 2016), it is critically important to ensure that the analysis of large biological datasets is reproducible. More often than not, it is difficult to reproduce graphs and results in publications, and this is largely due to incomplete methods (e.g. parameters missing for statistical methods used, manual curation of results, etc.), and the use of in-house scripts or software. Methods for increasing computational reproducibility include reporting code and documentation used, and automating research analyses (Piccolo & Frampton, 2016). Computational frameworks, including but not limited to Taverna (Hull et al., 2006; Wolstencroft et al., 2013), Galaxy (Goecks et al., 2010) and R markdown (Baumer & Udwin, 2015; Baumer et al., 2014), facilitate reproducibility and oftentimes create reports that record all parameters used during the analysis. In addition to usability, developers can thus take into account the importance of reproducibility and in talking with users, better understand which parameters and analysis information needs to be reported.\n\n\nISCB/GLBIO’2017 conference\n\nHosted by the University of Illinois at Chicago, International Society for Computational Biology affiliate meeting, Great Lakes Bioinformatics Conference (ISCB/GLBIO’2017), has attracted a record 347 registered participants, including ~60% graduate students and post-docs with a broad range of computational and experimental expertise. First convened in 2006 as the Ohio Collaborative Conferences on Bioinformatics (OCCBIO), since 2010 joining forces with ISCB, over the years GLBIO has established itself as an ideal conference for showcasing the latest developments in analysis approaches and tools that span many different fields, and is a venue that attracts both computational and bench scientists. As we are all aware though, communication between computational and bench scientists can be challenging, particularly during the initial introduction stages when the overlap in mutual interests is not clear, and the matchmaking session that we ran is a first attempt at promoting such communication.\n\nAs Dr. Funmi Olopade (University of Chicago) mentioned in her keynote speech, clinicians, basic researchers, and computational biologists must better communicate to advance research. This sentiment is generally shared in the biological sciences, yet each field has its own language and culture. Encouraging communication across different fields via a common theme (e.g. RNA-seq analysis, chromatin accessibility analysis, etc.) is precisely what our matchmaking session aimed to accomplish.\n\n\nMatchmaking for Computational and Experimental Biologists Session\n\nThe Matchmaking Session (Matchmaking@GLBIO session, #GenoMatch, #CompMatchBio) attracted over 40 participants, including 9 tool developers. The session, held at 8 am on the first day of the conference, kicked off with three short introductory talks, followed by multiple rounds of 4–5 minutes long small-table discussions led by individual tool developers, and then open discussion. Short (10 minutes each) introductory talks by Drs. Ben Busby (NCBI), James Chen (OSU) and Ewy Mathé (OSU) covered available NCBI tools for RNA-seq analyses, approaches in integration of clinical and genomic data, and chromatin accessibility analyses, respectively. The purpose of these talks was to introduce broad topics that pose current, relevant topics and challenges in computational biology, and to present developers that are working on tools to address these challenges.\n\nNext, small-table developer-led discussions were modeled after speed-dating. In each round, participants joined a table, listened to the developer’s pitch, asked questions, discovered common interests, exchanged contact information, and then moved on to the next table. Because these small-table discussions were timed (4–5 minutes each), each participant had an opportunity to visit all the tables. At the end of “speed-dating” small-table discussions, participants still had 30–45 minutes available for further discussion. At this point, most users had identified developers that were presenting tools useful to them, and thus had the opportunity to discuss their own data needs in more detail.\n\n\nTools and representatives of tool developing teams (developers)\n\nWhen planning the session, three main themes for tools were considered: analysis of RNA-seq, chromatin accessibility, and omics/multi-dimensional integration. A total 5 representatives of tool developer teams (Ben Busby, James Chen, Ewy Mathé, Arunima Srivastava, and Rick Farouni) were pre-registered for the session. However, at the start of the session, we asked whether other developers were interested in sharing their tool and, thus, were able to include 4 more developers. This near doubling of presenter-participants with a last minute change shows the level of interest that already exists in the community for sharing their tools. Table 1 lists all tools that were presented, with relevant reference information.\n\nEach developer had a chance to showcase their tool and to further discuss its usage with potential collaborators during the “speed-dating” small-table discussions.\n\n\nFeedback from presenters\n\nAs a follow-up to the session, developers were asked about their experiences afterwards, whether they had the sufficient opportunity to discuss their tools with potential users, and whether the subsequent interactions have occurred during the remainder of the conference. The majority of developers have found the session to be quite useful, in part due to the opportunity to network with many potential users, during the session or afterwards. Having time constraints for the matchmaking rounds have also allowed the session participants to quickly determine whether or not they were interested in learning about a specific tool in depth, and if the latter, move on to another tool.\n\nOf note, the 5-minutes rounds were sufficiently long to accommodate exchange of contact information for subsequent follow-up, which occurred later during the conference functions and/or after the conference was over. The primary aim of the session was to provide face-to-face interactions between users and developers, and to provide ample opportunities for contact information exchange. Per feedback we received afterwards, this aim appeared to be successfully accomplished.\n\n\nFuture matchmaking sessions\n\nWe plan to build up and expand on our successful experiment during GLBIO’2017, to offer similar matchmaking sessions at other ISCB venues, such as ISMB in Chicago in 2018, and GLBIO in 2019 Madison, WI. We have already run an informal session at the ISCB DC-RSG summer workshop in College Park, Maryland (July 12, 2017) with lightweight planning, enormous popularity, and a very positive response.\n\nIn the future, to broaden participation and improve participants’ experience, presenters/developers will be given the opportunity to prepare and present 1-2 slides about their tools at the beginning, similar to ‘flash talks’. This format will help developers to find other developers interested in solving similar problems. In our first matchmaking session, developers had little time to interact with each other during the session. In the future this flash talks-format could replace the broad, introductory topic-focused talks given at the beginning of the matchmaking session. Notably, though, these flash talks will not replace the small-table matchmaking portion of the session, which we believe is critical to foster communication between users and developers.\n\nLastly, it is important to note that this session was scheduled at 8 am at the start of the conference. While we had anticipated lower participation due to this scheduling (assuming that a number of participants would chose to come in later on the first day to avoid traveling the Sunday prior to the start of the conference), the timing of the session turned out to be advantageous. Indeed, having a discussion-promoting, interactive session as a start of the conference is a great way to engage participants and “break the ice” for subsequent interactions during the conference. Further, it provides ample time for attendees to find each other later during the conference and formalize potential collaborations.\n\n\nConclusions\n\nThe short-talk/“speed-dating” format provided a platform in which participants could learn about as many tools as possible in a short period of time, while making valuable connections across fields. Given the fast moving pace of Bioinformatics and the rapid advances across clinical/experimental biology fields, it is critical to keep the communication lines open between the communities. Our matchmaking session opened these communication lines by facilitating informal face-to-face interactions.\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\nBen Busby’s work on this project was supported by the Intramural Research Program of the National Institutes of Health (NIH)/National Library of Medicine (NLM)/NCBI.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank all the co-organizers and GLBIO participants for their contributions to the success of our session, and Belinda Hanson and Dr. Tandy Warnow for their help in developing the session. We would also like to thank developers that presented at the Matchmaking for Computational and Experimental Biologists Session, including Basel Abu-Jamous, Steven Kelly, Serdar Bozdag, James Chen, Alexandre Drouin, Rick Farouni, Lorena Pantano, and Arunima Srivastava.\n\n\nReferences\n\nAhmed Z, Zeeshan S, Dandekar T: Developing sustainable software solutions for bioinformatics by the “Butterfly” paradigm [version 1; referees: 2 approved with reservations]. F1000Res. 2014; 3: 71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAl-Ageel N, Al-Wabil A, Badr G, et al.: Human Factors in the Design and Evaluation of Bioinformatics Tools. Procedia Manufacturing. 2015; 3: 2003–2010. Publisher Full Text\n\nBaumer B, Cetinkaya-Rundel M, Bray A, et al.: R Markdown: Integrating a reproducible analysis tool into introductory statistics. arXiv preprint arXiv: 1402.1894. 2014. Reference Source\n\nBaumer B, Udwin D: R markdown. Wiley Interdisciplinary Reviews: Computational Statistics. 2015; 7(3): 167–177. Publisher Full Text\n\nBiospace: Why Social Networking Is Important for a Bioinformatics Developer. 2009; Retrieved on August 16, 2017. Reference Source\n\nBudd A, Corpas M, Brazas MD, et al.: A quick guide for building a successful bioinformatics community. PLoS Comput Biol. 2015; 11(2): e1003972. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClark TD: Science, lies and video-taped experiments. Nature. 2017; 542(7640): 139. PubMed Abstract | Publisher Full Text\n\nDuck G, Nenadic G, Filannino M, et al.: A Survey of Bioinformatics Database and Software Usage through Mining the Literature. PLoS One. 2016; 11(6): e0157989. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEditorial: Reality check on reproducibility. Nature. 2016; 533(7604): 437. PubMed Abstract | Publisher Full Text\n\nFuller JC, Khoueiry P, Dinkel H, et al.: Biggest challenges in bioinformatics. EMBO Rep. 2013; 14(4): 302–304. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoecks J, Nekrutenko A, Taylor J, et al.: Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010; 11(8): R86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang D, Lapp H: Software Engineering as Instrumentation for the Long Tail of Scientific Software. Figshare. 2013. Publisher Full Text\n\nHull D, Wolstencroft K, Stevens R, et al.: Taverna: a tool for building and running workflows of services. Nucleic Acids Res. 2006; 34(Web Server issue): W729–732. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumar S, Dudley J: Bioinformatics software for biologists in the genomics era. Bioinformatics. 2007; 23(14): 1713–7. PubMed Abstract | Publisher Full Text\n\nPavelin K, Cham JA, de Matos P, et al.: Bioinformatics meets user-centred design: a perspective. PLoS Comput Biol. 2012; 8(7): e1002554. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPiccolo SR, Frampton MB: Tools and techniques for computational reproducibility. Gigascience. 2016; 5(1): 30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaunders N, Beltrão P, Jensen L, et al.: Microblogging the ISMB: a new approach to conference reporting. PLoS Comput Biol. 2009; 5(1): e1000263. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTachibana C: A scientist's guide to social media. Science. 2014; 343(6174): 1032–1035. Publisher Full Text\n\nWolstencroft K, Haines R, Fellows D, et al.: The Taverna workflow suite: designing and executing workflows of Web Services on the desktop, web or in the cloud. Nucleic Acids Res. 2013; 41(Web Server issue): W557–561. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "30750",
"date": "26 Feb 2018",
"name": "Robert M. Blumenthal",
"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 summarizes experience and justification for a rapid developer-user meeting format, which was first implemented at the 2017 GLBIO-ISCB meeting. It is a useful summary and may stimulate others to try similar approaches. My comments are entirely on ways to clarify the writing, because the content is fine as is.\n\nP3 Para2: The heavy use of “e.g.” is distracting and unnecessary – suggest just leaving it out.\n\nP3 Para4: Top line, “fewer” should be “smaller”; 3rd line delete “an”; 4th line delete “often” (since you use the word “average”). Next column (same para), add a comma after “total usage”; near bottom of para replace “are” with “is” before “intuitive”.\n\nP3 Para5: 3rd line delete “e.g.”; 7th line, replace “since” with “for one or more of the following reasons:” and delete both occurrences of “and/or”; 12 lines from bottom replace “Second” with “In the second broad approach”; and 3 lines below that remove “an”.\n\nP3 Para7: replace “analysis” with “analytic”.\n\nP3 and throughout: Is it F1000Research style to capitalize “Bioinformatics” with every use?\n\nP4 Para2: top line add “the” before “International”; 3rd line remove “has”; 7th line add “and” before “since”.\n\nP4 Para3: remove “e.g.”\n\nP4 Para7: remove “have” before “also allowed the session”.\n\nP5 Para2: unclear what is meant by “lightweight” – please clarify.\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": "30752",
"date": "27 Mar 2018",
"name": "Guenter Tusch",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors discuss a unique experimental session that they initiated at the ISCB/GLBIO’2017 meeting in Chicago (May 15-17, 2017) in form of an opinion article. Based on the model of speed dating they teamed up interested parties with developers of bioinformatics software in order to connect those developers with potential users. The paper consists of roughly four parts. It starts with a brief introduction including a plea for the importance of face-to-face interactions at conferences and a description of options researchers have today to find the appropriate computer software to support their research projects. The next part describes the software development process for bioinformatics software as seen by the authors. They claim that there are basically two approaches that I would call developer-centric and research-centric. The first one seems to assume that developers develop a more general tool, but have difficulties to connect to potential users, while the other one apparently results in a program that suffers from a lack of general usability due to a too narrow focus on a specific biological problem. I’m not quite sure if this a based on the NCBI experience of one of the co-authors and how tools like Bioconductor would fit in here. There is certainly a problem for small scale software projects like those developed for one particular research project. That could be clarified with specific examples possibly from participants in the matchmaking session.\n\nThe next part of the paper describes and discusses the session at the conference, emphasizing the focus on face-to-face communication, setup, implementation, feedback of presenters, and future plans. I thought of this as the essential part of the paper. Finally, as a third part the authors included a table with the description of the presented software and contact information.\n\nI believe that this experimental session is a very interesting and important approach, and the authors make very valuable points about the setup and implementation of the session and the outcomes especially for younger researcher. From the success the authors had I hope there will be more sessions like that at future conferences. Of course, the conclusions need to be preliminary based on only one session, however the authors make strong points that many results can be generalized. While the introductory and the second part feel like a unit and are the only ones referred to in the conclusion, I feel like the second part and the table are not really integrated enough. They deal with important aspects of the topic, if the topic is not a mere description of the whereabouts of the session and the conclusions drawn by the authors. If the purpose is purely informative, it could be largely reduced, but if it is part of the argument – as I assume, see also my comments above -, it should be included in the discussion and conclusion, and that would strengthen the message. I also agree with the comments of the other reviewer.\n\nIn conclusion, the authors discuss a very interesting and promising approach to improve communication and personal connections especially for younger researcher in the bioinformatics community.\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/7-171
|
https://f1000research.com/articles/7-169/v1
|
09 Feb 18
|
{
"type": "Case Report",
"title": "Case Report: a rare case of PASH mimicking a lactational adenoma",
"authors": [
"Sam Hoggard",
"Natalie Meara",
"Sara Bundred",
"Natalie Meara",
"Sara Bundred"
],
"abstract": "Pseudoangiomatous stromal hyperplasia (PASH) is a rare, benign breast lesion that is usually discovered as an incidental finding in breast biopsies. We present a case report of a twenty three-year-old female who presented with a large central mass in the left breast 24 weeks into her pregnancy. An ultrasound-guided core biopsy was performed which was reported as a lactational adenoma and due to the significant size of the mass it was excised as a suspected giant lactational adenoma. The ultrasound appearance was of a mass with well-defined superficial and radial margins with multiple large gentle lobulations, and a thin echogenic pseudocapsule pointing towards a benign diagnosis. Multiple prominent internal vessels were visualised on doppler imaging; PASH lesions do not commonly have internal blood flow which therefore pointed away from the diagnosis in this case. It is likely the imaging features were confounded by the pregnant state. Macroscopically, the lesion consisted of a large lobulated red mass measuring 170 x 170 x 75 mm and weighing 838 g with a central area containing yellow cream-like material measuring 25 x 20 mm. Microscopically, the breast tissue showed prominent gynaecomastoid-like lobules with intervening oedematous stroma showing florid pseudoangiomatous hyperplasia. There was prominent but only patchy lactational change. PASH can often be an incidental finding and is commonly found in combination with other diagnoses. It is therefore possible for PASH to be overlooked in biopsy specimens, as in this case, and it is important to analyse the breast stroma carefully for evidence of PASH, even if the biopsy contains an alternative lesion that could account for the mass seen clinically. We feel this case highlights the potential for PASH to be overlooked in core biopsy specimens when a concurrent lesion is present and therefore not appropriately treated.",
"keywords": [
"Pseudoangiomatous stromal hyperplasia",
"pash",
"lactational adenoma",
"breast"
],
"content": "Case report\n\nPseudoangiomatous stromal hyperplasia (PASH) is a rare, benign breast lesion that is usually discovered as an incidental finding in breast biopsies1. Despite usually being an incidental finding, there have been many reports in the literature of PASH presenting clinically as a discrete mass, and several reports of it presenting as bilateral diffuse breast enlargement2. We present a case report of PASH presenting as a large clinically-evident breast mass in a pregnant woman which was initially diagnosed as a lactational adenoma on the core biopsy. The subsequent excision specimen demonstrated a large, discrete tumour composed of PASH with a central galactocele, as well as prominent but only patchy lactational change. Following this, PASH was retrospectively identified as being present in the initial core biopsy.\n\nClinically, PASH has a wide range of signs and symptoms, ranging from presenting as a large breast mass, to being an incidental finding. Indeed, non-tumour-forming PASH has been reported to be an incidental microscopic finding in 23% of breast biopsies3. Tumour-forming PASH occurs predominantly in premenopausal women and usually presents clinically as a palpable, mobile, firm, painless, intra-mammary mass4. Cases of tumour-forming PASH have also been described in post-menopausal women, men, adolescents and even in the paediatric age group5. In our case, the patient was a twenty three-year-old female who presented with a large central mass in the left breast 24 weeks into her pregnancy (Figure 1). She first noticed the mass at around week 10 of her pregnancy but it then massively increased in size around week 24. She had a history of a biopsy-proven lactational adenoma six years previously during her first pregnancy. An ultrasound-guided core biopsy was performed which was reported as a lactational adenoma, B2, and due to the significant size of the mass it was excised in week 25 of her pregnancy as a suspected giant lactational adenoma.\n\nThe patient first noticed a left breast mass in week 10 of her pregnancy but it then massively increased in size around week 24.\n\nSeveral publications of case series6–9 show the typical ultrasound appearance of PASH to be a well-circumscribed oval or round mass encompassed by a thin echogenic capsule and usually indistinguishable from a fibroadenoma, with size ranging from 3 to 70 mm. Internal echotexture is most commonly hypoechoic, sometimes isoechoic, and occasionally complex containing cysts or vascular channels. In this case, the 140 mm mass within a breast hypertrophied through pregnancy changes presented a challenge for imaging with conventional ultrasound due to the extremely large size. The subcutaneous breast tissue was oedematous and there was skin thickening present up to 5.5 mm in more dependant areas of the breast. The mass had well-defined superficial and radial margins with multiple large gentle lobulations, and a thin echogenic pseudocapsule pointing towards a benign diagnosis (Figure 2). The internal echotexture was predominantly bland, homogenous and hypoechoic. Multiple prominent internal vessels were visualised on doppler imaging; PASH lesions do not commonly have internal blood flow however, which therefore pointed away from the diagnosis in this case. It is likely the imaging features were confounded by the pregnant state. The differential diagnosis based on the ultrasound appearances included a fibroadenoma, a lactational adenoma and a phyllodes tumour.\n\nThe mass had a thin echogenic pseudocapsule pointing towards a benign diagnosis and the internal echotexture was predominantly bland, homogenous and hypoechoic.\n\nThe classical histological appearance of PASH is inter-anastomosing channels lined by slender spindle cells in the interlobular breast stroma10. As the name suggests, ultrastructural observations have determined that these channels are not true vascular spaces and the distinction from an angiosarcoma is obviously important11. Immunohistochemistry shows the cells are positive for vimentin and CD3412 but are negative for CD31. Macroscopically, the lesion in this case consisted of a large lobulated red mass measuring 170 x 170 x 75 mm and weighing 838 g with a central area containing yellow cream-like material measuring 25 x 20 mm (Figure 3). Histologically the breast tissue showed prominent gynaecomastoid-like lobules with intervening oedematous stroma showing florid pseudoangiomatous hyperplasia (Figure 4). There was prominent but only patchy lactational change. The central cyst was lined by a single layer of apocrine-like cells in keeping with a galactocoele. There was no evidence of atypical hyperplasia, in-situ or invasive carcinoma. The diagnosis was given as pseudoangiomatous stromal hyperplasia with prominent but only patchy lactational change and a galactocoele. Similar changes were seen in the initial core biopsy of this lesion and on review of the lesion cored during the first pregnancy.\n\nThe lesion consisted of a large lobulated red mass measuring 170 x 170 x 75 mm and weighing 838 g with a central area containing yellow cream-like material.\n\nThe breast tissue showed prominent gynaecomastoid-like lobules, with intervening oedematous stroma showing florid pseudoangiomatous hyperplasia and prominent but only patchy lactational change.\n\nPASH can often be an incidental finding and is commonly found in combination with other diagnoses. It is therefore possible for PASH to be overlooked in biopsy specimens and it is important to analyse the breast stroma carefully for evidence of PASH, even if the biopsy contains an alternative lesion that could account for the mass seen clinically. As highlighted well by this case, the presence of lactational change on its own in the core biopsy could have accounted for the mass lesion identified clinically and radiologically. This would have been further reinforced by the history of a previous biopsy-proven lactational adenoma during her last pregnancy. This highlights the potential for PASH to be overlooked in core biopsy specimens and therefore not appropriately treated. Fortunately, in this case the mass was so large and impacting upon the patient that it was excised anyway. PASH discovered incidentally does not require any specific additional treatment; however, tumour-forming PASH should be treated with local surgical excision and has an excellent prognosis with minimal risk of recurrence if adequately surgically excised according to some reports in the literature13. Other papers have reported recurrence rates between 15% and 22% after surgical excision14. The long-term prognosis for this patient would be expected to be excellent but we do not yet have long-term follow up data available. At her initial follow-up clinic appointment no problems were reported and her pregnancy was progressing as planned.\n\nIn summary we present a case report of tumour-forming PASH mimicking a giant lactational adenoma in a young pregnant patient. We feel it highlights the importance of looking carefully for PASH in core biopsy specimens, even if a concurrent lesion is also present which would account for the mass identified radiologically.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient according to the Declaration of Helsinki.",
"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\nPowell CM, Cranor ML, Rosen PP: Pseudoangiomatous stromal hyperplasia (PASH). A mammary stromal tumor with myofibroblastic differentiation. Am J Surg Pathol. 1995; 19(3): 270–7. PubMed Abstract | Publisher Full Text\n\nKrawczyk N, Fehm T, Ruckhäberle E, et al.: Bilateral Diffuse Pseudoangiomatous Stromal Hyperplasia (PASH) Causing Gigantomastia in a 33-Year-Old Pregnant Woman: Case Report. Breast Care (Basel). 2016; 11(5): 356–358. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIbrahim RE, Sciotto CG, Weidner N: Pseudoangiomatous hyperplasia of mammary stroma. Some observations regarding its clinicopathologic spectrum. Cancer. 1989; 63(6): 1154–60. PubMed Abstract | Publisher Full Text\n\nBowman E, Oprea G, Okoli J, et al.: Pseudoangiomatous Stromal Hyperplasia (PASH) of the Breast: A Series of 24 Patients. Breast J. 2012; 18(3): 242–247. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShehata BM, Fishman I, Collings MH, et al.: Pseudoangiomatous stromal hyperplasia of the breast in pediatric patients: an underrecognized entity. Pediatr Dev Pathol. 2009; 12(6): 450–4. PubMed Abstract | Publisher Full Text\n\nRaj SD, Sahani VG, Adrada BE, et al.: Pseudoangiomatous Stromal Hyperplasia of the Breast: Multimodality Review With Pathologic Correlation. Curr Probl Diagn Radiol. 2017; 46(2): 130–135. PubMed Abstract | Publisher Full Text\n\nHargaden GC, Yeh ED, Georgian-Smith D, et al.: Analysis of the Mammographic and Sonographic Features of Pseudoangiomatous Stromal Hyperplasia. AJR Am J Roentgenol. 2008; 191(2): 359–363. PubMed Abstract | Publisher Full Text\n\nJones KN, Glazebrook KN, Reynolds C: Pseudoangiomatous Stromal Hyperplasia: Imaging Findings with Pathologic and Clinical Correlation. AJR Am J Roentgenol. 2010; 195(4): 1036–1042. PubMed Abstract | Publisher Full Text\n\nStavros AT: Breast Ultrasound. First edition. Philadelphia. Lippincott Williams & Wilkins. 2004. Reference Source.\n\nSolomou E, Kraniotis P, Patriarcheas G: A case of a giant pseudoangiomatous stromal hyperplasia of the breast: magnetic resonance imaging findings. Rare Tumors. 2012; 4(2): e23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVuitch MF, Rosen PP, Erlandson RA: Pseudoangiomatous hyperplasia of mammary stroma. Hum Pathol. 1986; 17(2): 185–91. PubMed Abstract | Publisher Full Text\n\nFisher CJ, Hanby AM, Robinson L, et al.: Mammary hamartoma--a review of 35 cases. Histopathology. 1992; 20(2): 99–106. PubMed Abstract | Publisher Full Text\n\nVirk RK, Khan A: Pseudoangiomatous Stromal Hyperplasia: An Overview. Arch Pathol Lab Med. 2010; 134(7): 1070–1074. PubMed Abstract\n\nJung BK, Nahm JH, Lew DH, et al.: Treatment of Pseudoangiomatous Stromal Hyperplasia of the Breast: Implant-Based Reconstruction with a Vascularized Dermal Sling. Arch Plast Surg. 2015;42(5): 630–634. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "31465",
"date": "21 Mar 2018",
"name": "Malcolm M. Hayes",
"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 case that merits publication as a case report.\nI think the diagnosis is sound based on the description and single photograph.\nHowever, ideally, one would like to see a low-power picture of the lesion and a representative photograph of the epithelial elements exhibiting lactational and hyperplastic changes.\nAlso, the case is of interest because this lesion has a history of rapid growth - most likely induced by the pregnancy - a fact that should be stressed in the discussion to make radiologists and pathologists aware of the apparently alarming clinical evolution that may be encountered in PASH during the altered hormonal environment of pregnancy.\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": "39185",
"date": "15 Oct 2018",
"name": "Josko Bezic",
"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 PASH of the breast tissue is well known histological finding, firstly described by Vuitch et al. more than 30 years ago. However, some examples of PASH with unusual clinical, imaging, or pathohistological features may be worthy of publication. Here, Hoggard et al. presented the case of so-called nodular or tumorous PASH, which was associated with pregnancy and which on US guided core biopsy simulated a lactational adenoma.\nThe case is well presented on the clinical and radiological ground, but it is less persuasive in the histological part, mainly due to the only one presented micro-photograph of the lesion.\nMajor revision points:\n1.There is a strong evidence that the PASH is caused by hormonal stimulation of mammary myofibroblasts, with particular role of progesterone, which well explains the rapid enlargement of the lesion during pregnancy in the presented case. Curiously, I could not find any observation about the role of hormones in pathogenesis of PASH in the manuscript.\n2.The presented micro-photograph of the lesion (Figure 4) is insufficient in several ways:\nOnly the PASH is visible in the figure, and therefore the figure legend with the observations about visible gynecomastoid-like changes, oedematous stroma and lactational changes are completely inappropriate. This may be an unintentional error related to the article technical editing after the submission. Related to the previous point, it would be appropriate to show micro-photographs with visible lactational changes, as well as with visible gynecomastoid hyperplasia and central galactocoele. One low power micro-photograph of the nodule (or at least of the part of the nodule) with visible distribution of the PASH and epithelial elements is crucial for the correct pathohistological diagnosis (see the major revision point 3).\n\n3.The major differential diagnosis here is mammary hamartoma, and again without any notification about it in the text. The PASH is common finding in the stroma of the breast hamartoma, as well as gynecomastoid-like changes. The lactational changes are expected in the epithelium of hamartoma during pregnancy, as well as rapid enlargement of pre-existing small hamartomatous nodule. The authors must stress out in the discussion why that presented nodule better fits to the diagnosis of nodular PASH than to the diagnosis of mammary hamartoma.\nMinor revision point: 1.The abstract should be more concise.\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? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
},
{
"id": "39184",
"date": "19 Oct 2018",
"name": "Modupeola Omotara Samaila",
"expertise": [
"Reviewer Expertise Anatomic Pathologist with specialty in breast and gynaecologic pathology"
],
"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\nA well written manuscript with detailed ultrasound/radiological findings.\n\nThe abstract should only present the case without explanatory comments. “PASH lesions do not commonly have internal blood flow which pointed away from this diagnosis. It is likely the imaging study was confounded by the pregnant state” should be deleted from the abstract.\n\nIf there is no histological evidence of lactational adenoma in the initial core biopsy which was reviewed to PASH with lactational change and galactocele after surgical excision, then there is no mimicry in this case. There is a difference between lactational changes and lactational adenoma which is neoplastic. Could the pregnancy have induced PASH in view of the hormonal influences which have been well documented in literature. This should be clarified appropriately in the manuscript. How this pitfall of initial misdiagnosis can be avoided will be useful to clinicians if included in the discussion.\n\nPerhaps, the title should have reflected the radiological (ultrasound) potential of wrong diagnosis of PASH in pregnancy.\n\nFigure 3 should have been bisected to show the central galactocele and figure 4 shows PASH only with no areas of gynaecomastoid like changes.\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? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-169
|
https://f1000research.com/articles/7-168/v1
|
09 Feb 18
|
{
"type": "Review",
"title": "Missing the egocentric spatial reference: a blank on the map",
"authors": [
"Maria Concetta Miniaci",
"Elvira De Leonibus",
"Elvira De Leonibus"
],
"abstract": "Egocentric (self-centered) and allocentric (viewpoint independent) representations of space are essential for spatial navigation and wayfinding. Deficits in spatial memory come with age-related cognitive decline, are marked in mild cognitive impairment (MCI) and Alzheimer’s disease (AD), and are associated with cognitive deficits in autism. In most of these disorders, a change in the brain areas engaged in the spatial reference system processing has been documented. However, the spatial memory deficits observed during physiological and pathological aging are quite different. While patients with AD and MCI have a general spatial navigation impairment in both allocentric and egocentric strategies, healthy older adults are particularly limited in the allocentric navigation, but they can still count on egocentric navigation strategy to solve spatial tasks. Therefore, specific navigational tests should be considered for differential diagnosis between healthy and pathological aging conditions. Finally, more research is still needed to better understand the spatial abilities of autistic individuals.",
"keywords": [
"egocentric navigation",
"posterior parietal cortex",
"striatum",
"aging",
"Alzheimer’s disease",
"autism."
],
"content": "Introduction\n\nSuccessful navigation is a fundamental cognitive function that is crucial for survival. Humans, like other animals, must learn about the layout of their environments to return home, or move between known locations. The spatial reference frame used to locate positions and directions in a complex environment are commonly divided into two main systems: egocentric (subject-centered) and allocentric (object-centered)1,2. During navigation, information in both egocentric and allocentric reference frames can be integrated to provide a coherent representation of the environment and proper orientation.\n\nEgocentric frames define spatial positions using the body, or a specific part of the body (head or trunk) as a point of reference (Figure 1)3–5. Allocentric reference frame codes the position of the target relative to surrounding visual cues or landmarks and their spatial relationships, independently of the observer's current position6. Such information can be used to build a “cognitive map”, a sort of internal representation of the environment7.\n\nTypically, the egocentric strategy relies upon kinesthetic and vestibular sensory information as well as motor command efferent copies, and optokinetic flow information as the subject moves past surrounding objects8. Egocentric learning ability has been demonstrated in paradigms in which animals must repeat a sequence of responses or movements toward a target, e.g., turning to the left, or reaching a fixed goal from a fixed starting point. Egocentric spatial orientation is likely to occur in the absence of external allothetic visual cues—e.g., in total darkness or in water maze with high and opaque walls that do not allow the use of extramaze cues. However, learning occurs relatively slowly in the water maze in darkness9,10, due to cumulative errors of the vestibular system, which are not corrected by visual inputs11. Egocentric navigation is also associated with path integration, a strategy of spatial navigation that uses vestibular and proprioceptive cues generated during locomotion to keep track of position relative to a known starting point12. Path integration or dead reckoning is, for example, used by foraging animals, such as desert ants and honeybees, to search for food along novel routes extending hundreds of meters13. After reaching the site, those animals show an impressive level of accuracy at returning back to the nest using only the idiothetic cues generated by their movements. Environmental allothetic cues can be used to correct heading direction. Path integration can also be assessed in human subjects walking blindfolded to a previously seen target or asking them to estimate the distance and direction traveled while walking blindfolded14.\n\n\nNeuronal basis of egocentric navigation\n\nBehavioral and brain imaging studies indicate that egocentric and allocentric strategies are mediated by a different cognitive-neural system, with some degree of overlapping. Egocentric-based navigation relies mainly on the posterior parietal cortex (PPC; Figure 2) whereas hippocampus and parahippocampal cortex are crucial for allocentric navigation15,16. The activity in the caudated nucleus has also been associated with egocentric tasks requiring a response strategy, such as following a well-learned route in a virtual city17.\n\nThe importance of PPC in egocentric navigation has been demonstrated by lesion studies in rodents and humans as well as by functional MRI (fMRI) studies in healthy subjects. Rogers and Kesner18 have found that rats with selective lesions of the right posterior parietal cortex were impaired in the acquisition of the egocentric version of the water maze task, in the absence of extramaze cues, but had no difficulty in learning the allocentric version of the task, when extramaze cues were available. Rats with PPC lesions are also strongly impaired in path integration-based navigation under conditions in which visual inputs are irrelevant19. For example, they are unable to locate the escape platform in a water maze when tested in complete darkness or to return directly to the refuge after searching for a randomly located food reward on a circular arena surrounded by a curtain, suggesting that the PPC plays an important role in idiothetic processing during path integration. Furthermore, single-unit recording data have demonstrated that a substantial fraction of cells in PPC of rats responds selectively to head orientation or to locomotion such as forward motion to the left or right20–22.\n\nThe involvement of PPC in egocentric spatial processing has also been observed in humans as they navigated to goal destinations in the virtual simulation of London23. Analysis of the fMRI data revealed that the activity in bilateral PPC was significantly correlated with the egocentric direction to the goal. Additional evidence in support of a role of PPC in egocentric based spatial navigation comes from patients with PPC damage following cerebral infarction or hemorrhage in the right hemisphere24,25. Lesion of PPC can result in a contralateral neglect i.e. inattention to objects and space on the body opposite to the brain damage. Patients with neglect lose the ability to represent the location of objects (and landmarks) with respect to the self, though the landmarks are still recognizable. Indeed, they can get lost in their homes as they ignore left-hand turns or doorway. Neglect of representational space has been clearly described by Bisiach and Luzzatti26. They asked patients with left-sided neglect to recall the layout of the Piazza del Duomo in Milan, a place very familiar to them. When imagining themselves facing the cathedral in the middle of the piazza, such patients neglected the left side of the piazza, recalling only the right side; however, when asked to view the piazza from the opposite end, they recalled the previously neglected buildings. An impairment of egocentric processing of remote spatial memory has also been observed in patients with focal lesion of PPC without clinical signs of neglect25. In particular, when examined on mental navigation tasks in a very familiar environment (i.e., downtown Toronto), such patients were impaired on tasks that involved egocentric mental views of places such as describing an efficient route from one specific Toronto landmark to another. However, they showed preserved allocentric knowledge of the same environment, as they were able to indicate the location of landmarks on a map of Toronto or draw a map of the streets of Toronto.\n\nAnatomical studies support the important role of PPC in processing egocentric spatial information27. Indeed, PPC receives and integrates signals from cortical areas representing all main sensory modalities, such as vision, audition, proprioceptive, and vestibular. In addition, it is connected to cortical regions involved in goal-directed behavior such as the orbitofrontal, and medial prefrontal cortices and the striatum28. The parietal cortex is also reciprocally connected with the hippocampal formation via the retrosplenial and the postrhinal cortex29.\n\nPotegal30 was the first to suggest that the striatal complex is involved in processing egocentric information; he based his hypothesis on the observation that patients with Huntington’s disease, which are affected by neurodegeneration of striatal neurons, were impaired when using the egocentric frame of reference.\n\nThe striatal complex is part of the basal ganglia nuclei, which receive information from the whole cortical mantle (Figure 2). It was initially thought that these nuclei were selectively involved in the movement control, but more recent evidence clearly indicates that the basal ganglia, and in particular the striatum, also have important cognitive functions, including learning and memory31–36. Neuroanatomical evidence suggests that the striatal complex can be further distinguished in at least three different sub-regions including the dorsolateral and the dorsomedial striatum, corresponding to the putamen and the caudate in non-human and human primates, respectively, and the nucleus accumbens, which is located in the ventral striatum37. The role of these different components in spatial navigation seems to follow lateromedial and dorsoventral gradient. The dorsolateral striatum is involved in all forms of egocentric navigation strategies with little involvement in allocentric navigation32,33,38–43. These findings are in line with neuroanatomical evidence showing that this part of the striatal complex receives dense projections from the PPC and the dorsolateral prefrontal cortex, as well as from the vestibular system, which is crucial for egocentric spatial information processing44–46. Both the medial and the ventral striatum are involved in egocentric spatial processing. However, these brain regions seem to be recruited in egocentric spatial tasks only when complex visuospatial tasks require a flexible updating system, like locating the position of a displaced object based on its position related to the subject body axes33. The medio-ventral striatum receives a direct input from the hippocampal formation47,48 and has been particularly implicated in allocentric spatial information processing33,49.\n\nThe role of the dorsal striatum in egocentric spatial navigation has been attributed to its specific contribution to the formation of stimulus-response based habit learning50; however, more recent evidence in rodents using one-trial learning tasks, such as the identification of a displaced object based on the subject position, suggests that habit learning and egocentric spatial information processing are two distinct functions involving the dorsal striatum32,33. This dissociation has been confirmed using an outcome evaluation protocol; habit responses are, by definition, controlled by the releasing stimulus, and not by the outcome. For example, if the subject always takes the same route from home to work, it may happen that the subject continues to take the same route for days even after the goal has been changed (such as moving to another place). This situation has been modeled in rodent studies, applying an outcome devaluation protocol after a maze learning. Egocentric strategy is initially used to navigate in relatively constant environments in a flexible manner35; with increasing practice, the egocentric responses become habitual and resistant to changes in the environment and in the outcome value. Indeed, animals continued to turn right (or left) after learning even if their starting position has been changed by 180° or the food they obtained when turning right induced malaise. Furthermore, in the same study it was shown that deactivation of the dorsomedial striatum and the dorsolateral striatum mimicked and abolished, respectively, the effects of practice in the shift from egocentric to habit learning35.\n\nFunctional neuroimaging studies in humans have demonstrated that the cerebellum is activated during virtual navigation tasks that can be solved using either sequence-based strategy or place-based strategy51. However, the two strategies appeared to involve different cerebellar lobules and cortical areas: place-based responses revealed the activation of the left cerebellar lobule VIIA Crus I, the right hippocampus and the medial parietal cortex, whereas for sequence-based response, right lobule VIIA Crus I, left hippocampus and medial prefrontal cortex medial were coactivated and functionally connected.\n\nExperiments carried out in transgenic mouse model with selective inhibition of protein kinase C inhibitor (PKCI) in Purkinje cells, have brought new insights regarding the role of cerebellum in spatial navigation52,53. L7-PKCI transgenic mice lack parallel fiber–Purkinje cell long-term depression (LTD), which is considered the main neural correlate of cerebellar motor learning54–56. Interestingly, L7-PKCI mice exhibited disrupted hippocampal place cell properties when forced to use self-motion cues, such as navigation in total darkness. Consistently with their hippocampal place cell alteration, L7-PKCI mice were unable to navigate efficiently toward a goal in the dark, suggesting that the cerebellum may shape hippocampal activity during spatial navigation. The main hypothesis is that the cerebellum contributes to the formation of spatial representation by combining vestibular with proprioceptive inputs to generate appropriate information about body location in space57–59. The cerebellum appears to also be connected with the posterior parietal cortex, that encodes self-motion and acceleration22. The interaction between PPC and cerebellar lobules has been shown in humans and monkeys and is believed to play an important role in planning and execution of navigation behavior.\n\n\nEgocentric navigation in childhood and aging\n\nStudies on spatial abilities in childhood suggest that infants use mainly an egocentric reference system and that a gradual ability to use allocentric representations is acquired with age. Egocentric representation is considered a more elementary mean of representing the location of an object than an allocentric representation and, therefore, is already present early60,61. Acredolo and Evans62 found that 6-month-old infants, trained to anticipate the appearance of a face at either their left or right and then turned around, continued to look in the same egocentric direction after they were rotated to the opposite side of the room. The correct use of allocentric representations develops progressively with increasing age. A further study carried out in children of 5, 7, and 10-year-olds has demonstrated that a high percentage of children spontaneously used the egocentric strategy on the virtual reality adaptation of the StarMaze task, reproducing the same sequence of body turns during the probe trials as during the training trials63. The allocentric strategy, based on landmark guidance, was spontaneously used to solve the task in a few percentage of children at 7 and 10 years, but not at 5 years of age. However, when the allocentric strategy was imposed, the 5- year-olds were able to use allocentric behavior but their performance was below that of the 10-year-olds.\n\nThe shift from egocentric to allocentric strategy preference with increasing age is consistent with the heterogeneity in developmental trajectories of subcortical and cortical structures; decrease of subcortical and increase of cortical gray matter volumes have been described after age 764.\n\nAfter 60 years of age, there is a clear decline in spatial navigation abilities65. Such decline is often related to functional changes of the posteromedial, the medial-temporal and the frontal areas66,67. Older adults present spatial navigation deficits particularly in allocentric navigation, being less effective in forming and using the cognitive maps when examined in both virtual and real-life versions of the human Morris maze68,69. On the other hand, the egocentric spatial navigation and learning is preserved in older age. The same results have been confirmed in studies testing older adults (71–84 years old) in a real-space human analog of the Morris Water Maze70.\n\nBehavioral studies on rodents have confirmed the effect of aging on the spatial task performance requiring the use of allocentric strategy71. For example, aged mice were impaired in the water maze task in which they must remember the location of a submerged platform in relation to a series of extra-maze or distal cues, with the position of the starting point changing from trial to trial72. However, the aged mice performed correctly the egocentric spatial task in a T-maze, remembering a sequence of movements (such as rotation to the left). These results suggest that aging can affect the allocentric and egocentric processing of spatial information in a different way.\n\nThese findings are in line with an increased sensitivity of brain regions of the medial temporal lobe, including the hippocampus, to the insult of aging and associated neurodegenerative disorders such as AD.\n\n\nEgocentric spatial deficits in developmental disorders: the case of autism\n\nBased on neuropsychological literature showing that the use egocentric develops before allocentric spatial strategies73, altered egocentric spatial ability can be considered an early sign of neuro- and psychiatric developmental disorders. This issue is generally little explored. However, some studies have addressed egocentric spatial information processing in Autism spectrum disorder (ASD). ASD is a multifactorial neurodevelopmental disorder that can also be secondary to genetic syndromes, including DiGeorge syndrome, Optiz syndrome or Fragile X syndrome74. ASD manifests with impaired eye contact and communication skills, as well as impaired social interaction75. Furthermore, children with autism often present stereotyped behavior and self-injury. Visuo-spatial abilities in subjects with autism have been investigated. Although in one study, no impairment was observed76, more recent studies suggested that autistic adults show impaired performance in egocentric spatial tasks, when they have to use their body as reference frame77,78.\n\nThese deficits have never been directly associated to altered brain activation patterns. Interestingly, structural alteration of subcortical regions has been widely identified in autistic children. For example, the largest brain morphometric study in ASD to date shows that autism is associated with smaller subcortical volumes of the pallidum and putamen. Cerebellar cell loss and atrophy has also revealed in ASD children79–83.\n\nImpaired subcortical functions in animal models of autism have been mainly associated to social behavior impairment, with little attention to the possible effects of spatial information processing. Behavioral investigation of MID1-null mouse model of Opitz G/BBB syndrome (OS), a genetic disorder characterized by mental retardation and brain abnormalities such as hypoplasia of the anterior cerebellar vermis, has demonstrated that these mice can promptly learn to locate the food in a T-maze if the position of the food remains constant relatively to extra-maze cues (allocentric strategy), but they are impaired if the position of the food is anchored to the animal position in the maze (egocentric strategy)84. Impairment of egocentric spatial information processing has been also observed in children with Williams syndrome (WS), a neurodevelopmental disorder resulting from a hemizygous microdeletion of ~25 genes on chromosome 7q11.2373,85.\n\nEgocentric spatial abilities impairment correlates with altered social performance in ASD. Therefore, understanding the nature and the neuronal correlates of egocentric spatial abilities in ASD might be relevant to shed light on the nature of other cognitive and social deficits characterizing ASD, as evidenced by studies linking spatial and social cognitive abilities in ASD86,87.\n\n\nEgocentric impairments deficits in Alzheimer's disease\n\nAlzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangle accompanied by neuronal loss88. In the early phase of the disease, most patients show cognitive impairments, such as memory deficits, language impairment, poor judgment and decision making89–91. As the disease progresses, reasoning, visual-spatial skills, and sensory processing become increasingly affected. The cognitive impairment in patients with AD is closely associated with the progressive degeneration of medial temporal lobe, including hippocampal formation and entorhinal cortex, frontal and parietal cortex and the basal forebrain92,93. Spatial navigation deficits, such as becoming disoriented or feeling lost in familiar places, represent the first sign of AD, and have, therefore, an important clinical utility for the early detection of AD94,95. Several studies in recent years have focused on spatial deficits in patients with amnestic Mild Cognitive Impairment (aMCI), which is considered an intermediate stage between the expected cognitive deficit of normal aging and the serious decline of dementia. Patients with amnestic MCI, especially those with the hippocampal type of amnestic syndrome, are at very high risk of AD. The conversion rate from MCI to dementia range is about 10% per year; in contrast to conversion rate from healthy elderly subjects to dementia which is about 1–2 percent per year96. Interestingly, MCI patients are impaired in both allocentric and egocentric navigation, as documented by a series of study on virtual as well as real navigation. In one of the first studies, Hort and collaborators97 tested a group of AD and aMCI patients on a goal-directed navigation task within a circular arena. The goal was invisible and could be identified either by its position relative to the starting position (egocentric subtest) or relative to cues on the wall (allocentric subtest). Each subtest began with an overhead view of the arena on a computer monitor. Both AD and aMCI were similarly impaired in all types of experiments, suggesting that spatial navigation impairment is not limited to AD, but begins to decline earlier in MCI. To understand the neural mechanisms behind spatial navigation of AD, Weniger and collaborators98 examined a group of patients with aMCI on two virtual reality tasks, a virtual park containing several landmarks (allocentric memory) and a landmark-free virtual maze (egocentric memory) while undergoing an fMRI scan. They showed that aMCI patients were significantly impaired on both virtual reality tests, getting more frequently lost than controls and being unable to find a navigation strategy to learn the maze. Importantly, the allocentric and egocentric memory of aMCI patients were associated with size reduction of hippocampus, precuneus, and right-sided parietal cortex. These results have been recently confirmed by Boccia et al.99. In their studies, patients with aMCI were challenged on an intensive learning paradigm, during which participants were shown two paths using either video clips or maps of a real city. The video clips were used to encourage participants to develop an egocentric representation of the city, whereas the maps were used to encourage them to develop an allocentric representation. After learning, they were asked to retrieve each of these paths, using an allocentric or egocentric frame of reference, while undergoing fMRI scan. Behavioral results indicated that aMCI showed a reduction in the rate of learning on the allocentric task. In addition, patients with aMCI showed a selective impairment in retrieving topographical memory using an egocentric perspective. Imaging data suggest that this general decline was correlated with hypoactivation of the brain areas generally involved in spatial navigation, e.g. medial temporal lobe structures, angular gyrus, and orbitofrontal cortex.\n\nFurthermore, there is a strong evidence that patients with mild AD and aMCI are also impaired on path integration task, in which they are required to return to the starting point following an enclosed triangle pathway with a mask over their eyes100. Such deficit appeared to be correlated to a reduced size of hippocampus, entorhinal and parietal cortices.\n\nThese findings suggest that neuropsychological screening designed to assess navigational deficit may represent an important tool for detecting the prodromal symptoms of aMCI and early stage of AD, and therefore, allow appropriate diagnosis and intervention.\n\n\nConclusions\n\nMany interesting points emerge from spatial navigation studies presented in this review. First of all, it appears that, unlike allocentric, the egocentric spatial strategy is quite preserved under physiological aging. But there are conditions such as AD in which egocentric ability is also impaired and this determines devastating effects on spatial navigation so that individual get lost even in a familiar environment. Secondly, the egocentric spatial deficits in brain disorders such as AD are often correlated with a reduced size of brain areas involved in egocentric information processing. Overall, these findings suggest that neuropsychological screening designed to assess navigational deficit may represent an important approach for detecting neurological and psychiatric disease progression, thus allowing an appropriate intervention.",
"appendix": "Competing interests\n\n\n\nThere are no actual or potential conflicts of interest.\n\n\nGrant information\n\nThis study was supported by Bank of Italy grant #744460/13.\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\nO’Keefe J, Nadel L: The Hippocampus as a Cognitive Map. 1978; Oxford University Press. Reference Source\n\nBurgess N, Maguire EA, O'Keefe J: The human hippocampus and spatial and episodic memory. Neuron. 2002; 35(4): 625–641. PubMed Abstract | Publisher Full Text\n\nNadel L, Hardt O: The spatial brain. Neuropsychology. 2004; 18(3): 473–476. PubMed Abstract | Publisher Full Text\n\nBurgess N: Spatial memory: how egocentric and allocentric combine. Trends Cogn Sci. 2006; 10(12): 551–557. 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}
|
[
{
"id": "30711",
"date": "26 Feb 2018",
"name": "Sathyanarayanan V. Puthanveettil",
"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 beautifully written article by Miniaci and De Leonibus. Importantly this article is timely because more and more people are reported to have dementia and that both industry and academic research is struggling to produce therapeutics to cure dementia or for efficient long-term management of dementia. In this review article, Miniaci and De Leonibus discuss forms of spatial representations (allocentric and egocentric) in the brain and how these representations are altered in normal aging as well as in autism and Alzheimer's disease. The review is well written and images included are highly useful to the readers.\n\nFew suggestions:\nRevise the abstract so that it reads smoothly. If the cartoons drawn (including brain images) are modified versions of previous images, earlier work should be cited directly in the figure legend. If it is adapted from a database, authors need to include links to the image. It is not clear how the authors are trying to distinguish the egocentric/allocentric spatial deficits in mCI versus Alzheimer's disease. Conclusion section should also need comments on spatial deficits in ASD and comparison with AD.\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": "31042",
"date": "05 Mar 2018",
"name": "Sergio Chieffi",
"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 review, Miniaci and De Leonibus present a concise and balanced overview of the egocentric-based spatial navigation. In the first part, the authors focus on the brain areas mainly involved in egocentric information processing, such as the posterior parietal cortex and the striatum. Then, they review a series of studies showing that the egocentric spatial abilities are impaired in developmental disorders like autism, as well as under pathological aging conditions, such as those of Alzheimer's disease.\nThe authors provide a critical view of the summarized literature leaving also some important questions open. For example, they realize how little attention has been paid to the study of spatial processing in autism and associated brain activation patterns. Indeed, understanding the nature and the neuronal correlates of spatial processing in autism can shed light on the nature of cognitive and social deficits that characterize the autism spectrum disorder. In addition, neuropsychological screening designed to assess navigational deficit may represent an important tool for detecting the prodromal symptoms of amnestic mild cognitive impairment and early stage of Alzheimer’s disease. Overall, the paper is well written and the figures are clearly presented.\nMy recommendation to the authors is to improve the conclusions about the relevance of the egocentric spatial processing and its relationship with cognitive disorders.\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": "30709",
"date": "05 Mar 2018",
"name": "Silvia Middei",
"expertise": [
"Reviewer Expertise Neuroscience",
"Alzheimer disease"
],
"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 review paper on spatial reference abilities under physiological and pathological conditions. The manuscript is well written and clear, also it draws relevant, though in some instances not novel, conclusions. I fully support the publication of this paper, and I only have a couple of minor comments. First, I agree with the authors about the possibility to use spatial tasks for a pre-clinical screening of people at risk for AD. For this reason, I would suggest to also include in the discussion the (few) available studies that investigated the relationship between spatial navigation deficits and markers of AD, including for instance CSF levels of Abeta (see studies from Head and collaborators) or amyloid PET scan. Second, the sentence 'egocentric develops before allocentric spatial strategies' (and hence) 'altered egocentric abilities... can be considered an early sign' looks a bit confounding to me. If so, one would expect that deficits in allocentric spatial strategies may emerge after the ones in egocentric ones in ASD, which is not the case in the studies reported here. The general view of the paragraph is correct, then I would only suggest to re-phrase the first sentence.\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/7-168
|
https://f1000research.com/articles/7-162/v1
|
08 Feb 18
|
{
"type": "Research Article",
"title": "Integrated Biomedical System",
"authors": [
"Darrell O. Ricke",
"James Harper",
"Anna Shcherbina",
"Nelson Chiu",
"Tara Boettcher",
"James Harper",
"Anna Shcherbina",
"Nelson Chiu",
"Tara Boettcher"
],
"abstract": "Background: Capabilities for generating and storing large amounts of data relevant to individual health and performance are rapidly evolving and have the potential to accelerate progress toward quantitative and individualized understanding of many important issues in health and medicine. Recent advances in clinical and laboratory technologies provide increasingly complete and dynamic characterization of individual genomes, gene expression levels for genes, relative abundance of thousands of proteins, population levels for thousands of microbial species, quantitative imaging data, and more – all on the same individual. Personal and wearable electronic devices are increasingly enabling these same individuals to routinely and continuously capture vast amounts of quantitative data including activity, sleep, nutrition, environmental exposures, physiological signals, speech, and neurocognitive performance metrics at unprecedented temporal resolution and scales. While some of the companies offering these measurement technologies have begun to offer systems for integrating and displaying correlated individual data, these are either closed/proprietary platforms that provide limited access to sensor data or have limited scope that focus primarily on one data domain (e.g. steps/calories/activity, genetic data, etc.). Methods: The Integrated Biomedical System is developed as a Ruby on Rails application with a relational database. Results: Data from multiple wearable monitors for activity, sleep, and physiological measurements, phone GPS tracking, individual genomics, air quality monitoring, etc. have been integrated into the Integrated Biomedical System. Conclusions: The Integrated Biomedical System is being developed to demonstrate an adaptable open-source tool for reducing the burden associated with integrating heterogeneous genome, interactome, and exposome data from a constantly evolving landscape of biomedical data generating technologies. The Integrated Biomedical System provides a scalable and modular framework that can be extended to include support for numerous types of analyses and applications at scales ranging from personal users, communities and groups, to potentially large populations.",
"keywords": [
"genome",
"exposome",
"interactome",
"exposure",
"wearable",
"health tracker",
"fitness device",
"fitness tracker",
"sleep",
"heart rate"
],
"content": "Introduction\n\nHuman health and performance is understood to be affected by both nature (genome) and nurture (activities & environment). One notable example of the combined effects of genetics and the environment on health is the identification that the GRIN2A gene significantly modulates risk for developing Parkinson’s disease, but only in heavy coffee-drinkers1. This study provides proof that inclusion of quantitative measures of environmental factors can help identify important genes that would be otherwise missed in GWAS studies that ignore exposures. However, the challenges associated with designing and implementing broad quantitative studies of complex interactions at scales sufficient to achieve sufficient statistical power are considerable.\n\nThere are multiple efforts underway that are making progress toward addressing the challenges of integrating genome, interactome, and exposome2 data to support focused scientific studies. The Institute of Systems Biology’s Hundred Person Wellness Project3 and 100K Project4 are integrating genomics, monitors, and blood sampling to build on the pioneering N-of-one work conducted by Larry Smarr5 and Michael Snyder6,7 to articulate the vision and promise of predictive, preventative, personalized, and participatory (P4) medicine8. Orion Bionetworks9 is combining traits, genetics, and interactome with a focus on brain disorders. Sanchez et al.10 has also proposed exposome informatics integrating the genome, phenome, and exposome. Systems integrating personal sensors and exposome have been developed by Doherty & Oh11 and Nieuwenhuijsen et al.12. Other relevant available resources include PhysioNet13 and MOPED14. The Human Longevity project15 is examining genome, microbiomes, and metabolites of volunteers. Lifestyle affects human microbiomes16,17. While these projects all share the common elements of longitudinal integration of heterogeneous biomedically relevant data, each either focuses on a relatively narrow set of measurements or relies on custom data storage and analysis architectures that do not provide a scalable foundation for larger-scale integration across studies to enable meta-analysis of data from multiple studies.\n\nThe Integrated Biomedical System is being developed as an open source platform for integrating genome, interactome, and exposome data that provides a unifying model to promote more open data sharing and analysis. The software architecture with multi-scale operability design intended to scale from running on a single laptop/workstation as a standalone system with an embedded private local database, to a study platform, to large-scale implementations all using standard scalable web technology stacks.\n\n\nMethods\n\nThe Integrated Biomedical Project description and written consent form (Protocol # 1312006029) was reviewed and approved by the Massachusetts Institute of Technology (MIT) Committee on the Use of Humans as Experimental Subjects (COUHES) for the initial 20 volunteers and the expansion to 40 volunteers. COUHES is the MIT Institutional Review Board (IRB). This project used no recruitment. All volunteers learned about the project from other volunteers, typically by expressing interest in the devices being worn. Upon expressing interest to the principal investigator, the project was fully explained and a written consent form was provided and the project explained with multiple voluntary options. The project principal investigator and co-investigator were primary points of contacts for all volunteers. All volunteers signed the written consent form approved by the MIT COUHES and provided their signed form to the project principal investigator. Volunteers have full choice of all elements of the research project that they elect to participate in or not. Volunteers may elect to have all or any subset of their data removed from the system at any time. Volunteers either elect to opt-in or opt-out of notification of any possible data abnormalities detected.\n\nWritten informed consent was obtained from all volunteers.\n\nExtract, transform, and load (ETL) modules were developed for 23andMe SNPs18 files, SwissProt19 dat file, DrugBank20 XML file, NCBI Gene21 gene file, PharmGKB pathways22, and Protein Data Bank (PDB) protein structures23. After SwissProt sequences and PDB protein structures were loaded, the structure coordinates were mapped to sequence residues with the included lib/utilities/align_pdb.rb tool; this enables the visualization of residues and variants on structures. Interface modules were developed to allow individual or pooled variants to be visualized on protein structures with the integrated Jmol24 structure viewer.\n\nInteractome data included in the pilot collection described herein includes heart rate, interbeat interval (IBI), and electrocardiogram (ECG), skin temperature, skin conductance, galvanic skin response, and respiratory rate. These aggregated data were collected by a diverse collection of commercially available wearable physiological monitoring devices. All volunteers were offered a Basis B1 watch25 and Polar Loop H7 heart rate monitor26. A subset of volunteers are evaluating Hildago Equivital EQ-02-SEM27, Empatica E328, Mio Link29, and Zephyr BioHarness 330 devices. Data logging functionality was not built in for the Polar Loop and Mio Link heart rate monitors, so these data streams were wirelessly synced and stored continuously on co-worn Actigraph Actisleep device. ETL modules were developed for Basis B1 json files31, Actigraph heart rate csv or dat files (including Polar Loop and Mio Link), Empatica E3 zip files, Hidalgo Equivital SEM2 persisted summary csv files, Zephyr BioHarness summary csv files, vocal recordings and associated Matlab .mat files. Data displays include Ruby gems and JavaScript plugins: Google Maps32, jQuery33, lazy_high_charts34, Highstocks35, Data-Drive Documents (D3)36, FullCalendar37, rails3-jquery-autocomplete38, and more. The graphical user interface for “Data Loading” provides the ability to download data from the Basis web site and drag and drop interfaces for easy file uploads for each of the device ETL modules.\n\nActivity and sleep were monitored continuously using wearable and personal electronic devices that used algorithms to process raw data provided by built-in 3-axis accelerometers. Data describing daily nutrition, prescriptions, and over-the-counter medications were collected manually and provided by a subset of volunteers. Devices used by volunteers for continuous data collection included the Fitbit Flex, the Basis B1 watch, Actigraph ActiSleep monitors, basic Actigraph activity monitors GT3X+, Jawbone Up, and smart-phone apps including MyTracks, and Sleep Cycle. ETL modules were developed for Fitbit csv files, Jawbone csv files, Actigraph39 sleep csv files, MyTracks app40 csv files, and Sleep Cycle app41 csv files. Additionally to demonstrate the ability to integrate other publicly available data, modules were developed for integration of EPA AirData (daily and hourly csv files42), and foods43. Graphical user interfaces were developed for entering activities, events, meals, drinks, prescriptions, and over-the-counter medicines. Multiple volunteers submitted oral swab samples for metagenomics sequence analysis when sick (cued data collection).\n\nThe Integrated Biomedical System was developed on the Ruby on Rails44 platform with Ruby gems and JavaScript plugins. The Rails platform supports multiple SQL relational databases including MySQL and no SQL databases such as Mongo DB. MySQL, Oracle, Mongo DB, etc. all scale to over a billion records in a single table. The underlying architecture and approach can be extended to handle a variety of additional data sources. To facilitate data exchange between sites, global unique identifiers (guids) are used. The Integrated Biomedical System and Rails can be installed on computers ranging from stand-alone on a laptop/desktop to servers running Windows OS, Mac OS, Linux, or Unix. Individuals can install and run this system for personal use without needing to set up a web service; to facilitate this the default configuration uses the Sqlite3 database, which installs with the Rails setup. Switching to MySQL or Oracle requires database software installation and a 5-line update to the Rails database.yml configuration file with updated database instance details. To facilitate bulk loading of large numbers of data files, command line interfaces for each ETL module are included in the app/utilities folder.\n\nThe Integrated Biomedical System (version 1.0) is developed as a Ruby on Rails (versions 3 & 4) application. Current JavaScript libraries and versions are included in the Ruby on Rails Gemfile with the inclusion of JQuery, D3, FullCalendar, Highcharts, JSmol24 PDB structure viewer, and more. The Integrated Biomedical System can be optionally configured as a web site with Apache httpd web server plus Passenger (Phusion). The database schema is available in a MySQL Workbench schema in the docs folder for the application. The Integrated Biomedical System has been tested with both Sqlite3 and MySQL relational databases; it should work with most if not all Rails supported databases. The application Readme and GitHub site (https://github.com/doricke/ibio) list the 10 standard Rails application setup steps to setup this Rails application. Initial user accounts can be configured in the db/migrate/20131217194515_create_individuals.rb file.\n\nThe Integrated Biomedical System can be run as a local application with the “rails server” command and a web browser for http://localhost:3000/ or configured to run as a web application with Apache httpd server. The graphical user interface navigation control panel is a set of eight ovals containing text and image links to interfaces within the application, see top of Figure 1. Users can upload data through the web interface (Figure S1). A set of command line utilities are included for administrator loading of data (Table S1).\n\n(A) Screen shot of heart rate beats per minute measurements for a volunteer wearing Basis B1 watch, Empatica E3, Zephyr BioHarness, Hildago Equivital SEM2, and Mio Link devices. SEM2 values were filtered for minimum quality values of 70 with selection of median value; (B) Zoomed in view of heart rates illustrating measurements at different activity levels; and (c) Bland-Altman plots comparing measurements from the heart rate tracking devices with corresponding Pearson r correlation values.\n\n\nResults\n\nHeart rate monitoring. Heart rate monitoring devices provide heart rate, interbeat interval (IBI), and electrocardiogram (ECG) measurements. Heart rate measurements for multiple devices for an individual are shown in Figure 1. Hidalgo Equivital SEM2 and Zephyr BioHarness were typically worn only during more active periods. Lower Zephyr heart rate values observed on Aug. 29 likely resulted from the contact pads drying out during a period of extended wearing with low activity level. Some data gaps result from the need for device battery recharging (Empatica E3 - daily and Mio Link every 8 to 10 hours). Higher correlations of results are observed for periods of sleeping and light activity. This observation is consistent with previous anecdotal observations of data accuracy and coverage decreases for many wearable sensors during periods of high activity.\n\nSleep monitoring. Multiple devices tested provide top-level estimates of nightly time asleep and number of sleep interruptions. Some devices also attempt to break down the sleep time into sleep phases (light, deep, and rapid eye movement - REM sleep). This data was integrated to enable comparisons of sleep classifications assigned by these devices (investigation of the accuracy of these estimates vs. gold-standard polysomnography was beyond the scope of the present work). Example longitudinal measurements from a single individual collecting data in parallel using Jawbone Up, Basis B1, Fitbit, and ActiSleep are shown in Figure 2. Analytical modules enabling pairwise comparisons of unfiltered nightly time asleep estimates between different devices were developed and integrated into the Integrated Biomedical System. Simple comparisons of daily total time asleep reported across the range of devices revealed a lack of correlation for most device pairs as measured by Pearson r statistics. Likewise, finer-grained estimates of light sleep (provided by Basis and Jawbone) and deep sleep (Jawbone) compared to deep sleep plus REM sleep (Basis) were also poorly correlated. Only the two Actigraph algorithms, Sadeh and Cole-Kripke, which were run on the same raw Actigraph sensor data produced highly correlated results (r of 0.97).\n\n(A) Screen shot of daily total sleep measurements for a volunteer for Fitbit Flex, Jawbone Up, Basis B1 watch, and Actisleep. (B) Bland-Altman plots comparing measurements from the sleep tracking devices for this volunteer.\n\nGlobal Position System (GPS) tracking of outside activities available in the Integrated Biomedical System from smartphone or GPS data can provide continuous localization for an individual. This data enables a range of potentially useful correlations to be determined including correlations with data from nearby EPA or other air quality monitoring station(s) as an initial step toward quantitative tracking of individual exposures. Inferred exposure levels can be estimated from nearby sensors for a wide variety of measured pollutants, particulates42, and pollen levels45. Figure 3 illustrates NO2, PM2.5, carbon monoxide, and ozone exposures for an afternoon walk.\n\nExample visualization of activity data with estimated exposure levels from nearby EPA AirData monitoring site.\n\n\nDiscussion\n\nGenome, interactome, and exposome all influence an individual’s wellness. The Integrated Biomedical System was developed to demonstrate the ability to begin integrating these heterogeneous data sources in near real-time for individuals. This was accomplished using an architecture that can operate on a stand-alone laptop or desktop personal computer (PC) to provide additional privacy and security and can be connected seamlessly to voluntarily transfer selected data to centralized highly scalable systems built on the same data architecture that can integrate data from many thousands or even millions of individuals. This approach could provide a path to developing new crowd-sourced models for large-scale prospective/retrospective studies of how individual combinations of genomic and environmental factors correlate with a range of human health and performance traits. Individual monitoring devices, genetic data, blood biochemistries, nutrition, exposures, illnesses, vocal and additional data have been organized and integrated into a unified system. Using the same tools and architectures, additional quantitative lab results and diagnostic data like images and physiological monitoring system data can be added to further increase the research scope of the system. Incorporation of additional natural language processing tools and data architecture modifications can enable text-based metadata collections (e.g. regular symptoms logging from personal health blogs, social interaction details from social media platforms, information from electronic health records) to be included in future versions of the system. Furthermore, these personal datasets can be combined with relevant public datasets and other non-public data to provide new insights into health-associated effects to support detailed N-of-1 and population retrospective analyses.\n\nAs large-scale DNA sequencing costs continue to decrease, sequencing an individual’s DNA becomes more affordable and practical. Current costs enable exome sequencing of individuals for less than $1,000. In a few years, the costs for whole genome sequencing for individuals is projected to be below $1,000 for very large studies. The quality and completeness of results can be estimated by coverage, but room for improvement is illustrated by the Proton and Illumina exome results correlated with 23andMe SNP profiles. While tools exist to characterize variants (Polyphen2, SIFT, etc.), the potential to correlate variants with protein structure/function, physiology, molecular biomarkers, etc. typically is done manually and within studies with a single focus. Integrating genomic data with interactome and exposome data will help create new opportunities for turning data into new discoveries and knowledge. The Integrated Biomedical System also supports detailed analysis of variant analysis for genes, proteins, pathways, individual SNPs, and other variant types. Future inclusion of raw genomic sequencing data and connections with a variety of genome viewers is straightforward using this extendable data and software architecture. As advances in DNA sequencing technology enable more widespread access to genomic data for individuals, the ability to correlate that data with quantitative interactome and exposome data will become increasingly important. Together, these data can broadly enable efforts to elucidate the interplay between genomic and environmental factors that contribute to complex individual human traits and health.\n\nCognitive performance and health phenotypes can be assessed through a variety of indirect methods including analysis of biomarkers in blood, psychomotor vigilance task (PVT), profile of mood states (POMS), automated neuropsychological assessment metrics (ANAM), speech analysis, facial and eye movement tracking, electroencephalography (EEG), and similar approaches. These assessments and others have been developed and used quantitatively define progressions of important traits/symptoms in individuals experiencing a number of conditions including depression46, posttraumatic stress disorder (PTSD), and traumatic brain injury (TBI), as well as environmental stressors including sleep disruption, etc. Data streams produced from these assessments combined with traditional measurements of traits, molecular biomarkers, and clinical data to provide a new platform for gaining insight into the underlying physiology individual health, fitness, and well-being. Retrospective analysis of large-scale collections will provide future biomedical discoveries. Increasing proportions of future biomedical discoveries will be driven by the ability to effectively collect, manage, and interpret massive amounts of heterogeneous data. Enhancements to integrate additional interactome data types and analysis tools are currently underway and these features will be included in future releases.\n\nAsthma and COPD affect 18.7 and 6.8 million individuals in the United States47. Environmental exposures can exacerbate these conditions48. Asthma can be triggered by particulate matter, ozone, sulfur dioxide, nitrogen oxide, and pollens49. Devices, including smart phones, with GPS tracking ability enable the possibility of data integration with environmental monitoring data. Nearby monitoring stations and mobile monitoring devices provide weather and exposure estimates that can be correlated using time stamped GPS positional information. Monitoring stations track a rich variety of environmental exposure data42. While the current system provides incomplete coverage, it demonstrates a viable path to incorporation of additional sensor streams (including indoor air quality sensors, UV exposures, etc.) and activity-based estimates of indoor vs. outdoor exposures. It will be possible to provide increasingly complete individualized and integrated quantitative estimates of specific exposures that can be correlated with possible health effects, symptoms, and well-being. Larger and more complete data sets enabled by integrated systems like the one described here, can play a key enabling role for more quantitative genome vs. environment studies in the future.\n\n\nConclusions\n\nThe Integrated Biomedical System is being developed as an open source platform for individual health, fitness, and in the future wellness promotion. Data visualization, data mining, and new big data approaches will be integrated into the data analysis capabilities that will continue to expand over time. With the goal of creating an open data architecture that supports data exploitation and decision support, this system aims to provide useful information to individuals, medical personnel, researchers, and decision makers. Individuals can run this system on their home computer for use with their own data (and family members). This system will also support longitudinal studies integrating genome, interactome, and exposome heterogeneous data sources. Improving interfaces for user friendliness with valuable feedback and data visualization will be essential for user acceptance, continued use, and progress towards wellness promotion.\n\n\nData and software availability\n\nLatest source code: https://github.com/doricke/IBio.\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.115633150\n\nSoftware license: GNU General Public License, version 3.0.\n\nThe heart rate and sleep tracking data are included in Ricke, Darrell, 2017, “Integrated Biomedical System”, doi:10.7910/DVN/DEEHI251, Harvard Dataverse, V4.\n\nThe Equivital SEM data are included in Ricke, Darrell, 2018, \"Integrated Biomedical System Equivital SEM\", doi:10.7910/DVN/FD4B6C52, Harvard Dataverse, V1.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work is sponsored by the Assistant Secretary of Defense for Research & Engineering under Air Force Contract #FA8721-05-C-002. Opinions, interpretations, recommendations and conclusions are those of the author and are not necessarily endorsed by the United States Government.\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 acknowledge Carl Ricke, Freelance Illustrator at Wanderbots, for graphic artwork.\n\n\nSupplementary material\n\nFigure S1: Integrated Biomedical System web interface.\n\nClick here to access the data.\n\nTable S1: Integrated Biomedical System command line utilities for data loading. These extract, transform, and load (ETL) modules provide administrator tools capabilities for bulk loading of data files. 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}
|
[
{
"id": "30676",
"date": "26 Feb 2018",
"name": "Guillermo H. Lopez-Campos",
"expertise": [
"Reviewer Expertise Biomedical informatics",
"exposome informatics",
"translational bioinformatics"
],
"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 refers to the development of the “Integrated Biomedical System” (IBio) a system developed as a freely available Ruby on Rails application. The proposed system is multiplatform and capable of storing different data sources (genome, “interactome” and exposome) that can be used both at the individual level or to collect data from large populations. For this manuscript, the authors described the use of data gathered through different wearable devices to monitor some physiological parameters, such as heart rate or sleep patterns, and GPS data gathered from the MyTrack app and how they were uploaded into IBio. The development of systems similar to the one described in this manuscript is an interesting step for the future integration and analysis of multiple data sources.\nThe aim of the paper is to develop an open source platform to integrate different data sources “providing a unifying model to promote more open data sharing and analysis”. Unfortunately the manuscript fails to present evidence of having successfully achieved that aim or how this system would solve some of the problems mentioned in the introduction associated with other projects such as using “a relatively narrow set of measurements, or on custom data storage and analysis architectures that do not provide a scalable foundation for larger scale integration across studies to enable meta-analysisis of data from multiple studies” (Ricke DO et al. F1000Research 2018). The manuscript does not describe the suggested unifying model and it is limited (because it did not mention the use of any standards) to a set of “ad hoc” implemented scripts/modules to a limited set of devices and measurements and does not show any evidence of having used any \"genomic data\". It neither provides neither descriptions nor evidence of how IBio promotes more open data sharing and analysis. Data integration is limited to store the data in the same platform but keeping the three different data types separated from each other. The results section is mostly focused in comparisons between the different devices rather than in the system itself. Because of these reasons and despite working in an extremely interesting area this manuscript cannot be approved unless it undergoes major and extensive revisions in its design and its contents.\nMajor revisions 1. The article should provide a much better and extensive analysis of the existing literature and approaches and how these data are integrated or stored in the same repository. This analysis would facilitate identifying the existing alternatives, their deficiencies and therefore would put in context the contribution of the proposed solution. An example of relevant existing contributions would be the work developed by the National Center of Excellence for Mobile Sensor Data-to-Knowledge (MD2K) (1) but also other approaches such as those described in the references 2 to 7. In addition, other interesting approaches are the initiative known as “WikiLife” and the UK Biobank (http://www.ukbiobank.ac.uk). Wikilife aimed to manage and integrate different data sources and has left some software (https://github.com/wikilife-org), which could be compared with the system described in this manuscript. The UK Biobank also contains information similar to the one stored in IBio (It does not contain GPS data but does contain some other sources of geographical information that might be used to input exposure data) and its structure might be useful to compare both systems.\nFrom a technical and technological perspective for data integration and data sharing the manuscript did not mention any standardization initiatives or attempts. The authors should reflect upon this issue. A relevant example in this direction is the work developed by Open mHealth (http://www.openmhealth.org/) (8,9). These are all aspects that should be included in the paper and considered in the discussion or interpretation of the results as elements of comparison.\n2. The use of the term “interactome” in this manuscript is very confusing. It is widely accepted that the “interactome” is the set of interactions among proteins (10) whereas the authors have employed it in this manuscript in the context of physiological parameters or phenotypes, making it difficult to relate to the actual contents and data. Therefore, it should be replaced across the whole text with some other term that better describes these elements.\n3. The methods section provides some information about some of the data contained in the system, and how some volunteers gathered some of the data. Despite the manuscript mentioning the use of genome data by iBio, the methods section only reflects that they store information from other sources. The authors did not mention how the information from the different resources was integrated and related. This is critically relevant information that should be included in the manuscript. The manuscript did not provide any criteria for the selection or the inclusion of the different devices & data used to build the system. The “interactome” section describes elements designed to interact with the Basis Science website for the collection of these data however the company stopped offering their services on 31st December 2016 thus those modules are useless and therefore they can be removed from the text.\n4. The exposome subheading in the methods section refers mostly to sleep data gathered using some wearable devices (the some previously described in the “interactome” section). These data are not exposure data but physiological data. Other proper exposure data such as diet and prescriptions are poorly described and not referred at later stages with the exception of GPS data. Information about how these data are managed and stored in the system is relevant and should be provided. V.g. Were they entered just as free text?\n5. The results are not clearly related to the aims of the manuscript and should be revisited to better present the actual results of their research. A large part of the results presented actually relate to comparisons between the devices used by the participants rather than about the platform itself. The results section seems to focus more in the research question \"Do different devices produce the same results?\" than in the integration aspects which allegedly are driving the research and the manuscript. Methods section should contain information about the methodology used in these comparisons.\nAs the manuscript is describing a system based in a database the structure and contents of this database should have been presented in the results section. Results should have focused as well the interfaces developed for the system, user experience or the comparison with other existing platforms.\nOne of the main aspects of the manuscript is data integration, however with the results presented in the manuscript it is unclear the benefit of having the data in this platform or having three different platforms storing the same data\n6. The results section strikingly lacks results about the integration of any genomic data despite frequent mention of these data in the manuscript. For testing purposes the authors should at least enter some data (that could be collected from online resources) or could be simulated.\n7. Surprisingly, the authors distinguish between exposome and exposure data in the results section, what is the reason for that? By definition the exposome is the whole set of exposures of an individual. A detailed reading of this section shows that it just reflects about the possibility of including GPS data into the system that potentially could be linked with other sources such as the EPA. This is an interesting idea but sadly the manuscript lacks details about how this integration is performed and it should be included in the methods section.\n8. The discussion section is subdivided in four different elements, (vision, genome, interactome, exposome). Overall, the discussion very vaguely discusses the results presented in the paper, instead focusing on potential future applications and listing potential data sources. The results section should be revisited to actually focus on the discussion of the results.\nThe discussion starts with a vision that talks about genomic data, however as previously mentioned there is no evidence in the manuscript that any data of this kind has been uploaded into the system but these data are not mentioned anywhere in the manuscript other than in the methods section.\nThis paragraph also mentions that the system demonstrates the ability to integrate data in near real-time. This is not evident in the manuscript. As the system requires manual input of the data by the user it is unclear how this can be considered “near real-time”. These statements should be therefore corrected to better reflect the reality of the system as presented in this manuscript.\nThe “Genome” subheading in the results section must be removed or heavily amended. It provides a vague description of what can be done with genomic data and lacks evidence to support any other of the claims made. It is important to mention that the manuscript does not provide any references about how any genomic data can be uploaded into the system and therefore does not actually discuss any results. It also contains some vague references to some tools (that require a proper citation) that are not included in the system and there is an absolute lack of evidence that the system would be able to support any detailed analysis of any molecular data (genetic, protein, pathways…). In the current text, it is unclear what difference this system would make in terms of the analysis of the different variants in terms of automating these analyses or broadening their focus.\n9. The conclusions are vague and should be edited to better reflect the status of the system and the actual conclusions from the work presented. For example, according to the manuscript, it is unclear how without adequate integration this system would be able to provide useful information in medical environments (for medical personnel) or decision makers without analysing security or data sharing issues. Other conclusions are based in future improvements done in the system which are not available yet or haven't been described and in an integration that at the moment does not go any further than storing data together in the same platform without actually integrating them.\nThe authors themselves acknowledge that the system requires improvements in the user interface, aspects that should have been addressed much earlier in the manuscript and that are key for the success of these kind of applications.\n10. Surprisingly, a large number of the citations are incorrect and do not refer or match with the contents in the manuscript. (e.g. references 8, 15, 18,21,22,24,25,26,27….).\n\nMinor corrections 1. As a minor element in this aspect notice that reference 10 should read as Martin-Sanchez et al rather than Sanchez et al. 2. In the methods section authors said that in some cases the volunteers underwent a microbiome analysis but later on there are no mentions of this data nor how they can be incorporated therefore it should be removed from the manuscript. 3. In Figures 1A and S1 there are elements that are not mentioned at all in the manuscript or being part of the system (For example miRNAs, Ancestry, Pathogens). 4. Apparently, there is some sort of colour code used in the interface, it would be interesting to provide a description of that… 5. As previously indicated the “interactome” subheading in the discussion should be modified to better describe the results rather than vaguely talk about potential benefits derived from data integration.\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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "32216",
"date": "06 Apr 2018",
"name": "Wolfgang Kuchinke",
"expertise": [
"Reviewer Expertise Clinical research and IT infrastructures"
],
"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\nIntegrated Biomedical System\nThis article addresses a very important topic, the integration of different kinds of data from different domains for joint analysis. This is indeed the future of research to jointly analyze genomic, clinical, life style and environmental data. The authors claim to have developed an “Integrated Biomedical System”, a platform that collects and stores in a single place different data sources, like genome data (SNPs, NCBI), proteomic data (PDB, SwissProt), physiologic data (heart rate, skin conductance, respiratory rate derived from sensors), life style data (nutrition, prescriptions, other medications, sleep pattern) and air pollution data from EPA and used it for some kind of study. This is a considerable achievement, but it is presented in an unclear, incomplete and insufficient way.\nTo begin, what is this manuscript? A description of a new software tool, a study of the integration of sensor data, a pilot of a software tool? The purpose of the manuscript should be clearly stated; it determines how the results have to be presented.\nAn important aspect of such a study is how data is measured, treated and processed. There are different methods and algorithms used to analyze data from sensors used for monitoring. For example, sensor signals can be contaminated with noises or interferences. In addition, responses to these signals could be different for different persons. In this context, sensor data need calibration. All these issues are not discussed. In fact, the integration of data from diverse sources results in data with different data formats, data models, metadata schema, etc. and this must be addressed.\nEfforts of other groups working on the integration of sensor and physiological data should be mentioned. For example, Arturo Arino from the University of Navarra combines in the PAIRQURS project physiological data with location data and air pollution data measured with sensors attached to bicycles [1]. These sensors record the levels of selected atmospheric pollutants, like CO, NOx, Ozone, and airborne particles, together with auxiliary data (Temp, HR) and GPS coordinates and transmit processed packets via GPRS to a central database.\n\nIn detail:\nMethods\nThe Methods section is very weak. What is the Experimental Procedure? Protocol design and approvals are mentioned; but is it a medical study? What is the study design? What is the comparator? It is stated that the study was planned for 20, later 40 participants. But how many volunteers actually participated, what gender, age, number of drop-outs, ...?\nData privacy protection is a hot topic for medical data and GPS data. It should have been considered in the informed consent, because with GPS data identifiability of persons becomes a problem. Privacy protection should have been discussed.\n“Volunteers have full choice of all elements of the research project …” What does this mean? Can participants determine the study protocol?\nThere is sometimes redundance in the text: For example, \"COUHES approval\", \"Entering events, meals, prescriptions…” is mentioned 2 times\nIt seems that some terms are used arbitrarily. For example, the \"Genome part\" contains also Proteome data. \"Interactome\" means the whole set of molecular interactions in a cell, like the human protein-protein interactome. But in the manuscript Interactome contains even heart rate, ECG, respiratory rate, etc. The Exposome encompasses the totality of human environmental exposures. But in the manuscript it contains measurements of activity and sleep and nutrition.\nThe Integrated Biological System is obviously a software tool, including several databases. But no exact information about the structure and architecture of the system is given. How many tools / modules, what interfaces, how many databases, what data models, what standards, etc. Global unique identifiers are mentioned. How are these identifiers integrated in the system? A GUID is a number that the program generates to create a unique identity for an entity; why is this used for data exchange between sites (and between which sites).\nImplementation. This section contains partly redundant information with Integrated Biological System, describing that Ruby on Rails was used, some of the databases, etc. Better a list should be provided that describes all components of the application setup and their connections.\nOperation Concerns the use of the Integrated Biological System. “Users can upload data through the web interface”. This is very basic information, without any details. But what data, what format, how to use the interface,...?\nMeasurement procedures are missing. What are the Analytical Methods? Statistical Analyses? Some information is included in Fig. 1 (like, Bland-Altman plot is mentioned, but why use this plot?)\nResults The Results section has the same subtitles as the Methods section. But here the results of the pilot study should be presented in a standardized and comprehensive way. First, what is the primary result of the pilot study? The detailed numeric outcome could be summarized in a table.\nMany problems are mentioned, like devices worn only during active periods, contact pads drying out, battery recharging, etc. What was the effect on the data? This should be mentioned. In addition, an interpretation of Fig.1 is missing. The reason for comparing such a large number of different devices is missing. Were different devices worn by the same person, were multiple measurements done?\nSeveral rather vague statements, for example: “multiple devices were tested ..” how many? “Some devices also …” how many? \"This data was integrated to enable …” how? \"… provided by a subset of volunteers.” How many? \"Example longitudinal measurements from a single individual ..” how was this person selected? \"Analytical modules were developed …\" belongs to Methods \"GPS tracking of outside activities …\" how could outside activities be differentiated from inside activities? “…data from nearby EPA or other air quality monitoring stations.” More information is necessary. How were the monitoring stations selected, how far away are these stations from the participant, what kind of data is provided by the stations, what kind of calibration was used, …?\nDiscussion Here the results should be discussed in a critical way. This is completely missing in the manuscript. The vision should be put to the final end of the manuscript.\nIt is stated that individual monitoring devices, genetic data, blood biochemistries, nutrition, exposures, … were integrated into a unified system. But the integration was not complete, because the genetic data seems to be missing in the results section. Important is to discuss how good this integration was, and all results should be evaluated critically.\n\nReference https://eudat.eu/news/interview-with-arturo-ari%C3%B1o-from-pairqurs\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?\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": []
}
] | 1
|
https://f1000research.com/articles/7-162
|
https://f1000research.com/articles/7-52/v1
|
15 Jan 18
|
{
"type": "Research Article",
"title": "An explanatory model for the concept of mental health in Iranian youth",
"authors": [
"Ahdieh Chinekesh",
"Seyed Ali Hosseini",
"Farahnaz Mohammadi",
"Mohammad Esmael Motlagh",
"Monir Baradaran Eftekhari",
"Shirin Djalalinia",
"Gelayol Ardalan",
"Ahdieh Chinekesh",
"Farahnaz Mohammadi",
"Mohammad Esmael Motlagh",
"Monir Baradaran Eftekhari",
"Shirin Djalalinia",
"Gelayol Ardalan"
],
"abstract": "Background: Mental health is considered as an integral and essential component of overall health. Its determinants and related factors are one of the most important research priorities, especially in adolescents and young people. Using a qualitative approach, the present study aimed to identify factors affecting the mental health of youth in Iran. Methods: In 2017, following content analysis principles, and using semi-structured in-depth interviews, we conducted a qualitative study exploring the opinions of young people about mental health. A targeted sampling method was used, and participants were young volunteers aged 18 to 30 who were selected from Tehran province, Iran. Inclusion criteria for participants was willingness to participate in the study, and ability to express their experiences. Data collection was done with individual in-depth interviews. According to the explanatory model, the interviews were directed toward the concept of mental health and path of causality and auxiliary behaviors. Results: 21 young adults participated, who met the study inclusion criteria, of whom 12 participants were male. Their mean age was 24.4 ± 0.41 years and their education varied from primary school to Master’s degree. Mental health was considered as mental well-being and a sense of satisfaction and efficacy, not only the presence of a disease or mental disorder. Based on the opinions of the interviewees, three factors of personal characteristics, family and society are involved in mental health. Individual factors were associated with behavioral and physical problems. One of the most important issues was revealed as tensions in societal and family conflicts. Economic problems and unemployment of young people were also extracted from the social factor. Conclusion: In Iran, social factors such as jobs for the unemployed and job security are considered as important determinants in the mental health of young people.",
"keywords": [
"Mental health",
"youth",
"Explanatory model",
"Qualitative method",
"Iran"
],
"content": "Introduction\n\nMental health is a primary and principal component of health. Based on the definition of the World Health Organization, more than a lack of clinical mental illness, mental health is a sense of happiness that allows individuals to recognize their abilities, adapt to the natural tensions of life, and be efficient and productive resources for their communities1.\n\nMental health and well-being are essential for our personal and social abilities as human beings to think, feel excitement, interact with each other, earn money, and enjoy life. Accordingly, promoting and maintaining mental health can be considered an important and vital concern for individuals, societies, and communities around the world2.\n\nMany biological, social, and psychological factors influence the level and quality of health2. Based on recent evidence, more than 450 million people are suffering from some kind of mental disorder, and there are many psychological problems, around the world3.\n\nMany studies have focused on mental health promotion. Jorum and Wright, in their study in Melbourne, showed that young people believe that interventions for preventions and control of mental disorders could be effective4. There was broad agreement from young people and their parents about what interventions are likely to be helpful and these views applied across the range of disorders presented. These interventions could be described as general and informal sources of help, rather than as specialist mental health services. The most negative views were about psychiatric medications and admission to hospital.\n\nIn 2007, Barry and Jenkins discussed the impacts of many community participation factors in promoting the mental health5. In 2012, Francesco and colleagues examined the role of supporting colleagues in the Clubhouse model using a qualitative method. Through this study, four peer support levels emerged and stressed that peer support would improve6.\n\nIn 2004, in Iran, Norbala and colleagues, using the General Health Questionnaire, showed that 21% of the total population suffered from mental illness (25.9% of women and 14.6% of men)7. A related study in 2006 reported that about 2.2% of deaths in women and 1.2% of deaths in men could be attributed to mental disorders8. In 2009, Ebadifard and colleagues conducted a semi-experimental study. The results of this study showed that educational intervention was influenced by attitudes and healthy behaviors in adolescent girls through belief, attitude, and mental and constructive factors9.\n\nConsidering the high prevalence of mental health disorders and also their importance and role in the development of related disease10, it is necessary to design effective interventions to improve mental health. Many studies confirmed that improving mental health has considerable association with reducing community social harm11,12.\n\nIn the present study, we were interested in the mental health status of Iranian youth. To investigate this, we used the framework of the explanatory model13, which entails taking notes on a process, according to the concept of mental health, including causes and help-seeking behaviors. This model is mainly used to describe, organize, and manage determinants of welfare disorders14.\n\n\nMethods\n\nFor this study, to understand the world from youth’s perspectives and their experiences, and to uncover their lived worlds, we conducted individual interviews with young people15,16. We collected data from September 2016 to May 2017.\n\nFace-to-face in-depth interviews are an easy, flexible method for deeply understanding issues and are mostly used to collect data for explanatory studies17. In a qualitative study, the sample size is based on how many participants it takes to adequately answer the research question. With that aim, and considering the framework of the research question and purpose, the study time frame, and the available resources, we continued purposeful convenience sampling until the point of data saturation18 and no new categories, themes, or descriptions appeared.\n\nBased on the inclusion criteria, we selected participants of different ages from 18 to 30 years old, from different regions of the city, and with different socioeconomic and cultural statuses, using the register list of students in Tehran city universities. We invited participants through their recorded email addresses. This selection was the method of randomization. If participants had not Iranian nationality or if they were beyond the determined age range, they were excluded.\n\nInclusion criteria for the participants were (a) willingness to participate in the study and (b) ability to describe their experiences.\n\nThe interview guide questions were designed by the scientific committee in accordance with the relevant literature and the study objectives. Toward our aim, we searched for related documents and focused on tools and measurement methods from different approaches. In these regards we searched the main international data bases, Scopus, PubMed and ISI/WOS. These databases cover most multidisciplinary related fields of research and we aimed to access similar studies and research tools. We searched for qualitative studies, research measurements, and guide questionnaires and qualitative questionnaires. The reliability and validity of guide questionnaires are been confirmed by the trust and accuracy that mainly is considered in qualitative research19.\n\nWe conducted a pilot study of semi-structured interviews with five young adults, including introducing the entry criteria, and based on the pilot study, we further developed the interview questions. These participants were selected from the universities of Tehran city. We used the list of the university register and then emailed to ask to participate. Young people focused more on the help seeking of mental health problems. After this pilot study we revised some components of interview guide, mostly for more clearance and consistency.\n\nThe main interview guide consisted of six semi-open questions that guided the interview process and data collection (Supplementary File 1).\n\nWe collected the data for this study through deep, semi-structured, face-to-face interviews. At the beginning of the interviews, we explained the research goals and method to the participants. Each session lasted for 45–100 minutes (mean duration: 65 minutes). All interviews were conducted in peaceful environments such as special rooms of public libraries or parks with prior agreement of the participants.\n\nInterviews began through general and open-ended questions developed based on the goals of the study, for example, “What are your mental health needs?”; “Can you tell about your experiences of mental well-being?” or “What obstacles have stopped you from having mental health?” The interviews then continued with exploratory questions to clarify the concept and obtain more in-depth information. All conversations were audio recorded with the participants’ permission.\n\nWe performed content analysis of the data collected from the in-depth interviews,20 all of which had been transcribed. To facilitate data analysis, we used Dedoose (version 7.6.6; categorization, constant comparisons, and quotation retrieval). This web-based application, developed at UCLA, is mostly used for analyzing data in qualitative research21.\n\nWe read the transcripts to extract the “meaning units.” The coding scheme was theoretically consistent with the context of the study framework, with regard to the concept of mental health including causes and help-seeking behaviors. We extracted the themes and subthemes from the transcripts to generate new codes or modify codes by induction, and we categorized the inductive codes into meaningful topics22.\n\nTrustworthiness in qualitative research has been divided into credibility, dependability, conformability, and transferability23–30. In this study, we established credibility through the main researcher’s (AC, SAH) prolonged engagement with the subject matter and triangulation of data sources (interviews with youths).\n\nWe performed member checking by presenting each interviewee with a summary of the interview to check for accuracy. To address the issues of dependability and conformability throughout the entire research process, the main and local supervisors (Ahdieh Chinekesh, Seyed Ali Hosseini, Shirin Djalalinia) reviewed an audit trail of translated transcripts, related documents for data analysis, and member opinions. For transferability, a complete set of data analysis documents is available on request. This access to the research trail allows other researchers to share the results of this research with others or to repeat the research as closely as possible28–30.\n\nThe study was approved by the University of Social Welfare and Rehabilitation Sciences Ethics Committee in Tehran, Iran (IR.USWR.REC.1395.378). Participation in the study was voluntary, participants were ensured that they could withdraw from the study whenever they wanted, and we obtained written informed consent from all participants. At the beginning of the interviews, we explained the study research goals and methods to the participants and assured them of the confidentiality of the information. We respected the study ethical considerations, including data collection (recording and duplicating interviews), data analysis, and dissemination of results. We collected all information anonymously and used the results only for research purposes.\n\n\nResults\n\nThe participants consisted of 21 young adults who met the study inclusion criteria; among these, 12 participants were male. The participants’ mean age was 24.4 ± 0.41 years, and their education varied from primary school to master’s degrees. All participants resided in Tehran, although from different geographic regions of Tehran. Table 1 shows the participants’ demographic characteristics.\n\n* Employee: A person who is employed in the public sector; Worker: a person who works for a private sector and operates under an annual contract.\n\nThe results were analyzed under the following themes and subthemes:\n\nThe majority of participants believed that mental health is an inner emotional feeling of happiness. They stated that inner peace and satisfaction are among the most important signs of mental health. According to the participants, people who have high self-esteem and who are determined, tolerant, and realistic are satisfied.\n\nBased on the experiences of young people, good mental health cannot be recognized solely by the absence of illnesses and mental disorders such as depression or anxiety. Mental health is also the ability to adapt to one’s environment and surroundings. The participants in this study stated that mental health entailed having healthy individual and social functioning and that it includes skills such as coping, problem solving, communication skills, and stress management.\n\nWe classified the subthemes of the causes of mental health problems as social, family or personal.\n\nA. Social factors\n\nMost participants said unemployment or low-income employment was a major contributor to mental health problems. Unemployment, especially in men, lead to financial problems. On the other hand job insecurity is one of the main sources of stress. People who are unemployed or have low-income jobs are less likely to have mental well-being than do people who are gainfully employed, and they show higher rates of violence, anxiety, aggression, and depression31.\n\nYoung people included in this study were pointed out as having a bright future and reported marital problems and the tendency towards high-risk behaviors as important outcomes of unemployment.\n\nYoung people in this study highlighted that unemployment, lack of income and lack of entrepreneurship among young people lead to frustration and disappointment. The majority of participants noted the great attention by the authorities to creating jobs.\n\n‘If you are unemployed, you need to worry about your future and your living costs. Planning for the future is impossible. You’ll be disappointed after a while.’ (Boy, 26, single, bachelor’s degree)\n\n‘I am looking for a job so I can buy a house and a car.’ (Boy, 23, single, diploma)\n\n‘Most young people have no hope of finding right job.’ (Girl, 24, single, bachelor’s degree)\n\nYoung people in this study noted that having a job would prevent high-risk behaviors:\n\n“The constant presence of young people in the park is due to their unemployment, lead them to cigarettes and other tobacco products”. (Boy, 27, single, master’s degree)\n\nYoung people pointed to having enough income:\n\n“Having a job with enough income is one of the most important priorities of mental well-being.” (Girl, 26, married, bachelor’s degree)\n\n“Our problem is that we do not have a good salary system that has enough incentive work. When I started working for the first time, my salary was very low. I was disappointed, and I realized that I had to work two shifts”. (Boy, 30, married, master’s degree)\n\nSome young people pointed to gender discrimination in finding work:\n\n“Girls have less chance than boys for getting a job”. (Girl, 24, single, bachelor’s degree)\n\nSome young people expressed lack of confidence in the future as one of the most important factors in the lack of mental relaxation:\n\n“Society cannot provide this peace of mind for young people who, after completing their course, will find the right work, and we do not have a vision for the future”. (Boy, 26, single, master’s degree)\n\nThe most important issue raised by the young people was that economic problems lead to marital problems:\n\n“The lack of economic security in society and inflation have an important impact on the issue of marriage. My marriage has been destroyed by economic problems for five years, and we both are still engaged, we cannot solve our economic problems”. (Boy, 30, married, bachelor’s degree)\n\nB. Familial factors\n\nSome participants believed that lack of understanding and coherence in the family, lack of parental attention to young people’s needs, and tension in families such as parental violence can cause emotional difficulties and behavioral problems in children. The participants described the existence of disputes and differences between parents as influences on social security and the need for mental health services. The participants also stated that healthier, more secure family environments have positive effects on family members’ morale and that these favorable effects transfer to society.\n\n‘Controversy between parents makes me feel dizzy. When I go out of the house after my parent’s dispute, I feel like strangers. I feel like I’m separated. It’s very sad.’ (Boy, 22, single, bachelor’s degree)\n\n‘Our parents are our supporters, and when they fight and threaten each other, I get tired of life.’ (Girl, 19, single, diploma)\n\n‘I’m always worried, sometimes my mother speaks and suddenly everything goes wrong and there are arguments and arguments… I leave the house.’ (Boy, 20, single, university student)\n\nThe young people referred to factors such as high expectations and disobedience as causes of differences in families:\n\n‘Sometimes the expectations are too much. The main problem is that everyone has expectations from others, but they do not care what others expect from them. People think they have done well but have not seen anyone good.’ (Boy, 24, single, diploma)\n\nRelationships between family members also affect mental health. Study participants reported the existence of autarchy in the family in the form of patriarchy or matriarchy or collaborative decision-making and respect for the opinions of all family members as the two main types of families:\n\n‘I learned from childhood to consult, because all decisions in our family are made in consultation with family members.’ (Boy, 21, single, university student)\n\n‘In my family, my dad made all the decisions alone and did not consult with family members. Now I am married, but I have no ability to make decisions and I feel that I should obey my husband’s decisions.’ (Girl, 26, married, diploma)\n\nA large number of participants stated that the choice of spouse and the age of marriage played a role in promoting mental health:\n\n‘As you grow older, making a decision on marriage is harder, and choosing is difficult.’ (Girl, 25, single, master’s degree)\n\nC. Individual factors\n\nAccording to some participants, behavioral characteristics such as aggressiveness, lying, selfishness, jealousy, pride, irresponsibility are important in mental health problems. From the young people’s perspectives in this study, mentally relaxing requires “managing anger and anxiety” and “gaining happiness” and “hoping for a bright future.”\n\nMost participants stated that forgiveness, patience, anger control, and positive thinking are important for the mental health of young people. Acquiring emotion management skills, especially controlling anger and anxiety, was one of the most common and important mental health needs the young people discussed:\n\n‘We must not be angry in the tensions of life… learn to be calm. '(Girl, 22, single, university student)\n\n‘The anger in our society is too much. People argue with each other in accidents.’ (Boy, 24, single, diploma)\n\nThey also said that exercise is beneficial for mental health:\n\n‘When exercising, the person feels less jealous and humiliated. As a result, he has better mental health.’ (Girl, 21, single, university student)\n\nHelp seeking can refer to seeking counseling, having social support, or “strengthening the faith in God.” Participants stated that social support is important in personality development and mental health promotion, and they reported receiving social support from various sources such as parents and family members, peers, spouses, and community members. Participants suggested that engaging with friends and peers in activities such as entertainment, hobbies, traveling, and social gatherings were all effective strategies for changing the rhythm of life and facing depression:\n\n‘If I have a problem I will share with my mother. She understands me and is my best friend.’ (Girl, 21, single, university student)\n\n‘When I’m sad, I want to be with my friends. They help me and try to understand me.’ (Boy, 24, single, university student)\n\nMost participants referred to the role of religion in achieving peace of mind. These young people reported that they believed in God as a “safe beach” that could be trusted:\n\n‘When I’m sad, I want help from God. I believe in the existence of a supernatural power, and he helps me.’ (Girl, 21, single, university student)\n\nMost of the young participants in this study recommended learning life skills and communication skills, stating that skills such as stress management, problem solving, and decision-making are effective ways to promote mental health. Participants also recommended counseling during psychological crises, as well as herbal medicines, exercise, and travel as coping strategies:\n\n‘I think we need to learn how to manage our daily stress, learn the ability to solve a problem, learn communication skills, and learn how to say no. These should be in the form of courses at the school or university for young people.’ (Girl, 24, single, bachelor’s degree)\n\nAlthough most participants believed in counseling and its benefits, they did note that the cost was a primary barrier to seeking counseling services:\n\n‘I got help from a psychologist, but because of the high cost, I could not continue.’ (Boy, 21, single, University student)\n\n\nDiscussion\n\nThis was a qualitative study on the perceptions of young people in Tehran about mental health; most participants in this study equated mental health with mental well-being. Based on the results, mental health problems are caused primarily by social, individual, and family factors. Based on the participants’ comments, by controlling daily stress with the help of family and friends, you can manage mental problems at an early stage, but in acute cases, it is necessary to get help from psychologists. The participants also referred to the important role of religion in mental well-being.\n\nThe definition of mental health highlights mental well-being. Individuals with good mental health can recognize their abilities, cope with the everyday stress and challenges in life and participate effectively in society32. Health is a multidimensional concept that, in addition to lack of illness and disability, also involves a sense of happiness and well-being33, and the results of this study indicate that many young people are trying to explain mental health positively. They referred to feelings of happiness, hope for the future, and spirituality, which are among the most important indicators of positive mental health34. In various studies, there is a significant relationship between mental health and positive psychological factors such as happiness, hope, and spirituality35–40.\n\nOur study also showed that most youth attributed mental health to external factors in their social worlds, which was consistent with other studies of participants in Australia, Japan, and Kenya who believed in social causes of poor mental health such as difficult life events and circumstances, financial problems, and traumatic events41–43. Mental health problems can also be triggered by social circumstances44.\n\nIn our results, the young participants perceived psychological symptoms as transient reactions to external stressors. Therefore, young people should receive appropriate training on symptoms of mental illness to be able to recognize mental disorders according to symptoms. In addition, differing approaches to treatment could help professionals to choose appropriate and effective therapies for young people.\n\nWe also found that economic problems were one of the main causes of poor mental health among the young people in our study. Unemployment and lack of job security cause young people stress and increased tensions related to work, which negatively affect the mental health of young people45. Based on the project COS-RGO, in the field of mental health and economic development, economic changes can increase mental health problems46. Moreover, the results of a national project in Iran showed that economic growth and income inequality among economic variables had the most impact on mental health47.\n\nAmong families, the relationship between parents, tensions in the family environment, and divorce affect the mental health of family members48. A multi-method study in the UK showed that parental divorce had a negative effect on adult mental health48. Individual negative traits such as jealousy have been linked with low self-esteem. These behaviors refer to negative thoughts and sense of insecurity, worry, and anxiety49.\n\nThe results of this study indicate that help seeking begins with limiting daily stress and that resources such as social support from friends and family and counseling facilitate this process. In this context, religious beliefs are shown to have an important role in achieving peace of mind; reinforcing faith in God has been highlighted as one of the most important sources of peace. Levin et al. conducted a study of African Americans in 2005 and found that religion affected physical and mental health50.\n\nAs its main strength, we used for this study a well-developed methodology through which we extracted saturated data from in-depth interviews and analyzed the data precisely based on the defined protocol. During the study, we encountered some restrictions, such as the lack of cooperation of some of the invited participants, the diversity of participants' perceptions, and the limitations in generalizing the results.\n\nGiven the above, we suggest designing and evaluating participatory interventions for mental health promotion for future studies. Given the strategy outlined, further research on the determinants of mental health in young people in different populations is recommended.\n\n\nConclusion\n\nIn Iran, social factors such as jobs for the unemployed and job security for workers have essential roles in youth mental health. Based on the findings of this study, to promote mental health in young people, we propose participatory strategies. In this regard, the youth as a target group will engage with policy makers and researchers to design, develop, and implement mental health programs.\n\n\nData availability\n\nTranscripts of the 21 interviews performed with young people (in Persian) are available on request from the corresponding author (alihosse@gmail.com).",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe present article is sponsored by the Health Deputy, Ministry of Health and Medical Education of Iran (grant no. 12292).\n\nThe funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe present paper is based on a PhD thesis led by the University of Social Welfare and Rehabilitation Sciences of Tehran and with the financial support of the Deputy of Health, Ministry of Health and Medical Education of Iran. We appreciate the professors of the University of Social Welfare and Rehabilitation Sciences of Tehran, who provided necessary assistance in conducting this research.\n\n\nSupplementary material\n\nSupplementary File 1: Interview guide.\n\nClick here to access the data.\n\n\nReferences\n\nWorld Health Organization (WHO): Investing in mental health. Geneva. 2003. Reference Source\n\nWorld Health Organization (WHO): Mental health: strengthening our response (Fact sheet No. 220). Geneva, Switzerland; 2010; 68–9. Reference Source\n\nBurgermeister D, Kwasky A, Groh C: Promoting mental health concepts in a doctor of nursing practice curriculum: an integrated and global approach. Nurse Educ Pract. 2012; 12(3): 148–152. PubMed Abstract | Publisher Full Text\n\nJorum AF, Wright A: Beliefs of young people and their parents about the effectiveness of interventions for mental disorders. Aust N Z J Psychiatry. 2007; 41(8): 656–66. PubMed Abstract | Publisher Full Text\n\nBarry MM, Jenkins R: Implementing Mental Health Promotion. Philadelphia, USA: Churchill Livingstone. 2007. Reference Source\n\nConiglio F, Hancock N, Ellis L: Peer support within Clubhouse: a grounded theory study. Community Ment health J. 2012; 48(2): 153–60. PubMed Abstract | Publisher Full Text\n\nNoorbala AA, Bagheri Yazdi SA, Yasami MT, et al.: Mental health survey of the adult population in Iran. Br J Psychiatry. 2004; 184(1): 70–73. PubMed Abstract | Publisher Full Text\n\nHughes JR: Should criteria for drug dependence differ across drugs? Addiction. 2006; 101(Suppl 1): 134–41. PubMed Abstract | Publisher Full Text\n\nEbadifard AF, Solhi M, Roudbari M, et al.: Survey the effect of educational intervention through the BASNEF model on preventive behaviors according to mental health in girl adolescents. J Guilan Univ Med Sci. 2010; 19(73): 20–29. Reference Source\n\nBrown S, Inskip H, Barraclough B: Causes of the excess mortality of schizophrenia. Br J Psychiatry. 2000; 177(3): 212–17. PubMed Abstract | Publisher Full Text\n\nMohammadi F, Eftekhari MB, Dejman M: Seeking comfort: women mental health process in I. R. Iran: a grounded theory study. Int J Prev Med. 2014; 5(2): 217–23. PubMed Abstract | Free Full Text\n\nAnderson J, Huppert F, Rose G: Normality, deviance and minor psychiatric morbidity in the community. A population-based approach to General Health Questionnaire data in the Health and Lifestyle Survey. Psychol Med. 1993; 23(2): 475–85. PubMed Abstract | Publisher Full Text\n\nKleinman A: Culture and depression. N Engl J Med. 2004; 351(10): 951–3. PubMed Abstract | Publisher Full Text\n\nStuifbergen AK, Seraphine A, Roberts G: An explanatory model of health promotion and quality of life in chronic disabling conditions. Nurs Res. 2000; 49(3): 122–9. PubMed Abstract | Publisher Full Text\n\nKvale S: InterViews: An Introduction to Qualitative Research Interviewing. Thousand Oaks, Sage. California. 1996. Reference Source\n\nDahlgren L, Emmelin M, Winkvist A: Qualitative methodology for international public health. Department of Public Health and Clinical Medicine, Umea University, Umea. 2007. Reference Source\n\nBoyce C, Neale P: Conducting in-depth interviews: A guide for designing and conducting in-depth interviews for evaluation input. Pathfinder International Watertown, MA; 2006; 6–10. Reference Source\n\nPatton MQ: Qualitative evaluation and research methods. (3rd Ed.). Newbury Park CA: Sage Publications. 2001. Reference Source\n\nFlick U: An Introduction to Qualitative research. (3rd Ed.). Sage Publication LTD. 2006. Reference Source\n\nHsieh HF, Shannon SE: Three approaches to qualitative content analysis. Qual Health Res. 2005; 15(9): 1277–88. PubMed Abstract | Publisher Full Text\n\nDedoose introduction. 2017; [Last accessed on 2017 Aug. 21]. Reference Source\n\nGraneheim UH, Lundman B: Qualitative content analysis in nursing research: concepts, procedures and measures to achieve trustworthiness. Nurse Educ Today. 2004; 24(2): 105–12. PubMed Abstract | Publisher Full Text\n\nAPA: Diagnostic statistical manual of mental disorders (DSM-IV). American Psychiatry association, Washington C. 2000. Reference Source\n\nMaruish M: The Use of Psychological Testing for Treatment Planning and Outcomes Assessment: Instruments for Adults. Lawrence Erlbaum Associates. 2004. Reference Source\n\nKleinman A: Concepts and a model for the comparison of medical systems as cultural systems. Soc Sci Med. 1978; 12(2B): 85–92. PubMed Abstract | Publisher Full Text\n\nOkello ES, Neema S: Explanatory Models and Help-Seeking Behavior: Pathways to Psychiatric Care Among Patients Admitted for Depression in Mulago Hospital, Kampala, Uganda. Qual Health Res. 2007; 17(1): 14–25. PubMed Abstract | Publisher Full Text\n\nDejman M, Ekblad S, Forouzan AS, et al.: Explanatory Model of Help-Seeking and Coping Mechanisms among Depressed Women in Three Ethnic Groups of Fars, Kurdish, and Turkish in Iran. Arch Iran Med. 2008; 11(4): 397–406. PubMed Abstract\n\nElo S, Kyngas H: The qualitative content analysis process. J Adv Nurs. 2008; 62(1): 107–15. PubMed Abstract | Publisher Full Text\n\nLincoln YS, Guba EG: Naturalistic inquiry. Beverly Hills, CA: Sage. 1985. Reference Source\n\nMays N, Pope C: Qualitative research in health care. Assessing quality in qualitative research. BMJ. 2000; 320(7226): 50–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArcher J, Rhodes V: Bereavement and reactions to job loss: a comparative review. Br J Soc Psychol. 1987; 26(Pt 3): 211–24. PubMed Abstract | Publisher Full Text\n\nGreenberg J: Comprehensive Stress Management. 12th Edition, McGraw-Hill Humanities/Social Sciences/Languages©2006.\n\nPoursardar F, Sangari A, Abbaspour Z, et al.: The Effect of Happiness on Mental Health and Life Satisfaction: A psychological model of well-being. J Kermanshah Univ Med Sci. 2012; 16(2): 139–147. Reference Source\n\nEbrahimi A, Aarabi S, Khaluei MM: Comparing the Mental Health and Some Positive Psychologic Factors Including Happiness, Hope and Spirituality among Students of Medicine in Isfahan University of Medical Sciences, Iran, during Years of Education. J Isfahan Med Sch. 2014; 31(261): 1885–96. Reference Source\n\nJafari E, Dehshiri GR, Eskandari H, et al.: Spiritual well-being and mental health in university students. Procedia Soc Behav Sci. 2010; 5: 1477–81. Publisher Full Text\n\nKoenig HG, McCullough ME, Larson DB: Handbook of religion and health. Oxford, UK: Oxford University Press. 2000. Reference Source\n\nRew L, Wong YJ: A systematic review of associations among religiosity/spirituality and adolescent health attitudes and behaviors. J Adolesc Health. 2006; 38(4): 433–42. PubMed Abstract | Publisher Full Text\n\nAlberktsen G: Happiness and related factors in pregnant women Development of psychiatrist. Bangkok, Thailand: Faculty of Median, Chulalongkorn University. 2003.\n\nPerneger TV, Hudelson PM, Bovier PA: Health and happiness in young Swiss adults. Qual Life Res. 2004; 13(1): 171–8. PubMed Abstract | Publisher Full Text\n\nRodriguez-Hanley A, Sunder CR: The demise of hope: On losing positive thinking. In: Handbook of hope: Theory, measures, and applications. Snyder CR, editor. San Diego CA: Academic Press. 2000; 39–54. Publisher Full Text\n\nCinnirella M, Loewenthal KM: Religious and ethnic group influences on beliefs about mental illness: a qualitative interview study. Br J Med Psychol. 1999; 72(Pt 4): 505–24. PubMed Abstract | Publisher Full Text\n\nLawrence V, Murray J, Banerjee S, et al.: Concepts and causation of depression: a cross-cultural study of the beliefs of older adults. Gerontologist. 2006; 46(1): 23–32. PubMed Abstract | Publisher Full Text\n\nNakane Y, Jorm AF, Yoshioka K, et al.: Public beliefs about causes and risk factors for mental disorders: a comparison of Japan and Australia. BMC Psychiatry. 2005; 5(1): 33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO: Gender in Mental Health Research. Department of Gender, Women and Health Family and Community. World Health Organization, Geneva, 2004; 14–19, 7–9. Reference Source\n\nPaul KI, Moser K: Unemployment impairs mental health: Meta-analyses. J Vocat Behav. 2009; 74(3): 264–82. Publisher Full Text\n\nHerrman H, Saxena S, Moodie R: Promoting mental health: concepts, emerging evidence, practice, a report of the World Health Organization. 2005. Reference Source\n\nMehregan N, Ghasemifar S, Sohrabivafa H, et al.: The impact of economic and social conditions on mental health the provinces of Iran (1378–1391). Q Parliament Strat. 2016; 23(85): 85–106. Reference Source\n\nChase-Lansdale P, Cherlin AJ, Kiernan KE: The long-term effects of parental divorce on the mental health of young adults: a developmental perspective. Child Dev. 1995; 66(6): 1614–34. PubMed Abstract | Publisher Full Text\n\nJohnson RJ, Kaplan HB: Gender, aggression, and mental health intervention during early adolescence. J Health Soc Behav. 1988; 29(1): 53–64. PubMed Abstract | Publisher Full Text\n\nLevin J, Chatters LM, Taylor RJ: Religion, health and medicine in African Americans: implications for physicians. J Natl Med Assoc. 2005; 97(2): 237–49. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "29804",
"date": "23 Jan 2018",
"name": "Sara Esmaelzadeh Saeieh",
"expertise": [
"Reviewer Expertise Reproductive health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease correct grammar in the paper Please provide further details about time of sampling, situation and environment of the interview. Who is doing the interview? Was the interviewer an expert? We suggest that results of qualitative study include theme (category) sub them (sub category) and codes of study are mentioned in table. Please omit extra explanation about methods and only mention the methods of your study. Please explain about the type of content analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3379",
"date": "07 Feb 2018",
"name": "ahdieh chinekesh",
"role": "Author Response",
"response": "Dear chief editor of F1000researchThanks for your comments. All of them have been considered in revised upload formatted carefully. Ahdieh Chinekesh"
},
{
"c_id": "3399",
"date": "07 Feb 2018",
"name": "ahdieh chinekesh",
"role": "Author Response",
"response": "In the revised version of this article, we have included two sentences in the first paragraph of the ‘Interviews’ section, providing more detail on how they were conducted."
}
]
}
] | 1
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https://f1000research.com/articles/7-52
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https://f1000research.com/articles/6-2062/v1
|
29 Nov 17
|
{
"type": "Research Article",
"title": "Portable respiratory polygraphy monitoring of obese mothers the first night after caesarean section with bupivacaine/morphine/fentanyl spinal anaesthesia",
"authors": [
"Anette Hein",
"Jan G. Jakobsson",
"Anette Hein"
],
"abstract": "Background: Obesity, abdominal surgery, and intrathecal opioids are all factors associated with a risk for respiratory compromise. The aim of this observational study was to explore the use of portable respiratory polygraphy for monitoring of obese mothers for respiratory depression the first night after caesarean section (CS) with bupivacaine/morphine/fentanyl spinal anaesthesia. Methods: Consecutive obese (BMI >30 kg/m2) mothers, ≥18 years, scheduled for CS with bupivacaine/morphine/fentanyl spinal anaesthesia were monitored with a portable polygraphy device Embletta /NOX on the first postoperative night. The apnoea-hypopnea index (AHI) was identified by clinical algorithm and assessed in accordance to general guidelines. Results: Forty mothers were invited to participate: 27 consented, 23 were included, but polysomnography registration failed in 3. Among the 20 mothers: 11 had an AHI <5; 7, AHI 5-15; and 2, AHI >15. The oxygen desaturation index (ODI) was on average 4.4, and eight patients had an ODI >5. Those mothers with a high AHI (15.3 and 18.2) did not show high ODI or signs of hypercapnia on transcutaneous CO2 registration. Mean saturation was 94% (91-96), and four mothers had mean saturation between 90-94%, but none had a mean SpO2 <90%. Mean nadir saturation was 71% (range, 49-81%). None of the mothers showed clinical signs or symptoms of severe respiratory depression, shown by routine clinical monitoring. Conclusion: We found portable polygraphy registration during early post-CS in moderately obese mothers having had intrathecal morphine/fentanyl cumbersome and although episodes of oxygen saturation decrease were noticed, obstructive events and episodes of desaturation were commonly not synchronised. Upper airway obstructions seem not be of major importance in this clinical setting. Monitoring of respiratory rate, SpO2 and possibly transcutaneous CO2 in mothers at high risk of respiratory distress warrants further studies. Preoperative screening in obese patients, at risk for sleep breathing disorder, is of course of value.",
"keywords": [
"Caesarean section",
"obesity",
"intrathecal morphine",
"respiration",
"respiratory depression",
"apnoea/hypopnea"
],
"content": "Introduction\n\nIn caesarean section (CS), intrathecal morphine (ITM) is associated with better postoperative course with less pain and shorter time to mobilisation1. Respiratory depression associated with morphine is well known. Dahan et al. described the mechanism for the respiratory effects of opioids, defining the µ-receptor as the key target for the respiratory depressant effect and the sophisticated neuronal interaction in the ventral part of the brain stem2. Respiratory depression is the most feared adverse effect from intrathecal opioids, and although rare (0–0.9%), it is the reason for some anaesthesia units to withhold the addition of morphine to spinal anaesthesia for CS3–5. It has been suggested that ITM in doses <0.3 mg (300 µg) is associated with a lower risk of respiratory depression compared with doses >0.3 mg, and a tendency to a lower risk as compared to systemic opioids3,6. Palmer et al. found no analgesic benefit to exceed a 100 µg dose of intrathecal morphine, since incidence and severity of side effects increased1.\n\nRespiratory depression is, however, not well defined and there is still not a general definition of respiratory depression to opioids7. George et al. conducted a meta-analysis on the primary outcome risk of respiratory depression and commented that multiple definitions were used in the included studies8. Similarly, a review by Ko et al. concerning the “definitions of respiratory depression”, explicitly addressing ITM, found no common description9. They also clearly commented the need for further research for defining what is a clinically significant respiratory impairment caused by ITM and how it best can be monitored9. Shapiro et al. defined is solely as a respiratory rate (RR) of <10 breaths per minute10.\n\nBreathing disturbances postoperatively may be caused by several different factors. Residual effects from anaesthesia, abdominal surgery, obesity, immobilisation and opioid analgesics may all contribute to its occurrence7,11. Postoperative respiratory depression after CS, performed in spinal anaesthesia including neuroaxial morphine, is found in low frequency (0/5036, 8/856, 6/1915)4,12,13. Studies explicitly assessing the risk of respiratory compromise in obese patients, i.e. mothers with a high BMI (Body Mass Index), have not been previously conducted; apart from a retrospective study of 5036 mothers who had a CS with neuroaxial morphine, where 63% of patients were obese (BMI > 30 kg/m2), and the most commonly used morphine dose was 3 mg epidural and 0.15 mg spinal4. Crowgey et al. found no respiratory event with need for naloxone, defined as RR ≤8 breaths/min, oxygen saturation <90% or Richmond Agitation Sedation Scale <-2. Aboulish et al. published in 1991 the results from a study on ITM12. They found 8/856 cases of respiratory depression in women having intrathecal addition of 0.2 mg morphine in spinal analgesia for CS, all eight cases were obese and naloxone treatment did not reverse the respiratory depression during sleep12. Carvalho published a review in 2008 addressing the risk, monitoring and prevention of respiratory depression associated with neuroaxial morphine in the obstetric setting3. He summaries the risk and supports the need for observation up to 24 hours following neuraxial morphine, due to the duration of the depressed CO2 sensitivity. In addition, Carvalho commented on the present lack of efficient, simple and mother-friendly monitoring equipment; he supports RR, saturation and sedation monitoring3.\n\nPortable sleep test equipment and at-home polygraphy monitoring is commonly used for screening for obstructive sleep apnoea. This equipment assesses respiration, actual gas flow, and saturation, and transforms the information into indices: apnoea-hypopnea index (AHI) and oxygen desaturation index (ODI) using defined algorithms.\n\nThe aim of this observational study was to explore the use of portable respiratory polygraphy for monitoring of obese mothers for respiratory depression over the first night after CS with bupivacaine/morphine/fentanyl spinal anaesthesia.\n\n\nMethods\n\nThe study was approved by the Regional Ethical Review Board in Stockholm (2015/1257-31/2).\n\nA prospective postoperative observational study was conducted from December 2015 to October 2017. Parturients with BMI >30 kg/m2 at the first antenatal consultation, who planned for elective CS with low transverse incision performed using standard spinal anaesthesia, were included in the study by the anaesthetist, after informed verbal and written consent was obtained.\n\nPatients with language difficulties and known diagnosed obstructive sleep apnoea (OSA) receiving treatment, as those with continuous positive airway pressure or mouth guard, or any known contraindication to ITM were excluded.\n\nPatients were informed at the preanaesthetic consultation about spinal analgesia and that they would receive a mixture of local anaesthesia, bupivacaine, and two opioids, fentanyl and morphine, in order to optimize perioperative and postoperative anaesthesia, according to the standard routine at our department (Anaesthesia & Intensive Care Unit, Danderyds Hospital).\n\nSpinal anaesthesia was performed with the patient in sitting or left lateral position, at the anaesthetists’ preference, using a 25-gauge pencil-point needle. All patients received a mixture of heavy bupivacaine (11–12 mg), fentanyl (10 µg) and morphine (100 µg), according to standard routines for CS in our department. Preoperatively the patients received 1.5 g paracetamol orally, and paracetamol was continued postoperatively 1 g every six hours or 1.330g every eight hours. Ibuprofen (400 mg) was administered every eight hours. Additive medication to treat side effects, such as pain if numeric rating scale more than 3, nausea and vomiting or pruritus was administered according to the department's routines.\n\nRoutine monitoring in the postoperative and obstetric ward after spinal anaesthesia includes the following: checking for sedation, and if sedated counting RR every hour; pain by NRS/VAS; heart rate and blood pressure; control of bleeding; urine output; mobilization; and breast feeding. First mobilization, to stand by the bed, is usually encouraged at about 5–6 hours postoperatively. Urine catheter is normally removed after the first postoperative night.\n\nPatients were informed at the preanaesthetic consultation about extended postoperative monitoring in addition to routine monitoring: nasal catheter to measure expiration flow; finger probe to measure oxygen saturation; thoracic and abdominal strings to collect breathing movements for polygraphy registration; a portable OSAS breathing pattern monitor, Embletta (ResMed Sweden AB, Kista, Sweden)/Nox Sleep monitor (Nox Medical, Iceland); and a combined ear-probe for transcutaneous carbon dioxide (TcCO2)/oxygen saturation (SpO2) monitor (Tosca Radiometer Medical ApS, Denmark).\n\nApnoea was classified in accordance to the American Academy of Sleep Medicine (AASM) as a drop in the polygraphy peak signal excursion by ≥ 90% of pre-event baseline air-flow signal14. The breathing disturbance was classified as mild AHI 5–15, moderate 15–30 and severe >30. The duration of the ≥90% drop in sensor signal must be ≥10 seconds14. Hypopnea was classified by as a drop in the peak signal excursion by ≥30% of pre-event baseline14. The duration of the ≥30% drop in signal excursions must be ≥10 seconds14.\n\nNight-time respiratory monitoring device, that is polygraphy registration and Tosca as described above, was applied during rest/sleep during the first postoperative evening and night. For the 3–5 first hours postpartum, the patients were continuously observed awake in the postoperative department.\n\nAll patients answered a standardised ESS (Epworth Sleepiness Scale) questionnaire at time of enrollment.\n\nData is presented as the mean and standard deviation; categorical data are presented as frequencies. The study is explorative and observational, thus no power analysis has been conducted. Differences has been studied with Student’s t-test for continuous variables and Chi-squared test for categorical data. P<0.05 was considered significant. Data was analysed with StatView (v1.04) for MAC.\n\n\nResults\n\nForty mothers were invited to participate: 27 mothers consented but four of them had an early emergency CS delivery due to contractions, thus 23 mothers were included, but polysomnography registration failed in 3 (see Figure 1). Therefore, 20 mothers were included in analysis.\n\nIn all 20 mothers, the mean age was 35 ± 5 (24–43) years, mean BMI was 35 ± 4 (30–42), and mean ESS 6 ± 3 (0–12). For the ESS grade, 5 mothers had an ESS of <5, 12 had an ESS score between 5 and 10, and 3 scored >10.\n\nMean bed time during the polygraphic registration was 585 minutes (378–818). The mean registered SpO2 was 94 ± 1.3 (91–96) and mean nadir SPO2 71 ± 10 (49–81). In total, 4 mothers had a mean SpO2 <94 (91–93).\n\nMean AHI was 6.6 ± 5.2 (0–18.2) and mean ODI was 4.4 ± 3 (0–10.3). A total of 11 mothers had “normal” (<5) AHI, 7 had an AHI between 5 and 15, and 2 had an AHI 15–30. No mothers had an AHI >30. The longest apnoea duration was (mean) 30 ± 27 seconds, and mean longest hypopnea duration 55 ± 25 seconds.\n\nMean saturation was 94% (91–96) and four mothers had mean saturation between 90 and 94%, but no had a mean SpO2 < 90%. Nadir saturation was in mean 71% (49 – 81). In total, 11 mothers had an ODI <5, 8 had ODI between 5 to 10, and 1 mother had an ODI of 10.3. The 2 high AHI (15.3 and 18.2) mothers did not show high ODI or signs of hypercapnia on the transcutaneous CO2 registration.\n\nMean TcCO2 was 4.7 ± 0.3 (4.1–5.2) kPa, and mean of max TcCO2 was 5 ± 0.5 kPa. There were no TcCO2 >5.9 kPa. The pattern between AHI, ODI, BMI and ESS was overall scattered without correlation; Figure 2 describes the AHI and ODI pattern.\n\nNone of the mothers showed clinical signs or symptoms of severe respiratory depression as assessed by routine clinical monitoring.\n\n\nDiscussion\n\nWe found that two out of 20 mothers included in the present study had an AHI of >15 and none had an AHI defined as severe sleep apnoea. Mothers with a high AHI did not show typical high oxygen desaturation index or TcCO2 elevation. We did see frequent short episodes of oxygen saturation decrease, but we are unfortunately not able to assess whether these events were related to bradypnea or shallow breathing. We did not register any increase in TcCO2. The TcCO2 monitoring had a 15-minute averaging algorithm, thus it was not set for the detection of brief episodes of CO2 elevation. Respiratory depression typically progresses slowly3.\n\nStudies with polygraphy registration sleep apnoea signs, associated with ITM are sparsely performed. The effects of 30 mg oral morphine on patients with mild to moderate obstructive sleep apnoea has been investigated previously by Wang et al.15. They found that morphine may paradoxically improve sleep apnoea15. Similarly, Bernards et al. studied the effects of remifentanil infusion, and found a decrease in obstructive events, but a worsening in oxygenation during the infusion16. Cole et al. studied the respiratory effects of ITM (dose, 300 µg) in prospective randomized fashion among patients having knee replacement in spinal anaesthesia17. They found similarly to the present study that night time respiratory polygraphy is cumbersome and associated with high patient non-compliance17. Among the patients studied in that study, the incidence of apnoea and hypopnea episodes was not significantly different compared to the control group of patients that did not receive ITM17. Median mean oxygen saturation was, however, significantly lower among the ITM patients and the occurrence of “mild and moderate hypoxia” was also high in the ITM group. In another previous study, 45 obese patients undergoing elective bariatric surgery with general anaesthesia were monitored in a similar fashion with portable polygraphy equipment during the first postoperative night on the general ward; only two patients with an AHI >5 and only three with an ODI >5 was found18. Therefore, our findings, that registration during the first postoperative night following CS with spinal anaesthesia with a modest dose of morphine in obese mothers did not show any high incidence of AHI and ODI, might not be that surprising. It is also in line with a Cochrane review assessing the effects of opioid, hypnotic and sedating medications on sleep-disordered breathing in adults with obstructive sleep apnoea19. None of the studied drugs in the Cochrane review produced a significant increase in AHI or ODI and two trials have shown a beneficial effect on OSA19.\n\nHowever, Subramani et al. describes, in a recent paper in the British Journal of Anaesthesia, catastrophic events in patients related to obesity and sleep apnoea, stating that ‘Morbid obesity, male sex, undiagnosed OSA, partially treated/untreated OSA, opioids, sedatives, and lack of monitoring are risk factors for death or near-death events’20.\n\nWe cannot further comment on how and how long obese patients having ITM should be monitored. It seems still of importance to monitor respiration in patients at risk. Monitoring of AHI and ODI seems, however, not to be of major help. It may be that simple RR monitoring, TcCO2 measure and SpO2 are more feasible techniques21,22. Kopka et al. suggest that TcCO2 may be more effective in detecting respiratory depression compared to SpO2 when patients receive supplementary oxygen23. Ladha et al. studied oximetry after CS having 150 µg morphine intrathecal24. They found frequent mild desaturation events and increased risk in patients with obesity. Bauchat et al. studied TcCO2 tension and found that hypercapnia events (>50 mm Hg for ≥2-minute duration) occurred frequently in women receiving 150 μg ITM for post-caesarean analgesia; higher baseline TcCO2 readings were observed in women who had hypercapnia events25. Dalchow et al. studied both TcCO2 and SpO2 and found more frequent changes, hypercapnia as compared to desaturation26. They concluded, ‘The incidence of opioid-induced respiratory depression detected by TOSCA is higher than previously reported by other monitoring methods. TOSCA may have a role in detecting subclinical respiratory depression in the obstetric population. Further studies with a control population are needed’26. Patients at risk should of course be assessed prior to surgery/anaesthesia. Optimal screening method is however not well defined11.\n\nThere are several limitations with our study. It is merely an observational study, and we could only include 23 mothers. We had a high number of mothers that declined to participate after having been informed about the monitoring techniques. The portable polygraphy is intended for use in the home for sleep apnoea screening instead of being in-hospital for a full polysomnography. The equipment involves straps around the thorax and abdomen, nasal prongs and a pulseoximetry probe all connected with cables to the monitoring unit. We included mothers with a BMI between 30 and 42 (mean 35) and none of our mothers had a known sleep apnoea. Merely three had an ESS of more than 10. Higher BMI and higher number of patients, possibly with more signs and symptoms of sleep apnoea would have been of interest. We are not able to assess sleep time, whether the mothers studied were asleep or merely rested. A full polysomnography would be needed for further in depth analysis. One may however strongly question whether that is ethical in a mothers’ first night after caesarean section.\n\nIn conclusion, we found in this explorative study that portable polygraphy is cumbersome and many mothers decline its use. It seems also reasonable to conclude that although episodes of oxygen saturation decrease were not infrequently noticed, upper airway collapse, obstructive hypo/apnoea, role as risk factor for respiratory depression during the first night after caesarean section in spinal anaesthesia with addition of low dose intrathecal morphine even in obese mothers seems minor. However, further studies with a combination of RR monitoring, TcCO2 monitor and SpO2 seems warranted, especially in high risk mothers. Preoperative screening in obese patients, at risk for sleep breathing disorder, is of course of value.\n\n\nData availability\n\nDataset 1: Raw data for the study polygraphy on the first night after caesarean section in spinal anaesthesia with morphine in obese mothers by Hein et al. The three patients who were excluded from analysis are highlighted. doi, 10.5256/f1000research.13206.d18538827",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study has been supported by the Department of Anaesthesia, Danderyds Hospital. No external grants have been received.\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\nPalmer CM, Emerson S, Volgoropolous D, et al.: Dose-response relationship of intrathecal morphine for postcesarean analgesia. Anesthesiology. 1999; 90(2): 437–44. PubMed Abstract | Publisher Full Text\n\nDahan A, Aarts L, Smith TW: Incidence, Reversal, and Prevention of Opioid-induced Respiratory Depression. Anesthesiology. 2010; 112(1): 226–38. PubMed Abstract | Publisher Full Text\n\nCarvalho B: Respiratory depression after neuraxial opioids in the obstetric setting. Anesth Analg. 2008; 107(3): 956–61. PubMed Abstract | Publisher Full Text\n\nCrowgey TR, Dominguez JE, Peterson-Layne C, et al.: A retrospective assessment of the incidence of respiratory depression after neuraxial morphine administration for postcesarean delivery analgesia. Anesth Analg. 2013; 117(6): 1368–70. PubMed Abstract | Publisher Full Text\n\nHein A, Gillis-Haegerstrand C, Jakobsson JG: Neuraxial opioids as analgesia in labour, caesarean section and hysterectomy: A questionnaire survey in Sweden [version 2; referees: 2 approved]. F1000Res. 2017; 6: 133. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGehling M, Tryba M: Risks and side-effects of intrathecal morphine combined with spinal anaesthesia: a meta-analysis. Anaesthesia. 2009; 64(6): 643–51. PubMed Abstract | Publisher Full Text\n\nSultan P, Gutierrez MC, Carvalho B: Neuraxial morphine and respiratory depression: finding the right balance. Drugs. 2011; 71(14): 1807–19. PubMed Abstract | Publisher Full Text\n\nGeorge JA, Lin EE, Hanna MN, et al.: The effect of intravenous opioid patient-controlled analgesia with and without background infusion on respiratory depression: a meta-analysis. J Opioid Manag. 2010; 6(1): 47–54. PubMed Abstract | Publisher Full Text\n\nKo S, Goldstein DH, VanDenKerkhof EG: Definitions of “respiratory depression” with intrathecal morphine postoperative analgesia: a review of the literature. Can J Anaesth. 2003; 50(7): 679–88. PubMed Abstract | Publisher Full Text\n\nShapiro A, Zohar E, Zaslansky R, et al.: The frequency and timing of respiratory depression in 1524 postoperative patients treated with systemic or neuraxial morphine. J Clin Anesth. 2005; 17(7): 537–42. PubMed Abstract | Publisher Full Text\n\nde Raaff CA, De Vries N, van Wagensveld BA: Obstructive sleep apnea and bariatric surgical guidelines: summary and update. Current Opinion in Anesthesiology: Post Author Corrections: November 15, 2017. 2017. Publisher Full Text\n\nAbouleish E, Rawal N, Rashad MN: The addition of 0.2 mg subarachnoid morphine to hyperbaric bupivacaine for cesarean delivery: a prospective study of 856 cases. Reg Anesth. 1991; 16(3): 137–40. PubMed Abstract\n\nKato R, Shimamoto H, Terui K, et al.: Delayed respiratory depression associated with 0.15 mg intrathecal morphine for cesarean section: a review of 1915 cases. J Anesth. 2008; 22(2): 112–6. PubMed Abstract | Publisher Full Text\n\nBerry RB, Budhiraja R, Gottlieb DJ, et al.: Rules for scoring respiratory events in sleep: update of the 2007 AASM Manual for the Scoring of Sleep and Associated Events. Deliberations of the Sleep Apnea Definitions Task Force of the American Academy of Sleep Medicine. J Clin Sleep Med. 2012; 8(5): 597–619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang D, Somogyi AA, Yee BJ, et al.: The effects of a single mild dose of morphine on chemoreflexes and breathing in obstructive sleep apnea. Respir Physiol Neurobiol. 2013; 185(3): 526–32. PubMed Abstract | Publisher Full Text\n\nBernards CM, Knowlton SL, Schmidt DF, et al.: Respiratory and sleep effects of remifentanil in volunteers with moderate obstructive sleep apnea. Anesthesiology. 2009; 110(1): 41–9. PubMed Abstract | Publisher Full Text\n\nCole PJ, Craske DA, Wheatley RG: Efficacy and respiratory effects of low-dose spinal morphine for postoperative analgesia following knee arthroplasty. Br J Anaesth. 2000; 85(2): 233–7. PubMed Abstract | Publisher Full Text\n\nWickerts L, Forsberg S, Bouvier F, et al.: Monitoring respiration and oxygen saturation in patients during the first night after elective bariatric surgery: A cohort study [version 2; referees: 2 approved]. F1000Res. 2017; 6: 735. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMason M, Cates CJ, Smith I: Effects of opioid, hypnotic and sedating medications on sleep-disordered breathing in adults with obstructive sleep apnoea. Cochrane Database Syst Rev. 2015; (7): CD011090. PubMed Abstract | Publisher Full Text\n\nSubramani Y, Nagappa M, Wong J, et al.: Death or near-death in patients with obstructive sleep apnoea: a compendium of case reports of critical complications. Br J Anaesth. 2017; 119(5): 885–99. PubMed Abstract | Publisher Full Text\n\nFlisberg P, Jakobsson J, Lundberg J: Apnea and bradypnea in patients receiving epidural bupivacaine-morphine for postoperative pain relief as assessed by a new monitoring method. J Clin Anesth. 2002; 14(2): 129–34. PubMed Abstract | Publisher Full Text\n\nMcCormack JG, Kelly KP, Wedgwood J, et al.: The effects of different analgesic regimens on transcutaneous CO2 after major surgery. Anaesthesia. 2008; 63(8): 814–21. PubMed Abstract | Publisher Full Text\n\nKopka A, Wallace E, Reilly G, et al.: Observational study of perioperative PtcCO2 and SpO2 in non-ventilated patients receiving epidural infusion or patient-controlled analgesia using a single earlobe monitor (TOSCA). Br J Anaesth. 2007; 99(4): 567–71. PubMed Abstract | Publisher Full Text\n\nLadha KS, Kato R, Tsen LC, et al.: A prospective study of post-cesarean delivery hypoxia after spinal anesthesia with intrathecal morphine 150μg. Int J Obstet Anesth. 2017; 32: 48–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBauchat JR, McCarthy R, Fitzgerald P, et al.: Transcutaneous Carbon Dioxide Measurements in Women Receiving Intrathecal Morphine for Cesarean Delivery: A Prospective Observational Study. Anesth Analg. 2017; 124(3): 872–8. PubMed Abstract | Publisher Full Text\n\nDalchow S, Lubeigt O, Peters G, et al.: Transcutaneous carbon dioxide levels and oxygen saturation following caesarean section performed under spinal anaesthesia with intrathecal opioids. Int J Obstet Anesth. 2013; 22(3): 217–22. PubMed Abstract | Publisher Full Text\n\nHein A, Jakobsson JG: Dataset 1 in: Portable respiratory polygraphy monitoring of obese mothers the first night after caesarean section with bupivacaine/morphine/fentanyl spinal anaesthesia. F1000Research. 2017. Data Source"
}
|
[
{
"id": "28813",
"date": "19 Jan 2018",
"name": "Nina Stockfleth Buch",
"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 observational study by Hein and Jacobsen examines the use of a portable respiratory polygraphy device for monitoring of obese mothers for respiratory depression the first night after caesarean section performed with bupivacaine/morphine/fentanyl spinal anaesthesia. The topic is interesting and relevant, as an increasing number of parturients is expected to present with high antenatal BMI in the future. A total of 20 women were monitored the first night after caesarean section. The authors conclude that the use of the polygraphy device is cumbersome and that obstructive events and episodes of desaturation were not commonly synchronized.\n\nComments: Abstract: For the reader not familiar with this technique or the apnoe/hypopnea index (AHI), it is very difficult to interpret the results, e.g. AHI values of 5-15? And what does an ODI of 4.4 mean? For these reasons, the abstract is simply difficult to read. The introduction is a bit too long. The authors should consider omitting e.g. the sentence starting with Dahan in the first paragraph, and the second paragraph on definition of respiratory depression.\nMethods and results: Parts of the text could be written more clearly, e.g. page 4 top: Additive medication to treat side effects, such as pain..??? Is pain a side effect? The description of AHI, ODI etc. is very technical. Again, it is difficult for the reader not familiar with the technique, to interpret the values and relate them to the clinical setting. What is the clinical relevance of mild AHI/ ODI, mean nadir SpO2, mean and max TcCO2? The same applies to the ESS score: is a score of e.g. 5 good or bad? Did the women receive opioids during the study period? (Respiratory depression could be caused by ex. oral opioids).\nDiscussion: Could be shortened. Figure 2 is difficult to read. Could some of the results be presented in a table? The high number of abbreviations makes the paper more difficult to read. The cohort described/studied by Subramani et al. (reference 20) might not be comparable with the group studied in this paper.\nIn summary, a nice paper on an interesting and relevant topic but it would benefit from a revision.\n\nReview performed by MD Nina Stockfleth Buch, Department of Anesthesiology, Aarhus University Hospital, Denmark (approved by professor Lone Nikolajsen, Department of Anesthesiology, Aarhus University Hospital, Denmark).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3367",
"date": "23 Jan 2018",
"name": "Jan Jakobsson",
"role": "Author Response F1000Research Advisory Board Member",
"response": "Comments to reviewers at Department of Anesthesiology, Aarhus University Hospital, Denmark).Thank you for effective review of our paper and important comments and remarks. We used the America Academy of Sleep Medicine definitions, that the apnea/hypopnea index, the number of apnea/hypopnea events registered per hour are graded into three categories of Obstructive Sleep Apnea (OAS): Mild OSA: AHI of 5-15, Moderate OSA: AHI of 15-30 and Severe OSA: AHI of more than 30. Zero to 4 episodes, and AHI of<5 is commonly assessed as normal. According to the 2007 guidelines from the American Academy of Sleep Medicine, any event with a 3 percent drop in blood oxygen levels is counted as an oxygen desaturation, and the index is the number of desaturation episodes per hours. The screening and assessment for sleep apnea is updated in JAMA 2017 and criteria have not changed. The equipment software is screening for both the AHI and ODI. The program also provide each desaturation episode minimum blood oxygen saturation SpO2 level measured, the oxygen desaturation episode nadir.We consider our findings as signs of normal/mild breathing pattern; all but 2 mothers showed an AHI well within normal and mild ranges and 2 had values in the moderate zone (15.3 and 18.2, thus in the lower edge of moderate 15-39). The apnea/hypopenea episodes had duration of 11 to 111 seconds with a lowest SpO2 60 mean 73 % (60 – 81).None of the mothers showed a transcutaneous reading of above 5 kPa, thus none of our monitored mothers had a transcutaneous CO2 above normal.The questionnaires used ESS for screening has been the standard tool at our department. We used the recommended grading;0-5 Lower Normal Daytime Sleepiness6-10 Higher Normal Daytime Sleepiness11-12 Mild Excessive Daytime Sleepiness13-15 Moderate Excessive Daytime Sleepiness16-24 Severe Excessive Daytime SleepinessAlso the ESS scores were low 3 mothers scored more than 10 commonly assessed as “threshold value”. The description of pain and additional medication should be revised, to read easier.We provide our assessment of the “seemingly normal findings” from the polysomnography recordings in the first sentences of the discussion. We strongly believe that our study show that polysomnography is cumbersome and complex to use postoperatively and does not add valuable information. It is indeed intended for assessment of sleep apnea and is not primarily a monitoring device, it include no alarms to alert in case of major deviation it merely records and compile data for post recording assessment. Respiratory rate, SpO2 and CO2 monitoring seems of much more value. Preoperative assessment should of course be performed eventually including polysomnography in at risk patient . If positive findings CPAP should be recommended and used in conjunction to surgery/anesthesia.We will make a version 2 as soon as we have and additional referee report.Referenceshttps://aasm.org/resources/factsheets/sleepapnea.pdfhttp://epworthsleepinessscale.com/about-the-ess/ Jonas DE, Amick HR, Feltner C, Weber RP, Arvanitis M, Stine A, Lux L, Harris RP. Screening for Obstructive Sleep Apnea in Adults: Evidence Report and Systematic Review for the US Preventive Services Task Force. JAMA. 2017 Jan 24;317(4):415-433.Swedish Council on Health Technology Assessment. Obstructive Sleep Apnoea Syndrome: A Systematic Literature Review [Internet]. Stockholm: Swedish Council on Health Technology Assessment (SBU); 2007 Jun.Collop NA, Anderson WM, Boehlecke B, Claman D, Goldberg R, Gottlieb DJ, Hudgel D, Sateia M, Schwab R; Portable Monitoring Task Force of the American Academy of Sleep Medicine. Clinical guidelines for the use of unattended portable monitors in the diagnosis of obstructive sleep apnea in adult patients. Portable Monitoring Task Force of the American Academy of Sleep Medicine. J Clin Sleep Med. 2007 Dec 15;3(7):737-47.Madhusudan P, Wong J, Prasad A, Sadeghian E, Chung FF. An update on preoperative assessment and preparation of surgical patients with obstructive sleep apnea. Curr Opin Anaesthesiol. 2018 Feb;31(1):89-95.Roesslein M, Chung F. Obstructive sleep apnoea in adults: peri-operative considerations: A narrative review. Eur J Anaesthesiol. 2018 Jan 2. doi: 10.1097/EJA.0000000000000765. [Epub ahead of print]"
}
]
},
{
"id": "28494",
"date": "26 Jan 2018",
"name": "Sunil Kumar Chauhan",
"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\nThe aim of this study was to assess monitoring capabilities of portable respiratory polygraph device in detecting respiratory depression due to intrathecal morphine in parturients for lower segment caesarean section. Although the number of factors causing respiratory depression is not just limited to intrathecal morphine, obesity, residual effect of anaesthesia, immobilisation and abdominal surgery itself are contributors for respiratory depression. The device used for the study seems good for diagnosing respiratory depression in community for patients with OSA, but its usefulness in peri-operative setting is still to be proved.\nComments:\nThis study done on exploring the optimal monitoring device to diagnose respiratory depression with intrathecal morphine for LSCS in high BMI parturients, does not rule out undiagnosed OSA patients before abdominal surgery esp. when the patients were enrolled during the first antenatal visit and since that time till surgery patient had undergone changes in their BMI and usual respiratory physiological changes.\n\nThe device respirator is not adequately described esp. it’s functioning as well it’s methodology to diagnose respiratory depression is seriously questionable as it fails to show basic correlation between AHI and ODI.\n\nThe study design is prospective and observational, but would be of more use if it has compared with routine monitoring in subset of patients with intrathecal morphine for LSCS, to give idea of usefulness of the device.\n\nMean bedtime during which monitoring was done was never described as whether patient was asleep or was merely resting, which could seriously alter the results.\n\nThere was no mention of type of breakthrough analgesia (opioid or non opioid) used postoperatively which could alter the results.\n\nThe sample size to comment on correlation between AHI and ODI seems quite small to be conclusive and reproducible.\n\nThe results showed comparison with routine monitoring but data in support of routine monitoring was not provided.\n\nAgain high AHI and non reproducible ODI raises serious concerns about sensitivity of portable respirator.\n\nAs far as studies statistical significance I have limited knowledge for which input from statistician would be highly recommended.\n\nI agree with the conclusion by authors that device is cumbersome with no reproducible results until proven otherwise by studying with large sample size in patients with preoperative screening in obese parturients (with already decreased respiratory reserve ) at risk of OSA and comparing with routine monitoring.\n\nDiscussion:\n\nThis study has some minor limitations in view of enrollments during first antenatal visit It would be better if more clarity with data was shown for duration of sleep rather than rest.\n\nPost operative analgesia for breakthrough pain needs to be explained.\n\nAny study to quote for the efficacy of device would be of great value before using it in peri-operative setting.\n\nAny comorbidity like PIH or chest infections before surgery or any major fluid shift with any major blood losswas not ruled out or if it was then not declared in the study design which may easily alter the results.\n\nPost operative analgesia for breakthrough pain needs to be explained.\n\nAny study to quote for the efficacy of device would be of great value before using it in peri-opera- tive setting.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3374",
"date": "07 Feb 2018",
"name": "Jan Jakobsson",
"role": "Author Response F1000Research Advisory Board Member",
"response": "Dear Referee, thank you for important and adequate comments and criticism. The study is small and “explorative” – we still feel that it is of importance to present the available findings in structured fashion. We have tried to address all your comments and queries in Version 2 that has just been uploaded in the manuscript management system. Comments:This study done on exploring the optimal monitoring device to diagnose respiratory depression with intrathecal morphine for LSCS in high BMI parturients, does not rule out undiagnosed OSA patients before abdominal surgery esp. when the patients were enrolled during the first antenatal visit and since that time till surgery patient had undergone changes in their BMI and usual respiratory physiological changes.We have in version 2 put strong emphasis on the need for further studies in pregnant women per se and especially in obese mothers. Sleep apnea should be considered and assessed for. The device respirator is not adequately described esp. it’s functioning as well it’s methodology to diagnose respiratory depression is seriously questionable as it fails to show basic correlation between AHI and ODI.We did use a standard ambulatory monitor, the Embletta and the NOx and subsequent the ResMed Sweden AB, Kista, Sweden analysis program with manual screening. The accuracy in the program has been assessed as adequate by the Swedish Agency for Health Technology Assessment and Assessment of Social Services. We did see a discrepancy between ODI and AHI, the exact reason for this is to us unclear. The group may have had desaturation episodes due to low respiratory rate, atelectasis and thus alveolar hypoventilation, but we cannot exclude a monitoring error. The study design is prospective and observational, but would be of more use if it has compared with routine monitoring in subset of patients with intrathecal morphine for LSCS, to give idea of usefulness of the device.Agree, this would be of interest for further studies, but considering the cumbersomeness of the polygraphy I doubt it is worth the effort. I would much prefer preopstudies trying to find patients at risk during pregnancy. We have expanded on this is abstract, introduction and discussion. Mean bedtime during which monitoring was done was never described as whether patient was asleep or was merely resting, which could seriously alter the results.Indeed; this is also one of limitations, we do not have any objective measure of real sleep time, merely 1st night registration. There was no mention of type of breakthrough analgesia (opioid or non opioid) used postoperatively which could alter the results.The mothers provided rescue analgesics preferentially buprenorphine is added. All mothers had paracetamol regular prescribed. The sample size to comment on correlation between AHI and ODI seems quite small to be conclusive and reproducible.We have purposely not made any statistical assessment of AHI and OFI findings. This is a explorative observational study and we fully agree there is not data to make any firm statistical conclusions The results showed comparison with routine monitoring but data in support of routine monitoring was not provided. Again high AHI and non reproducible ODI raises serious concerns about sensitivity of portable respirator.We cannot give any firm explanation to the discrepancy observed. We have further commented on this finding and we feel that there may be patient factors to possibly explain the finding and there are other studies also discussing discrepancy between ODI/AHI. Novel parameters for evaluating severity of sleep disordered breathing and for supporting diagnosis of sleep apnea-hypopnea syndrome. Kulkas A, Tiihonen P, Eskola K, Julkunen P, Mervaala E, Töyräs J. J Med Eng Technol. 2013 Feb;37(2):135-43. As far as studies statistical significance I have limited knowledge for which input from statistician would be highly recommended.We have purposely not made any statistical assessment of AHI and OFI findings. This is a explorative observational study and we fully agree there is not data to make any firm statistical conclusionsI agree with the conclusion by authors that device is cumbersome with no reproducible results until proven otherwise by studying with large sample size in patients with preoperative screening in obese parturients (with already decreased respiratory reserve ) at risk of OSA and comparing with routine monitoring.Discussion: This study has some minor limitations in view of enrollments during first antenatal visit It would be better if more clarity with data was shown for duration of sleep rather than rest.Extensively commented in version 2Post operative analgesia for breakthrough pain needs to be explained.Added in version 2Any study to quote for the efficacy of device would be of great value before using it in peri-operative setting.Added in the Method sectionAny comorbidity like PIH or chest infections before surgery or any major fluid shift with any major blood loss was not ruled out or if it was then not declared in the study design which may easily alter the results.Mothers were considered “healthy” no obvious signs of infection or fluid overload was observed. Post operative analgesia for breakthrough pain needs to be explained.AddedAny study to quote for the efficacy of device would be of great value before using it in peri-opera- tive setting.On behalf of the authors Jan G Jakobsson"
}
]
}
] | 1
|
https://f1000research.com/articles/6-2062
|
https://f1000research.com/articles/7-156/v1
|
07 Feb 18
|
{
"type": "Research Article",
"title": "Comparing manual and mechanically assisted manipulations of the thoracic spine in neck pain patients: A pilot study",
"authors": [
"Anke Langenfeld",
"B. Kim Humphreys",
"Rob A. de Bie",
"Jaap Swanenburg",
"B. Kim Humphreys",
"Rob A. de Bie",
"Jaap Swanenburg"
],
"abstract": "Background: Manipulations of the thoracic spine are a common treatment option in patients with neck pain. This approach avoids the risk of cervical arterial dissection. Currently there are different options available which have been evaluated for their efficacy. The aim of this study was to assess short- and long-term effects of two different methods of manipulating the thoracic spine in combination with a standardized exercise program for neck pain. Methods: This pilot study included patients who were over 18 years of age, able to speak and read German or English, had acute or chronic neck pain, and had not previously undergone manual therapy for the thoracic spine. Patients were recruited from private physical therapy practices. Patients were randomly assigned to two treatment groups by using block randomization. The objective was to compare the effects of manually performed manipulations and electromechanical manipulations on the thoracic spine in patients with neck pain. Primary outcome was the visual analogue pain rating scale. Secondary outcomes included Neck Disability Index, European Quality of Life scale, and Patients’ Global Impression of Change Scale. Results: Ten patients were recruited. Five patients received manual manipulations, and five received electromechanical manipulations. Both groups showed an improvement in pain scores (VAS) (X2 (5) = 14.161, p = 0.015) with no difference between the groups. There were no significant changes in the secondary outcomes. The electromechanical (Impulse iQ®) manipulations group showed a clinically relevant reduction in the neck disability index. Conclusion: Both manual and electromechanical manipulations are well tolerated, and show that they can be both successful treatments for neck pain in combination with exercises. Thoracic manipulations seem to be beneficial for the patient’s complaint of neck pain, while electromechanical manipulations seem to be able to reduce neck disability.\nTrial registration: Current Controlled Trials ISRCTN88585962, registered in January 2013.",
"keywords": [
"Manipulation",
"Manual",
"Electromechanical",
"Thoracic spine",
"Neck pain"
],
"content": "Introduction\n\nNeck pain ranks fourth among the most common musculoskeletal complaints worldwide1. Although often not disabling, most patients with neck pain will not experience a complete resolution of their problem2. Furthermore, various possible causes for neck pain exist2. Neck pain symptoms are often temporary and can be treated successfully with nonsurgical care such as medication, physical therapy, and manipulations3–5. Best studied treatments are cervical spine manipulations in conjunction with exercise therapy6,7. Although there is conflicting evidence, cervical spinal manipulations are suspected to cause serious adverse events such as cervical artery dissection (CAD) (see The International Federation of Orthopaedic Manipulative Physical Therapists (IFOMPT) cervical framework)8–11. An alternative approach to cervical manipulations, which has been reviewed extensively, is manipulations of the thoracic spine12–23.\n\nManipulations may be delivered either manually (high velocity low amplitude) or mechanically using different spring-loaded or electromechanical devices24–27. However, manual forces given by the same practitioner may vary somewhat for each manipulation session and between practitioners28. Forces applied by an electromechanical device are more consistent24. Thus far, only three studies have compared between manual and mechanical manipulations26,27,29. In 2001, Wood and colleagues evaluated neck pain patients. They compared mechanical force, manually assisted (MFMA) adjustments (activator II adjusting instrument) to high velocity, and low-amplitude manipulation directed to a dysfunctional cervical spinal motion unit26. Shearar et al. (2005) evaluated patients with recent low back pain who were diagnosed with sacroiliac joint syndrome at the initial assessment. Patients were either treated with manually delivered chiropractic adjustments or an MFMA adjustment using an activator adjusting instrument27.\n\nSchneider et al. worked with acute low back pain patients in which one group received treatment by activator method proficiency-certified chiropractors while the other two groups used side posture thrust manipulation29. The abovementioned studies suggest that both ways of delivering manipulations are beneficial to reduce the patients’ pain and disability. The latest technical development of such an electromechanical device, the Impulse iQ®, incorporates a feedback loop to adjust the manipulation force to the resonance of the patient’s spine. Yet, no studies have investigated the effectiveness of this device.\n\nThe objective of our pilot study was to compare short- and long-term effects of two different ways of manipulating the thoracic spine in combination with a standardized exercise program for neck pain.\n\n\nMethods\n\nA pilot study was conducted to compare the effects of two different methods of manipulation (manual manipulation vs. electromechanical manipulation) on the thoracic spine for patients with neck pain. A complete study protocol for a randomized controlled trial was published in May 201530. The study received ethical approval from the Ethics Commission of the Canton of Zurich (2012-0248) and was registered at Current Controlled Trials (ISRCTN88585962). A completed CONSORT checklist can be found in Supplementary File 1.\n\nEvery participant received all relevant patient information in advance. When a potential participant had read the information and was willing to participate, he or she signed the informed consent form. Participants were recruited from, May 2013 until August 2015, in private physical therapy practices in Zurich, Switzerland. Inclusion criteria were the presence of acute or chronic neck pain grade I or II31, age 18 years or older, able to speak and read German or English, no previous manual therapy of the thoracic spine, and interest in participating in the study.\n\nPatients were excluded if they had severe disorders of the cervical spine, such as disk prolapse, spinal stenosis, postoperative conditions in neck and shoulder areas, a history of severe trauma, spasmodic torticollis, frequent migraine headaches, peripheral nerve entrapment, fibromyalgia, shoulder diseases (causing reduced mobility of the joint, e.g., fractures, adhesive capsulitis), inflammatory rheumatic diseases, osteoporosis, or cancer or if they were currently undergoing legal procedures related to their neck pain.\n\nAdditional evaluations (history taking and physical examination) of the participant were added after baseline measurements to confirm eligibility of the participant to be included in the study population30.\n\nA clinician not involved in the study randomized the participants independently into one of the two groups. Block randomization (20 blocks of 4) was carried out using a computer-based randomization program. The original list was stored in an opaque and sealed envelope, not accessible to the therapist delivering the manipulations. To reduce intervention bias, 80 individually numbered opaque and sealed envelopes were prepared, and the therapist received the assigned envelope immediately before each participant was to receive the manipulation. Additionally, after the participants completed the questionnaires, they were put into an opaque envelope that was individually and immediately sealed by the patient. The therapist was unaware of the outcome. The anonymous data were entered into the database and were untraceable to the participating patient.\n\nEach patient underwent three treatment sessions, with 4 days interval between each session. Additionally, during the first session, all participants were instructed to participate in an exercise program, which was to be completed as soon as possible. To evaluate compliance of the participants, they received a journal and were asked to mark the days that they had completed the required exercises. Follow-up assessments were conducted at 6 weeks, 3 months, and 6 months after the initial treatment. Patients received the questionnaires by mail, including prepaid envelopes, to facilitate return of the completed forms. An extensive explanation of the treatments can be found elsewhere30.\n\nFor the manually performed manipulations, the patient was supine. The therapist’s hand was placed under the thoracic spine using a pistol grip (the fingers positioned with the index finger straightened and fingers 3 to 5 flexed). The patient’s forearms were crossed in front of their chest. The force was applied against the therapist’s flexed fingers and the thenar eminence and slightly cranial to the transverse process of the caudal vertebrae of the segment to be treated32.\n\nFor the electromechanical manipulations, the Impulse iQ® (Neuromechanical Innovations, Chandler, AZ, USA) was used. The patient was prone on a treatment table, with arms parallel to the body, in a relaxed position. Before the treatment began, the patient was instructed that they would hear a rattling sound that indicated the treatment thrusts conducted by the device and a beep to signify the end of treatment. The Impulse iQ was placed onto the vertebrae previously identified as the painful segment. A double stylus and middle force setting (peak force = 200 N), as recommended by the manufacturer for the treatment of the thoracic spine, was used33. The device recorded and analyzed the spinal acceleration response each time a thrust was delivered by using the in-built firmware. To begin, the machine produced a series of repetitive thrusts while monitoring the acceleration response. If the response improved, the treatment thrusts continued up to 3 more seconds. If the acceleration response was negative (flat line or decrease), the thrusts delivered by the instrument ceased.\n\nAfter the first treatment session, all patients were introduced to the home exercise program and asked to perform this program once a day for 6 weeks. Compliance with the home exercise program was assessed using a training journal as follows: (1) retraining the craniocervical muscles (e.g., training the holding capacity of the deep neck flexors and head lift), (2) retraining the scapular muscles, and (3) eye/head coordination34,35. For a complete overview of the study`s course see Figure 1.\n\nBaseline measures included age, weight, height, and the European Quality of Life 5 Dimensions 5 Levels (EQ-5D-5L). Furthermore, each patient’s expectation of treatment outcome (the treatment he or she expected to be more beneficial) was assessed at the beginning of the study. The primary outcome measure was the Visual Analogue Pain rating scale (VAS) that assesses pain intensity level36–39. The VAS was completed before and after every treatment session and at all follow-up points. Secondary outcomes were the Neck Disability Index (NDI) and the Patients Global Impression of Change Scale (PGIC)40–44.\n\nThe NDI is a self-reported questionnaire to evaluate disability. It consists of 10 items that score from zero to five. The higher the total score, the higher is the disability level of the patient41. The NDI’s minimal clinical important difference (MCID) in patients with neck pain ranges from 3.5 to 9.5, which mainly depends on the patient characteristics45–47. Pereira et al. (2015) state that in patients with chronic neck pain, the score has to be above 5.5 points, otherwise it is clinically irrelevant45,48,49. Before the second, third treatments and all follow-up assessment, the patients were asked about their global impression of change, using the PGIC50. The PGIC is a valid outcome measure that is based on a seven-point Likert scale51. Its purpose is to obtain a patient’s report of improvement over time during treatment. The scale ranges from much better, better, somewhat better, no change, somewhat worse, worse, and much worse. Much better is rated as 7 and much worse as 144. It can be used to dichotomize participants into two groups: improved (ratings of 6 and 7) and not improved (ratings 1 to 5).\n\nThe data were stored and analyzed using the IBM SPSS 21 statistical software package (SPSS, Inc., Chicago, IL). Descriptive statistics were used to describe patient characteristics. Collected data were checked for normal distribution using the Shapiro-Wilk Test. The test identified that the scores for VAS and NDI were normally distributed. Because of outliers, the Friedman test was used as a nonparametric statistical test to retain all data points. The goal was to determine whether there were any statistically significant differences between the distributions of three or more related groups. Furthermore, pairwise comparisons were performed (SPSS Statistics, 2012) with a Bonferroni correction for multiple comparisons.\n\n\nResults\n\nThe study included two male and eight female participants. Because of unknown causes, one participant dropped out of the study after the first treatment. Nine participants had experienced chronic neck pain problems (>90 days), and one patient had acute problems (<90 days) (please see Table 4). Two participants had co-morbidities such as low back pain and coccydynia. Six participants believed that the conventional (manual) manipulation would be more successful, while four believed the electromechanical manipulation would be superior. Five participants received a manual treatment, and the remaining five received an electromechanical treatment. Three participants identified the third thoracic vertebra as the most painful and two identified the seventh thoracic vertebra. The second, fourth, fifth, sixth, and the tenth thoracic vertebrae were named by one patient as the most painful ones. Table 1 presents the demographic data and baseline measurements.\n\nBoth groups showed a significant improvement in pain scores (Friedman test, VAS Χ2 (5) = 14.161, p = 0.015). There were no statistically significantly differences between the two treatment groups: electromechanical X2(5) = 7.701, p = 0.174 / manual X2(5) = 7.774, p = 0.169. Bonferroni correction post hoc analysis revealed statistical differences in the rating from follow-up measurements at 3 months (median = 6.00) from the first appointment (median = 26.00) (p = 0.008). VAS scores at the baseline reduced by 11% at second treatment, 27% at third treatment, 54% after 6 weeks, 77% after 3 months, and 42% after 6 months. The % change of pain is shown in Table 2.\n\nBecause of outliers, the Friedman test was used for analyzing the NDI. There was no significant change (X2 (5) = 10.562, p = 0.061). No significant change in NDI was observed between the two groups (electromechanical X2 (5) = 5.234, p = 0.388 / manual group X2 (5) = 5.546, p = 0.232). The change over time (median) is shown in Table 3.\n\nAfter the first treatment, four out of nine patients rated themselves as improved, five patients after the second treatment, three patients after 6 weeks, two patients after 3 months, and three patients after 6 months. All other participants stated that they had no improvement, but the pain did not get worse. In total, there were 10 ratings of improvement in the electromechanical manipulation group and 7 in the manual manipulation group (see Table 4 for a detailed rating).\n\nOverall, four participants had additional treatment during the 6 months of follow-up. After 3 months of treatment, two participants visited a physical therapist and one participant received acupuncture once a week. At the 6 months survey, another participant received chiropractic treatment (three times) and one continued to visit a physical therapist, if the neck pain was too severe.\n\n\nDiscussion\n\nThe main goal of our pilot study was to compare short- and long-term effects of two different ways of manipulating the thoracic spine in combination with a standardized exercise program for patients with a main complaint of neck pain. In this study, the main outcome was pain intensity using the VAS. We found a significant reduction in neck pain for all patients, but not between the groups. Additionally, there was a tendency and a clinically relevant decrease in neck disability in accordance with the report of Pereira and colleagues that state a reduction of 5.5 points on the NDI in patients with chronic neck pain is a minimal clinically important difference49. In particular, the electromechanical group showed a decrease in their VAS scoring of 5.5 after 6 weeks and after 3 months. Although this did not reach statistical significance, we were able to show a tendency for improvement in this group.\n\nHowever, a wear off in VAS reduction was observed at 6 months. The follow-up of 6 months could have been too long. Patients may have forgotten the details of the interventions. Four of the patients had additional therapy during the follow-up period. Since our groups were small, this might have influenced the outcomes. One suggestion for further research is to use a paper-based or electronic diary to report all possible relevant incidents and interventions52,53.\n\nTo our knowledge this is the first study that investigated the short- and long-term outcome of electromechanical manipulation. None of the other studies, which were identified during literature review, assessed long-term outcomes of a device with a feedback loop26,27,29. Other studies had a follow-up period that lasted only 4 weeks28. Huggins and colleagues (2012) postulated the need for more studies evaluating activator adjusting instruments and their long-term outcomes54. Patients from previous studies received more treatment than those in our study. Our study had three treatment sessions as recommended by former clinical prediction guidelines55. Wood et al. (2001) and Schneider et al. (2011) had eight treatments sessions in 4 weeks26,29,58. Shearar et al. (2005) used four treatments over a 2-week period27. Even with the fewer treatment sessions, we could reduce pain significantly. However, in this study, an important difference compared to the other studies is the addition of exercises with the spinal manipulation treatment. The combination of exercise and manipulation is currently the best evidence-based approach for neck pain6,7.\n\nOne important reason for conducting this study relates to the question on cervical spinal manipulation and serious adverse events such as cervical artery dissection. (see Australian Physiotherapy Association guidelines for vertebrobasilar insufficiency)8,56. There is a debate in the literature referring to these events, and this serious complication should be considered if a patient is going to be treated with cervical spinal manipulation (see Australian Physiotherapy Association guidelines for vertebrobasilar insufficiency)8,57–63. In our study, no serious adverse events occurred. This suggests the treatment of patients with neck pain by manual or electromechanical manipulation of the thoracic spine may provide another treatment option.\n\nFrom the findings of our pilot study, we calculated the sample size for each time point of a RCT by using the formula 2 × [(1.96 + 0.842)2 × SD2]/12264,65. The calculation revealed that a sample size of 20 per treatment arm at the first time point would be sufficient to show significant differences in outcomes. However, the sample size requirements increased to 24 participants at 6 weeks and 57 participants at 6 months. For a two group pre-test / post-test / follow-up design, a total sample size of 114 participants will be sufficient to reach result with 80% power at an α-level of 0.05.\n\n\nLimitation\n\nOne obvious limitation was the small sample size of the study. One recruitment problem was to find patients who had not received previous manual therapy. Recruitment of chronic patients who were not already treated for their pain condition was therefore especially difficult. Furthermore, our exercise program was not tailored to the individual needs of every patient. This could have influenced the outcome because patients might have needed a more individualized exercise program to approach to their problem, although recent literature suggest, that an individualized treatment is not always superior66. Nevertheless, we could show that pain decreased during the intervention.\n\n\nConclusion\n\nNeck pain can be influenced by manipulations applied to the thoracic spine in combination with exercise therapy. Additionally, our study suggests that electromechanical manipulations may be able to decrease neck disability. However, caution is advised concerning these results because of the study’s small sample size. The findings of this pilot study allowed the power calculation of the actual sample size needed for a larger randomized controlled trial aimed at studying manipulations applied to the thoracic spine in combination with exercise therapy in patients with neck pain.\n\n\nData availability\n\nDataset 1: Complete set of all data recorded as part of this pilot study. File name - manual vs mechanically_RCT_f1000 10.5256/f1000research.13780.d19247867\n\n\nEthics approval\n\nThe study received ethical approval from the Ethics Commission of the Canton of Zurich (2012-0248) and was registered at Current Controlled Trials (ISRCTN88585962).",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe Impulse IQ® was provided by Neuromechanical Innovations, Chandler, AZ, USA.\n\nA disclaimer response was written and signed before the start of the study.\n\n\nSupplementary material\n\nSupplementary File 1: Completed CONSORT checklist.\n\nClick here to access the data.\n\n\nReferences\n\nGlobal Burden of Disease Study 2013 Collaborators: Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015; 386(9995): 743–800. 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PubMed Abstract | Publisher Full Text\n\nRichardson A: The health diary: an examination of its use as a data collection method. J Adv Nurs. 1994; 19(4): 782–791. PubMed Abstract | Publisher Full Text\n\nTakeuchi A, Mamorita N, Sakai F, et al.: Development of a comprehensive medical recorder on a cellphone. Comput Methods Programs Biomed. 2010; 97(1): 28–38. PubMed Abstract | Publisher Full Text\n\nHuggins T, Boras AL, Gleberzon BJ, et al.: Clinical effectiveness of the activator adjusting instrument in the management of musculoskeletal disorders: a systematic review of the literature. J Can Chiropr Assoc. 2012; 56(1): 49–57. PubMed Abstract | Free Full Text\n\nCleland JA, Childs JD, Fritz JM, et al.: Development of a clinical prediction rule for guiding treatment of a subgroup of patients with neck pain: use of thoracic spine manipulation, exercise, and patient education. Phys Ther. 2007; 87(1): 9–23. PubMed Abstract | Publisher Full Text\n\nHaldeman S, Kohlbeck FJ, McGregor M: Unpredictability of cerebrovascular ischemia associated with cervical spine manipulation therapy: a review of sixty-four cases after cervical spine manipulation. Spine (Phila Pa 1976). 2002; 27(1): 49–55. PubMed Abstract\n\nSweeney A, Doody C: Manual therapy for the cervical spine and reported adverse effects: a survey of Irish manipulative physiotherapists. Man Ther. 2010; 15(1): 32–6. PubMed Abstract | Publisher Full Text\n\nThiel HW, Bolton JE, Docherty S, et al.: Safety of chiropractic manipulation of the cervical spine: a prospective national survey. Spine (Phila Pa 1976). 2007; 32(21): 2375–8; discussion 2379. PubMed Abstract | Publisher Full Text\n\nMiley ML, Wellik KE, Wingerchuk DM, et al.: Does cervical manipulative therapy cause vertebral artery dissection and stroke? Neurologist. 2008; 14(1): 66–73. PubMed Abstract | Publisher Full Text\n\nThomas LC, Rivett DA, Attia JR, et al.: Risk factors and clinical features of craniocervical arterial dissection. Man Ther. 2011; 16(4): 351–6. PubMed Abstract | Publisher Full Text\n\nCassidy JD, Boyle E, Côté P, et al.: Risk of vertebrobasilar stroke and chiropractic care: results of a population-based case-control and case-crossover study. Spine (Phila Pa 1976). 2008; 36: 92; author reply 92.\n\nMarx P, Püschmann H, Haferkamp G, et al.: [Manipulative treatment of the cervical spine and stroke]. Fortschr Neurol Psychiatr. 2009; 77(2): 83–90. PubMed Abstract | Publisher Full Text\n\nMurphy DR: Current understanding of the relationship between cervical manipulation and stroke: what does it mean for the chiropractic profession? Chiropr Osteopat. 2010; 18: 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNoordzij M, Tripepi G, Dekker FW, et al.: Sample size calculations: basic principles and common pitfalls. Nephrol Dial Transplant. 2010; 25(5): 1388–93. PubMed Abstract | Publisher Full Text\n\nKelly AM: The minimum clinically significant difference in visual analogue scale pain score does not differ with severity of pain. Emerg Med J. 2001; 18(3): 205–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSvedmark Å, Djupsjöbacka M, Häger C, et al.: Is tailored treatment superior to non-tailored treatment for pain and disability in women with non-specific neck pain? A randomized controlled trial. BMC Musculoskelet Disord. 2016; 17(1): 408. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLangenfeld A, Humphreys BK, de Bie RA, et al.: Dataset 1 in: Comparing manual and mechanically assisted manipulations of the thoracic spine in neck pain patients: A pilot study. F1000Research. 2018. Data Source"
}
|
[
{
"id": "36000",
"date": "20 Jul 2018",
"name": "Arianne P. Verhagen",
"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\nThank you for asking me to review this manuscript. Although this RCTs is of very low power, I understand the need to publish it as it might be a kind of research waste not to publish it. Nevertheless I have a couple of comments: 1. this study aims to evaluate the difference, or maybe the absence of difference, between two forms of manual therapy. As these concern two active treatments it is essential to know whether the study aims at evaluating superiority of one intervention over another or aims at evaluating equivalence. The method is quite unclear about that, but I think this needs to be clear from the objective, please revise. 2. This study clearly aimed at including more people that they actually did. This information needs to be presented. Furthermore the reader needs to know where patient recruitment was performed, during which period and what actions have been done to improve recruitment. Please add this information. 3. In the result section (in the abstract as well as in the main text) the authors mix within group differences and between group differences. Any mention of within group differences are irrelevant as the study is set up to evaluate between group differences. I would strongly recommend to take out all mention of within group improvements as this is misleading. 4. In the discussion the authors mention as one of the main finding an improvement of all patients. This is a logical finding as, apart from one patient, all had acute neck pain, so natural course will probably be responsible for the improvement. This needs to be discussed! 5. The conclusions are way too firm given the low number of included people. The main conclusion (i.e. the first sentence) is even incorrect as all people improved, but we cannot state that manipulations can be held responsible for that, as I believe this is due to natural course. As long as there is not a no treatment group in this RCT one cannot make such a conclusion.\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? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-156
|
https://f1000research.com/articles/7-155/v1
|
07 Feb 18
|
{
"type": "Brief Report",
"title": "Genetic analysis of measles virus nucleocapsid gene identifies measles virus isolate of close similarity to Clade A viruses from Nigeria",
"authors": [
"Adedayo O. Faneye",
"Johnson A. Adeniji",
"Babatunde O. Motayo",
"Johnson A. Adeniji"
],
"abstract": "Previous studies on the molecular epidemiology of measles virus in Nigeria shows that genotype B3 clusters 1 and 2 are the circulating measles genotype. We report the isolation of measles virus strain of close similarity to reference genotype A measles virus strain from Ibadan, Nigeria. Molecular characterization of a measles virus isolate from a child presenting with fever and rash in a hospital in Ibadan, Nigeria, was done by measles virus isolation in Vero slam cell line, RT-PCR and direct sequencing of the COOH terminal of the nucleoprotein gene of the measles virus isolate. Phylogenetic analysis of the sequence shows that isolate MViIbadan, NIE/11.01 clustered with the reference strains of Clade A. Our current report shows evidence of another circulating MV genotype different from the previously reported genotype B3 in Nigeria. We advocate for expanded national molecular surveillance of measles virus as this will aid in achieving the country’s goal of control of the disease.",
"keywords": [
"Measles virus",
"Phylogeny",
"Clade A",
"Nigeria"
],
"content": "Introduction\n\nMeasles is an acute viral illness characterised by a prodromal illness of fever, coryza, cough, conjunctivitis and presence of Koplik spots followed by the appearance of a generalised maculopapular rash. The illness is either mild or severe depending on the immune and nutritional status of the infected person. It is caused by measles virus (MV), which is a negative sense single stranded RNA virus of the family Paramyxoviridae, genus Morbillivirus1. The virus is relatively antigenically stable and monotypic but has genetic variation in the hemagglutinin (H) and nucleoprotein (N) genes2. Measles is one of the leading causes of death among young children despite the availability of a safe and cost effective vaccine for over 40 years3. It accounted for over 145700 deaths in 2013 globally, 51% of which are from World Health Organization (WHO) defined African region4. Nigeria still ranks high among countries reporting measles infection yearly, with over 7000 suspected and over 3000 confirmed cases reported in 20144.\n\nThe 450 nucleotides that code for the COOH-terminus of the N protein is the single most variable part of the measles genome and analysis of this part of MV isolates or clinical specimens has helped to classify measles into eight clades (Clades A-H) and 23 genotypes5. Different measles genotypes and strains circulate in specific geographical regions5,6. Measles virus clades B3, D4, D5, D8 and H1 were isolated in the WHO Americas region between 2007 and 2009, genotypes D6 and D9, in addition to those found in the Americas, are also found in the European region, and genotype B3 is the major circulating strain in the Africa region in this period6.\n\nPrevious reports on circulating measles strains from Nigeria have indicated the circulation of only genotypes B3 cluster 1 and 2. However, due to the absence of integrated molecular surveillance in measles’ elimination programs, there has been only a few sequence data of Nigerian measles isolates, especially in recent times7,8. Molecular surveillance is essential in order to observe the changes in viral genotypes over time in a particular region. It is also an important tool in assessing the effectiveness of vaccination programs9. This study reports the molecular characterization of a previously unreported genotype of measles virus from Nigeria.\n\n\nMethods\n\nAs part of the standard care routine, a nasopharyngeal swab was collected from a 3-year-old child of suspected measles infection presenting with fever, maculopapular rash, cough and conjunctivitis at the Oni Memorial Children’s Hospital in Ibadan, Oyo State, Nigeria, in November 2010. The sample was transported to the lab in virus transport medium under reverse cold chain. The swab was inoculated into tissue culture flask of VeroSLAM cell line and observed for cytopathic signs for seven days.\n\nA nasopharyngeal swab was used as the sample type because it is the recommended site by the United States Centers for Disease Control and Prevention. In addition, measles virus is shed up to 10 day after the onset of rash and after the resolution of vireamia increasing the chances of viral nucleic acid detection up to the 14 day after the onset of rash.\n\nViral RNA was extracted from the throat swab collected and supernatant from the cell culture using QIAamp® Viral RNA kit by QIAGEN Valencia USA, according to the manufacturer’s instruction.\n\nExtracted RNA was synthesised to cDNA by reverse transcription using a commercial kit (SCRIPT DNA synthesis kit by Jena Bioscience® GmbH, Germany). Nested PCR was performed using two sets of primers: First round, fwd MN5 5-GCCATGGGAGTAGGATGGAAC-3; rev MN6 5-CTGGCGGGCTGTGTGTGGACCTG-3 and nested inner primers Nfla 5-CGGGCAAGAGATGGTAAGGAGGTCAG-3, Nr7a 5-AGGGTAGGCGGATGTTGGTTCTGG-3, as previously described by Kremer et al.8 using Applied Biosystems GeneAmp PCR System 9700 thermal cycler. Cycling conditions for both first and second reactions is as follows: 35 cycles of 94°C for 30 seconds, 55°C for 1 minute, 72°C for 1 min preceded by 94°C for 5 minutes and a final elongation at 72°C for 5 minutes8.\n\nPurified amplicons were sequenced at Jena Bioscience Laboratory (Germany) by Sanger sequencing method using the second round primer. Sequence data obtained was edited and assembled with Bioedit software version 7.0.5, sequence similarity was determined by Basic Local Alignment (BLAST). The query sequence was aligned with reference sequences downloaded from GenBank with the help of Measles Nucleotide Surveillance (MeaNS) database. Table 1 shows the names and GeneBank accession numbers of the reference sequences used for the analysis. The basis of sequence selection was predicated on selection of characterized isolates reported in publications from WHO European region laboratory7,8 using CLUSTALW software. Phylogenetic trees were constructed in Mega version 6.06 software using Maximum likelihood and Neighbor joining methods with p distance model and 1000 bootstrap replicates.\n\n\nResults and discussion\n\nBoth the swab and tissue culture sample showed the expected 560 base pairs after nested RT-PCR. Phylogenetic analysis of the measles sample MViIbadan/NIE/11.10 sequence (ccession no LN876569.1) gave a distinct BLAST search result (Supplementary File 1). From the search it was observed that the sequence with ascension no JF727650.1 Leningrad is the most closely related virus sequence with our Ibadan strain. The phylogenetic tree generated revealed that the Ibadan prototype Clade A strain co-clustered with sequences close to the vaccine Edmonston Zagreb stain with 98% bootstrap value (Figure 1), suggesting the possibility of common ancestral origin. Molecular Epidemiology of Measles virus in Nigeria classifies isolates into two distinct groups within genotype B3, B3 cluster 1 and B3 cluster 27,8. The B3 genotype was assigned after exhaustive genetic analysis of MV strains from Lagos and Ibadan in 19998.\n\nThe study isolate is shown in bold red font; clinical strains from Nigeria are shown in blue font. Clades are indicated beside the black horizontal lines. The GenBank accession numbers are indicated first in the sequence labels, bootstrap values are indicated if ≥ 50%, phylogenetic tree was constructed using the Neighbor joining algorithm in MEGA 6.0 with 1,000 bootstrap replicates.\n\nOur current study describes isolation of a different strain of measles from the previously described genotypes (MViIbadan/NIE/11.10). This virus was recovered from the throat swab of a 3 year old child with no documented history of vaccination at Oni Memorial Children Hospital in Ibadan Nigeria in November 2010.\n\nMeasles virus of genotype A has previously been detected in acute cases of measles in South and North America, China, Japan, Eastern Europe, Finland and the UK and South Africa over the last 40 years10, but there had not been any report of Clade A virus in West Africa. The virus isolated in this study is from a child who had never received any form of measles vaccination and there was no activities involving measles virus vaccine in the laboratory as at the time of virus isolation. Molecular epidemiologic information has revealed circulation of several measles genotype in Africa in recent times. Clade B viruses is reported to be endemic in central and western parts of sub-Saharan Africa while genotypes D2 and D4 has been continually detected southern and eastern parts of Africa and genotype C2 in northern Africa11–14 but no virus of Clade A has been reported in Africa recently.\n\nIn this study, although we did not independently investigate the immediate or remote factors that could have led to the introduction/emergence of this virus genotype in Nigeria, we postulate that the virus could have been probably imported from another continent or could have been an undetected circulating strain. This is because active molecular surveillance is not in place in Nigeria, and the last report of MV molecular epidemiology was in 20058. This leaves a huge gap in research information and multiple introductions of diverse measles genotypes might have taken place undetected over time. We therefore advocate for the establishment of an institution of a national surveillance program, and the inclusion of molecular epidemiology alongside national and global authorities, such as the WHO and Federal Ministry of Health Nigeria, to help achieve measles elimination goals in the country and globally.\n\n\nData availability\n\nThe sequence of the identified isolate has been deposited in GenBank under the accession number LN876569.1.\n\n\nEthical statement\n\nEthical approval was obtained from Oyo State, Hospital Management Board (Ibadan, Nigeria), approval number OY/HMB/REC-140010, to conduct this study. The parent of the infected child reported in this study also provided written informed consent for sequencing of the sample and use in research before the sample was collected.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were received in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to acknowledge the contributions of Prof D.O Olaleye and Dr G.N Odiabo during the course of this work. The effort of Mr B.A Olusola is also appreciated during the course of the field work. A.O. Faneye was a PhD student during the time of this study and this report is part of her PhD research project.\n\n\nSupplementary material\n\nSupplementary File 1: Blast search result for MViIbadan/NIE/11.10 Genbank no LN876569.1.\n\nClick here to access the data.\n\n\nReferences\n\nGriffin DE: Measles virus. In Fields Virology, 5th Edn eds Knipe DM, Howley PM, Griffin DE, et al:. editors. (Philadelphia: Lippincott Williams & Wilkins), 2007; 1551–1585.\n\nColf LA, Juo ZS, Garcia KC: Structure of the measles virus hemagglutinin. Nat Struct Mol Biol. 2007; 14(12): 1227–1228. PubMed Abstract | Publisher Full Text\n\nWHO: Measles fact sheet. 2015.\n\nWorld Health Organisation: Update of the nomenclature for describing the genetic characteristics of wild-type measles viruses: new genotypes and reference strains. Wkly Epidemiol Rec. 2003; 78(27): 229–323. PubMed Abstract\n\nRota PA, Bellini WJ: Update on the global distribution of genotypes of wild type measles viruses. J Infect Dis. 2003; 187 Suppl 1: S270–276. PubMed Abstract | Publisher Full Text\n\nRota PA, Brown K, Mankertz A, et al.: Global distribution of measles genotypes and measles molecular epidemiology. J Infect Dis. 2011; 204 Suppl 1: S514–523. PubMed Abstract | Publisher Full Text\n\nHanses F, Turong AT, Ammerlan W, et al.: Molecular epidemiology of Nigerian and Ghanaian measles virus isolates reveals a Genotype circulating widely in Western and Central Africa. J Gen Vir. 1999; 80(Pt 4): 871–7. PubMed Abstract | Publisher Full Text\n\nKremer JR, Nkwembe E, Bola Oyefolu AO, et al.: Measles virus strain diversity, Nigeria and Democratic republic of the Congo. Emerg Infec Dis. 2010; 16(11): 1724–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: Mannual for the diagnosis of Measles and Rubella infection. 2nd ed. Geneva, Swithzerland. WHO. 2007. Reference Source\n\nRiddell MA, Rota JS, Rota PA: Review of the temporal and geographical distribution of Measles virus genotypes in the prevaccine and postvaccine eras. Virol J. 2005; 2: 87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organisation: Nomenclature for describing the genetic characteristics of wild-type measles viruses(update). Part 1. Wkly Epidemiol Rec. 2001; 76(32): 242–247. PubMed Abstract\n\nWHO: New genotype of measles virus and update on global distribution of measles genotypes. Wkly Epidemiol Rec. 2005; 80(40): 347–351. PubMed Abstract\n\nKres S, Vadas E, Whistler T: Sequence analysis of the nucleocapsid gene of measles virus isolates from South Africa identifies a new genotype. J Gen Virol. 1997; 78(Pt 7): 1581–1587. PubMed Abstract | Publisher Full Text\n\nMuwonge A, Nanyuja M, Rota PA, et al.: New measles genotype, Uganda. Emerg Infect Dis. 2005; 11(10): 1522–6. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "31867",
"date": "09 Apr 2018",
"name": "Kevin E. Brown",
"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 appears to be a paper that was written in 2010 about the suggested identification of a genotype A virus from a child in Nigeria who was said to have not been vaccinated. Even a cursory glance of the literature would have provided more up to date information on the current status of measles epidemiology globally and in Africa.\nThere is a standard convention for naming and characterizing measles sequences that has not been adhered to. For genotyping there is a minimum 450 nucleotide window, and yet the sequence submitted in the Gnebank file is only 445 nucleotides long.\nExamination of that sequence file clearly indicates that there a problems with the sequence with a long run of ts followed by gs and then a second run of ts. This has never been seen in the coding region of measles viruses before and indicates a serious problem with the sequencing results. By only showing the similarity of sequences in the paper and in the supplementary file this problem is glossed over and is not discussed or commented on in the manuscript itself.\nFinally, although not as comprehensive as some countries there has nevertheless been a substantial number of sequences submitted from Nigeria since 2010. Their suggestion that a genotype A virus is circulating in Nigerai has never been substantiated by any other studies.",
"responses": []
},
{
"id": "32699",
"date": "08 May 2018",
"name": "Ismaila Shittu",
"expertise": [
"Reviewer Expertise Virology (RNA viruses)"
],
"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 study entitled “Genetic analysis of measles virus nucleocapsid gene identifies measles virus isolate of close similarity to Clade A viruses from Nigeria”, Faneye et al. analyze sequence data of the partial nucleocapsid gene of measles virus isolates recovered from a patient presenting with fever and rash in a hospital in Ibadan, Nigeria. The authors isolated measles virus and found evidence of a hitherto unreported measles virus, genotype A, in a child without previous vaccination history.\nThough the paper is clearly presented and provides seems to provide additional information on the circulating genotype of measles virus in Nigeria, the authors need to cite current literature to update readers on the molecular epidemiology of genotype A.\nMeasles viruses of genotype A are mostly vaccine-like, and according to the authors the Nigeria isolate clustered with the vaccine Edmoston-Zagreb. Since measles vaccination campaign in Nigeria is widespread, I will suggest that the authors use a real-time assay by Roy et al., 20171that can be used to differentiate a measles case from the commonly used measles vaccine to substantiate their claim of the uniqueness (wild type measles virus of genotype A) of the isolates from the unvaccinated child.\nAdditionally, the sequence of the virus used in the study had unusual insertions of nucleotides (Ts and Gs). It is however not stated the number of replicates of sequencing that was carried out. To clear the ambiguity on the clustering of the sequence as shown in the tree, I will suggest that an additional sequencing of the isolate be performed.\nIn conclusion, there are typographical errors across the manuscript that will require correction by the authors.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-155
|
https://f1000research.com/articles/7-148/v1
|
05 Feb 18
|
{
"type": "Opinion Article",
"title": "Ten steps to get started in Genome Assembly and Annotation",
"authors": [
"Victoria Dominguez Del Angel",
"Erik Hjerde",
"Lieven Sterck",
"Salvadors Capella-Gutierrez",
"Cederic Notredame",
"Olga Vinnere Pettersson",
"Joelle Amselem",
"Laurent Bouri",
"Stephanie Bocs",
"Christophe Klopp",
"Jean-Francois Gibrat",
"Anna Vlasova",
"Brane L. Leskosek",
"Lucile Soler",
"Mahesh Binzer-Panchal",
"Henrik Lantz",
"Victoria Dominguez Del Angel",
"Erik Hjerde",
"Lieven Sterck",
"Salvadors Capella-Gutierrez",
"Cederic Notredame",
"Olga Vinnere Pettersson",
"Joelle Amselem",
"Laurent Bouri",
"Stephanie Bocs",
"Christophe Klopp",
"Jean-Francois Gibrat",
"Anna Vlasova",
"Brane L. Leskosek",
"Lucile Soler",
"Mahesh Binzer-Panchal"
],
"abstract": "As a part of the ELIXIR-EXCELERATE efforts in capacity building, we present here 10 steps to facilitate researchers getting started in genome assembly and genome annotation. The guidelines given are broadly applicable, intended to be stable over time, and cover all aspects from start to finish of a general assembly and annotation project. Intrinsic properties of genomes are discussed, as is the importance of using high quality DNA. Different sequencing technologies and generally applicable workflows for genome assembly are also detailed. We cover structural and functional annotation and encourage readers to also annotate transposable elements, something that is often omitted from annotation workflows. The importance of data management is stressed, and we give advice on where to submit data and how to make your results Findable, Accessible, Interoperable, and Reusable (FAIR).",
"keywords": [
"Genome",
"Assembly",
"Annotation",
"FAIR",
"NGS",
"Workflows",
"DNA"
],
"content": "Introduction\n\nThe advice here presented is based on a need seen while working in the ELIXIR-EXCELERATE task “Capacity Building in Genome Assembly and Annotation”. In this capacity we have held courses and workshops in several European countries and have encountered many users in need of a document to support them when they plan and execute their projects. With these 10 steps we aim to fill this need.\n\nIn a de novo genome assembly and annotation project, the nucleotide sequence of a genome is first assembled, as completely as possible, and then annotated. The annotation process infers the structure and function of the assembled sequences. Protein-coding genes are often annotated first, but other features, such as non-coding RNAs or presence of regulatory or repetitive sequences, can also be annotated.\n\nWith the advances in sequencing technologies it has become much more feasible, and affordable, to assemble and annotate the genomic sequence of most organisms, including large eukaryote genomes1,2. However, high quality genome assembly and annotation still represent a major challenge. Considerable time and computational resources are often needed, and researchers have to be prepared to provide these resources in order to be successful. Assembly and annotation of small genomes e.g., bacterias and fungi, can often be performed with fairly small resources and a limited time commitment, but eukaryotic genome projects often take months or even years to finish, especially when no reference genomes can be used for these tasks. The mere running of assembly or annotation tools can take several weeks (see Section 3 for examples).\n\nConsidering the amount of time, knowledge, and resources required by these projects, an early question you need to ask yourself is: “Do I really need an assembled and annotated genome?” In many cases an assembled transcriptome, or perhaps a re-sequencing approach based on the genomic sequence of a related species, can be enough to answer your scientific questions. These two approaches both constitute solutions requiring much less resources, both in amount of sequencing data needed and in regards to compute hours, but are more limited and do not offer as many possibilities as an annotated genome does. In the event that a genome draft has a significant added value to address the problem, one should consider whether sufficient financial and computational resources are available to produce a genome of satisfactory quality.\n\nFor those that indeed have decided to embark upon a genome assembly and/or annotation project, we provide, here, a set of good practices intended to facilitate the project completion. The target audience is someone entering this field for the first time, and we strive to answer his/her beginner questions. We split the information up into different sections for the reader to easily find the parts that are of their particular interest. The guidelines are meant to be broadly applicable to multiple software pipelines and sequencing technologies and do not focus on specifics, as the field is rapidly changing and discussion on current tools could quickly become outdated.\n\nA checklist of things to keep in mind when starting a genome project:\n\nFor the DNA extraction, select an individual which is a good representative of the species, and able to provide enough DNA.\n\nExtract more DNA than you think you need, or save tissue to use for DNA extraction later. If you need to produce more data later, it is critical to be able to use the same DNA to make sure the data assembles together.\n\nRemember to extract RNA and order RNA-sequencing if you want to use assembled transcripts in your annotation (which is strongly recommended). If possible, extract RNA from the same individual as used in the DNA extraction to make sure that the RNA-seq reads will map well to your assembly.\n\nDecide early on which sequencing technology you will be using, and also consider which assembly tools you want to try. These two choices will greatly influence what kind of compute resources you will need, and you do not want to end in a situation where you have data that you cannot analyze anywhere. Plan compute resources accordingly.\n\n\n1. Investigate the properties of the genome you study\n\nEvery assembly or annotation project is different. Distinctive properties of the genome are the main reason behind this. To get an idea of the complexity of an assembly or annotation project, it is worth looking into these properties before starting. Here, we will discuss some genome properties, and how they influence the type and amount of data needed, as well as the complexity of analyses.\n\nTo assemble a genome, a certain amount of sequences (also called reads) is needed. For example, for Illumina sequencing (see Illumina Genome Assembly below), a number of >60x sequence depth is often mentioned. This means that the number of total nucleotides in the reads need to be at least 60 times the number of nucleotides in the genome. From this it follows that the bigger the genome, the more data is needed. You need to get an estimate of the genome size before ordering sequence data, perhaps from flow cytometry studies, or if no better data exists, by investigating what is the genome size of closely related and already assembled species. This is an important value to bring to the sequencing facility, as the genome size will greatly influence the amount of data that needs to be ordered. Available databases for approximate genome sizes are available for plants (http://data.kew.org/cvalues), for fungi (http://www.zbi.ee/fungal-genomesize), and for animals (http://www.genomesize.com).\n\nRepeats are regions of the genome that occur in multiple copies, potentially at different locations in the genome. Amount and distribution of repeats in a genome hugely influences the genome assembly results, simply because reads from these different repeats are very similar, and the assembly tools cannot distinguish between them. This can lead to mis-assemblies, where regions that are distant in the genome are assembled together, or an incorrect estimate of the size or number of copies of the repeats themselves3. Very often a high repeat content leads to a fragmented assembly, as the assembly tools cannot determine the correct assembly of these regions and simply stop extending the contigs at the border of the repeats4. To resolve the assembly of repeats, reads need to be long enough to also include the unique sequences flanking the repeats. It can therefore be a good idea to order data from a long-read technology, if you know that you are working with a genome with a high content in repeats.\n\nAssembly programs in general try to collapse allelic differences into one consensus sequence, so that the final assembly that is reported is haploid. If the genome is highly heterozygous, sequence reads from homologous alleles can be too different to be assembled together and these alleles will then be assembled separately. This means that heterozygous regions might be reported twice for diploid organisms, while less variable regions will only be reported once, or that the assembly simply fails at these variable regions5. Highly heterozygous genomes can lead to more fragmented assemblies, or create doubt about the homology of the contigs. Large population sizes tend to lead to high heterozygosity levels. For instance, marine organisms often have high heterozygosity levels and are often problematic to assemble. It is recommended to sequence inbred individuals, if possible.\n\nIf possible, it is better to sequence haploid tissues (true for bacteria and many fungi) since, this will essentially remove problems caused by heterozygosity. Diploid tissues, which will be the case for most animals and plants, is fine and usually manageable, while tetraploidy and above has the potential to greatly increase the number of present alleles, which likely will result in a more fragmented assembly (see heterozygosity above). Diploid-aware assemblers using long reads can help, but keep in mind that correct assembly of diploid genomes might require higher coverage.\n\nExtremely low or extremely high GC-content in a genomic region is known to cause a problem for Illumina sequencing, resulting in low or no coverage in those regions6. This can be compensated by an increased coverage, or the use of a sequencing technology that does not exhibit that bias (i.e., PacBio or Nanopore). If you are working with an organism with a known low or high GC-content, we would recommend using a sequencing technology that does not exhibit any bias in this regard.\n\n\n2. Extract high quality DNA\n\nIntrinsic properties of the genome are not the only consideration before sequencing. There are also other aspects that need careful planning. The extraction of high quality DNA is one such aspect that is of utmost importance. We discuss DNA extraction in some detail below, but also end this section with a short list of other pre-assembly considerations important to keep in mind when starting an assembly project.\n\nFew researchers are aware of the fact that to get a good reference genome one must start with good quality material. It must be immediately pointed out that PCR-quality DNA and NGS-quality DNA are two completely different things7.\n\nIn general, we recommend using long-read technologies (see also Section 3 below) when carrying out genome assembly. For these technologies, it is crucial to use best quality High Molecular Weight (HMW) DNA, which is obtained mainly from fresh material. The lack of a good starting material will limit the choice of sequencing technology and will affect the quality of obtained data.\n\nThe most important DNA quality parameters for NGS are chemical purity and structural integrity of the sample.\n\nDNA extracts often contain carry-over contaminants originating either from the starting material or from the DNA extraction procedure itself. Examples of sample-related contaminants are polysaccharides, proteoglycans, proteins, secondary metabolites, polyphenols, humic acids, pigments, etc. For instance, fungal, plant and bacterial samples can contain high levels of polysaccharides, plants are notorious for their polyphenols, and insect samples are usually contaminated by polysaccharides, proteins and pigments, and so on. All these contaminants can impair the efficacy of library preparation in any technology, but this is especially true for Illumina Mate Pair libraries and PCR-free libraries (both PacBio and ONT). For conventional short-read technology sequencing where a PCR step is involved in the library prep, this hurdle is partly overcome by the amplification step during the library construction. However, it can happen that the library complexity of a contaminated sample can be reduced due to lower efficacy of the reaction. It is widely known in the PacBio community that samples rich in contaminants can fail or underperform in the sequencing process, since there is no PCR step in the library preparation and sequencing workflow.\n\nThe way to address the contamination issue is to use an appropriate DNA extraction protocol taking into account the expected type of contaminants present in the sample (native contaminants). CTAB (cetyl trimethylammonium bromide) extraction is highly recommended for DNA extraction from fungi, mollusks and plants; at a certain salt concentration CTAB helps to differentially extract DNA from solutions containing high level of polysaccharides8 . For protein rich tissues, adding beta-mercaptoethanol (disrupting disulphide bonds in protein molecules) and optimization of Proteinase K treatment is recommended9. For plants, it is important to always use a combination of beta-mercaptoethanol (to prevent polyphenols from oxidizing and binding to DNA) and PVPP (polyvinyl polypyrrolidone; to absorb polyphenols and other aromatic compounds)10. For animal and human samples, it is advised to use tissues with low fat and connective tissue content.\n\nAside from native contaminants, phenol, ethanol and salts can be introduced during the DNA extraction procedure. Incomplete removal of phenol, or not using fresh phenol will harm DNA (e.g. introducing nicks making the nucleic acid more fragile); it can also impair enzymes used in downstream procedures, as can incompletely removed ethanol. High salt concentrations (e.g. EDTA carry-over) can potentially lower efficacy of any downstream enzymatic reactions.\n\nA second important issue is the DNA structural integrity, which is especially important for long-read sequencing technologies. DNA can become fragile due to nicking introduced during DNA extraction, or using storage buffer with inappropriate pH. Prolonged DNA storage in water and above -20°C is not recommended; it increases the DNA degradation risk due to hydrolysis. High molecular weight DNA is fragile; therefore using gentle handling (vortexing at minimal speed, pipetting with wide-bore pipette tips, transportation in a solid frozen stage) is advised. It is also advisable to keep the number of freeze-thaw cycles to a minimum, since ice-crystals can mechanically damage the DNA. For the same reason, one should avoid DNA extraction protocols involving harsh bead-beating treatment during tissue homogenization.\n\nIt must be also pointed out that RNA contamination of DNA samples must be avoided. Most NGS DNA library preps can only efficiently utilize double-stranded DNA. Having RNA contamination in the sample will overestimate the library nucleic acid molecules concentration. That is especially true for PacBio and 10X Chromium libraries.\n\nTo summarize, it is always worth investing time in getting a high quality DNA prep – it can potentially save lots of time and money that would otherwise be spent on sequencing troubleshooting, ordering more data, or, if ordering more data is not possible, trying to assemble a genome with a coverage that is lower than expected.\n\n\n\nPooling of individuals – For some organisms it can be difficult to extract a sufficient amount of DNA, and in these cases it might be tempting to pool several individuals before extraction. Note that this will increase the genetic variability of the extraction, and can lead to a more fragmented assembly, just like high levels of heterozygosity would. In general pooling should be avoided, but if it is done, using closely related and/or inbred individuals is recommended.\n\nWhole Genome Amplification (WGA) – In cases where perhaps only a few cells are available, the genomic DNA needs to be amplified to be sequenced. This will often result in uneven coverage, and in the case of amplification methods relying on multiple strand displacement, artificial so called chimeric sequences consisting of fused unrelated sequences can be created11. Be aware that this can cause mis-assemblies. If possible, use an assembly tool designed to work with amplified DNA, for example SPAdes12.\n\nPresence of other organisms – Contamination is always a risk when working with DNA. For genome assembly, contamination can be introduced in the lab at the DNA extraction stage, or other organisms can be present in the tissue used, e.g. contaminants and/or symbionts. Care should be taken to make sure that the DNA of other organisms does not occur in higher concentrations than the DNA of interest, as many reads will then be from the contaminant rather than the genome of the studied organism. Small amounts of contamination are rarely a problem as these reads can be filtered out at the read quality control step or after assembly, unless the contaminants are highly similar to the studied organism.\n\nOrganelle DNA - Some tissues are so rich in mitochondria or chloroplasts that the organelle DNA occurs in higher concentrations than the nuclear DNA. This can lead to lower coverage of the nuclear genome in your sequences. If you have a choice, choose a tissue with a higher ratio of nuclear over organelle DNA.\n\n\n3. Choose an appropriate sequencing technology\n\nThe choice of which sequencing technology to use is an important one (Figure 1). It will influence the cost and success of the assembly process to a large degree. In this section, we will discuss the currently available and most commonly used options, and also some supporting technologies. It is worth mentioning that assembly programs are often very specific in what type of data they accept, and might not be able to analyze reads from different sequencing technologies together. You should decide how to analyze your sequence data before you order it, to decrease the risk of needing to order, and wait for, more DNA/RNA material just to be able to perform your analyses.\n\nThe data is based on the throughput metrics for the different platforms since their first instrument version came out. The figure visualises the results by plotting throughput in raw bases versus read length. Data released under CC BY 4.0 International license. doi 10.6084/m9.figshare.100940.\n\nThese technologies started with the Sanger sequencing method developed by Frederick Sanger and colleagues in 1977. The method is based on selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. FGS technologies were the most widely used for approximately 30 years13,14.\n\nDuring the last decade, the Sanger method has been replaced by High-Throughput Sequencing platforms (HTS), in particular by Second-Generation Sequencing (SGS), which is much less expensive. However, the Sanger method remains widely used in smaller-scale projects and for closing gaps between contigs generated by HTS platforms.\n\nThe SGS have dominated the market, thanks to their ability to produce enormous volumes of data cheaply. Examples are the Illumina or Ion Torrent sequencers. Many remarkable projects like the 1000 Genomes Project15 and the Human Microbiome Project16 have been finished thanks to SGS technologies. However, some genes and important regions of interest are often not assembled correctly, mainly due to the presences of repeat elements in the sequences17. A promising solution is Third-Generation-Sequencing (TGS) based on long reads18. TGS technologies have been used for the reconstruction of highly contiguous regions in eukaryotic genomes19,20 and de novo microbial genomes with high precision21. In terms of resequencing, the TSG technology has generated detailed maps of the structural variations in multiple species and has covered many of the gaps in the human reference genome22,23.\n\nCurrently, the two most important third-generation DNA sequencing technologies are Pacific Biosciences (PacBio) Single Molecule Real Time (SMRT) and Oxford Nanopore Technology (ONT)24. These technologies can produce long reads averaging between 10,000 to 15,000bp, with some reads exceeding 100,000bp.\n\nHowever, these long reads exhibit per sequence error rates up to 10% to 15%, requiring a preliminary stage of correction before or after the assembly process. In fact, long read assembly has caused a paradigm shift in whole-genome assembly in terms of algorithms, software pipelines and supporting steps25.\n\nThere are also supporting technologies, most of which are used to improve the contiguity of already existing genome assemblies. These include optical mapping methods (e.g., BioNano), linked-read technologies (e.g., 10X Genomics Chromium system), or the genome folding-based approach of HiC26. In a rapidly changing field, it is difficult to recommend one of these technologies over the others. We advise researchers interested in assembling large genomes to read up on the current status of these methods when ordering sequence data, and remember to budget for them. For researchers interested in large-scale structural changes, the improvements of contiguity provided by these methods will be of extra interest.\n\nLong reads definitely have an advantage over shorter reads when used in genome assembly as they deal with repeats much better. In practice, this often leads to less fragmented assemblies, which is what most researchers are aiming for. The problems with third generation technologies are a higher price, a lack of availability in some countries, and sometimes higher requirements in terms of DNA amount and quality. Unless these complicating factors prevents the use of third generation long read technologies in your research project, we strongly recommend them over short read technologies. That being said, a combination of both might be even better, as the shorter reads have a different error profile and can be used to correct the longer ones27 (see Section 5).\n\n\n4. Estimate the necessary computational resources\n\nTo succeed in a genome assembly and annotation project you need to have sufficient compute resources. The resource demands are different between assembly and annotation, and different tools also have very different requirements, but some generalities can be observed (for examples, see Table 1).\n\nSPAdes is an assembler designed for the assembly of small genomes using short reads. Smartdenovo is a de novo assembler for PacBio and Oxford Nanopore (ONT) data. The REPET package is a software suite dedicated to detect, classify and annotate repeats. EuGene is an open integrative gene finder for eukaryotic and prokaryotic genomes. Processing time and RAM used will be affected by amount of input data, complexity of data, and genome size.\n\nFor genome assembly, running times and memory requirements will increase with the amount of data. As more data is needed for large genomes, there is thus also a correlation between genome size and running time/memory requirements. Only a small subset of available assembly programs can distribute the assembly into several processes and run them in parallel on several compute nodes. Tools that cannot do this tend to require a lot of memory on a single node, while programs that can split the process need less memory in each individual node, but do on the other work most efficiently when several nodes are available. It is therefore important to select the proper assembly tools early in a project, and make sure that there are enough available compute resources of the right type to run these tools.\n\nAnnotation has a different profile when it comes to computer resource use compared to assembly. When external data such as RNA-seq or protein sequences are used (something that is strongly recommended), mapping these sequences to the genome is a major part of the annotation process. Mapping is computationally intense, and it is highly preferable to use annotation tools that can run on several nodes in parallel.\n\nRegarding storage, usually no extra consideration needs to be taken for assembly or annotation projects compared to other NGS projects. Intermediate files are often much larger than the final results, but can often be safely deleted once the run is finished.\n\n\n5. Assemble your genome\n\nIn general, irrespective of the sequencing technology you choose, you would follow the same workflow (Figure 2). In the quality control (QC) stage the sequence reads are examined for overall quality and presence of adapters. Presence of contaminants can also be examined. In the assembly stage, several assemblers are often tried in parallel and the results are then compared in the assembly validation step, where mis-assemblies also can be identified and corrected. Often, assemblers are rerun with new parameters based on the results of the assembly validation. The aim is usually to create a genome assembly with the longest possible assembled sequences (least fragmented assembly) with the smallest number of mis-assemblies.\n\nInput and output data are indicated for each step.\n\nQuality control of reads and the actual genome assembly are different for the Illumina technology compared with long read technologies. These technologies will be discussed separately hereafter. We end this section with a discussion about assembly validation, which is similar for all technologies.\n\nThe most common approach to perform genome assemblies is de novo assembly, where the genome is reconstructed exclusively from the information of overlapping reads. For prokaryotes, it is also common to assemble with a reference genome, e.g., when complete strain collections are sequenced. The reference sequence can either be used as a template to 1) guide the mapping of reads, or 2) reorder the de novo assembled contigs.\n\nIn general, Illumina sequencing technology produces large amounts of high quality short sequence reads. The adapter and multiplex index sequences are screened for and removed after the base calling on the sequencing machine. However, it is highly recommended to assess the raw sequence data quality prior to assembly. Poor quality reads, ambiguous base calling, contamination, biases in the data and even technical issues on the sequencing chip, are some, but not all, possible technical errors that can be detected early and corrected28. Also, if the sequencing libraries contain very short fragments, it is likely that the sequencing reaction will continue past the DNA insert and into the adapter in the 3' end, a process known as adapter read-through, which may escape the adapter screening step on the sequencing machine29.\n\nAssessing the quality of the sequence data is important, as it may affect downstream applications and potentially lead to erroneous conclusions. Base calling accuracy measures the probability that a given base is called incorrectly, and is commonly measured by the Phred quality score (Q score). Several tools are available for the quality assessment. FastQC30 is a commonly used tool that can be run both from the command line or through an interactive graphical user interface (GUI). It produces plots and statistics showing, among others, the average and range of the sequence quality values across the reads, over-represented sequences and k-mers which in total can help the user interpret data quality. k-mers represent all subsequences of length k in a sequence read. Most methods for assembling or mapping reads are based on the use of k-mers. More in depth analysis of k-mers can also be performed, for example using KAT31 to identify error levels, biases and contamination, and this also comes highly recommended.\n\nAfter having investigated the sequence data quality, informed decisions on downstream operations can be made. We would in general recommend that adapters are removed, although there are also assemblers that prefer working with the raw data, including potential adapter sequences. It is highly recommended that the user studies the assembler documentation to determine whether the program requires quality-trimmed data or not. If trimming is required by the assembler, it would be sensible to omit poor quality data from further analysis by trimming low quality read ends and filtering of low quality reads. A variety of tools are available, such as PRINSEQ32, which offers a standalone command-line version, a version with a GUI and an online web based service, and Trimmomatic33.\n\nIllumina machines produce a wide range of read numbers, from 10 millions up to 20 billions (NovaSeq). Reducing the sequence coverage by subsampling for deeply sequenced genomes is recommended, as de Brujin assemblers work best around 60-80x coverage34. High coverage in a particular genome location will increase the probability that this location is seen as a sequencing error or sequencing errors can propagate and start to look like true sequence. BBnorm35, a member of the BBTools package, is a common kmer-based normalisation tool that can normalise highly covered regions to the expected coverage.\n\nFor the de novo assembly of short reads, the most commonly used algorithms are based on de Bruijn graphs, although other algorithms such as Overlap Layout Consensus (OLC)36 are still being used. One of the advantages of de Bruijn graph over OLC is that it consumes less computational time and memory. Depending on the complexity of the genome to be assembled such as size, repeat-content, polyploidy, a proper tool should be selected. Some assembly tools, such as SPAdes12, work best with smaller amounts of data and are thus well adapted for bacterial projects, while others handle large amounts of data well and can be used for any type of project. These include allpaths-LG37 and Masurca38. Note that with large amounts of data, available RAM will be a limiting factor.\n\nThe characteristics of the genomes being assembled have a greater impact on the results than the choice of the algorithm. Haploid genomes with no sequence repeats will be much easier to reconstruct than genomes of polyploids or genomes with many sequence repeats e.g. many plants species. The GAGE-B study39 showed that assembly software performing well on one organism, performed poorly on another organism. Hence, it is wise to test several approaches; different software, assembly with or without pre-processing of the sequence data, and also with different parameter settings. Another approach that will have impact on the assembly is the use of mate pair sequencing. This enables the generation of long-insert paired-end DNA libraries with fragments up to 15 kb, and can be particularly useful in de novo sequencing. The large inserts can span across regions problematic to the assembler such as repetitive elements, and anchor the paired reads in unique parts of the DNA, and reduce the number of contigs and scaffolds. Despite the enormous development in this field, it is still challenging to assemble large genomes from short reads. Further improvements, both in the assembly technology, but also in increasing read length and in fragment size is needed for more accurate reconstruction of genomes.\n\nTGS developed by Pacific Biosciences or Oxford Nanopore is able to produce long reads with average fragment lengths of over 10,000 base-pairs that can be advantageously used to improve the genome assembly40. In fact, long reads can span stretches of repetitive regions and thus produce a more contiguous reconstruction of the genome. However, raw long reads have a high rate of sequencing error (5–20%). As a result, some long read assemblers opt to correct these errors prior to assembly.\n\nThere are two main families of assemblers based on long reads:\n\nLong Reads Only assembler (LRO)\n\nShort and Long Reads combined assembler (SLR)\n\nIn general, LRO assemblers are based on the OLC algorithm. First, this algorithm produces alignments between long reads. Then it calculates the best overlap graph, and finally it generates the consensus sequence of the contigs from the graph. LRO assemblers require more sequencing coverage (minimum ~50X) from the long reads dataset than SLR assemblers. Schematically, SLR assemblers instead generate a de Bruijn graph pre-assembly using short reads, then the long reads are used to improve the pre-assembly by closing gaps, ordering contigs, and resolving repetitive regions. It is worth noting that some long reads assemblers require corrected long reads as input. Software to correct long reads are based on two strategies. The first strategy consists of aligning long reads against themselves. The second one uses short reads to correct long reads.\n\nA document with guideline practices for long-reads genome assemblies is available41. This document shows the performance of long read assembly benchmarked against 4 reference genomes: Acinetobacter DP1, Escherichia coli K12 MG1655, Saccharomyces cerevisae W303 and Caenorhabditis elegans (sequenced in different TGS platforms and under different conditions). Among the 11 tools that have been evaluated, 8 use only long reads as input data, while the 3 others can assemble genome using a mix of long and short reads. The tests show that it is strongly recommended to use a long read correction software before the assembly42.\n\nAlthough an error correction step may have been part of the assembler pipeline, errors can still be present in the assembly, particularly in long read assemblies. Polishing draft assemblies with either short or long reads can help to improve local base accuracy in particular correcting base calls and small insertion-deletion errors, and also resolve some mis-assemblies caused by poor reads alignment during the assembly43.\n\nIn scaffolding, assembled contigs are stitched together based on information from paired short reads. The unknown sequence between the contigs will be filled with Ns. If matching reads are instead used to join contigs together, for example long reads, actual sequence will fill in the gaps, and this is referred to as gap filling. In the case of an existing scaffolded assembly, long reads can also be used to replace the N-regions. Note that misassemblies in an existing assembly need to be broken prior to scaffolding in order to join the correct contigs together. Scaffolding and gap filling can be performed with low coverage44.\n\nDetermining if the assembly is ready for annotation is a key step towards successful genome annotation. Errors in assemblies occur for many reasons. Genomic regions can be incorrectly discarded as being fallacies or repeats. Others can be spliced together in the wrong places or in the wrong orientation. Unfortunately, there are few ways to distinguish what is real, what is missing, and what is an experimental artefact. There are, however, some statistics that often are used when choosing between assemblies, and some ways of identifying and removing potential problems.\n\nN50 is often used as a standard metric to evaluate an assembly45. N50 is the length of the smallest contig, after they have been ranked from longest to smallest, such that the sum of contig lengths up to it covers 50% of the total size of all contigs. It is thus a measure of contiguity, with higher numbers indicating lower levels of fragmentation. It is important to note that N50 is not a measure of correctness. So-called aggressive assemblers may produce longer contigs and scaffolds than conservative assemblers, but are also more likely to join regions in the wrong order and orientation. We recommend to compare the output from different assemblers (and of trimmed/filtered data). Assembly evaluation tools, such as Quast46, compare the metrics between assemblies, and allow the user to make educated choices to further improve and select the best assembly. If a reference sequence is available, Quast can also describe mis-assemblies and structural variations relative to the reference. If paired Illumina data is available, tools such as Reapr47 or FRCBam48 can be used to evaluate assemblies and to identify which assembly has the least amount of misassemblies. If other organisms were present in the reads (contaminants or symbionts) and have been assembled together with the other reads, these contigs can be identified using for example Blobtools49 and removed, if necessary. To determine how many protein coding genes have been assembled, BUSCO50 is very useful. This tool looks for genes that should be present in a genome of the investigated taxonomic lineage type, and reports the number of complete and fragmented genes found. Choosing the assembly with the highest percentage of complete genes could be given greater importance if the purpose of the genome project is to investigate protein coding genes.\n\nKnowing when to stop assembly and moving into annotation is one of the most difficult decisions to take in genome assembly projects. It is always possible to try one more tool or one more setting, and this wish of wanting to improve the assembly just a little bit more can delay these types of projects substantially. It is best to have a goal in mind before starting assembly, and to stop when that goal has been reached. If you feel that you can answer the questions you had before starting, then the assembly is good enough for your purposes and it is probably time to move into annotation. It is always possible to release a new and improved version of the genome later. Be aware that any changes to a genome assembly will most likely necessitate annotation to be re-started from scratch, and you should therefore be sure to “freeze” the assembly completely before starting annotation.\n\n\n6. Do not neglect to annotate Transposable Elements\n\nThe genome annotation stage starts with repeat identification and masking.\n\nThere are two different types of repeat sequences: ‘low-complexity’ sequences (such as homopolymeric runs of nucleotides) and transposable elements. Transposable Elements (TEs) are key contributors to genome structure of almost all eukaryotic genomes (animals, plants, fungi). Their abundance, up to 90% of some genomes such as wheat51, is usually correlated with genome size and organization. TEs ability to move and to accumulate in genomes, make them a major players of genome structure, plasticity, genetic variations and evolution. Interestingly, they can affect gene expression, structure and function when their insertion occurs in the vicinity of genes52 and sometimes through epigenetic mechanisms53.\n\nTEs are classified in two classes including subclasses, orders and superfamilies according to mechanistic and enzymatic criteria. These two classes are based on their mechanism of transposition using a copy-and-paste (Class I) or cut-and-paste mechanisms (Class II) through RNA or DNA intermediates respectively54.\n\nTE annotation is nowadays considered as a major task in genome projects and should be undertaken before any other genome annotation task such as gene prediction. Consequently, there has been a growing interest in developing new methods allowing an efficient computational detection, annotation, and analysis of these TEs, in particular when they are nested and degenerated. Many software have been developed to detect and annotate TEs55. One of the best known is RepeatMasker, which harnesses nhmmer, cross_match, ABBlast/WUBlast, RMBlast and Decypher as search engines and uses curated libraries of repeats, currently supporting Dfam (profile HMM library) and Repbase56,57.\n\nAnother important tool is the REPET package, one of the most used tools for large eukaryotic genomes with more than 50 genomes analyzed in the framework of international consortia. The REPET package is a suite of pipelines and tools designed to tackle biological issues at the genomic scale.\n\nREPET consists of two main pipelines: TEdenovo and TEannot. First, TEdenovo efficiently detects classified TEs (TEdenovo pipeline), then TEannot annotates TEs, including nested and degenerated copies58.\n\nDepending on the complexity and number of detected TEs, it might be possible that additional rounds of TEs identification and removal are needed once the initial gene set has been produced. It is a common practice to analyze the functional annotation of the initial gene set to detect those genes which are primarily annotated with terms associated to TEs activity. Those genes can be safely removed if they do not have homologous sequences in relative species and/or their homologous sequences have been annotated as TEs related59.\n\n\n7. Annotate genes with high quality experimental evidence\n\nA raw genomic sequence is to most biologists of no great value as such. Genome annotation consists of attaching biological meaningful information to genome sequences by analyzing their sequence structure and composition as well as to consider what we know from closely related species, which can be used as reference. While genome annotation involves characterizing a plethora of biologically significant elements in a genomic sequence, most of the attention is spent on the correct identification of protein coding genes. This is not because the other types of genetic elements are of lesser importance, far from actually, but mainly because the approaches to characterize them are either fairly straightforward (eg. INFERNAL60 and tRNAscan-se61 for non-coding RNA detection) or are the focus of more specialized analyses (eg. transcription factor binding sites).\n\nThe process of correctly determining the location and structure of the protein coding genes in a genome, “gene prediction”, is fairly well understood with many successful algorithms being developed over the past decades. In general, there are three main approaches to predict genes in a genome: intrinsic (or ab-initio), extrinsic and the combiners. Where the intrinsic approach focuses solely on information that can be extracted from the genomic sequence itself such as coding potential and splice site prediction, the extrinsic way uses similarity to other sequence types (e.g. transcripts and/or polypeptides) as information. There are inherent advantages and disadvantages to each of those.\n\nThe intrinsic approach is labor intensive as statistical models need to be built and software needs to be trained and optimized. Of prime importance for this approach is a good training set, i.e. a set of structurally well annotated genes used to build models and to train gene prediction software. As each genome is different, these models and software must be specific to each genome and thus need to be rebuilt and retrained for each new species. This is, however, also the big advantage of this approach, as it is capable of predicting fast evolving and species specific genes.\n\nThe extrinsic way, on the other hand, is much more universally applicable. A vast number of polypeptide sequences are already described and available in databases (eg. NCBI non-redundant protein, RefSeq, UniProt), which creates a wealth of information to be exploited in the gene prediction process. Transcript information, be it Sanger sequenced ESTs, RNA-Seq or even long read sequenced transcripts, plays an even bigger role in this approach. High quality protein sequences of other species provide good indication on the presence and location of genes and can be very useful to accurately predict the correct gene structure. Indeed, as polypeptide sequences often are more conserved than the underlying nucleotide sequences, they can still be aligned even from distantly related species. Although they are very useful to determine the presence of gene loci, they do not always provide accurate information on the exact structure of a gene. Transcripts on the other hand provide very accurate information for the correct prediction of the genes’ structure but are much less comprehensive and to some extent are noisier. Transcript information will not be available for all genes and sometime introns can still be present due to incomplete mRNA processing. Nonetheless, accurate alignment of the extrinsic data is key here: transcripts need to be splice-aligned (taking the exon-intron structure of eukaryotic genes into account) and protein sequences need to be compared to the six translation-frames of the nucleotide sequences. Moreover, it is a matter of thresholds: too stringent and less conserved genes will be missed, while too lenient will result in less specific information and introduce more false positives. These thresholds will depend on your objectives. A recommendation is to use lenient parameters in order to minimize the number of false negatives, as it is more difficult to create a new gene than to change the status of a false positive to obsolete. Then according to different confidence scores (e.g. coding potential, GO Evidence Codes), you can filter the gene set in order to provide, for instance, a high confidence gene set to train ab initio software, or a high confidence gene set to submit to a suitable repository and keep the full set for manual curation.\n\nThe combiners are probably the most popular and widely used gene prediction approach. They integrate the best of both worlds: they have an ab initio part that is then often complemented with extrinsic information (Figure 3). Especially, nowadays, with the advances of sequencing technologies, these approaches are increasingly used, reflecting the growing number of new tools and software trying to integrate RNA-Seq, protein or even intrinsic information. However, not all these combiners are the same. While some simply aim to pick the most appropriate model or build the consensus out of the provided input data (where an ab initio prediction tool might be one of them) for a given locus, others have a more integrated approach in which the intrinsic prediction can be modified by the given extrinsic data. The advantage of the latter is that they allow one type of information to overrule the other if this results in an overall more consistent prediction.\n\nOn the left, the diagram shows a typical assembly process. At the end of the process, scaffolds or chromosomes ready to be annotated are obtained. These scaffolds are then annotated using two different methods. The first method is called ab-initio and requires a known set of training genes. Once the ab initio tool has been trained it can be used to predict other similarly structured genes. The second similarity-based approach relies on experimental evidence such as CDSs, ESTs, or RNA-seq to build gene models. Combiners (such as Maker or Eugene) can then incorporate all of these results, eliminate incongruences, and present gene models best supported by all methods.\n\nApart from the choice of which tool to use, the choice of which data to integrate also has an influence on the final result. This is especially the case for the use of protein information. Error propagation is a real danger. Therefore, curated datasets, are preferred over the more general but less clean ones because it is vital that the provided information be as reliable as possible. The use of transcript information is less prone to error propagation although it is of importance that one realises what kind of data is being used. Short read RNA-Seq data is easily generated and is often an inherent part of a genome project. A downside is the short length of the reads. It will give accurate information on the location and existence of the exons but it will sometimes be more difficult to know how these exons are combined into a single gene structure. Therefore, it is becoming common to complement the short read transcript data with long read transcript information. Those will often contain the full set of exons into a single read and will as such provide unambiguous information on the complete gene structure and even alternative transcripts.\n\nWhen performing genome annotation, choices have to be made, not only what tools to use but equally important what kind of data to use. It is clear that the choice should go towards the more reliable but unfortunately sometimes less comprehensive data sources as the use of lower quality information will inevitably lead to an inferior gene prediction result.\n\nThe ultimate goal of the functional annotation process (Figure 4) is to assign biologically relevant information to predicted polypeptides, and to the features they derive from (e.g. gene, mRNA). This process is especially relevant nowadays in the context of the NGS era due to the capacity of sequencing, assembling, and annotating full genomes in short periods of time, e.g. less than a month. Functional elements could range from putative name and/or symbols for protein-coding genes, e.g. ADH to its putative biological function, e.g. alcohol dehydrogenase, associated gene ontology terms, e.g. GO:0004022, functional sites, e.g. METAL 47 47 Zinc 1, and domains, e.g. IPR002328, among other features. The function of predicted proteins can be computationally inferred based on the similarity between the sequence of interest and other sequences in different public repositories, e.g. BLASTP against Uniprot. Caution should be taken when assigning results merely based on sequence similarity as two evolutionary independent sequences which share some common domains could be considered homologs62. Thus, whenever possible, it is better to use orthologous sequences for annotation purposes rather than simply similar sequences63. With the growing number of sequences in those public repositories, it is possible to perform various searches and combine obtained results into a consensus annotation. The accurate assignment of the functional elements is a complex process, and the best annotation will involve manual curation.\n\nThis schema is showing a typical functional annotation pipeline, in which functional roles are assigned to coding sequences (CDSs) inferred in the gene prediction process. The process implements three parallel routes for the definition of functions. The first refers to proteins domains and motifs, the second for orthology search and finally the third is applied to homology search. At the end, the output from the three different sources is put together for more valuable predictions.\n\nThere are two main outcomes of the functional annotation process. The first is the assignment of functional elements to genes. Downstream analysis of these elements allow further understanding of specific genome properties, e.g. metabolic pathways, and similarities compared with closely related species. The second result of the functional annotation is the additional quality check for the predicted gene set. It is possible to identify problematic and/or suspicious genes by the presence of specific domains, suspicious orthology assignment and/or absence of other functional elements, e.g. functional completeness. These problematic genes can include those belonging to another species due to contamination, those detected as TEs, non-functional and/or artefactual genes annotated by error.\n\nThere are a number of tools available for functional annotation that allow users to obtain annotations for their gene set of interest via public databases in a high-throughput manner. These tools often start by sequence similarity search using tools like BLAST, HMMER or LAST against either non-redundant sequences database from NCBI GenBank and/or UniProt reference clusters (UniRef). After the initial homology search, candidate sequences can be assigned to one or more orthology groups using either best-reciprocal or tree-based methods63. Alternatively, users can make use of machine learning methods, such as Hidden Markov Models (HMM) or neural networks to predict particular patterns from a given input gene set. The majority of these tools are freely available for the academic users, working under Linux OS and are often part of large-scale annotation pipelines.\n\nFor those users who do not want to run individual tools and combine results, there are a few available workflows that provide the entire annotation process. These pipelines can either include installation of the required tools and corresponding databases, or users are required to make this installation on their own and the pipeline just provides a framework for the analysis.\n\n\n8. Use well-established output formats and submit your data to suitable repositories\n\nThe output of a genome annotation pipeline is almost always in GFF format. The information captured includes the structure and often the function of features of the genome, but usually not the actual sequence. Together with the Fasta file that was used in the annotation process, the sequence of these features can however easily be extracted. Other output formats are GTF, BED, Genbank, and EMBL, of which the last two include both sequence and annotation and are often used when submitting annotation results to sequence repositories. Some of these formats use controlled vocabularies and ontologies to guarantee interoperability between analysis and visualisation tools. We highly recommended the adoption of Fasta and GFF3 output formats. Both formats are compatible with the Genetic Model Organism Database (GMOD), a powerful suite of tools used for genome annotation, visualisation, and redistribution of genome data. By adhering to commonly used formats, you are making your results more useful to other researchers.\n\nTo improve the availability and findability of results from genome annotation projects, the annotated sequences have to be submitted to databases, such as Genbank at the National Center for Biotechnology Information (NCBI)64 or the European Nucleotide Archive (ENA)65. In these archives, the information relating to experimental workflows are captured and displayed. A typical workflow includes: 1) the isolation and preparation of material for sequencing, 2) a run of a sequencing machine in which sequencing data are produced, and 3) a subsequent bioinformatic analysis pipeline. ENA records this information in a data model that covers input information (sample, experimental setup, machine configuration), output machine data (sequence traces, reads and quality scores) and interpreted information (assembly, mapping, functional annotation).\n\nThere are also a growing number of theme-based genome databases. Human genome sequence projects are recommended to use the European Genome-phenome Archive (EGA)66. EGA is a service for permanently archiving and sharing data resulting from biomedical research projects, and all types of personally identifiable genetic and phenotypic can be included. This service provides the necessary security to control access and maintain the confidentiality of patient data, while providing access to researchers and authorized physicians to view the data. The data was collected from individuals whose consent agreements authorize the disclosure of data only for specific investigations.\n\n\n9. Ensure your methods are computationally repeatable and reproducible\n\nReproducibility and repeatability have been reported as a major scientific issue when it comes to large scale data analysis67. For genomics to fulfil its complete scientific and social potential, in silico analysis must be both repeatable, reproducible and traceable. Repeatability refers to the re-computation of an existing result with the original data and the original software. For instance, the authors report numerical instability arising from a mere change of Linux platforms, even when using exactly the same version of the genomic analysis tools.\n\nFortunately, solutions exist and along with their report of numerical instability, the authors did show that repeatability could be achieved through the efficient combination of containers technology and workflow tools. Containers can be described as a new generation of lightweight virtual machines whose deployment has limited impact on performances. Container methods, such as Docker and Singularity, make it possible to compile and deploy a software in a given environment, and to later re-deploy that same software in the same original environment while being hosted on a different host environment. Once encapsulated this way, analysis pipelines were shown to become entirely repeatable across platforms.\n\nSeveral workflow management systems, such as Nextflow, Toll and Galaxy, have recently been reported as having the capacity to use and deploy containers. These tools all share the same philosophy: they make it relatively easy to define and implement new pipelines, and they provide more or less extensive support for the massively parallel deployment of these pipelines across high performance computational (HPC) infrastructures or over the cloud.\n\nContainerization also provides a very powerful way of distributing tools in production mode. This makes it an integral part of the ongoing effort to standardise genome analysis tools. The wide availability of public software repositories, such as GitHub or Docker Hub provides a context in which the implementation of existing standards bring immediate benefits to the analysis, both in terms of costs, repeatability and dissemination across a wide variety of environments.\n\nThe choice of a workflow manager and the proper integration of the selected pipelines through a well thought containerization strategy can therefore be considered an integral part of the genome annotation process, especially if one expects annotation to keep being updated over time. This makes the adoption of good computational practices like the one described here an essential milestone for genomic analysis to become compliant with the new data paradigm. In order to carry this out, the first guidelines to make data “findable, accessible, interoperable and re-usable” (FAIR)68 was published in 2016. Even if FAIR principles were originally focused on data, they are sufficiently general so these high level concepts can be applied to any Digital Object such as software or pipelines.\n\nRepeatability is merely the most technical side of reproducibility. Reproducibility is a broader concept that encompasses any decision and bookkeeping procedure that could compromise the reproducibility of an established scientific result. For this reason the implementation of the FAIR principle also impacts higher level aspect of the genome annotation strategy and for a genomic project to be FAIR compliant, these good practices should be applied to both data, meta-data and software. This can be achieved as follows:\n\nFindable: Globally unique and persistent identifiers for data and metadata. Identifiers should persist across release and make it possible to trace back older analysis and relate them to the current annotation. Deprecated annotation should remain traceable. Even when data is not any longer available, meta-data should remain and provide a description of the original data.\n\nAccessible: Proper registration of data and metadata in suitable public, or self-maintained repository. All data should be properly indexed and searchable and accessible by identifier using standardized protocols\n\nInteroperable: Data and meta-data must be deposited using the most commonly used format\n\nReusable: Data and meta-data standards should insure that the data is sufficiently well characterized to be effectively reused in future analysis or to be challenged by novel evaluation methods. Licensing should be as little restrictive as possible.\n\nFindable: Software and pipelines should be deposited in an open source registries along with proper technical descriptions allowing their rapid identification.\n\nAccessible: Software should be deposited in public repositories such as GitHub, Docker Hub, so as to be available. Attempts should be made at having the licensing as little restrictive as possible. ELIXIR has taken the challenge to provide a long-term sustainable infrastructure to host software containers. Thus, this is the desirable solution to ensure software accessibility.\n\nInteroperable: Software should use the most common format and should be adequately documented. It should come along with a proper versioning for both the software and the reference biological databases they operate upon. The software behavior should also be adequately described using the right metadata, thus allowing programmatic interaction with other resources.\n\nReusable: Software should be distributed in open-source format so as to ensure possible long term maintenance by third parties. Software should be encapsulated within containers ensuring the permanent availability of production mode pipelines. Authors should be encouraged to develop their pipelines in commonly used workflow managers (Galaxy69, Nextflow70, Snakemake71). Decisions should be taken on the basis of a compromise between the level of usage of the selected workflow and its support of the required features. It should also contain meta-data describing which parameters have been used with the software in order to guarantee data reproducibility.\n\n\n10. Investigate, re-analyse, re-annotate\n\nSuccessful genome annotation projects do not just end with the publication of a paper; they should produce sustainable resources to promote, extend and improve the genome annotation life cycle.\n\nSome genome consortia choose to manually review and edit their annotation data sets via jamborees, for instance the BioInformatics Platform for Agroecosystem Arthropods. Although this process is time- and resource-intensive, it provides opportunities for community building, education and training. All these elements help to improve the annotation life cycle and are promoted by the International Society for Biocuration.\n\nManual and continuous annotation are critical to achieve accurate and reliable gene models, mRNA, TEs, regulatory sequences, among other elements. In addition, research communities will face the generation of a huge volume of new data including re-sequencing, transcriptomics, transcriptional regulation profiling, epigenetic studies, high-throughput genotyping and other related whole-genome functional studies. Thus, it is important to provide a software infrastructure to facilitate the updating of the genomic data.\n\nTools such as WebApollo72 from the GMOD project or web-portals like ORCAE73 are particularly useful. These tools allow groups of researchers to review, add and delete annotations in a collaborative approach. The applications are robust and flexible enough to allow the members of a group to work simultaneously or at different times. The administration of the server allows to initiate a session to a user and if it has the authorization, to edit the content.\n\nThanks to this system, annotations of genomes can be improved in a continuous cycle as data is collected and updated. In this way the annotations can always continue to improve.\n\nOther useful tools include Artemis74, a successful curation software from the European Sanger institute and Gencode75, which seems to succeed the Havana team’s Vega76.\n\n\nConcluding remarks/general recommendations\n\nGenome assembly and genome annotation are areas where there are no gold standards. Projects are often explorative, and knowing if your results are good or bad is often hard to determine. This is especially true if you are working with organisms only distantly related to already sequenced ones, which leaves you with little to compare with. Try to set an aim with your study, and stop working with the assembly and annotation once you have a result that allows you to reach that aim. Do not fall into the trap of wanting a “perfect” genome, as this tends to lead to a project that never ends. But also do not be afraid to start your own assembly and annotation project. With the development of new sequencing technologies it is more feasible than ever, and a well assembled and annotated genome will be a resource you can use for many years to follow.\n\nThe recommendations we give are broad guidelines, and we try not to force readers into explicit technologies or software. We do, however, present advantages of certain NGS technologies in specific cases, for example when looking at genome properties such genome size, complexity, or GC content. We also explain pitfalls to avoid throughout the whole assembly and annotation process. Finally, we also encourage the adoption of our guidelines regarding data deposition and reproducibility, as they offer a simple mechanism to improve the quality, findability, reusability and sustainability of results derived from genome assembly and genome annotation projects.",
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}
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[
{
"id": "31487",
"date": "09 Mar 2018",
"name": "Bruno Contreras-Moreira",
"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 opinion article by Victoria Dominguez Del Angel and collaborators is a well-written sort of broad tutorial which will certainly be of help for anyone trying to sequence and assemble a genome sequence with little previous experience. While it avoids details that are required when the real work is actually carried out (for instance, K-mer length), it discusses important questions that must be addressed before carrying out genomic projects, and choices that must be made along the way down to the point that data and procedures are published and released.\nNext I comment on specific parts of the text that I believe can be improved.\n0. Introduction\n0.1 Genomics in 2018 cannot possibly be done without a pan-genome perspective. This has been true for years in the area of microbiology, where projects now rarely assemble and annotate a single genome, but rather a few dozens related strains. In addition, this is becoming state-of-the-art also for genomic projects of crops and model plants, such as Brachypodium distachyon, as well as in human medicine. A couple of sentences should be added explaining that in this context a group of genomes are sequences, assembled and annotated in parallel, which makes it more challenging but also facilitates spotting and correcting errors.\n0.2 I would add to the checklist a literature survey to identify related genomes.\n\n1. Investigate the properties of the genomes you study\n1.1 Heterozygosity I would add a sentence about the outcrossing or selfing nature of species, which has a direct impact on the expected heterozygosity, and often limits the possibility to obtain inbred individuals. In plants double-haploids are used to this end (see for instance 1.\n1.2 Ploidy level Please note that it has been estimated that many plant species are polyploid 2-3. One strategy to solve these complex genomes is to first sequence the genomes of the expected/known parental species.\n\n2. Extract high quality DNA\n2.1 Organelle DNA I would add that frequently chloroplast genomes or plastomes are of high interest as they can provide a complementary, maternally-biased evolutionary history. This might be of particular interest for polyploid species as it might help determine which was the maternal and which the paternal genome donor. Even with a low ratio of organelle DNA in our experience is likely that complete chloroplasts can be assembled and annotated (see for instance 4) as a by-product of whole genome sequencing. Instead, mitochondria seem to be difficult to assemble in plants.\n\n4. Estimate the necessary computational resources\nI would add that the assembly tools selected at the time the proposal was written are likely to be replaced by others when the work is actually to be performed due to pace of innovation in this area.\n5. Assemble your genome\nIn the last left-side paragraph it is said that “misassemblies in an existing assembly need to be broken prior to scaffolding in order to join the correct contigs together.” . This is followed by another sentence later on “Unfortunately, there are few ways to distinguish what is real, what is missing, and what is an experimental artefact.”\nIn our experience many scaffolding errors can be spotted by mapping back the original sequence reads to the assembly and then visualizing the results with browsers such as IGV. Of course this can be cumbersome for very large assemblies, but tools such as SEQuel and Reapr can be really helpful for such tasks.\n5.1 BUSCO is shown as a way of evaluating assembly quality in page 10. I would add that in pan-genome projects this can be generalized so that assemblies, and subsequent annotations, can be evaluated in terms of the proportion of core-genes contained. Poor assemblies can be identified frequently for having less core-genes than expected.\n\n7. Annotate genes with high quality experimental evidence I feel this section can be improved by:\n7.1 Stressing that different annotation strategies often yield annotation sets that are implicitly biased. Therefore, if the user plans to compare its genome to others should make and effort to use a similar approach so that any conclusions regarding issues such as gene family expansions are not caused by the underlying methodology. Indeed we have seen this happening when annotating a microbial pan-genome and then comparing it to genomes in public databases.\n7.2 Adding a section on microbial genome annotation, mentioning popular tools such as PROKKA, RAST or NCBI Prokaryotic Genome Annotation Pipeline, and commenting on the annotation of bacterial features such as CRISPRs or plasmids.\n7.3 On page 12, 1st paragraph, is its stated that “Transcripts on the other hand provide very accurate information for the correct prediction of the genes” I think it should definitely be mentioned that, unlike HQ protein sequences, transcripts allow the annotation of unstranslated regions (UTR) and despite their noise and the isoform deluge can be used to define also gene promoters, which can then be annotated in terms of regulation.\n7.4 I miss a section on comparison of gene order/synteny\n\n8. Use well-established output formats and submit your data to suitable repositories\nI would add that soft/hard repeat-masked versions of the genome sequence can be made available in FASTA format, which are helpful for subsequent analyses of regulatory sequences.\nMinor edits:\nPage 3, 1st paragraph: “The advice here presented is based on a need seen while working in the ELIXIR-EXCELERATE task “Capacity Building in Genome Assembly and Annotation”. In this capacity we have”\ncan be changed to\n“The advice here presented is based on a need first seen while working in the ELIXIR-EXCELERATE task “Capacity Building in Genome Assembly and Annotation”. In this project we have”\nPage 6, 2nd paragraph Why is “or after” in bold?\nPage 14, left column Fasta format should be FASTA format\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": "4423",
"date": "16 Feb 2019",
"name": "Steven Sullivan",
"role": "Reader Comment",
"response": "Very useful, but it would be more useful still if section 7.2 on Functional Annotation, where it says, \"There are a number of tools available for functional annotation that allow users to obtain annotations for their gene set of interest via public databases in a high-throughput manner.\" was accompanied by a table listing tools and web servers used for this purpose. The more comprehensive the better."
}
]
},
{
"id": "30566",
"date": "12 Mar 2018",
"name": "Dave Clements",
"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 does a good job of covering the big picture of what's needed to assemble and annotate a genome. Its stated goal is to give guidelines that are \"broadly applicable\" and \"intended to be stable over time.\"\n\nThis paper achieves that goal, and all of my concerns are minor. However, \"stable over time\" was a frustrating goal for me. This means that comments on specific sequencing technologies and software are not as common as they would be in a review article on the state of the art. This is a fine line to walk.\nSpecific comments\nIntroduction\n1. Think about dropping or shortening the checklist at the end of the introduction. I believe that all of this is covered in detail in the individual sections.\nRepeats\n1. \"Contigs\" used for the first time, Not sure if these need to be explained. (Could reference figure 3.)\n\nChemical purity\n1. What ONT means is explained later. Move that explanation here.\n2. The text says:\n\nIt is widely known in the PacBio community that samples rich in contaminants...\nAre there any references for this? This highlights a larger question with the paper. It is an opinion article and it contains many opinions such as\n\nThe characteristics of the genomes being assembled have a greater impact on the results than the choice of the algorithm.\nI am not disputing any of these statements, but if and when references exist that support them, then those references should be included.\nLong read genome assembly This section says error rates are 5-20%. Elsewhere in the paper they are given as \"up to 10% or 15%.\"\nScaffolding and gap filling\nThe very end of this section states:\n\nBe aware that any changes to a genome assembly will most likely necessitate annotation to be re-started from scratch, and you should therefore be sure to “freeze” the assembly completely before starting annotation.\nI think \"restarted from scratch\" gives the wrong impression. Tools such as MAKER can do liftover from one version of an assembly to the next. Perhaps this could be clarified?\nEnsure your methods are computationally repeatable and reproducible\nToll -> Toil ?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-148
|
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