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https://f1000research.com/articles/6-1984/v1
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10 Nov 17
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{
"type": "Research Note",
"title": "Phenotype and genotype of lactic acid bacteria (LAB) isolated from the tiger grouper Epinephelus fuscoguttatus alimentary tract",
"authors": [
"Nursyirwani Nursyirwani",
"Widya Asmara",
"Agnesia Endang Tri Hastuti Wahyuni",
"Triyanto Triyanto",
"Muhammad Fauzi",
"Zainal A. Muchlisin",
"Widya Asmara",
"Agnesia Endang Tri Hastuti Wahyuni",
"Triyanto Triyanto",
"Muhammad Fauzi",
"Zainal A. Muchlisin"
],
"abstract": "Lactic acid bacteria (LAB) have been isolated successfully from the tiger grouper Epinephelus fuscoguttatus intestine. However, their genus or species have not been identified. Therefore, the objective of the present study was to characterize the three isolated LAB (KSBU-12C, KSBU-5Da, and KSBU-9) based on their phenotype and genotype. The LAB phenotype was examined by observing morphological features including cell morphology, spore production and motility. The physiological tests were performed in 6.5% NaCl at the temperatures of 10oC and 45oC, and the biochemical tests were evaluated based on the production of enzymes catalase, oxidase and arginine dehydrolase, following the Standard Analytical Profile Index, API 50 CH kit. The genotype was examined based on 16S rDNA gene sequence analysis , and the products were analyzed using the BLAST (Basic Local Alignment Search Tool) NCBI database. The three isolates (KSBU-5Da, KSBU-12C, and KSBU-9) were categorized into the genus Enterococcus. 16S rDNA sequence analysis indicated that the isolates had 99% similarity to E. hirae ATCC 9790, registered in GenBank with accession number NR_075022.1. It was concluded that the three LAB isolates taken from the tiger grouper Epinephelus fuscoguttatus are E. hirae.",
"keywords": [
"phenotype",
"genotype",
"characterization",
"lactic acid bacteria",
"tiger grouper"
],
"content": "Introduction\n\nLactic acid bacteria (LAB) have been investigated as probiotics in the feed of terrestrial and aquatic farmed animals1–7. This is based on the fact that LAB inhabit the human and animal gastrointestinal tract. This group of bacteria also convert lactose to acetic acid, thus decreasing gastrointestinal pH and naturally preventing colonization of harmful bacteria8. This is because they have the ability to produce the inhibitory materials that inhibit pathogen growth in the digestive tract9. Besides that, the probiotics can stimulate host immunity responses2,9,10.\n\nLAB are a group of gram-positive, non-sporulating bacteria, in cocci or rod form. They can live in aerobic or facultative anaerobic conditions. LAB are able to produce lactic acid through the fermentation process of carbohydrates11,12, and the optimum growth is at pH 5.5 to 6.0, so growth is restricted to neutral and alkaline conditions13. Those naturally occurring bacteria are non-pathogenic to humans and animals, hence being generally recognized as safe (GRAS) organisms14.\n\nAccording to Parada et al.15 and Carr et al.16 17l generas of LAB have been described worldwide. They are common in food products, for instance milk, meat, fruits, and vegetables, and also in genital and alimentary tracts of humans and animals17, including fish intestines18,19.\n\nMolecular approaches based on DNA such as 16S rRNA sequencing have been used to characterize the intestinal microflora, and this method is proven to be accurate and fast20. For instance, Balcazar et al.21 have identified LAB isolates from salmonid fish using 16S rRNA genes, and they found that there were 6 species of LAB, namely Lactobacillus curvatus, L. sakei, L. plantarum, Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, Carnobacterium maltaromaticum, and Leuconostoc mesenteroides. Sun et al.22 investigated microbiota from gastrointestinal tracts of the orange-spotted grouper Epinephelus coioides, using a standard isolation procedure in combination with analysis of 16S rRNA sequences, and found several bacterial species, namely Vibrio parahaemolyticus, V. harveyi, V. metschnikovi, V. alginolyticus, Delftia acidovorans, Pseudomonas putida, Acinetobacter baumannii, Burkholderia cepacia, Erwinia carotovora, S. aureus, L. lactis, L. casei, E. faecium, Bacillus pumilus, B. clausii, and Psychrobacter sp.\n\nNursyirwani et al.23 successfully isolated three LAB from the tiger grouper alimentary tract, and found that LAB have antibacterial activity against Vibrio alginolyticus. However, the taxonomic classification of these bacteria has not been examined. Therefore, the objective of the present study was to characterize the three LAB isolates from tiger grouper based on their phenotype and genotype.\n\n\nMethods\n\nThe experiment was carried out in compliance with the ethical guidelines provided by the Research Institution of Riau University (SOP/02/PL/LPPM/2016). Lactic acid bacteria (LAB) had been isolated from the tiger grouper (E. fuscoguttatus) alimentary tract following the procedure of Bucio et al24. The tiger grouper Epinephelus fuscoguttatus was taken from the aquaculture pond in Situbondo of East Java, Indonesia. The alimentary tract was removed from the fish, opened, and the gut content was removed. Then, the internal wall of the tract was scraped gently, and the mucus was collected in a sterile test tube containing 9 ml of phosphate buffer saline (PBS) solution at pH 7.2. The suspension was serial in PBS solution from 10-1 to 10-6. A volume of 0.1 ml of each serial dilution was inoculated in petri-dish containing deMan-Rogasa-Sharpe (MRS, Oxoid) agar medium. All inoculated dishes were incubated at 37°C for 24–48 hours. The bacterial colonies were reinoculated in new fresh MRS agar medium. Then, the grown colonies were inoculated on medium agar of glucose-yeast extract-pepton, supplemented with CaCO3 (GYP+CaCO3) agar and incubated at 37°C for 24 hours. The presence of LAB was indicated by the growth of white colonies surrounded by clear zones. The isolates were then examined for gram staining and catalase tests. Gram-positive and negative catalase isolates were selected and reinoculated in new fresh MRS agar and incubated at 37°C for 24 hours. The isolates were stored in a refrigerator at a temperature of -10°C before being used for the next tests.\n\nThe three LAB isolates (KSBU-12C, KSBU-5Da, and KSBU-9) previously isolated from the intestine of the tiger grouper were reinoculated in MRS broth (MRS, Merck). Phenotype characterization was based on cell morphology, physiology, and biochemical tests. Cell morphology involves observing the shape of the cells, spore production, and motility. Physiological tests were performed in 6.5% NaCl at 10°C and 45°C. Biochemical tests were based on the production of enzymes catalase, oxidase, and arginine dehydrolase following the Standard Analytical Profile Index, API 50 CH kit (BioMerieux SA, Marcy I’Etoil, France). Molecular characterization of 16S rDNA gene sequences allowed identification of the genotype of the LAB isolates.\n\nDNA of isolates KSBU-12C, KSBU-5Da, and KSBU-9 was extracted following the procedures of Ausubel et al25. Each of the isolates were reinoculated in 5ml of MRS broth and then incubated at 30°C for 48 hours.\n\nThe 1.5 ml culture was centrifuged at 13000 rpm for 2 minutes, the supernatant was discarded, the pellet was resuspended in 467 µL of TE buffer, and 30 µL of lysozyme was added and then incubated at 37°C for 30 minutes. The suspension was added to 30 µL of 10% SDS and mixed with 3 µL of K proteinase and incubated at 37°C for an hour. The mixture was added to 100 µL 5 M NaCl, mixed well, added to 80 µL of CTAB/NaCl solution, mixed thoroughly, and incubated for 10 minutes at 65°C. The mixture was further incubated at 65°C for 10 minutes, mixed by stirring up and down 100 times, and the same volume (0.7–0.8 ml) of phenol/chloroform/isoamyl-alcohol (PCIAA) was added, mixed well, and centrifuged for 5 minutes. The supernatant beneath the interphase was transferred to a new microtube, mixed with an equal volume of PCIAA, and centrifuged for 5 minutes.\n\nThe supernatant was placed into a new the microtube, then the isopropanol (60% of the volume) was added to the tube and centrifuged for two minutes. The supernatant was discarded, the DNA pellet was washed with 70% ethanol (±50 µL), centrifuged for 5 minutes, the ethanol was discarded, and the pellet was dried for 1 hour. Finally, the pellet was added to 100 µL TE buffer, vortexed, and stored at -20°C until it was used for further experiments.\n\nThe genome of bacterial 16S rDNA was amplified by PCR using a thermal cycler (Eppendorf, Mastercycler Personal). The PCR reaction consisted of 25 µL of final solution of 12.5 µL PCR mix (Promega), 1.0 µL of universal primer (1st base) 24F (5-‘AGAG TTT GAT CCT GGC T-3’) and 1.0 µL primer 1540R (5-‘AAG G AGGT GAT CC AG CC GCA-3’), 0.5 µL of DNA template, and 10.0 µL of dH2O.\n\nThe thermocycler program was run with the following conditions: pre-denaturation at 94°C for 4 minutes; 29 cycles of denaturation at 94°C for 3 minutes; annealing at 55°C for 1 minute; extension at 72°C for 1 minute and 30 seconds; and the final extension at 72°C for 10 minutes. The PCR product was detected by running electrophoresis in 1% of agarose gel, stained with ethidium bromide (1 µL/10 ml), and visualized under a UV lamp for 1500 base pairs of the target product.\n\nThe PCR product with clear bands was sent to 1st Base Laboratories in Singapore for sequencing. The sequencing program was performed by ABI PRISM 3730×L GENETIC ANALYZER (Applied Biosystems, USA). The sequenced products were blasted (NCBI Basic Local Alignment Search Tool)26, and the results were presented as homology (%) of bacterial DNA sequences to the database sequences27.\n\n\nResults\n\nThe three LAB isolates were categorized as cocci, gram-positive, non-motile, and non-spore forming. The LAB did not produce catalase, oxidase, and arginine dehydrolase, and were facultative anaerobes, with growth at 45°C at concentrations of 6.5% NaCl. However, differences were observed in the ability to produce acid from carbohydrates provided in the API 50 CH kit. Acid was not produced from substrates L-arabinose, D-raffinose, and D-xylose by the KSBU-12C isolate, meanwhile the KSBU-5Da isolate did not use L-arabinose, gluconate, glycerol, D-mannose, sorbitol, D-tagatose, and D-xylose; and the KSBU-9 isolate used L-arabinose, and D-xylose. The cell morphology, and the physiological and biochemical characteristics of the isolates are presented in detail in Table 1.\n\nNote: +, 90% or most strains are positive; -, 90% or most data are negative; O/F, oxidative or fermentative; nd, data not available; d, variable.\n\nThe 16S rRNA derived from the LAB isolates were amplified by PCR with two universal primers 24F (5-‘AGAG TTT GAT CCT GGC T-3’) and 1540R (5-‘AAG G AGGT GAT CC AG CC GCA-3’). The LAB isolates have a highly similar 16S rRNA sequence (99% similarity as shown in Table 2; those were 1510-bp for KSBU-12C and 1526-bp for KSBU-5Da and KSBU-9) (Figure 1). A phylogenetic tree was constructed for the LAB isolates sequences using Clustal W, followed by the Mega 5 neighbor-joining program as shown in Figure 2.\n\nScale 0.02 indicates sequence difference percentage. Escherichia coli strain EcPH2 was used as the outer group.\n\n\nDiscussion\n\nBased on morphological, physiological, and biochemical characteristics, the LAB isolates (KSBU-12C, KSBU-5Da, and KSBU-9) show similarity to three bacterial species of the Enterococcus genus found by Ludwig et al.28. The analysis of the 16S rDNA sequence indicated that KSBU-5Da, KSBU-12C, and KSBU-9 isolates were of the Enterococcus genus, and with high similarity to E. hirae ATCC-9790 registered in GeneBank with accession number NR_075022.1 (Table 2). Each of the LAB isolates indicated 99% homology with E. hirae ATCC 9790. Strains of bacteria with the same or more than 97% 16S rRNA gene sequence belonged to one species29.\n\nE. hirae ATCC 9790 is a gram-positive LAB used as a model organism in basic research for four decades30. E. hirae was first isolated in the intestines of pigs and chickens31. E. hirae strain NRIC 0101 was collected by the Japan Nodai Culture Collection Center, NCCC32. In addition, E.hirae C17456 was a species isolated from chickens33. In summary, Enterococcus hirae has been found in humans, poultry, foods for instant dosa batter, and in the environment34–37. Together with E. faecium, E. faecalis, E.casseliflavus, and E. mundtii they are enterococci which were predominant in coastal waters and sediments of South of California38. E. hirae was one of the homofermentative strains obtained from traditional fermentation products such as pla-som and pla-chom (fermented fish) in Thailand39. It has been shown that E. hirae K34, L. plantarum K39, and L. plantarum K50 isolated from Kung-Som (fermentative shrimp) display antimicrobial activity against pathogenic bacterial strains of Bacillus cereus, E. coli, Staphylococcus aureus, Salmonella typhimurium, Vibrio cholerae, and Listeria monocytogenes40.\n\nInformation on the use of E. hirae as a probiotic in aquaculture is still limited. Mazurkiewicz et al.41 reported that E. hirae isolated from the intestine of common carp (Cyprinus carpio L.) and applied into feed did not have a significant effect on the growth performance of the common carp. In contrary, another report by Adnan et al.42 and Carlos et al.43 showed that E. hirae from freshwater fish Catla catla and rainbow trout Oncorhynchus mykiss had a significant effect on inhibiting the growth of pathogens such as Escherichia coli, Staphylococcus aureus, Salmonella typhi and Pseudomonas spp.\n\nIn the present study, the role of E. hirae isolated from tiger grouper fish the growth performance of fish has not been evaluated yet. Therefore, further study will be needed to examine the effect of these bacteria on the feeding of cultured fish.\n\n\nConclusions\n\nThis research has successfully characterized three of the LAB isolates (KSBU-12C, KSBU-5Da, and KSBU-9) based on their phenotype and genotype. All isolates were determined to be E. hirae.\n\n\nData availability\n\nSequenced DNA of LAB isolates can be found in NCBI GenBank, with accession numbers MF977716 to MF977718.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was partially supported by Universitas Riau.\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 at the Microbiology Laboratory of the Fishery Department, Gadjah Mada University, Indonesia.\n\n\nReferences\n\nArisa II, Widanarni W, Yuhana M, et al.: The application of probiotics, prebiotics and synbiotics to enhance the immune responses of vannamei shrimp (Litopenaeus vannamei) to Vibrio harveyi infection. AACL Bioflux. 2015; 8(5): 772–778. Reference Source\n\nYulvizar C, Dewiyanti I, Defira CN, et al.: Pathogenicity assay of probiotic potential bacteria from the common carp Cyprinus carpio. AACL Bioflux. 2015; 8(5): 694–698. 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Reference Source\n\nAdnan M, Patel M, Hadi S: Functional and health promoting inherent attributes of Enterococcus hirae F2 as a novel probiotic isolated from the digestive tract of the freshwater fish Catla catla. PeerJ. 2017; 5: e3085. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAraújo C, Muñoz-Atienza E, Hernández PE, et al.: Evaluation of Enterococcus spp. from rainbow trout (Oncorhynchus mykiss, Walbaum), feed and rearing environment against fish pathogens. Foodborne Pathog Dis. 2015; 12(4): 311–322. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "27853",
"date": "14 Nov 2017",
"name": "Medana Zamfir",
"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 describes the identification of three LAB isolates from the alimentary tract of a tiger grouper. The identification was done using various phenotypic and genotypic methods. However, there are some remarks or suggestions that can be made:\n- First of all, the authors mention that LAB strains were previously isolated; why they included then in the Methods section and how they were isolated?; on the other hand, they do not mention from how many individuals they isolated these strains from? Were they isolated from one fish? Then, in fact, it is only one strain, since all three were identified in the end as Ent. hirae.\n\n- Introduction:\n\"According to Pareda et al. ... 17l genera...\" - that \"l\" doesn't belong there. I think the authors should use \"microbiota\" instead of \"microflora\".\n\n- Methods:\n\n- Collection of LAB isolates\n\"The suspension was serial in PBS...\" - I think \"diluted\" is missing. \"Gram positive and negative catalase isolates...\" should be \"Gram positive and catalase negative isolates...\" It is not clear how they kept the isolates, at -10 degrees, but on agar media? The subtitle \"Bacterial isolates\" should be \"Identification of the bacterial isolates\" since they are talking about identification. \"Physiological tests were performed in 6.5% NaCl...\" should be maybe \"... in the presence of 6.5% NaCl...\" \"... production of enzymes such as...\"\n\n- Amplification of 16S rDNA\n\n\"The genome of bacterial 16S rDNA...\" should be \" The 16S rDNA gene from the bacterial genome...\"\n\n- Results:\nIn Table 1 there is a discrepancy between the legend and the table. For instance, in the Table they have \"n.a\"., while in the legend is \"n.d\".; in the table they have both \"d\" and \"D\", and in the legend only \"d\". Moreover, there are many rows with data only for the Enterococcus species, but for the strains isolated by them, there are no data available. What is the point to show these things? Some information from the Methods is repeated in the Results, such as the primers used. The paragraph with the similarities among the sequences and 16S rDNA amplicons, with Fig. 1 and Table 2, is not very clear. Perhaps they should mention first that the amplicons obtained with the universal primers were very similar (and give their size and the reference to Fig. 1) and then they should mention about the high similarity among the sequences (99%) with the sequence of Ent. hirae (reference to Table 2).\n\n- Discussion:\n\nThis section can be improved, maybe with information about the source of this strain in the alimentary tract, the importance for the host, why was the only strain found in their samples, etc. There is a paragraph that I would suggest to be changed in: \" Strains... with the same or more than 97% homology of the 16S rRNA gene sequence may be considered to belong to the same species\". \"Ent. hirae was first isolated...\" - I think the word \"from\" is missing.\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? 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? Partly",
"responses": []
},
{
"id": "27851",
"date": "22 Nov 2017",
"name": "Delianis Pringgenies",
"expertise": [
"Reviewer Expertise Marine natural product"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 has excellent relevance value to both the development of marine science and is suitable for publication. This article should be indexed when minor improvements have been made.\nThe abstract shows that this research is interesting.\nHow many isolation works? Of all isolation, three isolates were selected. The method used in the study should be listed on the abstract. Show keywords, keywords generally refer from general to specific, and not mentioned in title or abstract.\nIntroduction: It needs to be rewritten more purposefully, including backgrounds on topic selection and importance. Writing in English should pay attention to diction and grammar.\nMethod: It needs to include the reference method used. The exposure method has not been written in a systematic or accurate way, so it can be repeated by others.\nResults and Discussion: There are still repeated discussions. References used are not current, need to use the latest references.\nAn annotated version of the Word document can be viewed here.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "28009",
"date": "04 Dec 2017",
"name": "Asus Maizar Suryanto Hertika",
"expertise": [
"Reviewer Expertise My research in the field of ecotoxicology. My focus is primarily about the expression of protein or certain enzymes as biomarkers due to pollution of the aquatic environment."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, here are some comments about the contents of the article.\n\nIn the background it has not been explained specifically why the characterization of lactic acid bacteria isolated from the grouper intestine. The question is that the bacteria will be mixed with fish feed in order to increase the digestibility of the food or the prevention of pathogens that attack groupers.\n\nProcedure of taking bacterial inoculum in the grouper's bowel. Is the fish fasted first? This is to know the bacteria do live in the intestines of grouper fish instead of bacterial contaminants derived from food given.\n\nIn the discussion section has not been submitted phenotype characteristics of three types of bacterial isolates LAB. These traits include color, colony shape, surface roughness of the colony and will be better when displayed images with given information.\n\nThe RNA sequence results also need to be displayed to make the right conclusions in the characterization process. It would be great if supported with comparative literature.\n\nIt would be great if the discussion section uses more literature to discuss especially the latest journals.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1984
|
https://f1000research.com/articles/5-2524/v1
|
17 Oct 16
|
{
"type": "Software Tool Article",
"title": "Creating, generating and comparing random network models with Network Randomizer",
"authors": [
"Gabriele Tosadori",
"Ivan Bestvina",
"Fausto Spoto",
"Carlo Laudanna",
"Giovanni Scardoni",
"Fausto Spoto",
"Carlo Laudanna",
"Giovanni Scardoni"
],
"abstract": "Biological networks are becoming a fundamental tool for the investigation of high-throughput data in several fields of biology and biotechnology. With the increasing amount of information, network-based models are gaining more and more interest and new techniques are required in order to mine the information and to validate the results. We have developed an app for the Cytoscape platform which allows the creation of randomized networks and the randomization of existing, real networks. Since there is a lack of tools for generating and randomizing networks, our app helps researchers to exploit different, well known random network models which could be used as a benchmark for validating real datasets. We also propose a novel methodology for creating random weighted networks starting from experimental data. Finally the app provides a statistical tool which compares real versus random attributes, in order to validate all the numerical findings. In summary, our app aims at creating a standardised methodology for the validation of the results in the context of the Cytoscape platform.",
"keywords": [
"random networks",
"cytoscape",
"network randomisation",
"network analysis",
"network generation"
],
"content": "Introduction\n\nIn network analysis many tools have been developed to address different problems related to the extraction of useful information from systems modeled as graphs1. Cytoscape2 is a very well known platform, that supports hundreds of apps, that simplifies the information mining in complex networks, with a specific focus on biological applications. By using Cytoscape it is possible to perform topological analysis, cluster and motifs retrieval, biological enrichment, draw nice graphs, search for ontologies, etc. As the number of apps increases, the possibilities of performing more and more complex analysis grows together with the amount of information that could be used and retrieved. The problem is that this analysis remain, in general, preliminary to further experimental validations and, in this sense, a sort of benchmark for an in-silico validation is required3. A possible solution to this issue may come from the biological background of the process, from the literature or from experimental data4. But this is not always possible since the network may represent some complex processes whose biology is yet not well understood, or that take advantage of some novel insights that require a different validation. In these cases, when biological evidences are missing or incomplete, an interesting approach allows comparing the real experiments with a set of randomly generated experiments to verify the robustness of the real data5. In network analysis this is achieved by comparing the networks we are interested into, with some randomly generated networks.\n\nIn this sense, our app was created to address the problem of creating a validation layer which allows simulating random experiments. With randomly created networks it becomes possible to compare, by using a specific statistical test, the numerical results that come from a common network analysis. To do so, our Network Randomizer allows randomizing existing networks by using a simple shuffling algorithm and a degree preserving algorithm. Moreover it allows creating Erdős–Rényi, Barabási–Albert, Watts-Strogatz, Lattice, and Community Affiliation models. We also implemented the Multiplication model which is designed to generate weighted networks where nodes are multiplied, i.e. represented in different copies which have the same topological characteristics, to fit quantitative data. Finally a statistical module, based on the two-sample Kolmogorov-Smirnov test, compares networks in order to check whether the values come from the same distribution or if they should be considered different. The original idea behind the app was to compare topological attributes, the so called centralities, computed by using CentiScaPe6 or with the Cytoscape’s built-in Network Analyzer, but it supports the comparison of all kinds of numerical attributes in order to be useful to all users.\n\n\nMethods\n\nNetwork Randomizer follows a modular structure, making it easy to add additional random network models in future releases. Each model is initialized with some user’s specified parameters and once the generation is done the app deploys the network into the Network Control Panel, making it instantly available to the user for future use. Since the user may want to generate a number of random networks, the network views are not created by the app, in order to avoid pop-ups of networks in the Cytoscape window during the algorithm’s computation.\n\nThe app is divided in several classes which allow a very flexible and modular structure. The CyActivator class runs the application and communicates with Cytoscape. The RandomizerCore class is used as a model of the current Cytoscape state (network handling etc.). The MenuAction class allows app initiation. The OptionsMenu class refers to the main GUI which is used to interact with the app. The ThreadEngine allows creating and handling threads and multithread tasks. Finally the AbstractModel is an abstract class which defines the basic random model and offers several methods that could be useful when defining a new model. The other classes in the app refer to the models we implemented. In order to add a new model, a new class should be instantiated by following the AbstractModel implementation. Then it is necessary to modify the GUI in order to let the new model become part of the app.\n\nThe statistical module that we implemented is a two-sample Kolmogorov–Smirnov test7. It takes each pair of real and random networks, and for each of the attributes used, it calculates the difference between their distribution. The K−S definition of the distribution difference is the maximum gap between the cumulative probability functions of those probabilities. Although it relies on the cumulative probability functions, they never need to be explicitly calculated. In fact, the algorithm only needs to sort the two lists of attribute’s values and then run through them in parallel, summing the normalized (divided by the sum) values along the way and comparing the two sums, saving only the largest difference.\n\nSystem requirements. To install and run the Network Randomizer app, the Cytoscape software must first be installed. Once this is done, no additional system requirements exist since the app does not use any external library nor does it significantly increase the memory consumption. An updated Java version is suggested, since the last Cytoscape version works only with Java 8.\n\nIt is important to note that, when using the Multiplication model, the number of nodes that are generated could result in a huge network. This is due to the model itself which, starting from an user defined attribute, creates an array of random weights which will result in a number of nodes. It works by creating a range between the minimum and the maximum values found in the file, hence in the worst case the algorithm will create a network with #max copies of nodes for each node. In numbers this means that if the attribute file varies in a range [0 − 100] and the real network is made of 10 nodes, then the algorithm in the worst case may generate a network with (100 ∗ 10) + 10 nodes. Memory usage problems may also arise within the other models, depending on the number of nodes the user selects for creating a random network.\n\nWorkflow. The usual Network Randomizer workflow consists of a few distinct steps. First of all, the user needs to load one or more real networks. Depending on whether one or multiple networks are loaded, the final output will be different. It consists of a file which shows the results of the statistical analysis. If there is only one real network, the output file will present more details about it. If there are multiple real networks, then the output file will be a summary of all the statistical tests that were carried out.\n\nAfter loading the networks, the randomization step creates random networks. The random networks may be created either by randomizing real networks, using the edge shuffle algorithm (degree preserving or completely random) (Figure 2), or by generating new random networks by using one of available models (Figure 3 and Figure 4).\n\nThe attribute to be compared must have the same name in all the networks that are selected (it is also case-sensitive).\n\nBoth of them are intended for randomizing an already existing networks.\n\nThey require some parameters which are inserted by the user. Some labels, in red, help the user in order to correctly fill the fields.\n\nThe Multiplication and the Community Affiliation require a file as input, in order to generate random networks.\n\nAfter real networks are loaded and random networks generated, it is possible to compare their shared attributes. Since the app was initially designed to be used in conjunction with centralities parameters, we recommend using the CentiScaPe app which is completely compatible with our Network Randomizer. It is important to note that the app allows comparing the attributes with the same name in all the networks that are selected within the statistical module. Basically, once the networks are selected, only the shared attributes across all networks would appear as possible attribute choices. If there are no attributes that are shared, the user should check the names in the Node Table panel of Cytoscape.\n\nFinally, once all the data are available, the statistical test (Figure 1) compares the selected attributes, pinpointing their differences and similarities and giving a textual file which summarizes the results. Each part of the app has a question mark button which provides help to the users, making the Network Randomizer easier to use, as well as appropriate for educational purposes.\n\n\nUse cases\n\nData preparation. In order to use the app we present a typical use case to guide the users through the network analysis process, using the Network Randomizer.\n\nThe first step when analyzing any network with Cytoscape apps is to import the network into the Cytoscape software. After that, as said, some information is required, and even though we focused on topological centralities, it is possible to use any kind of numerical information. There are multiple ways to fill in this information. It is possible to import a .csv file, to create a new attribute or to use a built-in network analyzing function.\n\nA more advanced approach is to use a popular centrality measures app, i.e. CentiScaPe, which can be downloaded from the Cytoscape Apps store. It provides the users (and consequentially the Network Randomizer itself) with more information about the networks, and, finally, results in a much wider perspective on differences between them. It is important to note that by using these apps one obtains standard names for the attributes. Caution should be taken when using user defined attributes, that the same attribute names are used for the real and for the random networks otherwise no attribute will appear while performing the statistical analysis.\n\nGenerating random networks. There are two main methodologies for generating random networks. By using the random network models or by randomizing current, real networks. For obtaining better results, random networks should be made as similar as possible to the real ones, as this will allow detecting the most important differences between those two groups.\n\nTo randomize an existing network, one needs to be chosen first. This is done by simply creating a View: right click on the network from the Network Control Panel, and choose Create View. Once the network is selected, there are two randomizing methods that can be used: simple edge shuffle, and degree preserving edge shuffle. Users can choose one or both models by checking the boxes (Figure 2). Once the module is selected, the Start button runs the randomization.\n\nApart from the randomization option, the main part of the Randomizer consists of multiple random network models. Currently implemented models are: Erdős–Rényi8, Watts–Strogatz9, Barabási–Albert10, lattices and Community Affiliation Graph11. There is also a new model which we called the Multiplication model.\n\nErdős–Rényi model (Figure 3) generates fully random networks by either uniformly choosing M pairs of nodes to connect, or by connecting each pair with probability p. Watts–Strogatz model (Figure 3) generates networks which show the so called small-world phenomenon. Here, although the network is not dense, the average shortest path is still significantly low. Networks generated by using the Barabási–Albert model (Figure 3) are scale-free networks, meaning that their degree distribution follows a power law distribution. The model uses the preferential attachment growth, hence each new node is connected to m other nodes, choosing them with a probability which is proportional to their degree. Once a model is selected the users are informed about the parameter constraints by a red label (Figure 3). The labels update their values, in order to be more helpful, once they are filled in correctly and after pressing the Enter button.\n\nMultiplication model (Figure 4) generates randomly weighted network (Figure 5). The algorithm generates a random array which defines a weight for each node belonging to an existing, user-defined network, starting from an attribute file which contains quantitative information about the nodes. Then the algorithm creates a new network which contains a number of copies that depend on the random weights that were assigned to each node. This random weighted network represents a possible experimental setup which derives from the attribute file. In this sense it is possible to generate a number of randomly weighted networks, in order to simulate a set of experiments. The new nodes, that are added as copies of existing nodes, have the same neighbours of the original node and share an edge with the original node and each of its copies.\n\nBy using the values in the random array a number of nodes, in green, are added to the original network, represented by the yellow nodes and some new networks are created.\n\nLattice model (Figure 4) generates regular, multidimensional lattices. Multidimensional lattices are grids of nodes, each of which is connected only to its first neighbours. For example, the one-dimensional lattice is a path graph, while the two-dimensional lattice is a square grid. Additionally, users have the option of generating torus-shaped lattices, where there are no end-nodes. For example, the one-dimensional torus lattice is a cycle graph.\n\nFinally, the Community Affiliation model (Figure 4) generates random networks from the community information: given a list of communities, their members, and the probability of having an edge between two members of each community, it randomly generates realistic social networks.\n\nSome models require special input files which define their behavior. The two models that currently require an input file are Multiplication and Community Affiliation. The Multiplication file must contain numerical values, one for each row. It is expected, but not mandatory, that the number of rows is equal to the number of nodes and the Randomizer will pop up a dialog which asks the user if the number of values found in the file is correct. The Community Affiliation model requires a file which contains a set of rows where each line represents a community. The rows starts with a p-value, i.e. the probability of an edge between two members of the community, and is followed by the names of the nodes inside that community, separated by spaces.\n\nOne last point concerns the fact that, once a model is selected in the app panel, every time the button Start Randomization is pressed each selected model is created in a number of copies chosen by the user (Figure 6). If the number of networks is not specified, by using the form, the algorithm will generate a network for each selected model. Otherwise the selected number of newtworks will be generated, for each selected model. This means that, after a computation, we suggest removing the check mark from all the models in order to avoid creating a number of not required new networks, every time Network Randomizer runs.\n\nComparing networks. Once random networks are generated, before comparing their attributes to the real ones, at least a numerical attribute should be present in all the networks that will be compared. To run the statistical comparison module, it is necessary to specify which networks it will use. To do this, users need to select the real networks in the Networks Control Panel and press Selected, and then do the same for the random ones. After selecting the networks, a list of all the node attributes shared within all the selected networks is provided. Users can now select the node attributes they would like to compare. If there is not a shared attribute then a dialog will appear to tell the user to check again the attributes. Selection is done using the left-click and additional keys, Ctrl for one-by-one multiple selection, Shift for range selection, and Ctrl+A to select all. Once the attributes are selected, output file name and directory need to be specified, and the comparison can be executed by clicking the Start Statistics button.\n\nInterpreting the results. Every output file consists of multiple comma-separated-values fields, each one beginning with an explanation marked by the > symbol. First few fields define the names of the networks used. The other fields differ, depending on whether only one, or multiple real networks are used.\n\nIf there is only one real network, output is either fragmented into centrality measures used, or into random networks to which the real one is compared. The first field indicates how different is each random network from the real one by using the average difference across all centralities. The second field represents the difference between the real network and its most similar random network, according to each centrality measure individually. The last field provides more in-depth information, specifying the difference between the real network and each random one, for each centrality measure.\n\nShowing all of the generated data to the user, in the case of multiple real networks, would result in a very unreadable output. To avoid this, only the most interesting points are chosen: either the pairs of real and random networks, or the pairs of real networks and centrality measures which show the least statistical differences. This way, users can check their networks for important non-random processes. The first field specifies the average difference across all centralities between real networks and their most similar random network. The second field shows the most similar random network with respect to the real one, for each real network and each centrality, and specifies their difference. The last field is the real-random distance matrix, with distances defined as average difference across all centralities. A value of 0 indicates that the distributions are completely the same, up to a normalization factor. Normalization factor, in this sense, is the number of elements in the series. So, for example, the series [1, 2, 3] and [1, 1, 2, 2, 3, 3] would be completely the same. A value which is close to 1 indicates the existence of an important difference between the distributions. This result is rarely achieved in real datasets, and it happens when the elements of one series are all greater than the elements of the other.\n\n\nSummary\n\nTo summarize, our app allows generating and creating random networks and is useful whenever a validation benchmark is required. Starting from real networks it is possible to compare their attributes with randomly generated attributes obtained by analyzing random networks. The main issue concerns the fact that there is not a specific model that should be suggested in a specific setup or with some specific kinds of data. It is up to the user to select which model best describes the network and its mathematical characteristics.\n\nConcluding, our app is designed to be as general as possible having a very wide range of applications and to be completely user-friendly, giving the possibility to perform a simple, but meaningful, statistical analysis and a readable output.\n\n\nSoftware availability\n\n1. http://apps.cytoscape.org/apps/networkrandomizer\n\n2. Source code as at the time of publication: https://github.com/gabrielet/Network-Randomizer authors Gabriele Tosadori and Ivan Bestvina\n\n3. 10.5281/zenodo.15927112\n\n4. Software license: Apache License, Version 2.0",
"appendix": "Author contributions\n\n\n\nGT defined and implemented the random multiplication model, designed and developed the app, and wrote the manuscript, IB designed and developed the app, and wrote the manuscript. Finally GS, FS and CL tested the app and gave interesting comments that improved our work.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by: Italian Association for Cancer Research (AIRC, IG16797), MIUR (PRIN 2009), Nanomedicine project University of Verona and Fondazione Cariverona to Carlo Laudanna.\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 acknowledge the National Resource for Network Biology (NRNB) Training Program which allowed the actual development of the app.\n\n\nReferences\n\nSaito R, Smoot ME, Ono K, et al.: A travel guide to Cytoscape plugins. Nat Methods. 2012; 9(11): 1069–1076. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCline MS, Smoot M, Cerami E, et al.: Integration of biological networks and gene expression data using Cytoscape. Nat Protoc. 2007; 2(10): 2366–2382. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSah P, Singh LO, Clauset A, et al.: Exploring community structure in biological networks with random graphs. BMC Bioinformatics. 2014; 15(1): 220. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaibe-Kains B, Emmert-Streib F: Quantitative assessment and validation of network inference methods in bioinformatics. Frontiers Media SA, 2015. Publisher Full Text\n\nNewman ME, Watts DJ, Strogatz SH: Random graph models of social networks. Proc Natl Acad Sci U S A. 2002; 99(suppl 1): 2566–2572. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScardoni G, Petterlini M, Laudanna C: Analyzing biological network parameters with CentiScaPe. Bioinformatics. 2009; 25(21): 2857–2859. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilcox RR: Some practical reasons for reconsidering the Kolmogorov-Smirnov test. Br J Math Stat Psychol. 1997; 50(1): 9–20. Publisher Full Text\n\nErdős P, Rényi A: On Random Graphs I. Publ Math Debrecen. 1959; 6: 290–297. Reference Source\n\nWatts DJ, Strogatz SH: Collective dynamics of ‘small-world’ networks. Nature. 1998; 393(6684): 440–442. PubMed Abstract | Publisher Full Text\n\nBarabasi AL, Albert R: Emergence of scaling in random networks. Science. 1999; 286(5439): 509–512. PubMed Abstract | Publisher Full Text\n\nYang J, Leskovec J: Community-affiliation graph model for overlapping network community detection. In Data Mining (ICDM), 2012 IEEE 12th International Conference on IEEE. 2012; 1170–1175. Publisher Full Text\n\nGabriele T, Bestvina I: F1000Research/Network-Randomizer: F1000Reserach/Network-Randomizer [Data set]. Zenodo. 2016. Data Source"
}
|
[
{
"id": "17046",
"date": "31 Oct 2016",
"name": "Giovanni Micale",
"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 paper, Tosadori et al. describe a Cytoscape app for creating random networks according to different random models and comparing them with one or more input networks on the basis of different topological metrics. The main goal of the app is to help the user in the creation of a benchmark for validating some properties of a real network or a dataset of real networks. I found the app very interesting and I think that the paper is suitable for being indexed. However, there are several major issues, especially regarding the potential applications of the tool and the statistical part of the app, that need to be addressed before indexing.\nThe actual usefulness of the app is not fully explained. In the Abstract and in the Introduction the authors point out that the app should serve as a framework to validate some numerical results of one or more input networks, but it is not clear how the results of comparing values of topological attributes of real and random networks can be exploited by the user to draw conclusions. I would suggest to insert (for example in the Introduction) one or more examples of usage of this tool. For instance, an interesting application of the tool could be the following: generate different random networks (one or more for each random model) with a similar number of nodes and edges of the real network, then compare these networks with the real one according to some statistics (for example clustering coefficient) in order to find the random model that best fits the input network. This can tell a lot about the features of the network and how it has been generated.\n\nThe statistical module of the app allows to compare two distribution of values on some topological property of the nodes (for example centrality) by using the two-sample Kolmogorov-Smirnov test. The aim of this comparison should be carefully clarified. Is the aim to establish if topological properties are maintained with respect to the random model used? Does it make sense when such a value is obtained comparing only a pair of networks, the real and the random ones?\nLet’s consider a set of N networks generated using the preferential attachment and compare them to a real network according to the values of some metric M. The reviewer expects that will be sensible variations on the Kolmogorov-Smirnov distance. It would be more reasonable to give the user the possibility to compare expected values of some metric in ensembles of real and random networks. In some context, this kind of analysis can be preferable and would result more robust.\n\nAmong the random models, the authors presented a new model called Multiplicative Model. It is not clear from the text how such a model works. There is a picture in the paper that shows three networks generated with three different weighted arrays, but it is not clear how these weighted arrays are generated. Which are the input parameters of the model? Please clarify how the weights are determined. It is not clear i) if the user has to provide a minimum value X and a maximum value Y and then weighted arrays are generated by randomly picking one value within interval [X,Y] for each node or ii) if just has to provide a text file with one weight for each node and generate a random network with exactly that sequence of weights. By using the app, it seems that a text file is required but it is not clear what should be its content. In general, authors should describe more clearly the model both in the paper and in the documentation of the app.\n\nIt looks like the Multiplication model is not properly working: I tried to generate a random network using the Multiplication Model starting from an input network of N nodes. As a parameter for the model, I loaded an input file with N rows and one number for each row (in each row there is 0 or 1 as weight). A window appeared saying which was the maximum number of edges admitted for the network. However, at the end of the process, a random network with exactly the same number of nodes and edges (maybe a copy) of the input network was created without any error or warning message. I expected as output a network with a different number of nodes and edges. Please clarifiy this point.\n\nIn the Abstract and in the Introduction authors claim that there are no tools for generating and randomizing networks. Actually, there are two Cytoscape apps that authors should cite in the paper: Randomnetworks plugin (http://apps.cytoscape.org/apps/randomnetworks) and NetMatch* (http://alpha.dmi.unict.it/netmatchstar/netmatchstar.html). Furthermore, since your app implements a comparison part between networks, I would also recommend to cite some other Cytoscape app to compare networks, such as GASOLINE (http://apps.cytoscape.org/apps/gasoline) and DyNet (http://apps.cytoscape.org/apps/dynet), remarking the different kind of approach to compare networks that you are following, which is the new research contribution.\n\nOn page 6, third paragraph, when authors talk about the two shuffling methods (with or without preservation of degrees) they say that users can choose one or both models. Authors should describe which is the effect when both options are selected.\n\nWhat is the maximum number of nodes in a network generated with the multiplication model? In the example at page 3 for a network of 10 nodes and weights in [0,100] is 100*10+10, but in the figure of page 6 it seems to be just the number of nodes multiplied by the maximum value of the range (in the figure of page 6 it seems 7*3 and not 7*3+7). Please clarify this point.\n\nIn the paper there is no indication of which sizes represent the Lattice Model (Figure 4). I finally deduced this information from the documentation. This should be reported with a sentence also in the main paper.\n\nThere is a difference between the panel of Multiplication Model depicted in Figure 4 and the Multiplication Model panel present in the Cytoscape App. In the app I found an option called “Graphical version” which is not reported in the paper. Please clarify and make the app and the paper coherent on such a point.\n\nWhen the user generates random networks or run the statistical analysis there is no indication on the status of the computation. It would be recommended to notify the user with logs and progress bars.\n\nI tried to execute a pipeline of statistical experiments with different input networks and/or random networks. However, after the first experiment, it was impossible to me to change the set of selected input or random networks for a second experiment. I had to close and open again the app to make other tests. Please fix this bug.\n\nIn the Statistical analysis panel there is a “Save as” button to select the path where to save output results. However, there is no text field next to the button that indicates the selected output folder. Please insert such a field.",
"responses": [
{
"c_id": "2776",
"date": "15 Jun 2017",
"name": "Gabriele Tosadori",
"role": "Author Response",
"response": "Dear Giovanni,Thank you for all the very useful comments. We amended the paper as you suggested and we hope it is now more clear and useful.I would like to answer you with respect to the points 10, 11 and 12.Point 10: we decided not to add the progress bar since the computation takes a very little time and we believe it is not a necessary feature. If you really think that it is needed, then we will add it for our newest release otherwise, we'll keep the app as it currently isPoint 11: we are not able to obtain the same bug you're mentioning. Please give us more feedback about this issue and we will see how to fix this.Point 12: we decided not to add the path to the folder since it was creating a lot of issuse with the Mac version of the Java library. In order to avoid bugs for the Mac users we removed this feature.If you have further questions and suggestions, we will be very happy to hear everything.Thank you,Gabriele Tosadori"
}
]
},
{
"id": "17030",
"date": "01 Nov 2016",
"name": "Francesco Russo",
"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 proposed a new Cytoscape App to generate random networks and compare them with real data. It consists of the most common approaches for network randomisation. Moreover, the authors proposed a novel method to generate random weighted networks.\nThere are few implemented apps available in Cytoscape for this purpose, so this app will be very useful and simple to use in a well known framework.\nThe manuscript is well written and clear. It could be nice and useful to have a figure representing the workflow of the proposed app, if it is compatible with the number of figures allowed for this journal.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-2524
|
https://f1000research.com/articles/6-1447/v1
|
14 Aug 17
|
{
"type": "Research Article",
"title": "An analysis of characteristics of post-authorisation studies registered on the ENCePP EU PAS Register",
"authors": [
"Robert Carroll",
"Sreeram V. Ramagopalan",
"Javier Cid-Ruzafa",
"Dimitra Lambrelli",
"Laura McDonald",
"Robert Carroll",
"Sreeram V. Ramagopalan",
"Javier Cid-Ruzafa",
"Dimitra Lambrelli"
],
"abstract": "Background: The objective of this study was to investigate the study design characteristics of Post-Authorisation Studies (PAS) requested by the European Medicines Agency which were recorded on the European Union (EU) PAS Register held by the European Network of Centres for Pharmacoepidemiology and Pharmacovigilance (ENCePP). Methods: We undertook a cross-sectional descriptive analysis of all studies registered on the EU PAS Register as of 18th October 2016. Results: We identified a total of 314 studies on the EU PAS Register, including 81 (26%) finalised, 160 (51%) ongoing and 73 (23%) planned. Of those studies identified, 205 (65%) included risk assessment in their scope, 133 (42%) included drug utilisation and 94 (30%) included effectiveness evaluation. Just over half of the studies (175; 56%) used primary data capture, 135 (43%) used secondary data and 4 (1%) used a hybrid design combining both approaches. Risk assessment and effectiveness studies were more likely to use primary data capture (60% and 85% respectively as compared to 39% and 14% respectively for secondary). The converse was true for drug utilisation studies where 59% were secondary vs. 39% for primary. For type 2 diabetes mellitus, database studies were more commonly used (80% vs 3% chart review, 3% hybrid and 13% primary data capture study designs) whereas for studies in oncology, primary data capture were more likely to be used (85% vs 4% chart review, and 11% database study designs). Conclusions: Results of this analysis show that study objectives and therapeutic area influence PAS design in terms of type of data capture used.",
"keywords": [
"drug safety",
"pharmacovigilance",
"post-authorisation studies",
"study design"
],
"content": "Introduction\n\nRandomised trials are considered the gold standard in evaluating the efficacy of new healthcare interventions. However, despite their ability to potentially provide causal estimates of the efficacy of new treatments, their generalisability to patients in the real world is often unclear. Strict criteria excluding patients with comorbidities or those above a certain age may lead to participants in trials differing from the general clinical population1,2. Observational studies offer a means of further characterising the safety and effectiveness of new healthcare interventions in real world clinical settings3.\n\nPost-authorisation studies (PAS) are an example of this type of research which aims to demonstrate the utilisation and safety profile of drugs following their regulatory approval. Historically, these studies have been criticized to be of poor quality and open to bias in order to increase sales at the cost of scientific rigor4. European pharmacovigilance legislation was introduced in an effort to increase transparency and ensure such studies are methodologically robust5.\n\nSince 2010 all PAS that are imposed as a condition of granting marketing authorisation are required to be published in a publicly available register. In Europe, the main register for PAS is the EU PAS Register and is held by the European Network of Centres for Pharmacoepidemiology and Pharmacovigilance (ENCePP). While one previous study has examined broad characteristics of studies registered on the EU PAS, the current study aimed to build on this work by further characterising studies requested by the EMA to understand trends in study design, data sources utilised, and the relationship to therapeutic area. As PAS are becoming increasingly mandated by the European Medicines Agency (EMA) and compliance is high6, approved PAS can likely provide insights into the successful conduct of studies.\n\n\nMethods\n\nAnalysis focused on all PAS requested by the EMA which were recorded on the EU PAS Register as of 18th October 2016. This included all studies in category 1 (imposed as a condition of marketing authorisation), category 2 (obligation of marketing authorisation), and category 3 (required by the risk management plans) on the EU PAS register.\n\nData from the Register on study status (finalised, ongoing or planned) were collected to allow comparison of studies across time. Information was also collected from the Register on medical condition to be studied, data sources used (if applicable), and study scope (effectiveness evaluation, drug utilisation or risk assessment studies). These data were collected from the publically available information published on the PAS register (http://www.encepp.eu/encepp/studiesDatabase.jsp).\n\nStudies were classified as either using primary data capture, secondary data capture, or a hybrid approach as used by Engel and colleagues7 who studied PAS protocols and assessments submitted from July 2012 to July 2015 to the EMA Pharmacovigilance Risk Assessment Committee (PRAC). Primary data capture refers to the collection of data specifically for the study. Secondary data is the use of data already collected for another purpose (e.g. administrative or claims databases; medical charts). Studies using a combination of primary and secondary approaches were classed as employing a hybrid approach.\n\nDescriptive analysis was performed. Counts and percentages were used for categorical variables.\n\n\nResults\n\nAs of 18th October 2016, a total of 314 studies were identified on the EU PAS register. These studies included 81 (26%) finalised, 160 (51%) ongoing and 73 (23%) planned (Table 1).\n\nOf the total studies identified, 205 (65%) included risk assessment in their scope, 133 (42%) included drug utilisation and 94 (30%) included effectiveness evaluation. The same study could cover more than one objective: 6 (2%) studies included drug utilisation and effectiveness evaluation, 36 (11%) drug utilisation and risk assessment, 32 (10%) risk assessment and effectiveness evaluation and 22 (7%) risk assessment, drug utilisation and effectiveness evaluation.\n\nFor the risk assessment studies, finalised studies were less common (42; 52% of all 81 finalised studies) than ongoing (112; 70% of all 160 ongoing studies) and planned (51; 70% of all 73 planned studies). For the effectiveness evaluation studies, again finalised studies were not as frequent (11; 14% of all 81 finalised studies) as ongoing (60; 38% of all 160 ongoing studies) or planned (23; 32% of all 73 planned studies). Finally, for the drug utilisation studies, finalised studies were more common (47; 58% of all 81 finalised studies) with 59 (37% of all 160 ongoing studies) ongoing and 27 (37% of all 73 planned studies) planned.\n\nJust over half of the studies (175, 56%) used a primary data capture design, 135 (43%) used a secondary data capture design and 4 (1%) used a hybrid design combining both approaches.\n\nMore primary data capture studies were ongoing 102 (64% of all 160 ongoing studies) or planned 49 (67% of all 73 planned studies) than finalised 24 (30% of all of all 81 finalised studies). For secondary data capture, finalised studies were more frequent (57; 70%), as compared to 55 (34%) ongoing and 23 (32%) planned.\n\nRisk assessment and effectiveness studies were more likely to use primary data capture (60% and 85% respectively for primary data capture as compared to 39% and 14% respectively for secondary data capture). The converse was true for drug utilisation studies, where 59% used secondary data capture vs. 39% for primary data capture.\n\nOf the secondary data capture studies, 117 (87%) used an existing claims or electronic medical record database (the remainder using a chart review approach). 93 (79%) studies used an existing real-world data source based in Europe alone, 9 (8%) studies used European and US data, 14 (12%) studies used US data alone and 1 (1%) study used Canadian data.\n\nA single database was used in 58 (50%) studies, with the remainder using two or more. Where more than one data source was used, this was always from two or more countries. The most frequent established data source used was the United Kingdom’s (UK) Clinical Practice Research Datalink (CPRD, 31%), followed by Nordic (Denmark, Finland, Norway or Sweden) National registries (29%), and The Health Improvement Network (THIN, UK, 18%).\n\nA total of 30 (10%) studies were in the field of type 2 diabetes mellitus, 29 (9%) in cardiovascular disease, 27 (9%) studies in oncology, 6 (2%) in chronic obstructive pulmonary disease (COPD) and 6 (2%) in multiple sclerosis (MS).\n\nFor type 2 diabetes mellitus, database studies were more commonly used (80% vs 3% chart review, 3% hybrid and 13% primary data capture). Similar patterns were seen for COPD (83% database vs 17% primary data capture) and cardiovascular disease (59% database vs 38% primary data capture and 3% hybrid design).\n\nFor studies in oncology, primary data capture was more likely to be used (85% vs 4% chart review, and 11% database study designs). A similar relationship was seen for MS (83% primary data capture vs 17% database study design).\n\n\nDiscussion\n\nOur analysis reveals a number of distinct characteristics of studies recorded on the register that can likely be used as a reference for future PAS designs.\n\nOverall there appears to have been an increase in the number of risk assessment studies, as planned and ongoing studies were far more likely to include a safety element than studies that had already been finalised.\n\nPrimary data capture study designs were the most commonly implemented study type, and again these were more frequently planned or ongoing, suggesting an increasing use or reflecting the longer nature in general to execute these types of studies. Furthermore, primary data capture were more commonly used for effectiveness and risk assessment studies. The predominance of primary data capture designs is similar to results observed by Engel et al., who studied 189 PAS protocols submitted to the EMA PRAC7.\n\nSecondary data capture was more likely to be used for drug utilisation studies, and this was also described by Engel et al.7 Of the secondary studies, the CPRD in the UK was used in nearly a third of all database PAS. Registry data from the Nordic countries was only slightly less commonly used, with the remainder of existing data sources (eg. THIN, in the UK) being less frequently used.\n\nOf the therapeutic areas investigated, type 2 diabetes mellitus, cardiovascular disease and COPD tended to use secondary data capture, whereas for oncology and MS, primary data capture studies were favoured.\n\nSome limiting characteristics of current data sources in Europe are that they may only cover a specific type of patient care (e.g. primary or secondary) and not the full patient pathway. They may not collect data on everything that is needed for PAS objectives, for example in-hospital treatments, clinical endpoints or markers of disease severity. Given PAS studies are often requested on the grounds of exploring sub-groups of patients who may have been treated outside of these settings8, this is a major limitation of some data sources. Indeed, this may explain why primary data capture study designs are becoming more frequent, especially in the field of oncology. Furthermore, that a handful of data sources are very commonly used highlights the limited data landscape in Europe, and the need to improve coverage and access to routinely collected medical data for observational research. On the other hand, given some similarity in data elements captured in currently available datasets, studies using only a single database may have potentially missed a chance to validate any findings or increase statistical power to detect relevant effects. Additionally, some data sources (eg. CPRD) can be linked to other datasets or have an integrated data collection component to provide additional data. Nevertheless, the tendency for effectiveness and risk assessment studies to use primary data capture is also likely a reflection of the fact that this methodology can potentially overcome problems such as confounding, often associated with some secondary designs due to a lack of data availability on potential confounders.\n\nIn summary, we show here that using data from the EU PAS Register, study scope and therapeutic area influences the choice of study design agreed by health authorities and therapeutic drug makers for the conduct of PAS. These results can contribute to the planning of future successful PAS.\n\n\nData availability\n\nDataset 1: The full dataset used for analysis, extracted from the PAS register (http://www.encepp.eu/encepp/studiesDatabase.jsp) on the 18th of October 2016.\n\nDOI, 10.5256/f1000research.12198.d1708649",
"appendix": "Competing interests\n\n\n\nRC, JC and DL are full-time employees of Evidera. SR and LM are full-time employees of Bristol-Myers Squibb.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKalata P, Martus P, Zettl H, et al.: Differences between clinical trial participants and patients in a population-based registry: the German Rectal Cancer Study vs. the Rostock Cancer Registry. Dis Colon Rectum. 2009; 52(3): 425–437. PubMed Abstract | Publisher Full Text\n\nMaasland L, Van Oostenbrugge RJ, Franke CF, et al.: Patients enrolled in large randomized clinical trials of antiplatelet treatment for prevention after transient ischemic attack or ischemic stroke are not representative of patients in clinical practice: the Netherlands Stroke Survey. Stroke. 2009; 40(8): 2662–2668. PubMed Abstract | Publisher Full Text\n\nMcDonald L, Lambrelli D, Wasiak R, et al.: Real-world data in the United Kingdom: opportunities and challenges. BMC Med. 2016; 14(1): 97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGale EA: Post-marketing studies of new insulins: sales or science? BMJ. 2012; 344: e3974. PubMed Abstract | Publisher Full Text\n\nBorg JJ, Aislaitner G, Pirozynski M, et al.: Strengthening and rationalizing pharmacovigilance in the EU: where is Europe heading to? A review of the new EU legislation on pharmacovigilance. Drug Saf. 2011; 34(3): 187–197. PubMed Abstract | Publisher Full Text\n\nBlake KV, Prilla S, Accadebled S, et al.: European Medicines Agency review of post-authorisation studies with implications for the European Network of Centres for Pharmacoepidemiology and Pharmacovigilance. Pharmacoepidemiol Drug Saf. 2011; 20(10): 1021–9. PubMed Abstract | Publisher Full Text\n\nEngel P, Almas MF, De Bruin ML, et al.: Lessons learned on the design and the conduct of Post-Authorization Safety Studies: review of 3 years of PRAC oversight. Br J Clin Pharmacol. 2017; 83(4): 884–893. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoekman J, Klamer TT, Mantel-Teeuwisse AK, et al.: Characteristics and follow-up of postmarketing studies of conditionally authorized medicines in the EU. Br J Clin Pharmacol. 2016; 82(1): 213–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRamagopalan S, McDonald L, Carroll R, et al.: Dataset 1 in: An Analysis of Characteristics of Post-Authorisation Studies Registered on the ENCePP EU PAS Register. F1000Research. 2017. Data Source"
}
|
[
{
"id": "26352",
"date": "28 Sep 2017",
"name": "Xavier Kurz",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well written and provides a descriptive analysis of the EU PAS Register. This type of research is welcome as an evaluation of the usefulness of the EU PAS Register. The article would benefit from a couple of clarifications:\n\nIntroduction\n\nThe EU PAS Register is accessible via the ENCePP website which is hosted and maintained by the European Medicines Agency (EMA).\n\nThe term post-authorisation study (PAS) is not exclusive to observational research and may include interventional study designs (e.g. see definition of post-authorisation safety study in GVP VIII), this should be made clear as the introduction refers to historical ‘criticism’ raised mainly in context of non-interventional research. The majority of studies in the EU PAS Register are non-interventional but also a few RCTs fall under the inclusion criteria applied by the authors for their descriptive study.\n\nThe EU pharmacovigilance legislation and the requirement to publish PAS imposed as a condition of marketing authorisation in a public register came into force in July 2012, not in 2010 (the new legislation was however published in December 2010).\n\nThe authors should explain what they mean by ‘approved PAS’. Regulatory approval of PAS is not a guarantee for successful conduct of a study. The EU pharmacovigilance legislation provides for a regulatory procedure to approve protocols of post-authorisation safety studies (PASS) imposed as a condition of marketing authorisation (EU RMP category 1 and 2 studies) before study start to ensure the objectives of the PASS are met and the protocol follows the format requirements. However, this needs to be distinguished from regulatory supervision of EU RMP category 3 studies where prior protocol approval is not required by law (but may be requested to the marketing authorisation holder voluntarily). The general statement above should be balanced, also in the discussion. Like for any other study, relevant scientific guidance should be considered by marketing authorisation holders and investigators for the development of study protocols, the conduct of studies and the writing of study reports (e.g. ENCePP Guide on Methodological Standards in Pharmacoepidemiology, Guidelines for Good Pharmacoepidemiology Practices of the International Society of Pharmacoepidemiology (ISPE GPP) etc.).\n\nMethods\nClarify if only studies requested by EMA or also by other EU or non-EU regulators have been included in the descriptive analysis. This is important for the discussion of the results on data sources.\n\nThe authors have chosen 3 distinct study scopes as inclusion criteria, but there might be other studies requested by a regulator which provide insight in trends in study design, data sources and therapeutic areas (a query performed on 27.09.2017 found more than 170 studies requested by a regulator with other scopes). The authors should explain this limitation.\n\nResults\nThe presentation of the results on study status, data sources and disease areas may benefit from a breakdown by EU RMP category to show any potential association of with the timelines for conducting studies.\n\nThe authors do not specify how the disease areas were defined (by ATC code?).\n\nDiscussion\nThe statement that planned and ongoing studies were far more likely to include a safety element is not clear from the presented results; any study in an EU RMP is linked to a safety concern, only with varying scope i.e. effectiveness of measures implemented to manage risks (e.g. educational materials), changes in drug utilisation (e.g. restricted indication) or risk quantification (e.g. frequency of adverse reactions)?\n\nA limitation of the study that may be worth explaining is that the registration of studies is voluntary (except for imposed PASS), and the entries are not verified as to their accuracy. For examples, it is not checked by ENCePP or EMA whether the RMP category entered is correct and corresponds to the outcome of the regulatory procedure. Therefore, there may be some misclassification. For PASS imposed by regulators, the RMP category field may have been incorrectly populated by the company.\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": [
{
"c_id": "3160",
"date": "10 Nov 2017",
"name": "Sreeram Ramagopalan",
"role": "Author Response",
"response": "We thank the reviewers for their comments. We respond specifically to their points below and hope they find our changes acceptable: The EU PAS Register is accessible via the ENCePP website which is hosted and maintained by the European Medicines Agency (EMA). Response: This has been clarified in the methods. The term post-authorisation study (PAS) is not exclusive to observational research and may include interventional study designs (e.g. see definition of post-authorisation safety study in GVP VIII), this should be made clear as the introduction refers to historical ‘criticism’ raised mainly in context of non-interventional research. The majority of studies in the EU PAS Register are non-interventional but also a few RCTs fall under the inclusion criteria applied by the authors for their descriptive study. Response: This point has been clarified in the introduction The EU pharmacovigilance legislation and the requirement to publish PAS imposed as a condition of marketing authorisation in a public register came into force in July 2012, not in 2010 (the new legislation was however published in December 2010). Response: Text corrected, thank you. The authors should explain what they mean by ‘approved PAS’. Regulatory approval of PAS is not a guarantee for successful conduct of a study. The EU pharmacovigilance legislation provides for a regulatory procedure to approve protocols of post-authorisation safety studies (PASS) imposed as a condition of marketing authorisation (EU RMP category 1 and 2 studies) before study start to ensure the objectives of the PASS are met and the protocol follows the format requirements. However, this needs to be distinguished from regulatory supervision of EU RMP category 3 studies where prior protocol approval is not required by law (but may be requested to the marketing authorisation holder voluntarily). The general statement above should be balanced, also in the discussion. Like for any other study, relevant scientific guidance should be considered by marketing authorisation holders and investigators for the development of study protocols, the conduct of studies and the writing of study reports (e.g. ENCePP Guide on Methodological Standards in Pharmacoepidemiology, Guidelines for Good Pharmacoepidemiology Practices of the International Society of Pharmacoepidemiology (ISPE GPP) etc.) Response: We have added text in the discussion to acknowledge this. Clarify if only studies requested by EMA or also by other EU or non-EU regulators have been included in the descriptive analysis. This is important for the discussion of the results on data sources. Response: This was just EMA only. The authors have chosen 3 distinct study scopes as inclusion criteria, but there might be other studies requested by a regulator which provide insight in trends in study design, data sources and therapeutic areas (a query performed on 27.09.2017 found more than 170 studies requested by a regulator with other scopes). The authors should explain this limitation. Reponse: This limitation has been notedResults The presentation of the results on study status, data sources and disease areas may benefit from a breakdown by EU RMP category to show any potential association of with the timelines for conducting studies. Response: Unfortunately we did not collect this data in our analysis but agree it would be of interest for future work. The authors do not specify how the disease areas were defined (by ATC code?). Response: The disease areas are listed in the EU PAS register. This has been clarified Discussion The statement that planned and ongoing studies were far more likely to include a safety element is not clear from the presented results; any study in an EU RMP is linked to a safety concern, only with varying scope i.e. effectiveness of measures implemented to manage risks (e.g. educational materials), changes in drug utilisation (e.g. restricted indication) or risk quantification (e.g. frequency of adverse reactions)? Response: Risk assessment is meant here (as opposed to drug utilisation, for instance). The text has been updated. A limitation of the study that may be worth explaining is that the registration of studies is voluntary (except for imposed PASS), and the entries are not verified as to their accuracy. For examples, it is not checked by ENCePP or EMA whether the RMP category entered is correct and corresponds to the outcome of the regulatory procedure. Therefore, there may be some misclassification. For PASS imposed by regulators, the RMP category field may have been incorrectly populated by the company. Response: This limitation has been noted"
}
]
},
{
"id": "26965",
"date": "23 Oct 2017",
"name": "Lex Bouter",
"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 contains a descriptive analysis of some characteristics of Post-Authorisation Studies registered in the ENCePP EU PAS Register. It’s clearly written and informative within the limited scope the authors set out to study. There are a number of issues that might benefit from further clarification.\n\nMajor issues\nThe 314 included studies were on 18th October 2016 contained in the ENCePP EU PAS Register AND concerned a PAS requested by EMA. That makes the reader wonder how many PAS requested by EMA (since a suitable date) were not contained in the Register. And also how many studies contained in the Register were not requested by EMA. In other words: how selective is the sample studied?\n\nThe conclusions come as a surprise because no explicit study questions or hypotheses were formulated. Furthermore, the conclusions are overstated in the sense that both the cross-sectional nature and the lack of a statistical (multivariable) data-analysis make a causal interpretation (‘influence’) unwarranted.\n\nA serious study limitation is that no checks are done (for the completed studies) for non-publication, and for selective publication and other discrepancies between protocol and publication for the published studies.\n\nMinor issues\nPlease explain better what is meant by PAS. Most readers will probably be more familiar with terms like ‘postmarketing surveillance’ and ‘phase IV trials’.\n\nI assume that entries in the Registry need to be made before the start of data collection. And also that later changes (amendments) to the protocol cannot be made without leaving traces (log file). But this is not clearly explained in the current version of the article.\n\nThe Introduction section suggests that the European pharmacovigilance legislation was introduced to foster transparency and methodological quality of the Post-Authorization Studies. That may be correct, but the current study says very little on transparency and nothing about the methodological quality of PAS.\n\nThe first sentence of the Discussion section suggests that this article will ‘likely be used as reference for future PAS designs’. It completely escapes me why this would be so.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "3161",
"date": "10 Nov 2017",
"name": "Sreeram Ramagopalan",
"role": "Author Response",
"response": "We thank the reviewers for their comments. We respond specifically to their points below and hope they find our changes acceptable: Major issuesThe 314 included studies were on 18th October 2016 contained in the ENCePP EU PAS Register AND concerned a PAS requested by EMA. That makes the reader wonder how many PAS requested by EMA (since a suitable date) were not contained in the Register. And also how many studies contained in the Register were not requested by EMA. In other words: how selective is the sample studied?Response: Looking at recent figures on the PAS register (06/11/17) 40% (482/1191) fit these criteria. The lack of generalisability of our findings to studies that are not requested by the EMA is now highlighted in the results The conclusions come as a surprise because no explicit study questions or hypotheses were formulated. Furthermore, the conclusions are overstated in the sense that both the cross-sectional nature and the lack of a statistical (multivariable) data-analysis make a causal interpretation (‘influence’) unwarranted.Response: We had not intended to claim causal inference, but simply describe associations/trends observed. We have removed wording relating to ‘influence’. A serious study limitation is that no checks are done (for the completed studies) for non-publication, and for selective publication and other discrepancies between protocol and publication for the published studies.Response: The reviewer highlights an important point, but one that is not covered by the aim of our study. We describe the design of studies on the PAS register, we are not addressing the question of whether researchers comply with their study protocols when publishing results (although this would be an interesting analysis!)Minor issuesPlease explain better what is meant by PAS. Most readers will probably be more familiar with terms like ‘postmarketing surveillance’ and ‘phase IV trials’.Response: These terms have been added to the manuscript where the concept of a PAS is first introduced I assume that entries in the Registry need to be made before the start of data collection. And also that later changes (amendments) to the protocol cannot be made without leaving traces (log file). But this is not clearly explained in the current version of the article.Response: We are not aware of a requirement for studies to have been entered on the register before data collection starts. Amendments can be made but no record of these amendments is madeThe Introduction section suggests that the European pharmacovig)ilance legislation was introduced to foster transparency and methodological quality of the Post-Authorization Studies. That may be correct, but the current study says very little on transparency and nothing about the methodological quality of PAS.Response: We agree, our study did not attempt to quantify transparency nor review the methodological rigor of studies, simply provide a overview of the study designs employed. The first sentence of the Discussion section suggests that this article will ‘likely be used as reference for future PAS designs’. It completely escapes me why this would be so.Response: The results presented on study objectives/study design could inform readers on designs most commonly employed to address certain research questions, and therefore potentially inform suitable designs for future studies."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1447
|
https://f1000research.com/articles/6-1980/v1
|
09 Nov 17
|
{
"type": "Method Article",
"title": "Estimation of the fractal network properties of multicellular life by cellular box-counting",
"authors": [
"József Prechl"
],
"abstract": "Multicellular life is based on the ability of cells to divide, differentiate, cooperate and die in a controlled and organised manner, generating and maintaining an organism. The temporal distribution of division, differentiation and death determines the cellular composition of the organism at any particular point in time. Like these ontogenetic events, phylogenetic development takes place with the changes in total cell numbers, the allocation of these cells to different tissues and the disappearance of certain tissues. Fractal properties of complex networks, a result of growth, can be estimated by box counting, whereby the topological properties of the network are mapped by changing the resolution of examination, that is changing the size of the boxes used to identify and group network components. Here we develop the concept of cellular box-counting, referring to the fact that cells can be grouped on various levels of hierarchy and these various levels can be interpreted as boxes of different linear sizes. We apply the method to data representing distinct stages and groups of evolution of life and interpret the network properties of brown algae, green plants and animals. The results are in agreement with previously established values of degree exponent of biological networks and provide clues to the differences in the network organization of multicellular life.",
"keywords": [
"network",
"fractal",
"complexity",
"multicellular",
"life"
],
"content": "Introduction\n\nMolecular pathways responsible for cellular complexity in a given multicellular organism are those that arose and have been selected during evolution leading to that organism. By going backwards in time, or alternatively by taking contemporary representative organisms of those backward steps, ancestor cells and tissues, of every organ and organ system of the examined organism can be traced. These tissues are the result of different expression patterns, different branches of molecular pathways. Going back far enough, a eukaryotic unicellular ancestor stage can be reached. By recording the relationships (lineage) and cellularity of these stages a biological network (Albert, 2005) representing the evolutionary development of multicellular organisms could be drawn. In this fractal network a node represents a cell belonging to a given tissue in the examined organism, the tissue being the virtual descendent of a single cell in an earlier organism, and the cell being the precursor of a tissue developing in an organism to appear later (Figure 1A). Expansion of a given cell type generates new nodes connected to the same hub, which represents a progenitor cell. Differentiation generates new hubs.\n\nA: Evolutionary fractal network. A molecular network represented by a single cell seeds the evolution of a novel tissue, which becomes an organ system with time. By using adjusted box sizes (lB=N) we normalize fractal box dimension dB according to organism size. Complexity of the organism is then defined by khub, degree of the most connected node at a subsequent time point in evolution. Ellipses represent networks: organisms organized at various levels, the basic unit of organization being a cell. With the development of novel molecular pathways new types of cells appear; further steps, brought about by duplication or alternative splicing, refine these pathways leading to subtypes of cells in ever more complex organisms. B: Difference of slopes of fractal dimension and degree exponent. Complete renormalization generates a single box, which represents the organism at lB=N. Here NB(lB)/N is 1/N and dB=1. Dots represent individual species (see Supplementary file), color lines are weighted regression lines representing groups as indicated. Double headed arrows indicate the difference in slopes (ds). Inset shows corresponding original approach described by Song et al. to define network fractality. C: Relationship between degree distribution and fractal dimension in different multicellular organisms. 95% confidence intervals of regression slope values are shown for the examined three different groups of multicellular life. Curves were generated online using fooplot.com.\n\nThe structure of such a fractal network can be described by rate of change of connectivity (node degree, kB) and the rate of change of node number (NB) while moving along the temporal dimension, represented by lB, the linear box size (Song et al., 2007; Song et al., 2006). Zooming in means decreasing lB, looking at pathways responsible for more and more specific cell types. Zooming out, called renormalization, means drawing simpler, more general, shared molecular networks, until reaching homeostatic networks shared by all cells in the multicellular organism that are present in the common ancestor. The number of hubs at various levels of development in an organism corresponds to cellular complexity, that is levels of organization as cells, tissues, organs, and organ systems.\n\n\nMethods\n\nThe properties of such a network can be estimated by using cellular complexity expressed as the number of different cells in an organism and total cell number (N) in an organism. The dataset compiled by Bell and Mooers (Bell, 1997) was supplemented with vertebrate data compiled by Schad et al. (Schad et al., 2011) to increase the representation of complex animals, estimating the cellularity of chordates based on average weight (see Supplementary file for dataset). The network scaling relations are interpreted after Song et al. as follows (Song et al., 2006; Song et al., 2005).\n\nBoxes provide a fractal dimension dB that describes how relative box numbers are changing with scaling:\n\nNB(lB)/N ∼ lB-dB 1)\n\nwhere NB(lB) is the number of boxes identified at linear box size lB in a network with N nodes. When lB is equal to N, the box contains all the nodes and NB equals 1. Consequently, plotting log(NB(lB)/N) against log(lB) for a number of different networks a straight line (Figure 1B) corresponding to dB=1 is obtained.\n\nBoxes have a degree exponent dk that describes the relationship of degrees outside and inside of the boxes (backwards and forwards in time) as scaling changes:\n\nkB(lB)/khub ∼ lB-dk 2)\n\nwhere kB(lB) is the degree of the most connected box and khub the degree of the most connected node inside the boxes. When lB is equal to N then the box has no links and kB(lB) is 1, while khub will be what is observed as complexity. Consequently,\n\n1/khub ∼ N-dk 3)\n\nAssuming that a single cell has a complexity of one and all the examined networks are descendants of this ancestral network the rule governing the generation of related (ideally linear descendant) networks can be identified by finding an average degree exponent dk for these networks. Here linear regression weighted with 1/N to compensate for the underestimation of complexity in organisms with very high cell numbers (Figure 1B) was used.\n\nThus, the average value of dk relative to dB can be obtained from the equation of the linear regression. This in turn provides the degree distribution exponent gamma:\n\nγ =1+dB/dk 4)\n\nusing\n\nγ = 1+dB/(dB-ds) 5)\n\nwhere ds stands for the difference of slopes obtained from the regression (Figure 1C).\n\n\nResults\n\nThe different relationships between fractal dimension and degree distribution exponent in three independently evolved multicellular groups should correspond to structural differences of cellular and molecular networks in these phylogenetic groups. At any value of gamma the corresponding fractal dimension is lower, in the order of brown algae, green algae and plants, and animals. Thus, the development of complexity corresponds to a decrease of difference between dB and dk, a trend towards γ = 2, which would represent maximal diversification with a hub linked to non-identical nodes. At any particular value of γ, plants show higher values of dB in agreement with the observed fractal anatomy of plants. Indeed, increasing complexity corresponds to decreasing self similarity. Curves representing steps of growing complexity gradually deviate from that representing fractal dimension:\n\ndB=(γ-1)/(γ-2) 6)\n\n(Kim et al., 2007), corresponding to:\n\nγ=1+dB/(dB-1) 7)\n\n(Figure 1C). The observed differences are not in agreement with observations on the metabolic network of bacteria and eukaryotes, where key parameters of network topology were found to be identical (Jeong et al., 2000). However, here we are not looking at conserved protein networks of metabolic activity, rather protein networks responsible for the multicellular organization of eukaryotes. Considering the dramatic differences in anatomy, physiology and metabolism in the examined groups the results are expectable.\n\n\nConclusions\n\nRelatively simple anatomical and histological data of phylogenetically related organisms can be used to get insight into the fundamental network organisation of cells and molecules responsible for multicellularity. This method is a simple top-down approach for the investigation of cellular and molecular networks, which complements bottom-up approaches used by proteomics, metabolomics and genomics.\n\nFurther elaboration of the methodology based on network science (Jin et al., 2013) and systems biology along with further refinement of phylogenetic groups, cell and cell type numbers can provide more accurate estimations for selected organisms. Incorporation of data on the cell numbers in various tissues will allow the estimation of dB, thereby the full description of fractal network properties. Finally, application of the method for ontologic data, examining the cellularity and complexity during the development of an organism can help draw the cell and molecular networks for any particular multicellular form of life.",
"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\nI wish to thank Krisztián Papp (MTA-ELTE Immunology Research Group, Budapest) for his help with data analysis. The book “A csodák logikája (The logic behind miracles)” by László Mérő gave me inspiration to search for the fractal properties of life.\n\n\nSupplementary material\n\nSupplementary File 1: Cellularity and complexity data. Data on the cellularity and complexity of a number of different organisms were compiled from the following two publications:\n\nNon-vertebrate data: Bell, G. 1997. Size and complexity among multicellular organisms. Biological Journal of the Linnean Society 60(3), pp. 345–363. https://doi.org/10.1006/bijl.1996.0108\n\nVertebrate data: Schad, E., Tompa, P. and Hegyi, H. 2011. The relationship between proteome size, structural disorder and organism complexity. Genome Biology 12(12), p. R120. https://doi.org/10.1186/gb-2011-12-12-r120.\n\nTaxonomical grouping follows what was used by Bell et al. Data on vertebrates in the first publication were replaced by more recent estimates in the second publication. Base 10 logarithm values were replaced by base 2 logarithm. Inverse values (1/complexity) of numbers describing cellular complexity were calculated in accordance with the proposed interpretation of khub. Cellularity of vertebrate organisms was calculated by using the average mass values shown and using conversion that assumes the mass of an average cell to be 7 nanograms (7×10-9 g). References, with first author and date, are shown for Bell et al’s dataset.\n\nClick here to access the data.\n\n\nReferences\n\nAlbert R: Scale-free networks in cell biology. J Cell Sci. 2005; 118(Pt 21): 4947–4957. PubMed Abstract | Publisher Full Text\n\nBell G: Size and complexity among multicellular organisms. Biol J Linn Soc. 1997; 60(3): 345–363. Publisher Full Text\n\nJeong H, Tombor B, Albert R, et al.: The large-scale organization of metabolic networks. Nature. 2000; 407(6804): 651–654. PubMed Abstract | Publisher Full Text\n\nJin Y, Turaev D, Weinmaier T, et al.: The evolutionary dynamics of protein-protein interaction networks inferred from the reconstruction of ancient networks. PLoS One. 2013; 8(3): e58134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim JS, Goh KI, Kahng B, et al.: Fractality and self-similarity in scale-free networks. New J Phys. 2007; 9(6): 177. Publisher Full Text\n\nSchad E, Tompa P, Hegyi H: The relationship between proteome size, structural disorder and organism complexity. Genome Biol. 2011; 12(12): R120. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSong C, Gallos LK, Havlin S, et al.: How to calculate the fractal dimension of a complex network: the box covering algorithm. Journal of Statistical Mechanics: Theory and Experiment. 2007; 2007(03): P03006. Publisher Full Text\n\nSong C, Havlin S, Makse HA: Origins of fractality in the growth of complex networks. Nat phys. 2006; 2(4): 275–281. Publisher Full Text\n\nSong C, Havlin S, Makse HA: Self-similarity of complex networks. Nature. 2005; 433(7024): 392–395. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "27994",
"date": "27 Nov 2017",
"name": "George W. Bassel",
"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 method to measure cellular complexity in different species by examining fractal network properties. This is potentially a very interesting method, yet there are several things which should be addressed:\nAs this is a methods paper, it would be nice to see how this particular method of measuring cellular complexity relates to others. There is no benchmark presented for the comparison of results.\n\nIt may not immediately be obvious to readers how fractals relate to complexity of an organism. The method illustrated in Fig. 1A could be more clear for readers who are not familiar with fractals, showing an example of how box-counting is actually carried out, and how this relates to complexity.\n\nThe results presented in Fig. 1 could be discussed in greater detail. It is not clear why there is a fairly noisy relationship between fractal dimension and degree exponent between species within different evolutionary backgrounds. Could this variance be due to the data that were used?\n\nThough it is possible to measure cellular complexity with this approach, it is not clear why this method is of particular value, and what purposes it would serve aside from the example presented. The addition of more relevant context in the introduction and conclusions could help with this.\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": []
},
{
"id": "29484",
"date": "17 Jan 2018",
"name": "Bela Suki",
"expertise": [
"Reviewer Expertise Biomechanics",
"mechanobiology",
"complexity"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes an analysis method and some results related to multicellular organization and complexity. While the study could be interesting, it is not well written. It is difficult to understand what is measured and what those measurements mean as described in the specific comments.\n\nSpecific comments:\n\nIn the introduction there are a few general statements that should be supported by general references.\n\nThe “eukaryotic unicellular ancestor” in the introduction represents a single cell, the ancestor of all eukaryotic cells. Is this the same as the Last Common Universal Ancestor, or LUCA? Maybe not. LUCA has been studied a lot and it appears as the ancestor of all cells including bacteria and archea. But is there a eukaryotic ancestor as well? Is it known that there was only one ancestor and only one transition from bacteria/archea to the eukaryotic cell? If so, is there any study that can back up such a statement?\n\nIs dB on the right hand side of equation 1 supposed to be d_B (i.e., B in subscript)? Similarly, dk is likely supposed to be d subscript k in equation 2.\n\nPlease explain where equation 4 comes from.\n\nPlease explain better what the data represent. There is a statement that N is the total number of cells in an organism. What is then N_B? Is it the number of different types of cell? And what is the network itself? There is a statement that “boxes have a degree exponent d_k” (page 2, right column, first line). But what is a box? Is it a cell itself? And what is the network inside the box? Or is it that the smallest box is a single cell and inside a box can only looked at when the box represent an organ? Yet towards the end of the Result, it is stated that “rather protein networks responsible for the multicellular organization of eukaryotes”. But I do not see any analysis on protein networks. All these questions arise because the methods is unclear and incomplete.\n\nIf complexity here means intercellular network complexity characterized by the degree distribution, then it is obviously different than the complexity of protein networks. It is not obvious why one should expect protein network to be self-similar with cell networks.\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? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1980
|
https://f1000research.com/articles/6-1974/v1
|
08 Nov 17
|
{
"type": "Case Report",
"title": "Case Report: A rare cause of oral bullae: Angina bullosa hemorrhagica",
"authors": [
"Salih Levent Cinar",
"Demet Kartal",
"Özlem Canöz",
"Murat Borlu",
"Ayten Ferahbas",
"Demet Kartal",
"Özlem Canöz",
"Murat Borlu",
"Ayten Ferahbas"
],
"abstract": "Angina bullosa hemorrhagica (ABH) is a benign disorder of the oral cavity. Clinically, oral, blood-filled blisters are seen. To give a proper diagnosis, one should rule out any other cause. We aim to present this case in order to emphasize this rare cause of oral bullae which is necessary to be differentiated from many serious dermatological and hematological disorders.",
"keywords": [
"Angina bullosa hemorrhagica",
"blister",
"oral mucosa"
],
"content": "Introduction\n\nMost oral bullae are caused by vesiculo-bullous disorders, blood dyscrasia and systemic diseases. One rare cause of oral bullae is angina bullosa hemorrhagica (ABH), a term which was first coined by Badham in 19671. Later, other synonyms like localized oral purpura and stomatopompholyx hemorrhagica were also used2.\n\nIn this case report our goal is to present a rare cause of oral blood-filled bullae, and to describe its differential diagnosis and treatment.\n\n\nCase report\n\nA 43 year-old male patient was admitted to our dermatology and venereology outpatient clinic with a complaint of a dark red, oral blister. He stated that his complaints started three years ago and since then he had experienced such episodes a few times each year. He had visited a few physicians but had not been able to get a proper diagnosis and because the blisters healed spontaneously he did not seek medical advice about his condition. He suspected that hot drinks and crispy food were the cause.\n\nOn dermatological examination we observed a tense, blood-filled bullae on his tongue with no additional dermatologic finding (Figure 1). He did not have any complaints of pain. On examination of the skin, there was nothing but a few seborrheic keratoses.\n\nHis biochemical markers and blood count, including platelet levels, were completely normal. Extra tests to rule out blood dyscrasia were performed and the results were normal.\n\nHe had no history of drug intake for the last few months, we therefore ruled out fixed drug eruption. Also he had not had any dental procedure or known oral trauma. One other possible diagnosis was a vesiculo-bullous disorder like pemphigus, pemphigoid, bullous lichen and acquired epidermolysis bullosa but he did not have any additional lesions elsewhere. A biopsy specimen was taken for histopathological examination. A subepithelial blister was observed. There were a few inflammatory cells and the subepithelial space was filled with erythrocytes (Figure 2). After the biopsy, a dark red erosion developed, which later healed without scarring.\n\nThe subepithelial space was filled with erythrocytes.\n\nAfter ruling out any possible disorder that can cause oral bullae, our diagnosis was angina bullosa hemorrhagica. Shortly after using an anti-bacterial mouthwash the complaints of the patient disappeared.\n\n\nDiscussion\n\nAngina bullosa hemorrhagica (ABH) is a benign oral cavity disorder with no known origin. The ABH term was first used in 1967 by Badham. However years ago, in 1933, this condition was described as traumatic oral hemophlyctenosis2. It usually affects the middle aged and elderly, with no sex discrimination. It is generally asymptomatic but sometimes pain or a sensation of choking can be reported. The term angina comes from this choking sensation. It can be solitary or multiple. Patients usually mention bullae forming during or shortly after a meal. Some patients report burning just before the blister onset, but pain and burning disappear after the bursting of the bullae3.\n\nAlthough ABH is accepted as a benign disorder, some authors reported a choking or gagging sensation when the lesions are in the posterior pharynx or in the epiglottis3. As a rare complication, acute upper airway obstruction was also reported4.\n\nThe cause of ABH is still unclear. No underlying hematological or immunological abnormalities have been identified. Trauma and personal predisposition seem to be necessary for the development of the disease. Increased fragility of the oral epithelial connective tissue can make the non-keratinized oral mucosa vulnerable to trauma. Thus, even a minor trauma can lead to breakage of the epithelial-connective tissue junction resulting hemorrhage from the superficial capillaries. The result is subepithelial hemorrhagic bullae5.\n\nHigh, in 1987, described the relationship between the chronic inhalation of steroids and ABH. In his study none of the 64 patients who used a steroid-free inhaler had a history of oral blistering whereas 15 patients out of 42 who used a steroid-based inhaler had oral blisters. The chronic use of steroid-based inhalers can affect collagen production causing epithelial and mucosal atrophy6. The proposal of Ferguson et al. that ABH could represent a localized amyloidosis failed after Congo red staining7.\n\nWhen the question is the histopathology of ABH, we see subepithelial separation from the lamina propria, with very few inflammatory cells. Parakeratosis in the surrounding tissue can also be seen. When the bullae bursts, the ulcerated epithelium with a chronic inflammatory infiltrate, mainly of lymphocytes, can be detected. Direct immunofluorescence staining for IgA, IgG, IgM and fibrin is negative8.\n\nIn our case the first few differential diagnoses which came to mind were erythema multiforme, bullous lichen planus, pemphigus, pemphigoid and fixed drug eruption along with blood dyscrasias. After some routine and specific blood tests we were able to rule out blood dyscrasias. As the blister was subepithelial and there were no additional mucosal and cutaneous lesions we eliminated pemphigus probability too.\n\nUnfortunately we were not able to perform direct immunofluorescence staining. However, with the help of the histopathology, we ruled out erythema multiforme as our patient had no recent history of herpes infection or drug intake. Again, with no recent drug intake history and absence of eosinophiles we ruled out fixed drug eruption. Pemphigoid and bullous lichen planus were not considered because our patient had a solitary lesion and this healed spontaneously in a week without any treatment.\n\nAs a result, in case of a blood filled bullae, the management should start with a detailed medical history and careful observation of the patient. Later on blood tests and biopsy, both histological and immunofluorescence, should be performed to differentiate it from other diseases. After the proper diagnosis has been made the patient must be informed about the disease. The benign nature of the disease must be emphasized to any individual with this diagnosis. However, any patient with ABH, must also be warned about some rare complications like acute airway obstruction in case of huge palatal or pharyngeal bullae as mentioned by Pahl et al4. Anti-inflammatory or anti-bacterial rinses/sprays can be used to prevent pain and secondary infection.\n\nAs dermatologists, we should be aware of one probable diagnosis, ABH, in case of oral blood-filled blisters. We should also keep in mind that many patients do not visit a physician due to the benign and self-limiting nature of the disease.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for publication of his clinical details and images.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBadham NJ: Blood blisters and the oesophageal cast. J Laryngol Otol. 1967; 81(7): 791–803. PubMed Abstract | Publisher Full Text\n\nDeblauwe BM, van der Waal I: Blood blisters of the oral mucosa (angina bullosa haemorrhagica). J Am Acad Dermatol. 1994; 31(2 Pt 2): 341–344. PubMed Abstract | Publisher Full Text\n\nGibson J: Oropharyngeal blood blisters are known as angina bullosa haemorrhagica. BMJ. 1997; 314(7094): 1625. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPahl C, Yarrow S, Steventon N, et al.: Angina bullosa haemorrhagica presenting as acute upper airway obstruction. Br J Anaesth. 2004; 92(2): 283–286. PubMed Abstract | Publisher Full Text\n\nHopkins R, Walker DM: Oral blood blisters: angina bullosa haemorrhagica. Br J Oral Maxillofac Surg. 1985; 23(1): 9–16. PubMed Abstract | Publisher Full Text\n\nHigh AS, Main DM: Angina bullosa haemorrhagica: a complication of long-term steroid inhaler use. Br Dent J. 1988; 165(5): 176–179. PubMed Abstract\n\nFerguson A, Johnston M, Leach IH, et al.: Angina bullosa haemorrhagica--a localized amyloidosis? J Eur Acad Dermatology Venereol. 2005; 19(4): 513–514. PubMed Abstract | Publisher Full Text\n\nHosain SI, Bounds G, Stanford J: Angina haemorrhagica bullosa causing respiratory obstruction postoperatively. Anaesthesia. 1991; 46(5): 422. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "27743",
"date": "14 Nov 2017",
"name": "Emine Tamer",
"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 dermatologists, we encounter oral mucosa pathologies very often. It is very important that we make differential diagnosis. This diagnosis must be kept in mind in oral mucosal pathologies. So that this self-limiting condition is not skipped. This article will raise our awareness on this subject. At the same time this article about angina bullosa hemorrhagica adequate and well-written.\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": "28829",
"date": "11 Dec 2017",
"name": "Wilfredo A. González-Arriagada",
"expertise": [
"Reviewer Expertise Oral Pathology and Medicine"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report of an angina bullosa hemorrhagica of the tongue. This is a rare condition of oral bullae, that is most frequent in hard palate and buccal mucosa. Authors said that the cause is unclear, however most of this bullae are of traumatic origin. For this reason it is important to mention to avoid any traumatic agent, like a malpositioned tooth or a restoration with defects, and the derivation to the dentist. Authors discard other common cause of these bullae that is the thrombocytopenic purpura.\nThis is a good article about angina bullosa hemorrhagica and it is well-written, but I think that they have to mention how important is to consult with a dentist, and to consider thrombocytopenic purpura as differential diagnosis, including the number of platelet count in the article (authors only said that is normal).\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1974
|
https://f1000research.com/articles/6-1971/v1
|
08 Nov 17
|
{
"type": "Software Tool Article",
"title": "NastyBugs: A simple method for extracting antimicrobial resistance information from metagenomes",
"authors": [
"Hsinyi Tsang",
"Matthew Moss",
"Greg Fedewa",
"Sharif Farag",
"Daniel Quang",
"Alexey V. Rakov",
"Ben Busby",
"Matthew Moss",
"Greg Fedewa",
"Sharif Farag",
"Daniel Quang",
"Alexey V. Rakov"
],
"abstract": "Multidrug resistant bacteria are becoming a major threat to global public health. While there are many possible causes for this, there have so far been few adequate solutions to this problem. One of the major causes is a lack of clinical tools for efficient selection of an antibiotic in a reliable way. NastyBugs is a new program that can identify what type of antimicrobial resistance is most likely present in a metagenomic sample, which will allow for both smarter drug selection by clinicians and faster research in an academic environment.",
"keywords": [
"Metagenome",
"microbiome",
"antibiotic",
"resistance",
"bacterial",
"genomic signature",
"MDR",
"workflow"
],
"content": "Introduction\n\nAntimicrobial resistance (AMR) of bacterial pathogens is a growing public health threat around the world. Most concerning are multidrug resistant (MDR) bacteria, which have become more prevalent in recent decades1. Well known examples of these pathogens include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus, extended spectrum beta-lactamase, and vancomycin-resistant Enterococcus. MRSA is prevalent in surgical and maternity hospitals and nursing homes, where it is often associated with hospital-acquired infection with high morbidity and mortality2. The current method of determining if a patient has a MDR infection is based on being able to culture a patient-derived sample in the presence of different antibiotic drugs3. This is a slow process that can take days to weeks, which can put the patient in danger of not receiving the correct antibiotic in time.\n\nThere are several known mechanisms by which bacteria acquire AMR. One common mechanism involves acquiring resistance through horizontal genetic transfer (HGT), which can include plasmid-, phage-, transposon-, and integron-mediated resistance4. The second major mechanism involves SNPs in chromosomal genes that can result in a change in antibiotic binding sites5.\n\nHigh-throughput whole genome sequencing (WGS) of microbiomes is a state-of-the-art method for studying complex microbial communities, such as the human gut. WGS creates large raw data sets, which must be processed quickly and efficiently to guide clinicians for the best and most efficient treatment strategy for the given patient. However, simple, clinically applicable bioinformatics methods that can provide a fast, reusable, reproducible, scalable pipeline to locate AMR genomic signatures in large metagenomics datasets from the NCBI Sequence Read Archive (SRA) and other public datasets are still lacking. Such pipelines could also be used for crowdsourcing of this analysis, such as with undergraduate students. The problem of determining efficient strategies for antibiotic usage is the keystone of the modern antibacterial therapy and prevention6.\n\nIn the last few years, a variety of different papers and tools have been developed that exploit AMR detection for both complete genomes and metagenomes. Some of the existing detection methods for AMR genomic signatures include: ResFinder7, PointFinder8, SSTAR9, DeepARG10, ARIBA11 and ResCap12. Another approach is Galaxy-based pipeline Amr++. All detection methods depend on the availability of collections of known AMR genomic signatures. These signatures are then directly searched for, or models are generated for the detection of novel AMR genes/loci. One of the most updated manually curated databases is the Comprehensive Antibiotic Resistance Database (CARD)13; others include ResFinder7, ARG-ANNOT14, and MegaRES15. Although some of these tools provide user-friendly web interfaces and use both FASTA and FASTQ files as input, they do not use the power of command line. Moreover, these solutions are not universal, e.g. ResFinder searches for only HGT-mediated resistance, whereas its successor PointFinder only looks for AMR caused by chromosomal point mutations. Other disadvantages of the existing solutions include an inability to work with big datasets or multiple raw sequence files, slow speed, and poor handling of metagenomic data.\n\nThe primary objective of this project is designing a reliable system for rapid diagnostics and prompt treatment of patients with MDR infections. At the heart of the system is a reusable, reproducible, scalable, and interoperable workflow to locate AMR genomic signatures in SRA shotgun sequencing (including metagenomics) datasets. To ease this task we used only RefSeq reference genomes for the bacterial pathogens important for public health, but the pipeline can be scalable to include databases for other microbes, viruses, and fungi. The result, NastyBugs, is a new program that can identify what type of antimicrobial resistance is most likely present in a metagenomic sample, which will allow for both smarter drug selection by clinicians and faster research done in an academic environment. NastyBugs is a framework created during the National Center of Biotechnology Information Hackathon in August 2017.\n\n\nMethods\n\nThe detailed workflow to extract antimicrobial resistance gene signatures is described in Figure 1.\n\nThree BLAST databases for downstream analysis were created using the latest versions of: 1) RefSeq human genome assembly GRCh37/UCSC hg19 (RefSeq accession no. GCF_000001405.37); 2) RefSeq bacterial genomes; 3) CARD13. For comparison purposes, CARD was used as two databases: one consisted of genes and another of SNPs.\n\nFurther analysis consists of three steps: 1) Host (human) reads removal; 2) Antimicrobial resistance signature identification; 3) Bacterial identification and characterization. Steps 2 and 3 were performed in parallel. Input data is SRA accession numbers (ERR or SRR) of the metagenome of interest. Another option is using FASTQ files from local storage.\n\nUsing STAR16 or Magic-BLAST, all reads mapped to human genome GRCh37/hg19 were removed, and unmapped non-human reads (considered as bacterial) were collected using SAMtools for further analysis.\n\nTo remove adapters/linkers/barcodes we used FASTX Clipper and Trimmer. The non-human reads are mapped again using Magic-BLAST; however this time they are mapped to bacterial genes/SNPs from the CARD. This allows for the identification of genes and SNPs that can lead to antimicrobial resistance in the bacterial population. Obtained reads were sorted and the sum of read abundance was calculated.\n\nThe identification of bacterial species and abundance is carried out in parallel. For that we again used Magic-BLAST and the database of NCBI RefSeq reference bacterial genomes. The resulting list of species was visualized using Krona17.\n\nThe output TAB-delimited formatted file contains data in five columns: 1) RefSeq accession number; 2) genus; 3) resistance gene; 4) ARO (Antibiotic Resistance Ontology) accession number; 5) score (number of mapped reads per 1kb). The data can be used for constructing a scatter plot showing relative abundance of antimicrobial resistance and the corresponding bacterial species in metagenomic sample analysed.\n\nThe documented workflow contains the script with containerized tools in Docker.\n\nWe used the following dependencies: 1) Magic-BLAST v. 1.3, a novel tool allowing mapping of large next-generation RNA or DNA sequencing runs against a whole genome; 2) SAMtools v. 1.3.1, a popular suite of programs for interacting with HTS data; 3) FASTX-Toolkit v. 0.0.13, a collection of command line tools for Short-Reads FASTA/FASTQ files preprocessing; 4) STAR v. 2.5.3a, RNA-seq aligner; and 5) Krona v. 2.7, a tool for metagenomic pie chart visualization.\n\n\nUse case\n\nTo validate our pipeline we used two human gut metagenomic datasets, SRA acc. no. ERR1600439 and SRR5239736. The SRR5239736 sample was used for comparison of our results with the results obtained by ResCap12. For metagenome sample SRR5239736, 24% of reads were mapped to the gene database of CARD and 1.6% reads were mapped to the SNP database of CARD.\n\nMagic-BLAST, a novel program from the BLAST family, can provide a faster and more accurate way to align reads of interest with reference sequences. A quick comparison of STAR and Magic-BLAST showed at least a 10-fold difference in speed increase with Magic-BLAST for mapping of SRA reads to human genome compared to STAR. For that reason, we chose not to use STAR in the pipeline.\n\n\nConclusion and next steps\n\nObtained results showed high efficiency of identification of antibiotic signatures in the studied samples. However, the presented workflow may be improved. Planned improvements will include: 1) optimization of the pipeline; 2) additional large-scale validation for different metagenomic samples; 3) representation of results with more information; 4) adding information about proteins participating in AMR; and 5) prediction of novel resistance genes using Hidden Markov Model.\n\nMoreover, implementation of machine learning analysis may provide additional capabilities.\n\nThe pipeline can be used to efficiently identify the presence of antimicrobial resistant genes, which in turn can be used as features for further downstream machine learning analysis. One useful application of machine learning in antimicrobial resistance is the prediction of the appropriate antimicrobial therapy to apply to a critically ill patient. For these patients, the time taken to administer an appropriate antibiotic agent inversely correlates with improved patient outcomes18. Whole genome sequencing of microbial isolates, followed by antimicrobial resistance genes identification and machine learning prediction provides an attractive solution to this problem. A previous application in this regard applied a simple rules-based approach and a logistic regression model19. More sophisticated, non-linear supervised machine learning methods, such as random forests, gradient boosting, and artificial neural networks may play a key role in producing accurate predictions for clinical use. Artificial neural networks, such as convolutional and recurrent neural networks, are particularly interesting as they may be able to extract novel features.\n\n\nData and software availability\n\nThe code for the pipeline is publically available on GitHub: https://github.com/NCBI-Hackathons/MetagenomicAntibioticResistance.\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.102026620\n\nLicense: MIT",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nBen Busby was funded by the Intramural Research Program of the NLM. Hsinyi Tsang was funded by NCI Contract #D14PD00826.\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 NCBI for hosting the hackathon, Grzegorz Boratyn, Sean Davis and Lisa Federer for technical discussions.\n\n\nReferences\n\nFriedman ND, Temkin E, Carmeli Y: The negative impact of antibiotic resistance. Clin Microbiol Infect. 2016; 22(5): 416–22. PubMed Abstract | Publisher Full Text\n\nBoucher HW, Corey GR: Epidemiology of Methicillin-Resistant Staphylococcus aureus. Clin Infect Dis. 2008; 46(Suppl 5): S344–49. PubMed Abstract | Publisher Full Text\n\nBonev B, Hooper J, Parisot J: Principles of assessing bacterial susceptibility to antibiotics using the agar diffusion method. J Antimicrob Chemother. 2008; 61(6): 1295–301. PubMed Abstract | Publisher Full Text\n\nGyles C, Boerlin P: Horizontally transferred genetic elements and their role in pathogenesis of bacterial disease. Vet Pathol. 2014; 51(2): 328–40. PubMed Abstract | Publisher Full Text\n\nWoodford N, Ellington MJ: The emergence of antibiotic resistance by mutation. Clin. Microbiol. Infect. 2007; 13(1): 5–18. PubMed Abstract | Publisher Full Text\n\nLee CR, Cho IH, Jeong BC, et al.: Strategies to minimize antibiotic resistance. Int J Environ Res Public Health. 2013; 10(9): 4274–305. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZankari E, Hasman H, Cosentino S, et al.: Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012; 67(11): 2640–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZankari E, Allesøe R, Joensen KG: PointFinder: a novel web tool for WGS-based detection of antimicrobial resistance associated with chromosomal point mutations in bacterial pathogens. J Antimicrob Chemother. 2017; 72(10): 2764–8. Publisher Full Text\n\nde Man TJ, Limbago BM: SSTAR, a Stand-Alone Easy-To-Use Antimicrobial Resistance Gene Predictor. mSphere. 2016; 1(1): pii: e00050-15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArango-Argoty GA, Garner E, Pruden A, et al.: DeepARG: A deep learning approach for predicting antibiotic resistance genes from metagenomic data. bioRxiv. 2017; 149328. Publisher Full Text\n\nHunt M, Mather AE, Sánchez-Busó L, et al.: ARIBA: rapid antimicrobial resistance genotyping directly from sequencing reads. Microbial Genomics. 2017. Publisher Full Text\n\nLanza VF, Baquero F, Martinez JL, et al.: In-depth resistome analysis by targeted metagenomics. bioRxiv. 2017; 104224. Publisher Full Text\n\nJia B, Raphenya AR, Alcock B, et al.: CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res. 2017; 45(D1): D566–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGupta SK, Padmanabhan BR, Diene SM, et al.: ARG-ANNOT, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes. Antimicrob Agents Chemother. 2014; 58(1): 212–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLakin SM, Dean C, Noyes NR, et al.: MEGARes: an antimicrobial resistance database for high throughput sequencing. Nucleic Acids Res. 2017; 45(D1): D574–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDobin A, Davis CA, Schlesinger F, et al.: STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1): 15–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOndov BD, Bergman NH, Phillippy AM: Interactive metagenomic visualization in a Web browser. BMC Bioinformatics. 2011; 12(1): 385. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumar A, Roberts D, Wood KE, et al.: Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med. 2006; 34(6): 1589–96. PubMed Abstract | Publisher Full Text\n\nPesesky MW, Hussain T, Wallace M, et al.: Evaluation of Machine Learning and Rules-Based Approaches for Predicting Antimicrobial Resistance Profiles in Gram-negative Bacilli from Whole Genome Sequence Data. Front Microbiol. 2016; 7: 1887. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRakov A; harper357, DCGenomics, Tsang S, et al.: NCBI-Hackathons/MetagenomicAntibioticResistance: Nastybugs. Zenodo. 2017. Data Source"
}
|
[
{
"id": "27750",
"date": "20 Nov 2017",
"name": "Torsten Seemann",
"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\n\"A Software Tool Article should include the rationale for the development of the tool and details of the code used for its construction. The article should provide examples of suitable input data sets and include an example of the output that can be expected from the tool and how this output should be interpreted.\"\nThe authors present a software tool which claims to detect AMR genes from raw metagenomic FASTQ reads. However, there are several problems with the manuscript and the source code.\nThe discussion of existing tools, methods and databases is confusing. The term \"AMR genomic signature\" is not defined. ResCap is mentioned, but that is a wet lab set of probes, not a bioinformatics method per se. CARD does have a command line tool, it is called \"RGI\". This section would benefit from a table of methods which explain if they are a tool, a database, or both; whether they work with contigs or FASTQ reads; and whether they work on bacterial isolates or metagenomes.\nThe algorithm is poorly explained. Why was Magic-BLAST chosen as an aligner? Did you turn splicing off (as bacteria don't have introns). It is stated that reads were removed of adaptors etc using Fastx_Clipper but no details of which adaptor files were used. The database used was CARD, but not explaination of how it was split into HGT and SNP/Variant parts. Alignment to Bacterial RefSeq is also done to get a species distribution, but it is unclear why or how, and how it links to the genes found. No examples of output results are provided, nor is it described how you identify the SNPs.\nI was unable to install and run the software. The github site https://github.com/NCBI-Hackathons/MetagenomicAntibioticResistance is very confusing also. It seems I have to use Docker to just get MagicBlast, but it doesn't include all the other dependencies. The primary script is called \"main.h\" but it appears to have lots of hard-coded parameters (eg. -threads 12) and refers to reference database files that do not exist. It also has bash syntax errors like \"var= wc -l file\" which should probably be \"var=`wc -l file`\" or \"var=$(wc -l file)\" - it appears something has lost characters in the file?The docs say \"The pipeline use three databases that should be downloaded with the script\". But there is no such script. I tried hard to find more info, but I was unable to.\nIn summary, more detail is needed, and the software needs to be installable and have some basic tests.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? No\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? 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": []
},
{
"id": "27751",
"date": "08 Dec 2017",
"name": "Tom J. B. de Man",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a new tool that is able to detect antimicrobial resistance (AMR) genes and identify bacterial species composition directly from metagenomic datasets. This pipeline removes the need to culture bacterial specimens and perform PCR for AMR gene detection and could potentially guide patient antimicrobial therapy.\n\nIntroduction comments: Extended-spectrum beta-lactamase (ESBL) is associated with what bacteria? (e.g., Enterobacteriaceae?) Please define the acronym ‘SNP’ (Single Nucleotide Polymorphism) the first time it is used within the manuscript. PointFinder is not the successor of ResFinder, they serve different purposes.\n\nMethods comments: It is not immediately clear when STAR or Magic-Blast will be selected for read mapping. Or is STAR not used in the current pipeline? There seems to be conflicting comments about the use of STAR in the manuscript (“Methods” versus “Use Case”).\n\nThe pipeline screens out human reads prior to further analysis; however, the authors might also consider removing PhiX reads in order to get cleaner output in your Krona plots.\nBacterial species composition is determined using Bacterial RefSeq and Magic-Blast. How does this compare to tools like Kraken? Did the authors consider using a different tool than Magic-Blast for determining bacterial species composition? Is there a way to link bacterial species to identified AMR genes?\n\nThe paper does not mention what cut-offs are being used for reporting AMR genes in terms of coverage and percentage sequence similarity. Is this something that can be adjusted by the user?\n\nOutput format comments: It would be useful for the reader to have a figure that illustrates and explains the output generated by this pipeline.\n\nGitHub repository comments: KRONA is missing from the dependency list on the authors GitHub repo\n\nThe “workflow method” section on the GitHub page does not contain any STAR aligner commands, only Magic-Blast. Users will be interested in the STAR parameters as well. Or is STAR not part of the pipeline anymore?\n\nOverall comments:\nTried to run the pipeline but it does not work. The pipeline, by default, is not able to find RefSeq database files. It would be a good solution to let the user choose the location of databases using a parameter option.\n\nPipeline has no help function or usage string when executed without any input parameters.\n\nDisclaimer: The findings and conclusions in this review are those of the author and do not necessarily represent the views of the Centers for Disease Control and Prevention.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? No\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? 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": []
}
] | 1
|
https://f1000research.com/articles/6-1971
|
https://f1000research.com/articles/6-1960/v1
|
06 Nov 17
|
{
"type": "Opinion Article",
"title": "Amending published articles: time to rethink retractions and corrections?",
"authors": [
"Virginia Barbour",
"Theodora Bloom",
"Jennifer Lin",
"Elizabeth Moylan ",
"Theodora Bloom",
"Jennifer Lin",
"Elizabeth Moylan "
],
"abstract": "Academic publishing is evolving and our current system of correcting research post-publication is failing, both ideologically and practically. It does not encourage researchers to engage in necessary post-publication changes in a consistent way. Worse yet, post-publication ‘updates’ can be misconstrued as punishments or admissions of misconduct.\n\nWe propose a different model that publishers of research can apply to the content they publish, ensuring that any post-publication amendments are seamless, transparent and propagated to all the countless places online where descriptions of research appear. At the center of our proposal is use of the neutral term “amendment” to describe all forms of post-publication change to an article.\n\nWe lay out a straightforward and consistent process that applies to each of three types of amendment that differ only in the extent to which the study is amended: minor, major, and complete. This proposed system supports the dynamic nature of the research process itself as researchers continue to refine or extend the work, and removes the emotive climate particularly associated with retractions and corrections to published work. It allows researchers to cite and share the most up-to-date and complete versions of articles with certainty, and gives decision makers access to the most up-to-date information. Crucially, however, we do not underestimate the importance of investigations of potential misconduct. This proposal allows two interrelated processes - amendment of articles and investigation of misconduct - to be uncoupled temporally, allowing a more rapid correction of the literature at a journal while institutional investigations take place, without either having to follow the others’ timeline.",
"keywords": [
"Post-publication amendments",
"corrections",
"retractions",
"expressions of concern."
],
"content": "Introduction\n\nAcademic publishing is evolving. It is no longer the case that, once published, articles remain unchanged for ever. It is also no longer the case that the final published version is the only version that is made public. Increasingly, preprints, datasets and authors' accepted versions (and revised versions) of manuscripts, are made available via a variety of mechanisms. A key question that needs to be addressed in the context of this evolving landscape is: are we well-served by the notion of a ‘version of record’ that is static post-publication?\n\nThis article reviews current ‘best practices’ for amending published articles and discusses problems that are encountered as a result. We suggest an alternative system, first proposed in a preprint1 which challenges current thinking but proposes a future solution. We highlight seven key principles that we believe do, and should continue to, apply to the integrity of the literature, the approach to authors suspected of misconduct, and how best to resolve these potentially conflicting issues (Figure 1).\n\nThe main guideline for journals, publishers and other publishing organisations handling retractions is the Committee on Publication Ethics (COPE) Retraction Guidelines2, published in 2009. Although the guidelines have been helpful, their consistent implementation has proved more difficult as publishing has evolved. Nonetheless, the guidelines, combined with regular discussion between editors, have provided a core framework for handling retractions that now covers many disciplines and countries. Of particular importance has been the repeated assertion of the overarching intention of the guidelines to assist in correction of the literature, whatever the cause. Thus, a first key principle is that the publisher’s role is to maintain the integrity of the literature. A second principle enshrined in the COPE’s Retraction Guidelines is that in cases where misconduct is suspected, it is the role of the authors’ institution or employer to investigate and provide a ruling. As in all legal and quasi-judicial systems, the accused are considered innocent until guilt is proven. Currently, in our experience many retractions occur only after such an investigation has been completed, and investigations can take many months or even years. The need to be fair to the authors and the need to maintain the integrity of the literature are therefore in opposition during the period that an investigation is underway.\n\nWe are by no means downplaying the need for rigorous investigation and, if needed, sanctions for potential misconduct. By contrast, we believe that our proposal would strengthen such investigations by ensuring that amendments to the literature and investigations can happen independently. This procedure is standard for many other areas where rapid notification of an incident is required; the notification for example of a consumer product failure happens as quickly as possible; the investigation of the cause and the application of any sanctions happens after due process has occurred, often much later.\n\n\nCurrent and evolving best practices\n\nThe traditional publishing workflow was originally established to facilitate robust peer review within a print publishing paradigm and is carried out with very little variation amongst conventional publishers (Figure 2). Following publication, however, the traditional scholarly communications process gives way to “best practices.” For concerns raised about an article, or author-initiated changes, there are a number of approaches that can be taken to correcting an article, depending on the scale of the issue. Small errors which do not undermine the findings of the published article can be resolved with a ‘correction’. Historically, some printed journals used to distinguish between errata and corrigenda, according to whether the author or the journal introduced the error - a now meaningless and poorly understood distinction. In other more recent situations, a comment, editorial or blog (or sometimes many tweets and blogs4) have been helpful in providing commentary with or without a correction to the article itself. Letters to the editors also have a long tradition as a place for signed criticism, (e.g. rapid responses and letters at The BMJ5, although many journals do not allow letters. PubMed Commons6 also offers a place for any qualified individual to comment on any article that is indexed in PubMed.\n\nWhere an article is so seriously flawed or erroneous that the findings can no longer be relied on, then the method of correction is typically wholesale i.e. the article is retracted. COPE guidelines on retraction1 advise retracting articles if the main findings are found to be unreliable, redundant, plagiarised or if the authors have reported unethical research or failed to disclose a major competing interest which could influence the interpretation of the article. COPE’s intention was to offer practical guidance and not be overly prescriptive (for example the guidelines deliberately did not contain information about the process of retraction and the wording to be used). The guidelines also do not offer guidance on what is to be done after a retraction. For example, some publishers are now experimenting with retracting and replacing an article in its entirety, for example, “retract and replace” by the JAMA network7. In other situations, where it is unclear whether a retraction will be the final outcome, ‘Expressions of Concern’ typically flag issues that do not yet have a final resolution2,7,8. A recent study has made plain the inconsistent and variable way ‘Expressions of Concern’ have been used in the past 30 years9.\n\nThese approaches were designed to help resolve issues with published articles while maintaining the integrity of the research literature - preserving the original article for the record. But increasingly, such approaches are inconsistently adopted by researchers and editors because they seem a less than perfect response to an evolving literature in the digital age. In parallel, readers are becoming accustomed to news sites and blogs posting rapid corrections when errors occur. Also, sites that encourage anonymous discussion of research, e.g. PubPeer10 have provided a route for all readers to comment in an only lightly moderated way on research articles, and to continue commenting while institutions attempt to investigate. Thus a third principle is the need to be able to correct rapidly, while implying no fault on the part of the authors until or unless any case against them is proven.\n\nTo date, we have observed a bewildering variety of notices on articles posted after publication. We have captured nine most commonly used and point to examples in Table 1. Furthermore, many of these are implemented differently by different publishers. No standard taxonomy of updates exists for publishers to adopt. This leads to inconsistencies from journal to journal and potential confusion for the reader.\n\nMoreover, a lack of willingness to engage in proper post-publication correction and amendment of the literature is further exacerbated when any type of post-publication ‘updates’ are misconstrued as punishments or admissions of guilt. This is particularly the case with retraction, a term which many feel has come to be loaded with blame and recrimination. It is fair to say that no one who has been involved with the retraction of an article – either as an editor, publisher, reviewer or author - has ever walked away from the process feeling wholeheartedly good about the experience. This is the case even if a retraction is done for the best of reasons – a genuine, no fault mistake11,12. As a result, we believe a ‘retraction’ will never be fully embraced as a positive outcome by researchers.\n\nThere is a fundamental misconception that retractions are ‘bad’ without pausing to ask why the retraction took place. A fourth principle is, therefore, that we need neutral terminology for the method of correcting published work that implies no fault on any party. In order to provide this, it is important to distinguish the correction of the published record from any investigation or description of misconduct that has occurred. If misconduct or fraud has occurred, this should be reported on, but such reporting should be considered as distinct from the process of correcting the literature. Such a separation is especially important as those who are likely to be responsible for an investigation into misconduct will be different from those issuing any correction of the published record, and the two may need to happen on quite different time frames. During this time, we feel it is important to alert readers to the possible issues with the published work, and to update the literature without awaiting the final outcome of a lengthy investigation13. Once any investigation is complete, however, its outcome should be recorded on any it has considered.\n\nAlthough corrections may not be as universally disliked as retractions, once an article has more than one or two corrections, in the current publishing system it is difficult to track what has happened. Tracking corrections is even more challenging when they are documented outside of the publication itself. For such ‘external corrections’ tracking is arguably even more crucial, as articles (as well as references to them) increasingly propagate across the internet and often do not link back to one version of record. However, developments such as Crossmark14 are beginning to address this issue. Furthermore, there are no universal guidelines for corrections, and editors and publishers often act on a case-by-case basis. Many editors and publishers struggle with the need for a correction notice for a very minor amendment to an article (such as a typographical error that has no effect on meaning), while many readers feel that all amendments post-publication need a clear audit trail. Our fifth key principle is that all changes made post-publication should be traceable and all versions of an article should be preserved. The circumstances in which an article should be removed (for ethical, legal or safety reasons) are extremely rare, and even in these cases a metadata record should remain, alongside an explanation.\n\nThere are better approaches for amending scholarly communications post-publication. Examples from newspapers and blogs [e.g. 15] take a simple approach to corrections which are effective, speedy and user-friendly. However, these types of outlet rarely require any cross-referencing to external articles and hence the process is much simpler than for academic research articles.\n\nWhile we reaffirm the importance of preserving the integrity of the published literature, we equally strongly affirm that research outputs are now dynamic objects online. Our sixth key principle is that the idea of the journal article as a monolithic object that will stand for all time unless formally retracted has gone. Rather we are seeing calls for articles to be viewed as organic publications or “living articles”16. If this idea is to be accepted, it is critical to ensure that updates to the scholarly record of a publication are appropriately made and that they properly link the latest update to the original record. Readers need a complete history of changes to an article. We now have the technical tools at hand to do this, and indeed a number of publishers now publish successive versions. These versions can be elegantly handled for readers online by making it clear which version is being displayed and by employing links to help readers navigate between versions (e.g. F1000Research17). Crossmark also provides a vital insurance mechanism, as it displays the publication history consistently across publishers18. Our seventh key principle is that both human and machine readers should be able to trace the full history of an article with ease, and should by default be taken to the most recent version of any article.\n\nWe now have the technical tools to ensure there is clear notation of version history in the citation record. The digital object identifier (DOI) is central to this as a unique alphanumeric string that identifies the content and provides a persistent link to the location of each resource on the Internet. It is an actionable identifier as it resolves to (i.e., takes the user to) the corresponding resource online. It is also descriptive as it binds the DOI to specific metadata about the digital object.\n\nTo support versioning, publishers could assign a new DOI (or, more logically a DOI with a suffix), to each version, and link to the previous version in the metadata record. Crossref ensures that all versions are linked through the relationship metadata included in the record. Once deposited with Crossref19, all versions could be threaded together20 through their DOIs and made available to systems across the research ecosystem. This practice provides specificity and precision to the citation record that has not been possible before. Researchers can thus cite a specific version, rather than, unintentionally, the original one. Essentially, we can now, technologically, think beyond the article of record to a number of versions, whether they are preprints or postprints or institutional copies. The primary challenge, however, is facilitating publisher (and perhaps institutional) adoption and implementation of practice to deposit and update metadata records as changes occur.\n\nIf a fundamental change in how we amend published articles is to be successful, we need both the technology to make it happen (as outlined above) and the will and support from the community to embrace the change.\n\n\nA proposal for the future\n\nIn order to facilitate a more reliable and useful scholarly literature, we propose the following components of handling changes to published articles. The changes can be implemented by the same journal (or other publishing venue) that published the original research and be linked to the original article. We propose removing the terms correction and retraction altogether and instead using the term “amendment” to describe all forms of post-publication change to an article.\n\nThe term “amendment” carries a neutral tone and is generic enough to apply to a wide array of cases, including the smallest instances such as a typographical error all the way to wholesale withdrawal of an article. By employing a uniform term, we hope to remove any associated stigma in the context of scholarly literature. When readers encounter each amendment, they can read the notice for details on each change, and can judge the article and its revisions on their own terms.\n\nWe considered alternative names such as “update” but we felt they imply progress or addition. In particular, we strongly feel that retaining the word “retraction” even for the most egregious instances of scientific malpractice would further perpetuate the problem of stigma and is thus not desirable.\n\nWe propose an amendment model made up of three different types: minor, major, and complete (Figure 3). These are distinguished by the scale of change entailed. Minor amendments cover small changes such as typographical errors, or other minor amendments to the content or metadata that has no effect on the substance of the article. Issues currently worded as corrections belong as Major amendments, but might also include clarifications and addenda (which are not currently easy to make under most current publishing workflows). Amendments of this type would make changes to one or two small parts of the article but not its whole message. Examples are changes in authorship, correction of one figure or method, or additional data or discussion. Lastly, Complete amendments covers situations where the article as a whole is considered unreliable in its current form. There may be elements that remain ‘correct’ but large proportions are not. Instead of “retract and replace” as currently practiced by some publishers, we would recommend “retract and republish” with a new DOI that resolves on the newer version and makes plain the chain of events. In cases where authors and/or journal may wish to dissociate themselves from the original article completely, this can be noted with a complete description in the associated narrative and no attempt to insert new text or other content. We would emphasise the importance of using the single neutral term ‘complete amendment’ to cover all types of retraction including those for honest errors retractions and misconduct. The narrative should include explanation of why the amendment is being made, and can report on any misconduct allegations once they are known and proven. However, an amendment could nevertheless be made initially without awaiting the outcome of any misconduct allegation or investigation.\n\nThe notice (for all amendment types) is comprised of a declaration followed by key details. The declaration is posted at the forefront of the document stating that: “The authors and/or the journal wish to make the following amendment to the published article [article full reference].” We envisage this declaration being authored most often by the authors of the original article, in consultation with the journal or publisher, but the format allows for the possibility that a journal or publisher can amend an article without an author’s consent if that proves necessary. In either case the source of the amendment will be clearly visible to readers.\n\nEvery amendment notice would then include the following:\n\na) Who is issuing the notice (an author, all authors, editor, journal, publisher, institution), and whether any of this group dissents from the notice. The CReDIT taxonomy might be applied to specify the role entailed in crafting the amendment.\n\nb) The type of amendment (as above)\n\nc) Link to the article that is being amended as well as other relevant links to associated resources\n\nd) Date\n\ne) Associated narrative (optional for minor amendments). This is particularly critical in the case of removal of an article without replacement: there needs to be some narrative notice that indicates the reason. This should be updated as needed with links to any investigation if that is publicly available\n\nThe process of the amendment within the publication lifecycle is straightforward and consistent for all types of amendment. Whether the incident at hand merits a minor, major, or complete amendment, the publisher can issue the notice, assign a new DOI to it, register it with Crossref and link to the target publication. Moreover, the same process is also consistent and streamlined to apply at a higher level to include all the various versions of the publication from the original publication to the posting of amendments (Figure 4).\n\nPublishers register all amendments and versions of the paper as they currently do now with Crossref. Each amendment is assigned a DOI, and its metadata should contain a link to its associated article. This links the amendment notice and specific version of the paper in both directions. When a subsequent version of a paper is published, the publisher registers it with Crossref with a new DOI so that it can contain its own, independent set of metadata. The metadata should contain a link to the previous version, thereby creating a sequential chain across multiple updates.\n\nThis version of scholarly communications supports the dynamic nature of the research process, as researchers continue to refine or extend the work. They can publish updates along the way, sharing their latest findings, analysis, and conclusions. For each version of an article an amendment can be issued. In each case, the publisher assigns a new DOI and deposits the metadata with Crossref so that researchers can cite with clarity and specificity.\n\nWhile the proposed amendment model simplifies the process by which published results are shared and updated, it also increases the potential number of components that might be published from a single set of research results. As such, linking amendments to their associated articles and across individual versions needs to be carefully implemented online so readers can easily navigate between versions.\n\nSince every publisher employs their own specific design approaches to content delivery, we recommend the following linking and display strategies to ensure that amendment display fully supports editorial intent (Figure 5). We also illustrate the proposed amendment model, to a generalised case in Supplementary file 1 that contains elements of a number of COPE cases21.\n\nLinking. Link from each amendment to the respective article it amends and vice versa, so that the reader can easily navigate back and forth between the notice and the research itself. Also link general amendments to their respective article version to ensure that the amendment notice links back to the specific article version to which it amends. And in the event of a correction to a figure legend, the link should direct users to the latest version of the article, complete with the correct figure legend. Readers can then go back and look at it with the wrong legend as they wish (i.e. be transparent about the change). This is akin to the journalistic model and is much cleaner.\n\nArticle versioning. Each article version has its own DOI and URL, which persists even with the publication of subsequent versions. Where the reader is on an outdated version, clearly communicate its date on the article and provide a prominent link to the most up-to-date version. Provide a clean and simple way for readers to navigate to and from each version. In cases where the publisher is linking to the article in general (e.g. from a journal home page, etc.), directing users to the latest version is recommended.\n\nDOI handling. Although any two DOIs can be linked via Crossref to explain the relationship between versions of an article, or between an article and an amendment, we suggest that the relationship between these would be more obvious to human readers if the original article’s DOI were given a suffix (e.g. https://doi.org/10.1136/bmj.j1072.1; https://doi.org/10.1136/bmj.j1072.2, etc). Furthermore, The DOI should direct users directly to the specific document at hand, which corresponds to the precise version at hand. (This goes hand in hand with the recommendation to assign a new DOI to each version.)\n\nAll amendments must be evident to readers and machine harvesters of the literature. They must be inextricably and permanently linked to the original article and also propagated to systems that index articles. Some instances of complete amendments would mean publication of a new article, with a new DOI linked to the original article.\n\nFor publication updates to reach all the places online where articles are read, indexed, shared, discussed, recommended, etc. publishers need to make the metadata available in a central store where the data is freely available for systems and applications to consume. Crossref currently provides this facility and their metadata framework fully supports full disclosure of amendments. Once metadata updates are available from publishers, systems need to apply the latest information wherever the publications affected may appear online. All metadata are openly licensed for reuse, propagated through a variety of interfaces and formats via Crossref APIs22.\n\nAnyone can search the published literature (humans and machines) to find the latest updates for any publication regardless of origin in the Crossref corpus (85+ million publications at time of writing). Publishers can flag this not only in their own content (online and PDF versions) via Crossmark, but also in the references of papers they publish. They can propagate these notices through other delivery channels offered such as email alerts, RSS feeds, recommendations, and so on. Non-publisher platforms such as indexers, reference managers, recommendation systems, social bookmarking tools, researcher profile systems, and others, can apply the update information and bibliographic metadata to the content they display as well. This information is also potentially useful for research information systems used by funders and research institutions, which also track scholarly outputs.\n\n\nConclusion\n\nOur current system of correcting research post-publication is failing both ideologically and practically. We propose a model that publishers of research can apply to the content they publish which ensures that any post-publication amendments are seamless, transparent and propagated to all the numerous places online where descriptions of research appear. We believe that this proposal puts in place a system which both incorporates new technological thinking and removes the emotive climate now associated with retractions and corrections to published work. It also exploits the opportunities of new technologies to allow researchers to cite and share the correct versions of articles with certainty. Furthermore, it allows readers to have the most up to date information in order to support academic research. We recognise that there are aspects of the model that need to be further refined, including for example how to handle changes in authorship. We look forward to initiating a pilot test of our model to learn how it could work in practice, please contact us if you are interesting in testing this model.\n\nThere is a growing openness in various aspects of research and movements towards linked documentation of the methods, results and discussions derived from such research. We envisage a future with a fully seamless means of publishing that starts with protocols and registered experiments then moves to results publication and data sharing and finally onto revisions, with version control. This system incorporates easy amendments, which are themselves integrated with the articles they amend; articles may have multiple versions. “No fault” amendments will be enabled and encouraged, and reporting on the changes that need to be made to an article may be separated in time from the reason the changes happened. The degree of reliability of a study will be separated from the notion that the author and/or a prestigious journal provides an absolute guarantee for the work. In all cases by default a human or machine reader will see the most recent and up-to-date version, but they will also be able to navigate to previous versions. There will be full disclosure of publication history and metadata that is made freely available to humans and machine applications.",
"appendix": "Competing interests\n\n\n\nVB is the Director of the Australasian Open Access Strategy Group. She also works part-time for Queensland University of Technology (QUT), Brisbane as a Professor in the Office of Research Ethics & Integrity and in the Division of Technology, Information and Library Services. She was the Chair of COPE until May 2017 and was a COPE Trustee until November 2017. She is also an Editorial Board Member for Research Integrity and Peer Review. TB is Executive Editor of The BMJ. She chairs the scientific advisory board of EMBL-EBI Literature Services. JL is Director of Product Management at Crossref. She serves on the Dryad digital repository board. ECM is Senior Editor (Peer Review Strategy and Innovation) at BMC (part of Springer Nature). She is also a COPE Council Member, an Editorial Board Member for Research Integrity and Peer Review, a member of the Advisory Board for EnTIRE (an EU proposal for Mapping the research ethics and research integrity framework) and mentor for MiRoR (Methods in Research on Research).\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nProvenance\n\nThis work was conducted as part of an ongoing open discussion within an initial working group and a wider consultation. COPE convened the working group that wrote this paper. This is one of the ways that COPE helps foster high-quality and progressive discussions that, one day, may be reflected by changes in practice. This paper and the ideas presented within it exemplify COPE's commitment to collegial discussion and debate.\n\n\nAcknowledgments\n\nWe are grateful to COPE for facilitating this discussion. We are also grateful for comments and feedback on the preprint from Geoffrey Bilder, Peter Doshi, Andy Collings, Michaela Torkar and Liz Wager. We are also grateful to others who commented on the preprint versions1 and have attempted to incorporate the feedback into this paper.\n\n\nReferences\n\nBarbour V, Bloom T, Lin J, et al.: Amending Published Articles: Time To Rethink Retractions And Corrections? bioRxiv. 2017; 118356. Publisher Full Text\n\nWager E, Barbour V, Yentis S, et al.: Committee on Publication Ethics Retraction Guidelines. 2009. Reference Source\n\nPublishing Outline from HEFCE. Reference Source\n\nYeo SK, Liang X, Brossard D, et al.: The case of #arseniclife: Blogs and Twitter in informal peer review. Public Underst Sci. 2017; 26(8): 937–952. PubMed Abstract | Publisher Full Text\n\nResponding to articles. Reference Source\n\nPubMed Commons. Reference Source\n\nHeckers S, Bauchner H, Flanagin A: Retracting, Replacing, and Correcting the Literature for Pervasive Error in Which the Results Change but the Underlying Science Is Still Reliable. JAMA Psychiatry. 2015; 72(12): 1170–1171. PubMed Abstract | Publisher Full Text\n\nCOPE: Lack of trial registration leads to new concerns about study conduct and ethical review/approval. 2011. Reference Source\n\nVaught M, Jordan DC, Bastian H: Concern noted: A descriptive study of editorial expressions of concern in PubMed and PubMed Central. Res Integr Peer Rev. 2017; 2: pii:10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPubpeer. Reference Source\n\nMann R: Rethinking Retractions. 2013. Reference Source\n\nRetraction Watch: Pamela Ronald does the right thing again, retracting a Science paper. 2013. Reference Source\n\nhttp://blogs.plos.org/biologue/2013/11/11/so-tell-me-what-would-you-do/.\n\nCrossmark. Reference Source\n\nMoylan E: What does transparency in peer review mean to you?2017. Reference Source\n\nShanahan DR: A living document: reincarnating the research article. Trials. 2015; 16: 151. PubMed Abstract | Publisher Full Text | Free Full Text\n\nF1000Research. Reference Source\n\nCrossref. Crossmark. Reference Source\n\nCrossref. Reference Source\n\nShanahan D, Meddings K: Clinical trial data and articles linked for the first time. 2016. Reference Source\n\nCOPE cases. Reference Source\n\nCrossref Metadata Delivery APIs. Reference Source"
}
|
[
{
"id": "27668",
"date": "27 Dec 2017",
"name": "Lex Bouter",
"expertise": [
"Reviewer Expertise Epidemiology",
"methodology",
"research ethics",
"research integrity"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 opinion article on a very important topic: how to make necessary post-publication changes to a scientific article as quickly as possible and in a way that will not confuse readers. It argues that the current methods (retraction, replacement, expression of concern, correction, erratum, comment) are slow and confusing. Instead a proposal is launched to separate making the changes needed from the fact finding about why it went wrong and if someone is to blame and should be sanctioned. While developing their proposal the authors opt for more neutral language and suggest to let the term amendment replace emotionally laded terms like retraction.\nI applaud the authors for raising the issue, but I should say that I’m more convinced by their analysis of the problem than by the solution they propose. The piece is well written and quite clear in explaining its core ideas, but I would like to raise a number of points with the single purpose of inviting the authors to broaden the scope of the article a bit and to rethink some of the elements of their proposal.\nThe current manuscript is in fact a quite technical policy paper that contains many details on how all versions, amendment notices and metadata could be linked together with a view to make all changes transparent and traceable. While it indeed is important to be able to follow the history of changes to a published article, the technical details are only relevant for the small audience that would be responsible for ‘making it happen’ if the proposal is implemented. I assume that many readers will not be interested in the technicalities and will also find them difficult to follow.\n\nI was puzzled by the fact that the example that explains how the amendment model works in practice was buried in a supplement. This needs to be the core of the main text at the expense of the technical details that would be more fitting for a supplement.\n\nThe example in the supplement illustrates my main concern by assuming that in cases of alleged research misconduct it’s clear what is wrong and needs to be rectified before a full investigation is concluded. That may be the case in some instances of plagiarism like massive text recycling and clumsy image duplication. But in my experience most allegations of research misconduct are not so clear and almost always the accused does not only deny that he or she did something wrong, but also contests that it is wrong. Consequently authors will usually resist any amendment of their published work before - and often also after - the conclusion of the full investigation.\n\nI agree that the amendment model will probably help to make necessary changes more quickly and more clearly if the authors request them or agree to requests made by others. But editors and publishers have a track record of doing nothing without having consent from the authors. And I cannot see why this would be different under the new paradigm. This needs to be discussed in the opinion article.\n\nWho should make the final decision about publishing an amendment? The current manuscript seems to suggest that this is the role of the publisher. But I assume that most scientific journals have some form of editorial independency from its publisher and that consequently the editor-in-chief will decide. Of course the publisher has the role of providing technical assistance and legal advice.\n\nI would have expected some remarks that make clear that the authors are in favour of evidence-based policy and meta-research. That could for example imply a proposal to perform a Delphi study among editors and authors with the purpose to gain a better understanding of what these stakeholders think about the problem and which solution they would prefer. Similarly I would have expected a plea to thoroughly evaluate the intervention proposed when it is piloted.\n\nI rather like the fact that at the end of their piece the authors link the amendment model to the broader picture of transparency of the publication process. The role of pre-prints, pre-registration, publication of data and data-analysis plans, post-publication peer review, and anonymous commenting (e.g. on PubPeer) are all very briefly mentioned. But the article might improve if they explain in a bit more detail how these phenomena may help or hinder the implementation of the amendment model.\n\nThere is one technical detail that puzzles me. How can we make references to outdated versions go away? Authors that write a new paper often use their collection of PDFs or their own often quite primitive system for storing (the metadata of) relevant publications. This may be the main reason why retracted papers are still cited. Maybe the list of references of a new manuscript can be checked whether the latest version is referred to by some clever piece of software. And I wonder whether it would also be feasible to update the references of articles that are already published.\n\nI’m not convinced that the term retraction can be avoided completely. The authors propose to substitute this with complete amendment. But when for instance the data were fabricated or no mandatory informed consent was obtained, there is nothing to amend in the sense of providing a better version of the paper. The publication in these instances just needs to be disqualified and will not be substituted by a new version. Retraction seems to be by far the best word for it. Complete amendment will probably work well if the authors for instance acknowledge that huge mistakes have been made in the data-analysis and want to replace their article by a version in which that issue is solved. Complete amendment might then indeed be a better term than honourable retraction as proposed by Daniele Fanelli.\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": []
},
{
"id": "27671",
"date": "02 Jan 2018",
"name": "C.K. Gunsalus",
"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 proposal for an amendment system is an important contribution on a topic central to research integrity. It is a thought-provoking and strong proposal that has merit and could be an advance if adopted broadly with a central refinement to exclude documented misconduct. A no-fault system that focuses on the integrity of the literature is positive and could have some positive effects. The attempt to encompass all post-publication change into three categories stretches a good idea too far. It overlooks impulses contributing to the current situation that features a “bewildering variety of notices” and “inconsistencies” across journals illustrated in Table 1, namely that humans seek proportional responses and labels for documented lapses.\nAn analogy can be found in criminal justice systems worldwide, which typically differentiate conduct that has resulted in death with gradations of responsibility that factor in severity, intent, and surrounding circumstances. Findings can range from self-defense to involuntary manslaughter (criminally reckless or negligent behavior resulting in death) to voluntary manslaughter (performed in the heat of the moment) up through degrees of “murder,” a label that generally implies premeditation. Collapsing all post-publication anomalies into a no-fault system is likely to give rise over time to the creation of embellishments through additional labels that allow for degrees of fault, causing more confusion.\n\nRather than seeking to avoid all differentiation, the amendment process could be a improvement if it focuses on documenting changes over time, and reserves another category entirely for the most serious cases of willful, intentional conduct or bad practice, cases in which the article should be disavowed completely. “Complete amendment” will be valuable for circumstances described in present proposals for “honorable retractions” and the like, as there is room for a complete replacement in circumstances that do not imply fault. If the goal is the integrity of the literature, then no-fault terms are a strong advantage in a large set of—though not all—circumstances.\n\nThe crux of the matter seems to be that the authors believe that “If misconduct or fraud has occurred, this should be reported on, but such reporting should be considered as distinct from the process of correcting the literature.” The authors also state the “we strongly feel that retaining the word retraction even for the most egregious instances of scientific malpractice would further perpetuate the problem of stigma and thus is not desirable.” I disagree. Stigma for egregious malpractice is warranted. If the integrity of the literature is at issue, then notorious cases of substantiated misconduct should be labeled as such, in no small part to address the continued citation of such work. At the same time, given current uses of “retraction,” indeed, a new term may be needed to differentiate from historical usages that have not been restricted to misconduct. A middle ground is possible: if the amendment process is used for all revisions short of proven misconduct, a hybrid system could exist and be valuable: the amendment system for changes post-publication, and a different label for publications containing adjudicated misconduct.\nThe proposal is strong where it focuses on its key principles and the mechanisms in place that now allow for clear adoption of a linking system and versioning documentation. It seems muddier when it attempts to correct vexing problems journals face when institutional review of misconduct allegations are underway. Several statements in the effort to make this case seem problematic:\n\n“The need to be fair to the authors and the need to maintain the integrity of the literature are therefore in opposition during the period that an investigation is underway.” It is not axiomatic that they “are” in opposition and this tension exists beyond the investigation period.\n\n“Thus a third principle is the need to be able to correct rapidly, while implying no fault on the part of the authors until or unless any case against them is proven.” The proposal doesn’t provide for labels of fault once a case is proven, apparently seeking to divorce the integrity of the research literature from institutional findings.\n\n“As a result, we believe a ‘retraction’ will never be fully embraced as a positive outcome by researchers.” True as far as it goes, and yet could be remedied if a category is created and reserved for documented misconduct.\n\n“…removes the emotive climate associated with retractions and corrections to published work.” This seems unlikely. Amendments post-publication are never likely to be any researcher’s choice of outcome. It could reduce the overly broad associations with post-publication notices that are indeed confusing in their lack of consistency as currently applied.\n\nFinally, the illustration in Supplementary File 1 is confusing. It begins “An institution finds misconduct from one of their senior scientists who has reused the same images to show controls in many figures.” The publisher is notified and opens an investigation into the three affected publications, as apparently the institution will not share its findings, so the illustration involves great exertions on behalf of the journal and its editorial board members to parse the appropriate label to apply.\n\nThis illustration seems at odds with the descriptions of how principles two and three in the proposal will improve the current system. First, the journal’s process here hasn’t even started until after the institution has already notified it that there has been a finding, so the amendment to the literature is not independent of that process at all. Further, the authors are integrally involved in the case involved in the illustration. This is counter to my experience, where more often authors protest and will not cooperate in any corrections or retraction, and the process more often involves the institution and the journal (and their respective lawyers). The proposal does not address what happens when the authors are not cooperating, which seems an oversight, and again argues for application of the amendment process in situations absent findings of misconduct. There is nothing that would prevent amendments before and the revised/new label after such a finding.\n\nAn especially strong feature of the proposal is the key principle calling for clear audit trails and full disclosure of publication history and metadata. A revised amendment system is worthy of our attention and the adoption of such a system should be considered.\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": []
}
] | 1
|
https://f1000research.com/articles/6-1960
|
https://f1000research.com/articles/6-1708/v1
|
19 Sep 17
|
{
"type": "Research Note",
"title": "A review of data sharing statements in observational studies published in the BMJ: A cross-sectional study",
"authors": [
"Laura McDonald",
"Anna Schultze",
"Alex Simpson",
"Sophie Graham",
"Radek Wasiak",
"Sreeram V. Ramagopalan",
"Laura McDonald",
"Anna Schultze",
"Alex Simpson",
"Sophie Graham",
"Radek Wasiak"
],
"abstract": "In order to understand the current state of data sharing in observational research studies, we reviewed data sharing statements of observational studies published in a general medical journal, the British Medical Journal. We found that the majority (63%) of observational studies published between 2015 and 2017 included a statement that implied that data used in the study could not be shared. If the findings of our exploratory study are confirmed, room for improvement in the sharing of real-world or observational research data exists.",
"keywords": [
"Data-sharing",
"real-world data",
"observational"
],
"content": "Introduction\n\nOver the recent years, a number of articles and movements have called for the sharing of clinical trial data1–3. However the access to and sharing of real-world/observational data receives little and arguably insufficient attention. In this study we sought to assess the current state of data sharing in published observational studies in a general medical journal, namely the British Medical Journal (BMJ). The BMJ was chosen as the journal does not enforce a commitment of data sharing for observational studies4, but all research articles are required to contain a data sharing statement5.\n\n\nMethods\n\nAll observational research articles published in the BMJ between 1st January 2015 and 31st August 2017 were investigated. These dates were chosen as it provides a reasonable sample size for analysis and recent data post-dating some of the articles regarding clinical trial data sharing2. Observational research articles were defined as cohort, case-control and cross-sectional studies, as well as case series. Meta-analyses, systematic reviews, randomised controlled trials and genetic/Mendelian randomisation studies were excluded. The data sharing statements of these studies were reviewed. If the statement written was “no additional data available”5 or that data was not publicly available, the study data was classed as not shared. Statements alluding to data being available from the corresponding author, or a referral to access policies of the data source the study data was classed as shared. Where statements alluded to code or technical appendix being available, but no reference to data specifically being available, the study data was classed as not shared.\n\n\nResults\n\nTwo hundred and thirty seven observational studies were included. A review of the data sharing statements of these studies revealed that 149 (63%) studies had a statement implying that the data underlying the study could not be shared.\n\n\nConclusions\n\nIn our review of the data sharing statements of observational studies published in the BMJ, we identified that the majority of studies did not share data. Whilst there are likely many reasons for this, including patient confidentiality concerns and the access possibilities of the data source, the lack of data sharing in observational research is a potential cause for concern. The key limitation of our study was the scope of the data. We only reviewed the data sharing statements of one medical journal and therefore the generalisability of our results is unclear. Our analysis should be seen as exploratory rather than definitive. Further studies are needed, in greater depth, to confirm or refute our findings. If consistent findings are seen, the lack of sharing of observational research data is an area that warrants further attention.\n\n\nData availability\n\nDataset 1: Raw data showing the studies identified from the BMJ and whether the data sharing statements indicated data was not/were available. doi, 10.5256/f1000research.12673.d1778716",
"appendix": "Competing interests\n\n\n\nLM and SR are employees of Bristol-Myers Squibb Company. AS, AS, SG and RW are employees of Evidera Inc.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGoldacre B, Lane S, Mahtani KR, et al.: Pharmaceutical companies’ policies on access to trial data, results, and methods: audit study. BMJ. 2017; 358: j3334. PubMed Abstract | Publisher Full Text\n\nKrumholz HM, Peterson ED: Open Access to Clinical Trials Data. JAMA. 2014; 312(10): 1002–1003. PubMed Abstract | Publisher Full Text\n\nAllTrials: All Trials Registered. All Results Reported. AllTrials. Reference Source\n\nResearch | The BMJ. (Accessed: 2nd September 2017). Reference Source\n\nGroves T: Sharing the raw data from medical research [corrected]. BMJ. 2009; 338: b1252. PubMed Abstract | Publisher Full Text\n\nMcDonald L, Schultze A, Simpson A, et al.: Dataset 1 in: A review of data sharing statements in observational studies published in the BMJ: A cross-sectional study. F1000Research. 2017. Data Source"
}
|
[
{
"id": "26085",
"date": "26 Sep 2017",
"name": "Laurie A. Tomlinson",
"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 study that draws attention to an important area - open access to the data underpinning research papers allowing assessment of reproducibility. It is of course limited by it's focus on one journal as data-sharing policies vary between journals. In addition, requested statements by journals often become formulaic as the tendency is to copy what other papers have said previously. Some journals have tried new approaches in an effort to encourage data-sharing such as the NEJM 'Sprint Challenge'.\nHowever, the major limitation is a lack of further data on why data was not shared. Some providers such as the UK primary care data repository CPRD do not allow data-sharing, even if the authors of articles are keen proponents of open data-access. More in depth work will be required to understand the true barriers and way ahead for data-sharing.\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": "26439",
"date": "02 Nov 2017",
"name": "Robin E. Champieux",
"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 exploratory analysis contributes to the literature on biomedical data sharing practices, and demonstrates the importance of the further understanding of data sharing policies, practices, and barriers for domain and type specific data sets, such as those associated with observational studies.\nWhile it does not impact the soundness of this short report, I would recommend that the authors expand on their reasoning for choosing BMJ for their analysis. Specifically, while BMJ requires data sharing statements, its data sharing policy is relatively general (sharing is encouraged, but specific requirements are not communicated). It would be helpful for the authors to discuss (potentially as a follow-up analyses), how the robustness of a journal's data sharing policy could influence the rate of data sharing.\nRegarding the sampling methodology, the authors write, \"All observational research articles published in the BMJ between 1st January 2015 and 31st August 2017 were investigated. These dates were chosen as it provides a reasonable sample size for analysis and recent data post-dating some of the articles regarding clinical trial data sharing.\" In my view, this statement is insufficient. The authors should provide more detailed and quantitative information about the strength and the nature of the sample.\nFinally, the authors write \"A review of the data sharing statements of these studies revealed that 149 (63%) studies had a statement implying that the data underlying the study could not be share\". However the methodology does not indicate if the data \"could\" be shared, only if it was shared. I would urge the authors to revise this statement be be more precise.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3153",
"date": "06 Nov 2017",
"name": "Sreeram Ramagopalan",
"role": "Author Response",
"response": "We thank the reviewer for their comments. We focussed on the BMJ because of the journal being a key backer of the all trials initiative but we agree with the reviewers point and have updated the discussion. We have updated the methods to include a precision calculation. We corrected the results sentence."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1708
|
https://f1000research.com/articles/6-1957/v1
|
06 Nov 17
|
{
"type": "Research Article",
"title": "Prospective observational research on the clinical profile and outcome analysis among a cohort of patients sustaining traumatic cervical spine and cord injury in a peripheral tertiary spine care centre in Nepal",
"authors": [
"Sunil Munakomi",
"Binod Bhattarai",
"Iype Cherian",
"Binod Bhattarai",
"Iype Cherian"
],
"abstract": "Background: In developing nations like Nepal, spinal cord injury has multispectral consequences for both the patient and their family members. It has the tendency to cripple and handicap the patients, and burn out their caretakers, both physically and mentally. Furthermore, the centralization of health care with only a handful of dedicated rehabilitation centers throughout Nepal further places patients into disarray. This study was carried out as a pilot study to determine the modes of injury, age groups affected, clinical profiles and patterns of injury sustained, as well as the efficacy of managing a subset of patients, who have sustained cervical spine and cord injuries. Methods: This was a prospective cohort study comprising of 163 patients enrolled over a period of three years that were managed in the spine unit of College of Medical Sciences, Bharatpur, Nepal. Results: Road traffic accidents were implicated in 51% of these patients. 65% of them were in the age group of 30-39 years. Traumatic subluxation occurred in 73 patients with maximum involvement of the C4/5 region (28.76%). Good outcome was seen in patients with ASIA ‘C’ and ‘D’ with 55% of patients showed improvement from ‘C’ to ‘D’ and 95% of patients showed improvement from ‘D’ to ‘E’ at 1 year follow up. The overall mortality in the patients undergoing operative interventions was only 1.98%. Conclusions: The prevalence of cervical spine injuries in the outreach area is still significant. The outcome of managing these patients, even in the context of a resource limited setup in a spine unit outside the capital city of a developing nation, can be as equally as effective and efficient compared to the outcome from a well-equipped and dedicated spine unit elsewhere.",
"keywords": [
"Trauma",
"Cervical spine",
"Spinal cord",
"Outcome"
],
"content": "Introduction\n\nSpinal cord injury (SCI) remains one of the most devastating incidents to happen to an individual1. This not only has multispectral negative impacts to the affected individual, but also has an ill effect on the individual’s family members, society and nation as a whole.\n\nThe United Nations has recently implemented the “Decade of Action for Road Safety” with an aim of reducing this problem globally2. In Nepal road traffic accidents area major cause of spine injuries. Therefore, globally there are certain reforms being applied to reduce the incidence of SCI, such as the implementations of regular traffic checkups, laws on the use of seat belts while driving and increasing public awareness through media3.\n\nAccording to a report by the World Health Organization, 82% of the victims with SCI are male, with the majority of them (56%) in the age group of 16–30 years. To make the matter worse, 50–60% of them remain unemployed following the tragedy4. Such injuries have tremendous consequences on the overall resource allocations in many developing nations.\n\nStudies have shown that hospital acquired pneumonia and wound infection propagate disability and mortality in patients with SCI5. Therefore, these complications bear negative impacts on patients’ overall functional outcome and their quality of lives6. Re-admission rates within a year for such patients have been found to be as high as 27.5%7. One cross-sectional study from the US Healthcare System found out that 95.6% of SCI patients had at least one medical complication at the time of their routine annual check-up5.\n\nThere has been a recent suggestion of incorporating multifamily group interventions and active educations to improve the overall outlook of SCI patients8. This approach also helps minimize burn out among the care givers who are encountering a new role. Most often, there is only the manpower available for providing necessary care for sustaining critical support for these patients8.\n\nMost patients with SCI have problems achieving a positive outlook and perceiving a sense of self-efficacy9. Confidence or self-efficacy in managing SCI in many community-living people with SCI is suboptimal10. This means that the caretaking aspect becomes an “unexpected career”, and they have to enter this new role without any preparation or specialized training11. There is also “post-injury shift in relationship dynamics” from family members to that of a care provider12. High levels of caregiver burden adds to physical and emotional stress, burnout, fatigue, anger, resentment and depression among caregivers13,14. Having a community peer support service for individuals with SCI provides psychological and emotional support by a person with a SCI, advice on living with a SCI, practical advice and information, and ongoing support and friendship to the patients and their care-providers as well15.\n\nThere are difficulties in managing patients in Nepal due to certain limitations16. The foremost being the poor financial aspects of our people; Nepal has an annual per-capita health expenditure of just $4017. The next hurdle is that of bureaucracy involved in the custom offices while clearing the ambulances, since the ambulances come from other countries and have to cross international borders. Other hindrances pertain to infrastructure, e.g. road conditions: only 43% of the population has access to all-weather roads, and the inaccessibility of adequate transportation results in delays in providing timely health care18. Logistical (e.g. frequent strikes) and cultural (e.g. public behavior and response to emergency vehicles) problems are also other relevant hurdles.\n\nQualified professionals are often unwilling to work in low-resource settings given the lack of incentives, thereby there is decentralization of manpower and lack of health facilities outside the capital city19. There is only one truly dedicated spine rehabilitation centre in the whole of Nepal, which is situated in the capital city (Kathmandu). The concept of a peer support group is almost not heard off here. Therefore, SCI patients and their care providers often become neglected, and become separated from society.\n\nThis study was carried out to determine the clinical profile of patients presenting with cervical spine and cord injuries at our centre in Nepal (the first centre with a complete armamentarium for managing almost every SCI case scenario outside of the capital city) and also to evaluate the patient’s outcome from the management provided. This study is the first to make a small initial step, thereby motivating others to make a giant leap in decentralizing efficient and effective patient care.\n\n\nMethods\n\nThis was a prospective observational cohort study of all patients with documented traumatic injury to the cervical spine or its cord, presenting to the Spine Unit at the College of Medical Sciences, Chitwan from March 2013 to March 2016.\n\nAll patients who presented to our department, either primarily or following their referral from other centers, and were diagnosed of having traumatic cervical spine and cord injuries, were eligible for inclusion in our study. They were enrolled in our cohort study following obtaining their written consent for participation in the study.\n\nExclusion criteria consisted of any patients with significant poly-trauma or significant medical co-morbidities, those failing to provide written consent for inclusion in the study, patients who left the hospital against medical advice.\n\nImaging of the injury. The immobilization of the neck was first secured. National Emergency X-Radiography Utilization Study (NEXUS) criteria20 and Canadian C-spine Rule21 was utilized as guidelines in forming algorithms to obtaining X-ray images (Figure 1). Further necessary imaging with Computerized Tomography (CT) or Magnetic Resonance Imaging (MRI) of the spine was carried out as and when necessary. CT images helped us in assessing fracture, degree of subluxation and the integrity of the facet joints. MRI images provided us with information on the status of the disc, associated hematomas, degree of compression of the cord, associated cord contusions and the integrity of the posterior ligamentous complex.\n\nPatient assessment. Neurological assessment was first carried out and documented as American Spinal Injury Association (ASIA) grading22. Sensory and motor findings were thoroughly assessed by evaluating single breath count and the presence of Horner’s syndrome was also checked so as to aid in clinical localization. Anal tone and the presence of priapism were also documented. In order to avoid the confounding bias of neurogenic shock, final recordings of the neurological assessment were undertaken 72 hours after the injury, especially in patients with ASIA ‘A’ and ‘B’ grading so as to avoid the confounding bias of spinal shock. In the presence of any deficits, Methylprednisolone was initiated as per the National Acute Spinal Cord Injury Study protocol in all patients presenting within 8 hours of the injury23.\n\nFurther management was undertaken as per the lesions revealed from radio imaging and the clinical assessment of the patients.\n\nTraumatic subluxation. In cases of traumatic subluxation, classification was done per Meyerding grading (Figure 2)24.\n\nIn all cases of Meyerding grade 4 subluxation and spondyloptosis, as well as in cases planned for occipito-cervical fusion and C1 lateral mass screw fixation, CT angiography was also carried out to assess the course of the vertebral artery. Incentive chest spirometric, as well as limb physiotherapy was initiated in all these patients. Guarded traction was applied for reduction in all the patients, with frequent monitoring to prevent over distraction (Figure 3).\n\nIf there was good realignment following traction, the patients were managed by either discectomy or median corpectomy followed by in situ strut iliac bone graft with plate and screw fixations (anterior cervical approach) (Figure 4).\n\nSometimes, in patients with poor financial status, unassisted bone graft placement was also performed followed by hard cervical collar application for at least 6 weeks. In cases where no reduction was possible with traction (locked facets), then the reduction was tried under anesthesia with muscle relaxants. If reduction was possible, only an anterior approach was taken (Figure 5).\n\nHowever, if the reduction was still not possible, then in patients with ASIA ‘A’ and ‘B’ status, only anatomical fixation was ensured by performing the inter-spinous wiring through the posterior approach (Figure 6).\n\nThe main rational was to allow patients with early mobilization in wheelchairs. In patients with ASIA grade of ‘C’, ’D’ and ‘E’, if there was no significant disc seen in the MRI, then posterior was taken first for unlocking the jammed facets. The posterior instrumentation was then carried out by placement of lateral mass screws with occasional usage of trans-laminar screws and inter-spinous wire placement (Figure 7). Sometimes, we had to resort to placement of inter-spinous wiring only. This was followed by discectomy or corpectomy and graft with plate and screw fixation from the anterior approach (Figure 8). However, if there was presence of a significant disc, discectomy or corpectomy was first carried out, then unlocking of the facets with posterior instrumentation was done, followed by placement of the graft with plate and screw fixation from the anterior approach (global approach) (Figure 9 and Figure 10)25.\n\nHangman’s fracture. In cases of Hangman’s fracture in young patients, C1 and C3 lateral mass screw and rod placement was undertaken. However, in older patients, above 65 years, occipito-cervical fusion was carried out (Figure 11)26.\n\nOdontoid fracture. We classified odontoid fractures into three subgroups depending on the displacement of the fractured odontoid segment in relation to the C2 body: anterior displaced, neutral and posterior displaced (Figure 12). Realignment was achieved with careful and judicious dorsal or ventral movement of the neck under anesthesia. We have also designed a surgical technique that helps placement of odontoid screws in resource limited settings; with high accuracy27. We create a longus colli gutter so as to completely expose the body of C3. Following a C2-C3 median discectomy, a median gutter is created in the upper body of C3 (Figure 13).\n\nThis has many benefits. Firstly, it ensures midline projection of the screw, thereby minimizing the use of C-arm or O-arm, which reduces the risk of excessive radiation hazards. Secondly, it ensures adequate banking of the screw in the cortex of C2, thereby minimized screw pullout. The gutter in C3 homes the head of the screw, thereby minimizing the chances of post-operative discomfort in the patient (Figure 14).\n\nWhenever possible, MRI compatible titanium plates and screws were used in the surgery (cost, $700). In poorer patients, we opted for alloy implants (cost, $350), and sometimes even steel implants (cost $80).\n\nRemaining pathologies. In patients with central cord syndrome, instability was ruled out by performing dynamic X-ray of the spine. The neck was immobilized in a hard cervical collar. These patients were also started on citicoline (oral tablet, 500 mg three times daily).\n\nAll the patients with C1 arch fractures, dear drop fractures and C7 spinous process fracture were stable, and therefore managed conservatively.\n\nAll operated patients were aimed for early mobilization in a wheelchair with rigorous chest and limb physiotherapy. The relatives were also taught necessary care protocols28.\n\nAll these patients were advised for follow-ups at 2 weeks, 1 month, 3 months, 6 months and then yearly following discharge. Use of telephone interviews and even video calls using social media were used, in order to inquire as to the current status of the patients.\n\nData gathered about these patients included their clinical profile, ASIA grading, nature and level of their injuries, mode of management, any associated complications and subsequent outcome in their follow up. These data were recorded by residents and were discussed monthly and evaluated by the respective consultants of the Spine Unit. All the patients were prospectively followed up to assess the management undertaken on them and their subsequent outcome in their follow up visits. The records of the patients were stored in the central record store of our hospital and later descriptive analysis was carried out for our study purpose.\n\n\nResults\n\nDuring the study period (March 2013 to March 2016), a total of 163 patients were enrolled and then followed up in our cohort study.\n\nThe age of the patients in our cohort ranged between 2 and 80 years, with 65% of them in the age group 30–39, 19.85% in age group of 40–49, and 17.73% in the age group of 20–29.\n\nOnly 36% of patients had been treated with a cervical collar for neck immobilization at the time of their arrival to the hospital. In addition, only 16% of patients in ASIA ‘D’ or below, presented within 8 hours from the time of injury. One missed, and thereby neglected, case presented 4 years after the injury with a severe swan neck deformity with features of gross myelopathy (Figure 15).\n\nMode of injury. Road traffic incidences were implicated in 51% of the cases, followed by fall related incidents for 41% of cases in this cohort group. Minor remaining cases were related to physical assault, playground injuries, animal attacks, earthquake-related incidents and gas explosions.\n\nTraumatic subluxation was seen in 73 patients, followed by odontoid fractures in 24 patients (Table 1).\n\nA total of 73 patients had traumatic subluxation of cervical spine with maximum involvement in the C4/5 (28.76%) followed by C5/6 (24.65%) region. Most of them had Meyerding Type 1 injury (35.6%) and were in the ASIA ‘D’ neurological status (Table 2).\n\nThere were 24 cases of odontoid fractures in our study (Table 3). Anteriorly displaced variant was seen in 41.66%, neutral type was seen in 41.66% followed by posteriorly displaced variant seen in the rest 16.66% of cases. In patients with central cord syndrome, 60% of them were centered in the C4/5 region, with 58% of these patients presenting in ASIA ‘C’ status.\n\nThere was 1 screw pullout seen in a case with occipito-cervical fixation in Hangman’s fracture (Figure 16). The implant was removed after ensuring good fusion at the fracture site. A graft extrusion occurred in a case that underwent an unassisted graft owing to financial restrain. It was managed by replacement of the graft with support from simple steel plate and screws (Figure 17). One patient had post-operative hematoma in the surgical site requiring its evacuation. Two patients developed superficial surgical site infections, which were both managed conservatively.\n\nTwo patients had trachea-esophageal fistula. One patient was managed conservatively with Ryle’s tube insertion and was healed after a month (Figure 18). The other patient died of severe mediastinitis despite multiple attempts to repair it.\n\nOne patient undergoing odontoid screw fixation had the wrong lateral projection of the screw requiring reinsertion. Another patient with odontoid fracture died following inferior wall myocardial infarction in the fourth post-operative day.\n\nIn total, 2 out of 13 patients in ASIA ‘A’ showed improvement to ASIA ‘B’ at 6 months; none of the patients showed improvement from ‘B’ to ‘C’ at 6 months; 55% of the patients showed improvement from ‘C’ to ‘D’ at 1 year; 95% of patients showed improvement from ‘D’ to ‘E’ at 1 year. Only 45% of patients in ASIA ‘A’ and ‘B’ were able to be followed beyond 6 months; 100% of them had developed pressure sores.\n\n\nDiscussion\n\nThe cervical spine remains the most common level for SCI, representing 55% of all SCIs29. People in the low- and middle-income countries experience 80% of fall related mortality worldwide30.\n\nWithout appropriate preventive action road traffic accidents (RTA) are predicted to be the third leading contributor to the global burden of disease and injury by 202031. Studies have shown that falls and land transport account for more than 75% of traumatic SCI cases, with almost 30% of them resulting in tetraplegia32.\n\nA prospective observational study conducted in a Tertiary Hospital in India found that RTA caused 62.5% cases, with 21.8% sustaining a C5 level injury33. Another observational study based on autopsy, death due to cervical spinal cord injury, found that men made up 89.4% cases, and young adults (20–39 years) were 63.8% cases. C3-C4 (37.3%) was most commonly involved with 56.6% of the victims dying even before reaching nearby hospitals. The mode of injury was RTA (52.2%) followed by fall from a height (25.0%)34. In our study, 65% of the patients were in the age group of 30–39 years; RTA was the most common cause of injury (51%) followed by fall injury in 41%.\n\nThere have been very few studies carried out on traumatic spinal cord injuries in Nepal35,36. One of these studies reported 149 injuries in the cervical region over a period of three years35. The most commonly involved age group was between 30 and 49 years (44%), with a male to female ratio of 4:1. Fall-related injury was the commonest mode of injury (60%). In addition, 81% of these patients were transported without any neck protection, and the C5 vertebra was the most commonly injured vertebra. In our study, 36% of patients had their neck immobilized with hard collar application. In our study, C4/5 was involved in 28.76% of cases followed by C5/6 in 24.76% of all cases with traumatic subluxation (44.78% of all cases). The same previous study found mostly men were injured with an average age at 40 years, with almost 58% being the sole bread earner being involved in the injury. Another study found that patients presented late for clinical treatment (mean time of almost 40 hours) after the injury37. Only 16% of the patients in our study presented within 8 hours of injury as well. This may be due to us being one of the referral centers for spinal injuries, thereby mostly those selective cases requiring operative interventions were only transferred to us. The remaining cases requiring conservative management would have been managed in other centers as well. This was also true with regards to the ASIA status of the patients presenting to us. Only a few patients with ASIA ‘A’ presented to us, as most of these patients and their relatives were already counseled of the poor prognosis in other centers beforehand and therefore they were not interested in carrying out further treatment. Only patients having some preserved neurological status either in terms of sensory or motor modalities were more likely to seek further expert opinion, and thereby more likely to present to our care center.\n\nAnother study from Nepal found 80% of wheelchair users not able to enter their homes independently and 74% of those using mobility aids having maximal difficulties due to physical terrain. 50% of them had no income, and almost half of them did not have easy access to toilet, water source or roads to their home38.\n\nPrevious studies have also found that patients with incomplete cervical injuries (Grades C and D) or with edema in MR studies had a better clinical improvement39. This may be the reason for improvement in 80% of the cases with central cord syndrome presenting to our unit, by at least one ASIA score.\n\nIn one large series of such patients, surgical mortality was 2.3%, and neurological long-term results were good, with 51% improvement in AIS grade40. The surgical mortality in our cohort study was 1.98%. In our study, 2.5% of patients in ASIA ‘A’, 7.5% of patients in ASIA ‘B’, 55% of patients in ASIA ‘C’ and 95% of patients in ASIA ‘D’ showed neurological improvement.\n\nThere are certain fields that need to be monitored in our quest to minimize RTA. The provision for legislation, strict adherence to seat belt use and awareness of safe traffic behaviors can be the stepping stone in this regard41,42. A study by Dandona et al. also noted that enforcing traffic laws, strengthening the driving licensing system, and providing periodic conditioning of vehicles minimized RTA43.\n\nIn total, around 16,600 deaths are due to fall-related incidents in Nepal annually44. Awareness among ambulance drivers or even lay persons about the need of immobilization of the neck and back during transportation of patients can prevent many secondary catastrophes. There is now an utmost need to decentralize manpower, and equip centers, as well as providing dedicated spinal rehabilitation centers outside the capital city45.\n\n\nConclusion\n\nIn our setting, spinal cord injury has multispectral negative impacts from the patient to society as a whole. The facility for pre-hospital care is not even in its infancy. Furthermore, poor patient transport, e.g. difficult roads, due to the centralization of manpower and treatment centers puts these medical hazards into further disarray. However, small steps in managing patients may prove to be a giant leap in our attempt to provide healthcare management to patients with spinal cord injuries with equally effective therapeutic benefits even in a peripheral set up.\n\n\nEthical statement\n\nThis study was cleared by the Ethical Review Board at the College of Medical Sciences, Nepal. Written informed consent was obtained from all patients (sometimes their caregivers provided consent, when patients were not able to do so), regarding their participation in the study and the publication of their relevant clinical data and clinical images.\n\n\nData availability\n\nDataset 1: Spreadsheet containing the data underlying the results for all 163 patients. doi, 10.5256/f1000research.12911.d18265346",
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PubMed Abstract | Publisher Full Text | Free Full Text\n\nMunakomi S, Tamrakar K, Chaudhary PK, et al.: Anterior single odontoid screw placement for type II odontoid fractures: our modified surgical technique and initial results in a cohort study of 15 patients [version 2; referees: 2 approved]. F1000Res. 2016; 5: 1681. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRodger S: Care of spinal cord injury in non-specialist settings. Nurs Times. 2016; 112(26): 12–15. PubMed Abstract\n\nSekhon LH, Fehlings MG: Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine (Phila Pa 1976). 2001; 26(24 Suppl): S2–12. PubMed Abstract | Publisher Full Text\n\nGupta S, Gupta SK, Devkota S, et al.: Fall Injuries in Nepal: A Countrywide Population-based Survey. Ann Glob Health. 2015; 81(4): 487–94. PubMed Abstract | Publisher Full Text\n\nMurray CJ, Lopez AD: The Global burden of disease: A comprehensive assessment of mortality and disability from diseases, injuries and risk factors in 1990 and projected to 2020. Cambridge, MA: Harvard School of Public Health on behalf of the World Health Organization and the World Bank. 1996; 52. : 247–293. Reference Source\n\nLee BB, Cripps RA, Fitzharris M, et al.: The global map for traumatic spinal cord injury epidemiology: update 2011, global incidence rate. Spinal Cord. 2014; 52(2): 110–116. PubMed Abstract | Publisher Full Text\n\nWills SJ, Pandian GR, Bhanu TK, et al.: Clinical profile of patients with traumatic cervical spine injury in the emergency department of a tertiary care hospital. J Emerg Trauma Shock. 2016; 9(1): 43–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDas S, Datta PP, Das M, et al.: Epidemiology of cervical spinal cord injury in eastern India: An autopsy-based study. N Z Med J. 2013; 126(1377): 30–40. PubMed Abstract\n\nShrestha D, Garg M, Singh GK, et al.: Cervical spine injuries in a teaching hospital of eastern region of Nepal: A clinico-epidemiological study. JNMA J Nepal Med Assoc. 2007; 46(167): 107–11. PubMed Abstract\n\nLakhey S, Jha N, Shrestha BP, et al.: Aetioepidemiological profile of spinal injury patients in Eastern Nepal. Trop Doct. 2005; 35(4): 231–3. PubMed Abstract | Publisher Full Text\n\nBajracharya S, Singh M, Singh GK, et al.: Clinico-epidemiological study of spinal injuries in a predominantly rural population of eastern Nepal: a 10 years’ analysis. Indian J Orthop. 2007; 41(4): 286–289. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScovil CY, Ranabhat MK, Craighead IB, et al.: Follow-up study of spinal cord injured patients after discharge from inpatient rehabilitation in Nepal in 2007. Spinal Cord. 2012; 50(3): 232–7. PubMed Abstract | Publisher Full Text\n\nSrinivas BH, Rajesh A, Purohit AK: Factors affecting outcome of acute cervical spine injury: A prospective study. Asian J Neurosurg. 2017; 12(3): 416–423. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFredø HL, Rizvi SA, Rezai M, et al.: Complications and long-term outcomes after open surgery for traumatic subaxial cervical spine fractures: a consecutive series of 303 patients. BMC Surgery. 2016; 16(1): 56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nToroyan T, Peden M: Youth and road safety. Geneva: World Health Organization, 2007. Reference Source\n\nSharma R, Grover VL, Chaturvedi S: Health-risk behaviors related to road safety among adolescent students. Indian J Med Sci. 2007; 61(12): 656–662. PubMed Abstract | Publisher Full Text\n\nDandona R, Kumar GA, Dandndona L: Risky behavior of drivers of motorized two wheeled vehicles in India. J Safety Res. 2006; 37(2): 149–158. PubMed Abstract | Publisher Full Text\n\nGupta S, Gupta SK, Devkota S, et al.: Fall Injuries in Nepal: A Countrywide Population-based Survey. Ann Glob Health. 2015; 81(4): 487–94. PubMed Abstract | Publisher Full Text\n\nShrestha D: Traumatic spinal cord injury in Nepal. Kathmandu Univ Med J (KUMJ). 2014; 12(47): 161–2. PubMed Abstract | Publisher Full Text\n\nMunakomi S, Bhattarai B, Cherian I: Dataset 1 in: Prospective observational research on the clinical profile and outcome analysis among a cohort of patients sustaining traumatic cervical spine and cord injury in a peripheral tertiary spine care centre in Nepal. F1000Research. 2017. Data Source"
}
|
[
{
"id": "27617",
"date": "13 Nov 2017",
"name": "Juha Hernesniemi",
"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 important study. I recommend it to be published. My only remarks are the use of percentages: I would use only numbers like 54 per cent/ not 54.54 per cent.\nOtherwise it is ready to be accepted and published!\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "27619",
"date": "23 Nov 2017",
"name": "Nikolay Peev",
"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 contributes for the understanding of the problem in a tertiary centre in Nepal. The cervical spinal cord injury is a condition associated with significant mobility and severe invalidity. In that respect a proper understanding and management of the patients suffering with spinal cord injury is of utmost importance.\n\nI recommend publishing\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/6-1957
|
https://f1000research.com/articles/6-1953/v1
|
03 Nov 17
|
{
"type": "Research Article",
"title": "Dysglycemic states and hypertension: A relationship dependent on low-grade inflammation",
"authors": [
"Juan Salazar",
"Valmore Bermúdez",
"Wheeler Torres",
"Víctor Arias",
"María Sofía Martínez",
"Mervin Chavez-Castillo",
"Sandra Wilches-Durán",
"Modesto Graterol-Rivas",
"Nelson Villasmil",
"Carla Navarro",
"Rosemily Graterol-Silva",
"Rosanna D´Addosio",
"Kyle Hoedebecke",
"Manuel Velasco",
"Joselyn Rojas",
"Juan Salazar",
"Wheeler Torres",
"Víctor Arias",
"María Sofía Martínez",
"Mervin Chavez-Castillo",
"Sandra Wilches-Durán",
"Modesto Graterol-Rivas",
"Nelson Villasmil",
"Carla Navarro",
"Rosemily Graterol-Silva",
"Rosanna D´Addosio",
"Manuel Velasco",
"Joselyn Rojas"
],
"abstract": "Background: Hypertension (HTN) is a prominent cardiovascular risk factor, affecting over 1 billion people worldwide. Identification of closely associated cardiometabolic conditions may be crucial for its early detection. The objective of this study was to identify factors associated with HTN and prehypertension (PHT) in an adult population sample from Maracaibo City, Venezuela. Methods: A randomized multi-staged sampling cross-sectional study was performed in 2230 individuals from Maracaibo City Metabolic Syndrome Prevalence Study database. PHT and HTN were defined according to JNC-7 criteria. Multiple logistic regression analysis was used to assess the main risk factors for each condition. Results: 52.6% (n=1172) of the subjects were female, the prevalence of HTN was 32% (n=714), while the prevalence of PHT was 31.1% (n=693). The main risk factors for HTN were age ≥60 years (odds ratio [OR]: 40.99; 95%CI: 16.94-99.19; p<0.001) and the local indigenous ethnic group (OR: 3.06; 95%CI: 1.09-8.62; p=0.03). Adjustment for high sensitivity C-reactive protein levels increased the OR of these factors and diminished the impact of other factors. Meanwhile, age ≥60 years (OR: 3.39; 95%CI: 1.41-8.18; p=0.007) and alcohol consumption (OR: 1.49; 95%CI: 1.06-2.00; p=0.02) were the main risk factors for PHT. Conclusion: There are significant differences in the risk factor profiles for HTN and PHT. Additionally, low-grade inflammation appears to link multiple metabolic factors and preexisting vascular characteristics.",
"keywords": [
"hypertension",
"prehypertension",
"risk factors",
"inflammation",
"C-reactive protein",
"Hispanic"
],
"content": "Introduction\n\nHypertension (HTN) is the leading risk factor contributing to mortality worldwide, associated with approximately 9.4 million deaths every year1. In addition, it is the main modifiable risk factor for the development of cardiovascular disease (CVD)2, which causes the greatest numbers of death globally, with 17.5 million deaths in 20123 and projections of up to 23.3 million deaths annually by 20304. Given its complex etiology, HTN has been classified as primary when appearing without any identifiable cause (90% of all cases) and secondary HTN when resulting as a direct consequence of another known preexisting condition, such as cardiovascular or kidney disease5.\n\nResearch on primary HTN has focused on identifying different risk factors, which could help define its specific etiology6. Various genetic and environmental conditions have been categorized as modifiable and non-modifiable risk factors: Family history of HTN is considered to be one of the main non-modifiable risk factors7, along with age, sex and race8. On the other hand, high sodium intake, hypercaloric diets, smoking, alcohol consumption, obesity, and a sedentary lifestyle all represent modifiable risk factors9,10.\n\nLikewise, low-grade inflammation has been suggested to play a great role in the pathophysiology of HTN, due to the stimulation of the proliferation of endothelial vascular smooth muscle cells in addition to vascular remodeling and endothelial dysfunction11. Several epidemiological studies have reported high levels of inflammatory markers, such as C-reactive protein (CRP) and proinflammatory cytokines in patients with HTN. Furthermore, these findings correlate with a higher risk of developing HTN, as well as target organ damage in healthy individuals12.\n\nConsidering the high prevalence of HTN in the Venezuelan population13, it becomes imperative to identify the conditions associated with both HTN and prehypertension (PHT) in order to aid in early diagnosis. The objective of this study was to determine the main associated risk factors for HTN and PHT in adult patients from Maracaibo City, Venezuela.\n\n\nMethods\n\nThis study was approved by the Bioethics Committee of the Endocrine and Metabolic Research Center – University of Zulia (approval number: BEC-006-0305). All participants enrolled in the study signed a written consent before any questioning, intervention, or physical examination. This ethical approval includes all future studies using this data set.\n\nThis report is part of the Maracaibo City Metabolic Syndrome Prevalence Study14, a cross-sectional study whose purpose is to identify metabolic syndrome and cardiovascular risk factors in the adult population of Maracaibo, the second largest city of Venezuela. The study sample was calculated based on city population estimates by the National Institute of Statistics: (1,428,043 inhabitants for the year 2007), amounting to 1,986 individuals. An additional 244 subjects (12%) were included for oversampling in order to increase accuracy of the estimates obtained from smaller subgroups within the overall sample, totaling 2,230 individuals14.\n\nMaracaibo City is divided into parishes, where each parish was proportionally sampled through a multi-staged cluster selection method. In the first stage, clusters were represented by sectors from each of the 18 parishes with proportional representation. Four sectors were selected from each parish by simple randomized sampling. In the second phase, the clusters were represented by city blocks within each sector, which were selected by simple randomized sampling using a random number generator. From the overall population, 2,026 individuals were selected on the basis of availability of insulin determination. Further details of the sampling process have been previously published14.\n\nA full medical history and blood samples for biochemical evaluation were obtained from each participant by trained personnel. A full physical examination was also performed. Collected data included age, gender, ethnic group, socioeconomic status (according to the method of Graffar modified by Mendez Castellano15), smoking habit and family history of HTN. High risk alcohol intake was defined as those consuming ≥1 gram of alcohol daily.\n\nPhysical activity (PA) was measured using the International PA Questionnaire-Long Form (IPAQ-LF)16. Each domain of the questionnaire was clustered in quintiles according to the calculated metabolic equivalents (METs) and classified according to the PA pattern in one of six categories: very low, low, moderate, high, and very high in addition to a category for inactive individuals or those whose METs equaled zero.\n\nIn addition, an anthropometric evaluation was performed: weight was determined using a digital scale (Tanita, TBF-310 GS Body Composition Analyzer, Tokyo-Japan) while height was measured with a previously calibrated stadiometer on a flat surface. Body Mass Index (BMI) was calculated with the Quetelec’s formula [Weight/(Height)2] and individuals were categorized based on the World Health Organization (WHO) classification17. Waist circumference was measured with a plastic measuring tape graduated in centimeters and millimeters, taking an equidistant point between the costal margin and the anterior superior iliac spine as an anatomical reference, according to the protocol proposed by the US National Health Institute (NHI)18.\n\nBlood pressure (BP) was taken through auscultation with subjects sitting down and feet on the floor following 15 minutes of rest, determined with a calibrated mercury sphygmomanometer. Korotkoff’s phases I and V were identified as systolic and diastolic BP, respectively. BP was determined 3 times with 15 minutes intervals between each registration on two different days. BP was categorized according to the Seventh Joint National Committee on Prevention, Detection, Evaluation and Treatment of Hypertension (JNC-7) criteria19. PHT is classified as systolic BP between 120 mmHg and 139 mmHg or diastolic BP between 80 mmHg to 89 mmHg. HTN is classified as systolic BP greater than 140 mmHg or diastolic BP over 90 mmHg.\n\nOvernight fasting determination of glucose, total cholesterol, triacylglycerides (TAG), and HDL-C was performed with an automated analyzer (Human Gesellschaft für Biochemica und Diagnostica mbH, Germany); the intra-assay variation coefficients for total cholesterol, TAG, and HDL-C were 3%, 5%, and 5%, respectively. Serum hs-CRP was quantified through immunoturbidimetric assays (Human Gesellschaft für Biochemica und Diagnostica mbH, Germany). Insulin was quantified using ultrasensitive ELISA double-sandwich methodology (DRG Instruments GmbH, Germany, Inc.).\n\nHOMA2-IR was utilized for the evaluation of insulin resistance (IR) as proposed by Levy et al.20 computed with the HOMA-Calculator v2.2.2 software application with IR defined as HOMA2-IR ≥221. Elevated hs-CRP was defined as levels ≥0.765 mg/L22; hypertriacylglyceridemia was defined as fasting TAG ≥150 mg/dL; and low HDL-C as fasting HDL-C <50 mg/dL in females or <40 mg/dL in males23. Regarding glycemic state, subjects were classified as: a) Normoglycemic, individuals with fasting glucose <100 mg/dL; b) Impaired Fasting Glucose (IFG), those with fasting glycemia between 100–126 mg/dL; and c) Type 2 diabetes mellitus (DM2), those with fasting glycemia >126 mg/dL or a previously established diagnosis24. Likewise, waist circumference cutoff points ≥90 cm (females) and ≥95 cm (males) for the definition of abdominal obesity were used25. We followed the methods similar to previous analyses with other risk factors26.\n\nVariables with non-normal distribution underwent logarithmic transformation, showing a normal distribution after the Geary test, qualitative variables were expressed in absolute and relative frequencies, evaluating association through the χ2 test. Multiple logistic regression analysis was used to study the risk factors associated with PHT and HTN. Two models were devised for the latter, where the first model was adjusted by sex, age groups, ethnic groups, family history of hypertension, smoking, alcohol consumption, household physical activity, leisure time physical activity, hypertriglyceridemia, low HDL-C, glycemic status, BMI categories and elevated waist circumference. The second model was adjusted for all these factors and high serum hs-CRP levels. The multiple logistic regression model for PHT was adjusted for the same factors as the second model. Results were shown in Odds Ratios (95%CI) being statistically significant when p<0.05. Data analysis was performed using the Statistical Software SPSS v.19 for Windows (IBM inc. Chicago, IL).\n\n\nResults\n\nNormotensive individuals comprised 36.9% of the sample (n=823). The prevalence of PHT was 31.1% (n=693), while 32% (n=714) of the subjects were hypertensive. Therefore, a combined total of 63.1% of the sample showed altered BP levels (Figure 1).\n\n2016.\n\nTable 1 and Table 2 show the distribution of subjects according to the presence of PHT and HTN. Age (χ2=438.82; p<0.0001) and home PA (χ2=39.53 p<0.001) were the main sociodemographic and psychobiologic factors associated with these conditions, respectively; whereas BMI was the main clinical-metabolic factor associated with HTN and PHT in the univariate analysis (χ2=264.36; p<0.0001).\n\nMaracaibo, Venezuela. 2016.\n\nHTN= Hypertension\n\n¥ Drinker defined as intake ≥1 gram of alcohol per day.\n\nMaracaibo, Venezuela. 2016.\n\nBMI= Body Mass Index; DM2= Type 2 Diabete Mellitus; hs-CRP=high sensitivity C-Reactive Protein\n\nIn the first model (Table 3), the main risk factors for HTN were age ≥60 years (OR=33.79; 95%CI: 17.15-66.61; p<0.01), and the diagnosis of DM2 (OR=2.92; 95%CI: 1.33-6.41; p<0.01). After adjustment for elevated hs-CRP levels in the second model, the impact of age ≥60 years (OR=40.99; 95%CI: 16.94-99.19; p<0.01) and family history of HTN (OR=2.70; 95%CI: 1.77-4.13; p<0.01) increased; whereas characteristics such as the indigenous ethnic group (OR=3.06; 95%CI: 1.09-8.62; p=0.03) and IR (OR=1.50; 95%CI: 1.00-2.25; p=0.05) became significant risk factors, while conditions like hypertriacylglyceridemia, IFG, DM2 lost significance.\n\n2016.\n\na Confidence Interval (95%); b Significance level; c Triglycerides ≥150 mg/dL; d HOMA2-IR≥2\n\n* Model 1: Adjusted for sex, age groups, ethnic groups, socioeconomic status, family history of hypertension, smoking habit, alcohol intake, home physical activity per the IPAQ-LF, leisure physical activity per the IPAQ-LF, low HDL-C, hypertriacylglyceridemia, glycemic status, BMI categories, insulin resistance, elevated waist circumference (≥95cm for males; ≥90cm for females).\n\n** Model 2: Adjusted for sex, age groups, ethnic groups, socioeconomic status, family history of hypertension, smoking habit, alcohol intake, home physical activity per the IPAQ-LF, leisure physical activity per the IPAQ-LF, low HDL-C, hypertriacylglyceridemia, glycemic status, BMI categories, insulin resistance, elevated waist circumference (≥95cm for males; ≥90cm for females); AND elevated hs-CRP levels.\n\nTable 4 depicts the multivariate analysis for factors associated with PHT. The main risk factors were age ≥60 years (OR=3.39; 95%CI: 1.41-8.18; p<0.01) and age 30-59 years (OR=1.85; 95%CI: 1.29-2.65; p<0.01); as well as alcohol intake ≥1 g/day (OR=1.49; 95%CI: 1.06-2.10; p=0.02) However, moderate PA demonstrated a protective effect (OR=0.62; 95%CI: 0.38-1.01; p=0.05).\n\n2016.\n\na Confidence Interval (95%); b Significance level; c Triglycerides ≥150 mg/dL; d HOMA2-IR≥2\n\nModel adjusted for sex, age groups, ethnic groups, socioeconomic status, family history of hypertension, smoking habit, alcohol intake, home physical activity per the IPAQ-LF, leisure physical activity per the IPAQ-LF, low HDL-C, hypertriacylglyceridemia, glycemic status, BMI categories, insulin resistance, elevated waist circumference (≥95cm for males; ≥90cm for females); AND elevated hs-CRP levels.\n\n\nDiscussion\n\nHTN represents the main modifiable risk factor for the development of CVD2, which is why it is alarming that only 4 of 10 individuals in this sample exhibit normal BP levels. According to the WHO, the prevalence of HTN in the Americas is 35%27, similar to those in our study at 32%. In a cross-sectional study by Wang and Wang28, based on the data obtained in the 1999–2000 National Health and Nutrition Examination Survey (NHANES) and using JNC-7 classification, the prevalence of HTN was slightly lower than ours (27.1%), while also reporting a similar prevalence for PHT (31%). Following the same methodology, Erem et al.29 assessed a population of 4809 individuals in Turkey and reported results markedly differing from ours, with a prevalence of 44% for HTN and 14.5% for PHT. Lastly, another study by Do et al.30 on a population of 17,199 Vietnamese individuals described a vastly different picture, where the prevalence of HTN was lower than PHT (20.7% vs 41.8%), which illustrates the heterogeneous distribution of this disease around the world.\n\nIn regards to the non-modifiable risk factors for HTN, our multivariate models showed that men had a higher risk of developing this disease, similar to several studies in Turkey29, China31 and Guatemala32. However, age appears to be the main risk factor in our sample. In this context, a cross-sectional study by Awoke et al.33 on 679 adult individuals aged ≥35 years ascertained greater HTN risk for individuals aged ≥55 years in comparison to those aged 35–44 years. Similarly, Katulanda et al.34 and Mc Donald Posso et al.35 found higher risk in individuals aged ≥70 years and ≥60 years, respectively. This association could be explained by the development of atherosclerosis with age36. Family history of HTN was also a significant risk factor in our study. Other studies around the world, such as the one published by Ranasinghe et al.37, have reported a higher risk in adult individuals with family history of HTN, similarly to Awoke et al.33; these findings support the relevance of the genetic component in the pathogenesis of HTN7.\n\nAmong the modifiable risk factors assessed, both hypertriacylglyceridemia and dysglycemic states showed a close link with HTN, in harmony with previous reports38. Nevertheless, it should be noted that both of these factors lost significance in the multivariate models after adjusting for hs-CRP, underlining the dependent relationship between low-grade inflammation and HTN in regards to alterations of carbohydrate and lipid metabolism. This link was ascertained notwithstanding adjustment for IR, the hallmark etiopathogenic mechanism found in the metabolic syndrome39.\n\nConcerning obesity, BMI-classified obese and overweight individuals. as well as those with increased waist circumference. showed a higher risk for HTN even after adjustment for hs-CRP. Similarly, Guwatudde et al.40 studied 3906 individuals in sub-Saharan Africa and reported BMI as the only modifiable risk factor for HTN with a higher risk for overweight and obese individuals. Indeed, the predictive value of waist circumference as a risk factor for HTN tends to vary in a population-specific fashion. In a Croatian cohort of 9070 subjects, Ivičević Uhernik et al.41 ascertained greater HTN risk in individuals with waist circumference ≥94 cm for males, and ≥88 cm for females. These results coincide with previous investigations that show the propensity of HTN to present alongside other cardiometabolic disorders42,43. In this regard, IR has been suggested to play a paramount role as the common denominator connecting the pathophysiology of these conditions44, promoting vascular alterations independently of the impact of concurrent low-grade inflammation, including increased expression of vascular cell adherence molecules, oxidative stress, and a decrease in nitric oxide availability45. On the other hand, an increase in adiposity has also been associated with higher angiotensin II and aldosterone levels46,47, postulating obesity as a state of inappropriate activation of the renin angiotensin aldosterone system; thus, participating in conjunction with IR in the pathogenesis of HTN and other associated cardiometabolic diseases46.\n\nThe link between immunity, inflammation, and the development of HTN has been a subject of intense research in recent decades12. Increased levels of inflammatory biomarkers, such hs-CRP, have been independently associated with higher cardiovascular risk48. In this study, after adjusting for hs-CRP levels and several other variables, indigenous populations were shown to be at a higher risk for HTN in contrast with the data analyzed by NHANES 2011–201249. Afro-Venezuelan individuals had the highest prevalence for HTN when compared to non-Hispanic white individuals, Hispanic, and Asian groups. This may be explained by the fact that this ethnic group often suffer from lower socioeconomic status in addition to chronic stress, worse healthcare, or discrimination50.\n\nIR also behaved as a risk factor after adjusting for hs-CRP, comparable to results of El Bcheraoui et al.51, reported after studying 10,735 individuals aged ≥15 years where subjects with DM2 had a higher risk for HTN. The risk in prediabetes, however, was not significant. Similarly, a study by Sung et al.52 in 8347 individuals showed that increased hs-CRP levels were an independent risk factor for the development of HTN, analogous to our findings. CRP is involved in the innate immune response with diverse functions that include stimulating monocytes for the release of pro-inflammatory cytokines, such as interleukin(IL)-6, IL-1β and tumor necrosis factor alpha, as well as the expression of intercellular adhesion molecules (ICAM-1) and vascular cell adherence molecules53. These effects underlie the link between inflammation and the development of HTN.\n\nAge, alcohol consumption and low HDL-C were identified as risk factors for PHT in this study, echoing the findings by Wu et al.54 where individuals with PHT were older and had low HDL-C. Furthermore, Singh et al.55 reported a higher risk for PHT with older age in both males and females, while the risk associated with alcohol consumption was only significant for men. On the other hand, PA at home was determined to be a protective factor for PHT according to our model, in contrast to the higher risk reported for sedentary individuals56. Poor lifestyle habits have been previously highlighted as the main determinants of PHT19, possibly able to modify early pathophysiologic alterations found in these subjects, such as high levels of IL-1757.\n\nOur findings reflect the importance of focusing efforts towards decreasing the presence of various risk factors in the adult population of Maracaibo City, particularly considering the high prevalence of PHT and HTN and the differences in risk profiles for each state. The key modulating role of low-grade inflammation was also prominent in our study.\n\nThe limitations of this study include its cross-sectional design, which prevents the assessment of causality. However, the data analysis is comparable to current literature and provides important information about non-communicable diseases in Latin American communities - allowing for the design and implementation of primary care programs to detect individuals at risk of PHT and HTN in this context.\n\n\nData availability\n\nDataset 1: Raw data for the 2231 patient sample used in this study. Doi, 10.5256/f1000research.12531.d18235558",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by research grant N° CC-0437-10-21-09-10 from the Technological, Humanistic, and Scientific Development Council (CONDES), University of Zulia, and research grant N° FZ-0058-2007 from Fundacite-Zulia.\n\n\nReferences\n\nLim SS, Vos T, Flaxman A, et al.: A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012; 380(9859): 2224–2260. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLewington S, Clarke R, Qizilbash N, et al.: Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies. Lancet. 2002; 360(9349): 1903–1913. PubMed Abstract | Publisher Full Text\n\nWHO.int: OMS | Enfermedades cardiovasculares [Internet]. 2015, [cited 2 December 2015]. Reference Source\n\nMathers CD, Loncar D: Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med. 2006; 3(11): e442. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMesserli FH, Williams B, Ritz E: Essential hypertension. Lancet. 2007; 370(9587): 591–603. PubMed Abstract | Publisher Full Text\n\nCarretero OA, Oparil S: Essential hypertension. Part I: definition and etiology. Circulation. 2000; 101(3): 329–335. PubMed Abstract | Publisher Full Text\n\nBarlassina C, Lanzani C, Manunta P, et al.: Genetics of essential hypertension: from families to genes. J Am Soc Nephrol. 2002; 13(Suppl 3): 155S–164. PubMed Abstract | Publisher Full Text\n\nSimsolo RB, Romo MM, Rabinovich L, et al.: Family history of essential hypertension versus obesity as risk factors for hypertension in adolescents. Am J Hypertens. 1999; 12(3): 260–263. PubMed Abstract | Publisher Full Text\n\nIbekwe R: Modifiable Risk factors of Hypertension and Socio-demographic Profile in Oghara, Delta State; Prevalence and Correlates. Ann Med Health Sci Res. 2015; 5(1): 71–7. PubMed Abstract | Free Full Text\n\nDevi P, Rao M, Sigamani A, et al.: Prevalence, risk factors and awareness of hypertension in India: a systematic review. J Hum Hypertens. 2013; 27(5): 281–7. PubMed Abstract | Publisher Full Text\n\nTsounis D, Bouras G, Giannopoulos G, et al.: Inflammation markers in essential hypertension. Med Chem. 2014; 10(7): 672–681. PubMed Abstract | Publisher Full Text\n\nHarrison DG, Guzik TJ, Lob HE, et al.: Inflammation, immunity, and hypertension. Hypertension. 2011; 57(2): 132–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBermudez V, Rojas J, Añez R, et al.: Prevalence, awareness, management of hypertension and association with metabolic abnormalities: the Maracaibo city metabolic syndrome prevalence study. Revista Latinoamericana de Hipertensión. 2012; 7(4): 71–79. Reference Source\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\nMéndez Castellano H, de Méndez MC: Estratificación social y biología humana: método Graffar modificado. Arch Ven Pueric Pediatr. 1986; 49: 93–104. Reference Source\n\nInternational Physical Activity Questionnaire [Internet]. 2015, [cited 5 December 2015]. Reference Source\n\nWorld Health Organization: Obesity: preventing and managing the global epidemic. Report of a WHO Consultation on Obesity. Geneva: The Organization; (WHO Technical Report Series, No. 894). 2000. Reference Source\n\nCDC.gov: NHANES - NHANES III - Reports and Reference Manuals [Internet]. 2015. Reference Source\n\nChobanian AV, Bakris GL, Black HR, et al.: The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure: the JNC 7 report. JAMA. 2003; 289(19): 2560–2572. PubMed Abstract | Publisher Full Text\n\nLevy JC, Matthews DR, Hermans MP: Correct homeostasis model assessment (HOMA) evaluation uses the computer program. Diabetes Care. 1998; 21(12): 2191–92. 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\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\nAlberti KG, Eckel RH, Grundy SM, et al.: Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation. 2009; 120(16): 1640–1645. PubMed Abstract | Publisher Full Text\n\nChamberlain JJ, Rhinehart AS, Shaefer CF Jr, et al.: Diagnosis and Management of Diabetes: Synopsis of the 2016 American Diabetes Association Standards of Medical Care in Diabetes. Ann Intern Med. 2016; 164(8): 542–52. PubMed Abstract | Publisher Full Text\n\nBermudez V, Rojas J, Salazar J, et al.: Optimal Waist Circumference Cut-Off Point for Multiple Risk Factor Aggregation: Results from the Maracaibo City Metabolic Syndrome Prevalence Study. Epidemiol Res Int. 2014; 2014: 718571. Publisher Full Text\n\nBermúdez V, Salazar J, González R, et al.: Prevalence and Risk Factors associated with Impaired Fasting Glucose in Adults from Maracaibo City, Venezuela. J Diabetes Metab. 2016; 7: 683. Publisher Full Text\n\nWHO.int: WHO | Raised blood pressure [Internet]. 2015. Reference Source\n\nWang Y, Wang QJ: The prevalence of prehypertension and hypertension among US adults according to the new joint national committee guidelines: new challenges of the old problem. Arch Intern Med. 2004; 164(19): 2126–34. PubMed Abstract | Publisher Full Text\n\nErem C, Hacihasanoglu A, Kocak M, et al.: Prevalence of prehypertension and hypertension and associated risk factors among Turkish adults: Trabzon Hypertension Study. J Public Health (Oxf). 2009; 31(1): 47–58. PubMed Abstract | Publisher Full Text\n\nDo HT, Geleijnse JM, Le MB, et al.: National prevalence and associated risk factors of hypertension and prehypertension among Vietnamese adults. Am J Hypertens. 2014; 28(1): 89–97. PubMed Abstract | Publisher Full Text\n\nWei Q, Sun J, Huang J, et al.: Prevalence of hypertension and associated risk factors in Dehui City of Jilin Province in China. J Hum Hypertens. 2015; 29(1): 64–68. PubMed Abstract | Publisher Full Text\n\nOrellana-Barrios MA, Nuggent KM, Sanchez-Barrientos H, et al.: Prevalence of hypertension and associated anthropometric risk factors in indigenous adults of Guatemala. J Prim Care Community Health. 2015; 6(1): 16–20. PubMed Abstract | Publisher Full Text\n\nAwoke A, Awoke T, Alemu S, et al.: Prevalence and associated factors of hypertension among adults in Gondar, Northwest Ethiopia: a community based cross-sectional study. BMC Cardiovasc Disord. 2012; 12(1): 113. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatulanda P, Ranasinghe P, Jayawardena R, et al.: The prevalence, predictors and associations of hypertension in Sri Lanka: a cross-sectional population based national survey. Clin Exp Hypertens. 2014; 36(7): 484–491. PubMed Abstract | Publisher Full Text\n\nMc Donald Posso AJ, Motta Borrel JA, Fontes F, et al.: High Blood Pressure in Panama: prevalence, sociodemographic and biologic profile, treatment, and control (STROBE). Medicine (Baltimore). 2014; 93(22): e101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFranklin SS, Gustin W 4th, Wong ND, et al.: Hemodynamic patterns of age-related changes in blood pressure. The Framingham Heart Study. Circulation. 1997; 96(1): 308–15. PubMed Abstract | Publisher Full Text\n\nRanasinghe P, Cooray DN, Jayawardena R, et al.: The influence of family history of Hypertension on disease prevalence and associated metabolic risk factors among Sri Lankan adults. BMC Public Health. 2015; 15(1): 576. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaldmäe M, Viigimaa M, Zemtsovskaja G, et al.: Prevalence and determinants of hypertension in Estonian adults. Scand J Public Health. 2014; 42(6): 504–510. PubMed Abstract | Publisher Full Text\n\nIsezuo SA, Sabir AA, Ohwovorilole AE, et al.: Prevalence, associated factors and relationship between prehypertension and hypertension: a study of two ethnic African populations in Northern Nigeria. J Hum Hypertens. 2011; 25(4): 224–230. PubMed Abstract | Publisher Full Text\n\nGuwatudde D, Mutungi G, Wesonga R, et al.: The Epidemiology of Hypertension in Uganda: Findings from the National Non-Communicable Diseases Risk Factor Survey. PLoS One. 2015; 10(9): e0138991. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUhernik AI, Milanović SM: Anthropometric indices of obesity and hypertension in different age and gender groups of Croatian population. Coll Antropol. 2009; 33(Suppl 1): 75–80. PubMed Abstract\n\nGoel R, Misra A, Agarwal SK, et al.: Correlates of hypertension among urban Asian Indian adolescents. Arch Dis Child. 2010; 95(12): 992–997. PubMed Abstract | Publisher Full Text\n\nNguyen NT, Magno CP, Lane KT, et al.: Association of hypertension, diabetes, dyslipidemia, and metabolic syndrome with obesity: findings from the National Health and Nutrition Examination Survey, 1999 to 2004. J Am Coll Surg. 2008; 207(6): 928–934. PubMed Abstract | Publisher Full Text\n\nSemenkovich CF: Insulin resistance and atherosclerosis. J Clin Invest. 2006; 116(7): 1813–1822. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuniyappa R, Sowers JR: Endothelial insulin and IGF-1 receptors: when yes means NO. Diabetes. 2012; 61(9): 2225–2227. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYiannikouris F, Gupte M, Putnam K, et al.: Adipocyte deficiency of angiotensinogen prevents obesity-induced hypertension in male mice. Hypertension. 2012; 60(6): 1524–1530. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhaley-Connell A, Sowers JR: Aldosterone and Risk for Insulin Resistance. Hypertension. 2011; 58(6): 998–1000. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDanesh J, Whincup P, Walker M, et al.: Low grade inflammation and coronary heart disease: prospective study and updated meta-analyses. BMJ. 2000; 321(7255): 199–204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNwankwo T, Yoon SS, Burt V, et al.: Hypertension among adults in the United States: National Health and Nutrition Examination Survey, 2011-2012. NCHS Data Brief. 2013; (133): 1–8. PubMed Abstract\n\nWilliams DR: Race, socioeconomic status, and health. The added effects of racism and discrimination. Ann N Y Acad Sci. 1999; 896: 173–188. PubMed Abstract | Publisher Full Text\n\nEl Bcheraoui C, Memish ZA, Tuffaha M, et al.: Hypertension and its associated risk factors in the kingdom of saudi arabia, 2013: a national survey. Int J Hypertens. 2014; 2014: 564679. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSung KC, Suh JY, Kim BS, et al.: High sensitivity C-reactive protein as an independent risk factor for essential hypertension. Am J Hypertens. 2003; 16(6): 429–433. PubMed Abstract | Publisher Full Text\n\nPasceri J, Willerson T, Yeh ET: Direct proinflammatory effect of C-reactive protein on human endothelial cells. Circulation. 2000; 102(18): 2165–8. PubMed Abstract | Publisher Full Text\n\nWu S, Huang Z, Yang X, et al.: Cardiovascular events in a prehypertensive Chinese population: four-year follow-up study. Int J Cardiol. 2013; 167(5): 2196–2199. PubMed Abstract | Publisher Full Text\n\nSingh RB, Fedacko J, Pella D, et al.: Prevalence and risk factors for prehypertension and hypertension in five Indian cities. Acta Cardiol. 2011; 66(1): 29. PubMed Abstract | Publisher Full Text\n\nYao W, Sun Y, Wang X, et al.: Elevated serum level of interleukin 17 in a population with prehypertension. J Clin Hypertens (Greenwich). 2015; 17(10): 770–774. PubMed Abstract | Publisher Full Text\n\nDinh QN, Drummond GR, Sobey CG, et al.: Roles of inflammation, oxidative stress, and vascular dysfunction in hypertension. Biomed Res Int. 2014; 2014: 406960. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalazar J, Bermúdez V, Torres W, et al.: Dataset 1 in: Dysglycemic states and hypertension: A relationship dependent on low-grade inflammation. F1000Research. 2017. Data Source"
}
|
[
{
"id": "30374",
"date": "05 Feb 2018",
"name": "Luis M. Ruilope",
"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 contains data clearly presented showing the relevance of inflammation in dysglycemic states. The group investigated is adequate in size and selection of the population in the city of Maracaibo. It is interesting the demonstration of different factors promoting prehypertension and sustained arterial hypertension and how inflammation acts when blood pressure is above 140/90 mmHg. The article in its present format is scientifically sound albeit the only caveat is that it refers only to the population of a single city.\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": "38699",
"date": "30 Nov 2018",
"name": "Harry HX Wang",
"expertise": [
"Reviewer Expertise Primary health care",
"cardiovascular diseases"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis was a cross-sectional study that explored factors associated with hypertension (HT) and pre-hypertension (Pre-HT), respectively, among a sample of population in a single city in Venezuela. The merits of this paper included a very detailed description of the sampling methodology and a clear demonstration of main results.\n\nHowever, factors that contribute to increasing level of high blood pressure (BP) have already been well reported in many existing literature, and thus the novelty of the research question in the current study seems to be limited.\n\nAs the study identified different sets of factors associated with HT and Pre-HT in the two separate logistic regression models, I would suggest conducting a sensitivity analysis using the BP values as dependent variable in a multiple linear regression model. This could provide further evidence to illustrate a picture showing how variables currently identified for both HT and Pre-HT are linked with BP values.\n\nThe seventh paragraph in the Discussion section (page 9 of 13) argued that household physical activity was found as a protective factor for pre-HT. However in Table 4, the variable was leisure time physical activity (p=0.05). On this particular variable, the authors can add more texts to describe what is defined as a \"moderate physical activity\" so that people who are at risk for higher BP can be benefited in adjusting their physical activity level from the findings of this study.\n\nThe title of the paper highlighted dysglycemic states and HT; however, this was not closely reflected by the main results 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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1953
|
https://f1000research.com/articles/6-1937/v1
|
02 Nov 17
|
{
"type": "Research Note",
"title": "Detection of illicit online sales of fentanyls via Twitter",
"authors": [
"Tim K. Mackey",
"Janani Kalyanam",
"Janani Kalyanam"
],
"abstract": "A counterfeit fentanyl crisis is currently underway in the United States. Counterfeit versions of commonly abused prescription drugs laced with fentanyl are being manufactured, distributed, and sold globally, leading to an increase in overdose and death in countries like the United States and Canada. Despite concerns from the U.S. Drug Enforcement Agency regarding covert and overt sale of fentanyls online, no study has examined the role of the Internet and social media on fentanyl illegal marketing and direct-to-consumer access. In response, this study collected and analyzed five months of Twitter data (from June-November 2015) filtered for the keyword “fentanyl” using Amazon Web Services. We then analyzed 28,711 fentanyl-related tweets using text filtering and a machine learning approach called a Biterm Topic Model (BTM) to detect underlying latent patterns or “topics” present in the corpus of tweets. Using this approach we detected a subset of 771 tweets marketing the sale of fentanyls online and then filtered this down to nine unique tweets containing hyperlinks to external websites. Six hyperlinks were associated with online fentanyl classified ads, 2 with illicit online pharmacies, and 1 could not be classified due to traffic redirection. Importantly, the one illicit online pharmacy detected was still accessible and offered the sale of fentanyls and other controlled substances direct-to-consumers with no prescription required at the time of publication of this study.\n\nOverall, we detected a relatively small sample of Tweets promoting illegal online sale of fentanyls. However, the detection of even a few online sellers represents a public health danger and a direct violation of law that demands further study.",
"keywords": [
"fentanyl",
"prescription drug abuse",
"digital surveillance",
"fentanyl",
"counterfeit",
"Twitter",
"social media",
"internet pharmacies"
],
"content": "Introduction\n\nA fentanyl crisis is currently underway in the United States, characterized by an influx of counterfeit fentanyl-laced prescription drugs (e.g. Xanax®, Norco®, OxyContin®, and Oxycodone) now being advertised, sold and consumed by the public1. The result of this counterfeit infiltration into the U.S. drug supply chain has been an alarming increase in fentanyl-related overdose, deaths and seizures, due to illicitly produced products containing undeclared fentanyls and related fentanyl analogues/variants (e.g. acetyl fentanyl, butyrfentanyl, and furanylfentanyl.)1–3\n\nFueling this public health emergency is a global network of fentanyl producers and traffickers including countries such as China (where powdered fentanyls and other synthetic opiates are sold with pill presses or sold as precursors used to manufacture fentanyl), Mexico (where fentanyls are manufactured in clandestine laboratories and smuggled into the USA) and Canada (where fentanyls are also manufactured and sold locally)1. Unfortunately, criminals see an opportunity to sell cheap and deadly versions of fentanyl-laced pills to satiate demand driven by a national prescription opioid and heroin epidemic, with the U.S. Drug Enforcement Agency (DEA) estimating that a kilogram of fentanyl could generate $5–20 million in retail counterfeit sales1.\n\nOther studies have explored the public health consequences of counterfeit fentanyl and have argued for better surveillance and harm reduction approaches to curb consumption and demand2,3. However, the potential impact and easy accessibility of fentanyls sold direct-to-consumer via the Internet and social media has not been examined. Other studies have identified illegal marketing and direct sales of other prescription controlled substances by illicit online pharmacies and the use of popular social media sites to promote these services4–10.\n\nNo study has assessed the role of Internet in fentanyl product promotion, despite concerns from the DEA about documented covert and overt sale of fentanyls online1. To better characterize these risks, we conducted a social media surveillance study using big data approaches to identify fentanyl promotion and sale via the popular microblogging platform Twitter.\n\n\nMethods\n\nOur data collection was part of a larger study exploring user behavior characteristics of illicit prescription drug abuse mediated by Twitter, with tweets collected over a five-month period (June–November 2015) filtered for the prescription drug abuse-related keywords, including the term “fentanyl.”5,6,10 Data collection and analysis was carried out using a methodology combining Amazon Web Services (AWS) cloud computing virtual instances for data collection and machine learning algorithms to analyze tweets using both assisted and unassisted protocols used in previously published studies5,6,10.\n\nSpecifically, we used a Biterm Topic Model (BTM) that can identify underlying latent patterns or “topics” present in a large corpus of tweets6,11. Given the input corpus of text, and a predefined number k, the model outputs a set of k latent topics present in the corpus. Each topic represents an underlying pattern associated with fentanyl twitter conversations that were then isolated and coded using human annotation.\n\nThe number of topics to be detected was set to 40 and the alpha and beta parameters of BTM were set to 1 and 0.01 respectively. The output of the BTM was set to display the top 10 words with the highest weight for each topic. Each topic was manually reviewed based on these words and classified into “news” and “online pharmacy” themes (results discussed below). We also used a keyword filtering process using common terms included in the text of tweets associated with illegal online drug promotion, also known as “selling arguments,” (e.g. “buy”, “discount”, “price”) in conjunction with the BTM outputs. Code used in this study is available from https://github.com/kjanani/health_topicmodeling.\n\n\nResults\n\nOur study collected 28,711 fentanyl-related tweets during 2015, a period when the fentanyl crisis was escalating6,11. The majority of topics detected in the whole corpus of fentanyl-related tweets were news-related (97.3%, n=27,940), detailing counterfeit fentanyl dangers reported in national and local media outlets.\n\nAfter isolating Tweets related to news reports, we used a keyword filtering process in conjunction with machine learning to detect the subset of tweets associated with illegal online drug promotion. Using this approach, we detected 771 (<1% of total) tweets promoting the marketing and sale of fentanyls and other controlled substances online. These tweets were then manually annotated by the authors (inter-coder reliability kappa=0.98) to assess if they included a hyperlink enabling direct-to-consumer sale and purchase of illegal fentanyls. Nine unique tweets (not duplicates or retweets) and their associated hyperlinks were then identified for further analysis.\n\nWhen examining website content of hyperlinks, 6 were associated with online classified ads, 2 with illicit online pharmacies, and 1 could not be classified due to traffic redirection. These results indicate some interesting trends. First, it appears that individual drug dealers use online classifieds ads to digitally advertise “street buys” of controlled substances (Example A, Figure 1). Additionally, one illicit online pharmacy that we detected and which is currently was still accessible at the time of initial publication of this study. offers the sale of fentanyls and other controlled substances direct-to-consumers with no prescription required (Example B). Its website purportedly offers Abstral® 800mg (fentanyl brand name) for $3.00 per tablet, and based on further inspection of WHOIS data, has its internet domain registration identity and location masked by an Internet privacy service company.\n\nWe note that there are certain limitations to this study. First the study only examined online promotion and availability at a single point-of-time, as websites of this nature are often removed or become inactive. Additionally, because of the illegal nature of the websites identified, we did not purchase fentanyls and test them for authenticity and potency. Buying controlled substances and making payments to an illicit online pharmacy raises serious legal concerns and is generally illegal.\n\n\nDiscussion\n\nOur results indicate that in our entire corpus of fentanyl-related tweets, direct-to-consumer sale of fentanyls occurred infrequently. This is not surprising, given the deadly nature and high potency of fentanyl, and that many victims may not actively seek its purchase1,2. However, the presence of even a few online sellers is concerning, as these actions represent a clear violation of Federal law (e.g. Controlled Substances Act and the Ryan Haight Online Pharmacy Consumer Protection Act) and directly endanger the public. Importantly, these sites may only be the tip of the iceberg, as hyperlinks in one result directed us to online user forums (Google Groups) actively selling fentanyl online (Example C).\n\nOur data collection occurred in late 2015, a period arguably at an early stage of the fentanyl crisis epidemiological curve, which may limit the generalizability of the results. Hence, this indicates that more research is needed to accurately characterize the dangers of the online environment in relation to counterfeit fentanyls. This includes longer term follow-up studies examining online promotion and access to fentanyl and interrogating other online platforms (including other social media sites, chat rooms/user forums, the deep web, and private sales facilitated by online and mobile technology).\n\n\nData availability\n\nRaw datasets have not been made available per concerns regarding user information and confidentiality of publicly available processed data. This data is stored at the Global Health Policy Institute and is available upon request in a de-identified and aggregated dataset. Please contact the institute’s general contact email (ghpolicyinstitute@gmail.com) for further information.\n\nThe code for BTM can be found at https://github.com/kjanani/health_topicmodeling.\n\nArchived code: https://doi.org/10.5281/zenodo.103817712",
"appendix": "Competing interests\n\n\n\nTM is a non-compensated member of the academic advisory panel of the Alliance for Safe Online Pharmacies (ASOP), a 501(c)(4) social welfare organization engaged on the issue of illicit online pharmacies. There was no involvement of anyone other than the authors in the conception, design, collection, planning, conduct, analysis, interpretation, writing, and discussion to submit this work. Authors report no other financial relationships with any organizations that might have an interest in the submitted work.\n\n\nGrant information\n\nTM received funding for the data collection phase of this study from an ASOP pilot research grant exploring prescription drug abuse risks online. Though this pilot research grant supported the overall process of capturing data, the aims of the grant were not related to this study.\n\n\nReferences\n\nDEA: Counterfeit Prescription Pills Containing Fentanyls: A Global Threat [Internet]. dea.gov. 2016; [cited 2017 Feb 17]. Reference Source\n\nGreen TC, Gilbert M: Counterfeit Medications and Fentanyl. JAMA Intern Med. American Medical Association, 2016; 176(10): 1555–7. PubMed Abstract | Publisher Full Text\n\nFrank RG, Pollack HA: Addressing the Fentanyl Threat to Public Health. N Engl J Med. 2017; 376(7): 605–7. PubMed Abstract | Publisher Full Text\n\nMackey TK, Liang BA, Strathdee SA: Digital social media, youth, and nonmedical use of prescription drugs: the need for reform. J Med Internet Res. 2013; 15(7): e143. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatsuki T, Mackey TK, Cuomo R: Establishing a Link Between Prescription Drug Abuse and Illicit Online Pharmacies: Analysis of Twitter Data. J Med Internet Res. 2015; 17(12): e280. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKalyanam J, Katsuki T, Lanckriet G, et al.: Exploring trends of nonmedical use of prescription drugs and polydrug abuse in the Twittersphere using unsupervised machine learning. Addict Behav. 2017; 65: 289–95. PubMed Abstract | Publisher Full Text\n\nRaine C, Webb DJ, Maxwell SR: The availability of prescription-only analgesics purchased from the internet in the UK. Br J Clin Pharmacol. Blackwell Publishing Ltd, 2009; 67(2): 250–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nForman RF, Block LG: The Marketing of Opioid Medications without Prescription over the Internet. J Public Policy Mark. 2006; 25: 133–46. Publisher Full Text\n\nU S Government Accountability Office HGG: INTERNET PHARMACIES: Federal Agencies and States Face Challenges Combating Rogue Sites, Particularly Those Abroad [Internet]. gao.gov. 2013, [cited 2016 Jan 11]. Reference Source\n\nMackey TK, Kalyanam J, Katsuki T, et al.: Machine Learning to Detect Prescription Opioid Abuse Promotion and Access via Twitter. Am J Public Health: published online before print October 19, 2017. e1–e6. PubMed Abstract | Publisher Full Text\n\nYan X, Guo J, Lan Y, et al.: A biterm topic model for short texts. The 22nd international conference. New York, New York, USA: ACM, 2013; 12. Publisher Full Text\n\nMackey T, Kalyanam J: Detection of Illicit Online Sales of Fentanyls via Twitter. Zenodo. 2017. Data Source"
}
|
[
{
"id": "27555",
"date": "30 Nov 2017",
"name": "Marvin D. Shepherd",
"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\nMy main concern is the discontinuity between the introduction and the research conducted. The introduction focused on problems of pharmaceutical purchased which were laced with counterfeit fentanyl. This is true and is a major health hazard as pointed out by the authors. Fentanyls are a deadly pharmaceuticals and again this was emphasized by the authors. However, the paper's research focus is on tweets from people who are interested in purchasing fentanyl illegally. It wasn't on the counterfeit fentanyl laced products. I suggest that the intro be rewritten and I mention the legitimate uses of fentanyl. Please mention that one of fentanyl's major uses is as a pain reliever in hospital surgical suites and must be carefully controlled by medical practitioners. To reiterate, the introduction doesn't match well the research content.\nThe methodology approach was was appropriate and innovative. I know they provided a link for a full description of the search terms, but in reading it I suggest that they provided at least a partial list of the names used i.e. different spellings of fentanyl, different names for the same product (foreign names). Searching tweets was original, but it didn't produce huge number of tweets. This is worth reporting in that few people tweet to purchase fentanyl. The first thing I thought of was why tweet when you can do a google search and get right to the web sites? This needs to be put in the discussion as to why the results were so low. I am sure there are other methods to find the product.\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? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "28806",
"date": "11 Dec 2017",
"name": "Róbert G. Vida",
"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\nAn up-to-date and innovative methodological approach of a possible global problem of illegal trade of pharmaceuticals via the Internet is absolutely useful. The main messages are in accordance with the scientific literature.\nThe concern highlighted by the Dr. Marvin D. Shepherd regarding the dissonance between the introduction and the other parts of the publication is real and similarly I suggest the reconsideration and rewrite of that part. Also it would be advisable to include the comparison of the legal and illicit use and highlight the sources of fentanyl products or fentanyl containing products (outpatient and inpatient settings). Beside the counterfeit fentanyl crisis another possible public health danger is hitting the health care system of the United States is the shortage of multiple drugs including opioid drugs as well (e.g.: fentanyl) necessitate the use of alternative supply channels (e.g.: importation of drugs outside the U.S., internet pharmacies) make the health sector more susceptible to illegal and counterfeit products.\nBeside the network science the Big Data type of analysis is a novel and hopefully in the future a routinely used methodology to identify and monitor illegal vendors and their connections in the field of illegal internet sale of pharmaceuticals. The more detailed description of the method or even the representation with a figure would help the understanding of it.\nThe characterisation (e.g.: ordering is possible, LegitScript or VIPPS NABP legitimacy, products being sold, prescription requirements) of the identified online pharmacies would have highlighted the illegal internet sale of fentanyl products more accurately.\nMoreover the inclusion of comparison of the results with the literature data regarding other social media platforms or the simple google search methodology would give a more complete picture of this public health danger.\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? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "28804",
"date": "18 Dec 2017",
"name": "Emilio Ferrara",
"expertise": [
"Reviewer Expertise social networks",
"data science",
"machine learning",
"AI"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 report on a timely and important research topic, directly connected to the current US opioid epidemic. Overall, the methodology is sound and the findings are interesting. One limitation of the data collection that could be addressed in future studies is the focus on one single keyword (fentanyl): a quick search using brand names (e.g., oxycodone, oxycontin) and slang terms / street names usually associated with this drug (e.g., ox, ocs) produce many additional hits, including at times associated with illicit person-to-person sales. The authors carried out a scrupulous investigation of the clearnet domains associated with the links discovered on Twitter – I wonder if a similar analysis could be carried out on the Dark Web (often associated with the illicit sale of drugs): this would require significant changes in the data collection infrastructure thus I can only expect it could be done in a follow up 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? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1937
|
https://f1000research.com/articles/6-1935/v1
|
02 Nov 17
|
{
"type": "Method Article",
"title": "Clinical protocol for the registration of individual translucent zones of frontal teeth",
"authors": [
"Rangel Todorov",
"Georgi Todorov",
"Stefan Zlatev",
"Georgi Todorov",
"Stefan Zlatev"
],
"abstract": "In contemporary dentistry, a successful restoration should not only restore the shape and function of the impaired dentition, but also contribute to the overall aesthetic appearance. In order to achieve this goal, the dental team should possess complete information about the different optical dimensions of the tooth structure. Besides the standard properties of the color, it is important to define, register and interpret the shape and position of the individual translucent zone. With the help of a device and software developed by the authors, a five step clinical protocol has been developed by the authors for individual translucent zone registration, with the help of trans-illumination. The translucent areas are then shown in a digital photograph of the teeth, analyzed and interpreted. The translucent zone is finally visualized as a ‘translucent map’, which is clearly defined, and is easily interpreted and used by the dental team.",
"keywords": [
"trans-illumination",
"translucent zone",
"frontal teeth"
],
"content": "Introduction\n\nThe term trans-illumination depicts tissue diagnostics with a high intensity light beam projected through an object. The object that is studied is positioned between the light source and the observer. The method is quick, accurate, precise, and can determine the current state of the tissues, which makes it a valuable tool1–3.\n\nTrans-illumination is a well-known diagnostic concept used in dentistry. The index of light transmission of healthy tooth tissues is higher than those with caries, and differs from dental calculus. If a tooth is illuminated with sufficient intensity, those clinical findings would appear as dark spots surrounded by light, healthy tooth substance3,4. Using this method fractures lines and orifices of molars, and premolars can be visualized5,6. These are among the reasons why trans-illumination is most frequently applied in operative dentistry. The two devices commonly used are FOTI (fiber-optical trans-illumination) and DIFOTI (digital fiber-optic trans-illumination)7,8.\n\nCurrently for trans-illumination, white LED light that is transmitted to the surveyed field through a fiber-optical handpiece is used. This ensures direct contact, without the risk of overheating the structures or causing discomfort to the patient8,9. The small dimensions of the hand-piece allows direct access to all surfaces of the teeth – lingual, vestibular, occlusal - and the ability to assess hard to reach areas, for example the distal parts of the dentition3.\n\nIn comparison to X-ray imaging, the teeth can be lit from different angles, which enables the operator to assess more viewpoints. Another advantage of the method is the absence of distortion, which occurs in X-ray images because of the translation of three dimensional objects to two dimensions10. In addition, trans-illumination has better acceptance among patients because of its noninvasive nature11.\n\nIn prosthetic dentistry, trans-illumination is used as a proof tool for diagnosis of cracks in ceramic masses after sintering. In cases of full ceramic crowns, the light beam is projected from the intaglio surface of the construction, whereas in cases of metal-ceramic restorations, a tangential direction is used3,6.\n\nIn dentistry, translucency is defined as the gradient between transparent and opaque12. Furthermore, the translucent zones, especially in the upper frontal teeth, play a major role in the aesthetic perception and have an impact on the vital appearance of artificial prosthetic constructions. Therefore, their correct in-office registration and recreation by the dental technician are important prerequisites for successful treatment outcome. In standard clinical conditions these areas cannot be assessed. The only commercially available device for individual translucent zone registration currently is the MHT SpectroShade (Verona, Italy), which is based on spectrophotometry. The device presents the individual translucent zone as a scale, which was developed by the manufacturers13.\n\nTherefore, the objective of this article is to propose a clinical protocol for obtaining the individual localization of the translucent zone of frontal teeth with the use of trans-illumination.\n\n\nMethods\n\nAll experimental work associated with the current project was approved by the Ethical Committee of Plovdiv Medical University (protocol number P-1546; approved on 13th March 2014). Written informed consent was obtained from all individuals participating in this method, which has been used in clinical practice as part of routine care.\n\nIn order to utilize trans-illumination for individual transparent zone registration, a device called ‘Apparatus for registration of translucent individual zone’ (ARTIZ; shown in Figure 1) was developed by the authors. The main components of the apparatus are a power module and emitting module. The main component of the emitting module is a 10W, LZ4-00MA10 emitter (type RGBA LED, Led Engine, USA). The power module is constructed with separate drivers for each LED, based on ZXLD1360 (ZETEX) controller.\n\nThe proposed protocol for clinical registration of the individual transparent zone is separated into five steps:\n\n\n\n1. Ensuring access to the structures undergoing translucent zone diagnosis\n\n● Intraoral examination and removal of calculus/tartar, debris and food leftovers;\n\n● Choice of appropriate lip and cheek retractor.\n\n2. Powering and tuning of the apparatus\n\n3. Positioning of the hand-piece\n\n4. Registration of the observed translucent zone with a digital photograph\n\n5. Creation of a ‘translucent map’ with the use of custom-made software developed by the authors – ARTIZ-soft.\n\nThe steps of the clinical protocol are visualized in Figure 1–Figure 5. Step 2, as well as different technical peculiarities are further mentioned in the next sections.\n\nStep 1 (Figure 1)\n\nThe teeth undergoing translucent zone registration should be polished with a soft silicone brush and pumice prior to the procedure. If calculus, food leftovers or debris are present, a cleaning procedure is advised. The lip and cheek retractor should enable the operator to position the hand piece in the desired orientation and ensure direct visibility of the surveyed teeth.\n\nSteps 2 and 3 (Figure 2)\n\nThe optimal settings of the ARTIZ apparatus are white light with intensity 1 for all potentiometers. The orientation of the hand-piece depends on the position of the tooth undergoing translucent zone registration, and is up to 50° tangential to the long axis. The optimal angle is between 30–45° tangential to the lingual surface of the tooth, pointed apically.\n\nStep 4 (Figure 3–Figure 5)\n\nThe camera is fixed to the reflector of the dental unit with a standard super-clamp at an angle approximately perpendicular to the vestibular surface. This will minimize the dimensional distortion caused by the transition of a 3D object to 2D. A remote shooter is used to negate vibration and movement during the image acquisition phase. We found out that the optimal camera settings for the Canon camera are as follows:\n\nExposition time: 1/260 to 1/500 s.;\n\nISO: 200;\n\nFocal length: 6–7 cm from the vestibular surface of the diagnosed tooth.\n\nStep 5 (Figure 6)\n\nThe image is then transported into ARTIZ-soft. After some in-software image manipulation, e.g. cropping, a template representing the tooth and the shape of the translucent zone is chosen (ARTIZ-soft includes digitized templates of the different types of translucent zones according to the classification of Ralin Ralev14) in order to create a ‘translucent map’ of the tooth.\n\nThe use of ARTIZ-soft is optional, since there are image manipulation tools freely available in common open-source graphical software, e.g. Inkscape, Gimp, ImageMagick, and using a free-draw tool the translucent zone can be recreated from Ralev’s classification, as depicted in Figure 5.\n\n\nDiscussion\n\nThe translucent zone visualized with the help of the proposed method is clearly defined and easily visible. The registration itself is fast, straightforward and takes 5 to 10 minutes. As a result, a digital image of the individual translucent zone of the tooth is created. With the help of the custom developed software ARTIZ-soft, it is possible to transform the photograph into a ‘translucent map’, which can be used as a guideline for different restorative procedures.\n\nIn addition to the established concept of horizontal distribution of the translucent zone, parallel to the incisal edge, with the help of trans-illumination a vertical translucent zone is visualized. This finding complements the overall color registration procedure and gives the practitioner a more detailed information, making restorative procedures predictable.\n\n\nSoftware availability\n\nARTIZ-soft is currently still in development, with a release date of early 2018. However, the method can be performed with other freely available manipulation tools, as detailed above.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was funded with a MU Plovdiv PhD Grant (DP-05/2013).\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\nCôrtes DF, Ekstrand KR, Elias-Boneta AR, et al.: An in vitro comparison of the ability of fibre-optic transillumination, visual inspection and radiographs to detect occlusal caries and evaluate lesion depth. Caries Res. 2000; 34(6): 443–7. PubMed Abstract | Publisher Full Text\n\nDavies GM, Worthington HV, Clarkson JE, et al.: The use of fibre-optic transillumination in general dental practice. Br Dent J. 2001; 191(3): 145–147. PubMed Abstract | Publisher Full Text\n\nPretty IA: Caries detection and diagnosis: novel technologies. J Dent. 2006; 34(10): 727–39. PubMed Abstract | Publisher Full Text\n\nAnusavice KJ: Present and future approaches for the control of caries. J Dent Educ. 2005; 69(5): 538–54. PubMed Abstract\n\nAilor JE Jr: Managing incomplete tooth fractures. J Am Dent Assoc. 2000; 131(8): 1168–74. PubMed Abstract | Publisher Full Text\n\nStrassler HE, Pitel ML: Using fiber-optic transillumination as a diagnostic aid in dental practice. Compend Contin Educ Dent. 2014; 35(2): 80–8. PubMed Abstract\n\nPine CM: Fibre-optic Transillumination (FOTI) in Caries Diagnosis. In Stookey G., Proceedings of the First Annual Indiana Conference: Early Detection of Dental Caries. Indiana: Indiana University, 1996.\n\nSchneiderman A, Elbaum M, Shultz T, et al.: Assessment of dental caries with Digital Imaging Fiber-Optic TransIllumination (DIFOTI): in vitro study. Caries Res. 1997; 31(2): 103–10. PubMed Abstract | Publisher Full Text\n\nVaarkamp J, ten Bosch JJ, Verdonschot EH, et al.: The real performance of bitewing radiography and fiber-optic transillumination in approximal caries diagnosis. J Dent Res. 2000; 79(10): 1747–51. PubMed Abstract | Publisher Full Text\n\nHintze H, Wenzel A, Danielsen B, et al.: Reliability of visual examination, fibre-optic transillumination, and bite-wing radiography, and reproducibility of direct visual examination following tooth separation for the identification of cavitated carious lesions in contacting approximal surfaces. Caries Res. 1998; 32(3): 204–209. PubMed Abstract | Publisher Full Text\n\nSmith RN, Brook AH, Elcock C: The quantification of dental plaque using an image analysis system: reliability and validation. J Clin Periodontol. 2001; 28(12): 1158–62. PubMed Abstract | Publisher Full Text\n\nPensler AV: Shade selection: problems and solutions. Compend Contin Educ Dent. 1998; 19(4): 387–90, 392–4, 396; quiz 398. PubMed Abstract\n\nGuerra F, Mazur M, Corridore D, et al.: Evaluation of the esthetic properties of developmental defects of enamel: a spectrophotometric clinical study. ScientificWorldJournal. 2015; 2015: 878235. PubMed Abstract | Publisher Full Text | Free Full Text\n\nРалев Р: Естетика на съзъбието Анфас. София, Quintessence BG. 1993; 141–55. Ralev R., Aesthetics of dentition. Full face. Sofia, Quintessence BG, 1993; p.141-55. Reference Source"
}
|
[
{
"id": "29488",
"date": "25 Jan 2018",
"name": "Livia Ottolenghi",
"expertise": [
"Reviewer Expertise Preventive and community dentistry",
"cariology",
"minimally invasive procedures",
"technology-aided caries diagnosis",
"spectrophotometry applied to colourimetric evaluation of sound enamel",
"caries and developmental defects of enamel."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nQuestion 3 The presented method is not really reproducible by others, as the “apparatus” described by the Authors is not yet on the market.\n\nQuestion 4 The results should be presented differently, as there is a lack of standardization. Please present the results in a more effective way, because they are unclear and difficult to be followed.\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? Yes",
"responses": []
},
{
"id": "32558",
"date": "18 Apr 2018",
"name": "Yong-Keun Lee",
"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 tries to provide clinical protocols for the registration translucent zone of teeth. Study design is good and paper preparation was accurate.\nI am wondering whether you have some idea on the opalescence of teeth correlated with translucency of teeth. In Figs 5 and 6, you might have found opalescence property of teeth. If that is possible, we can map the translucency and opalescence of teeth simultaneously.\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/6-1935
|
https://f1000research.com/articles/6-1933/v1
|
01 Nov 17
|
{
"type": "Method Article",
"title": "Continuous outcome logistic regression for analyzing body mass index distributions",
"authors": [
"Tina Lohse",
"Sabine Rohrmann",
"David Faeh",
"Torsten Hothorn",
"Tina Lohse",
"Sabine Rohrmann",
"David Faeh"
],
"abstract": "Body mass indices (BMIs) are applied to monitor weight status and associated health risks in populations. Binary or multinomial logistic regression models are commonly applied in this context, but are only applicable to BMI values categorized within a small set of defined ad hoc BMI categories. This approach precludes comparisons with studies and models based on different categories. In addition, ad hoc categorization of BMI values prevents the estimation and analysis of the underlying continuous BMI distribution and leads to information loss. As an alternative to multinomial regression following ad hoc categorization, we propose a continuous outcome logistic regression model for the estimation of a continuous BMI distribution. Parameters of interest, such as odds ratios for specific categories, can be extracted from this model post hoc in a general way. A continuous BMI logistic regression that describes BMI distributions avoids the necessity of ad hoc and post hoc category choice and simplifies between-study comparisons and pooling of studies for joint analyses. The method was evaluated empirically using data from the Swiss Health Survey.",
"keywords": [
"Distribution regression",
"transformation model",
"conditional distribution",
"odds ratio",
"smoking"
],
"content": "Introduction\n\nBody mass index (BMI) is an anthropometric measure that is relatively easy to capture in epidemiological studies. Thus, it is widely used for describing underweight, overweight, and obesity1,2. The most prominent standard BMI categories, underweight, normal weight, overweight, and obesity as defined by the World Health Organization [WHO, 3], are commonly applied to ensure comparability and reproducibility of statistical analyses across epidemiological studies4,5. Such international standards are important for the communication of scientific results, for risk factor assessment and monitoring in populations, and for providing information to the general public. However, categorization of BMI values inevitably leads to information loss because an individual’s weight and height can be measured precisely using simple tools1, but this precision is lost in statistical analyses by such an ad hoc categorization6. The most important problem, however, is the lack of comparability across studies that rely on different categorization schemes. Even more troublesome is the problem of comparability of studies and findings over time because the WHO categories can be expected to be updated to better reflect contemporary BMI distributions. Only roughly half of the studies published up to 2000 that used BMI as a risk factor for death used the WHO categories; the other half relied on a variety of different alternative schemes4,5. The same problem occurs when the primary interest in a statistical analysis is the comparison of BMI distributions between different risk groups. In this latter situation, we advocate post hoc categorization of model outputs instead of ad hoc categorization of BMI measurements to better combine measurement precision, ease of communication, comparability, and reproducibility. Specifically, we propose that statistical analyses should be based on precise BMI measurements without ad hoc categorization, and then parameters and interesting contrasts thereof should then be categorized post hoc. Such results would be interpretable and universally comparable between studies using any type of category.\n\nConceptually, the traditional approaches to the analysis of BMI can be understood as regression models for the conditional distribution of BMI, given exposure, sex, and covariates7–13. Treating smoking as the only exposure variable in the following, a generic logistic regression model for BMI, conditional on smoking status, sex, and covariates x of the form\n\nlogit(ℙ(BMI ≤ b | smk, sex, x)) = r(b | smk, sex, x) (1)\n\nhelps to understand the properties of specific models for BMI and connections between them. Specific classical models, such as binary logistic regression or polytomous logistic regression, are implemented via a specific regression function r; details will be given in the next section. The majority of published BMI analyses relied on a small number of ad-hoc cut-off points b. After such an ad hoc categorization, only the conditional distribution of BMI at the corresponding cut-off points b can be evaluated. The core idea of continuous outcome logistic regression is to model the entire conditional distribution of BMI for all reasonable BMI values simultaneously. This requires that the parameterization of the regression function r is a smooth and monotonically increasing function of b. The statistical underpinnings of such models were developed only recently14,15. In models treating BMI as a continuous outcome, the exposure smoking status, sex, and covariates x then have an impact on the regression function r and thus on conceptually all moments (mean, variance, skewness, kurtosis, etc.) of the conditional continuous BMI distribution. Although such models are more complex, the interpretation of parameters and contrasts thereof remains as simple as in models based on specific categories. For example, the difference between r(b | former smoker, female, x) and r(b | never smoker, female, x) is the log-odds ratio of the event BMI ≤ b of former female smokers compared to females who never smoked, both of which share the same covariate status x. After traditional ad hoc categorization, this odds ratio can only be evaluated for the small set of cut-off points b that define the categories. For continuous outcome logistic regression, the odds ratio can be evaluated for all potential BMI values b > 0, which allows the associations for different categorization schemes to be interpreted post hoc. This feature ensures comparability and reproducibility independent of any ad hoc choice of categories.\n\nThe continuous outcome logistic regression model can be estimated by maximum likelihood for BMI measurements recorded at different scales15. The likelihood contribution of an individual with a BMI value in the interval (b, b] is simply the probability, in light of some specific regression function r, of observing a BMI within this interval16\n\nℙ(b < BMI ≤b | smk, sex, x) (2)\n\n= ℙ(BMI ≤ b | smk, sex, x) – ℙ(BMI ≤ b | smk, sex, x)\n\n= expit(r(b | smk, sex, x)) – expit(r(b | smk, sex, x)).\n\nThe BMI measurement (b, b] can be a narrow numeric interval based on precise measurements of height and weight, or a wide interval corresponding to some standard or non-standard categorization scheme. Thus, continuous outcome logistic regression is applicable to studies that implement different BMI measurement scales or categorization schemes, or even a mixture of those. The procedure thus directly addresses the conceptual problem of lack of comparability between different studies. The aim of our study was to propose a continuous outcome logistic regression model for BMI that is independent of both the BMI measurement scale and cut-offs used for ad hoc categorization, which would allow tailored categorized parameters and contrasts to be extracted, compared, and communicated post hoc. We expected the model to be insensitive to the BMI measurement scales, in light of both the estimated conditional BMI distributions and the covariate model parameters. We evaluated this hypothesis empirically by analyzing the association of smoking status and BMI using data from the Swiss Health Survey 201217 while controlling for important covariates, such as age, alcohol intake, diet, physical activity, and socio-economic variables. We compared models fitted to a cascade of increasingly precise BMI values, starting with the four WHO categories and ending with the “exact” BMI values. This allowed an understanding of the impact of the measurement scale on the resulting models. We also expected the results of the novel continuous outcome logistic model for BMI to be comparable to previously reported associations of smoking and BMI, and evaluated this hypothesis for the Swiss Health Survey 2012.\n\n\nMethods\n\nPopulation for empirical evaluation. The Swiss Health Survey (SHS) is a population-based cross-sectional survey. Since 1992, it has been conducted every five years by the Swiss Federal Statistical Office17. For this study, we restricted the sample from the 2012 survey to 16,427 individuals aged between 18 and 74 years. Height and weight were self-reported by telephone interview. Records with extreme values of height or weight were excluded (highest and lowest percentile by sex). Smoking status was categorized into never smoked, former smokers, light smokers (1 – 9 cigarettes per day), moderate smokers (10 – 19), and heavy smokers (> 19). Individuals who never smoked stated that they did not currently smoke and never regularly smoked for longer than a six-month period; former smokers had quit smoking but had smoked for more than 6 months during their life. One cigarillo or pipe was counted as two cigarettes, and one cigar was counted as four cigarettes. The following adjustment variables were included: fruit and vegetable consumption, physical activity, and alcohol intake. Information on the number of days per week fruits and vegetables were consumed was available. We chose to categorize as close to the “5-a-day” recommendation as possible18. Fruit and vegetable consumption was combined in one binary variable that comprised the information on whether both fruits and vegetables were consumed daily or not. The variable describing physical activity was defined as the number of days per week a subject started to sweat during leisure time physical activity and was categorized as > 2 days, 1 – 2 days, or none. Alcohol intake was included using the continuous variable grams per day. Education was included as highest degree obtained and was categorized as mandatory (International Standard Classification of Education, ISCED 1-2), secondary II (ISCED 3-4), or tertiary (ISCED 5-8)19. Nationality had the two categories: Swiss and foreign. Language region reflecting cultural differences within Switzerland was categorized as German/Romansh, French, or Italian.\n\nModels for BMI distributions. Binary logistic regression, ordered, and unordered polytomous logistic regression20 were previously applied to the analysis of BMI distributions based on ad hoc categorized BMI values. We will review the corresponding parameterizations and compare the model parameters in the common framework of model (1) before introducing the novel continuous outcome logistic regression for the analysis of BMI distributions.\n\nBinary logistic regression For a binary outcome, such as non-obesity vs. obesity (BMI30 = I(BMI ≤ 30)), the regression function is defined for non-obese individuals only\n\nr(30 | smk, sex, x) = α30 + γsmk:sex + x⊤β,\n\nwith intercept α30, main and interaction parameters γ of smoking and sex, and regression coefficients or covariate parameters β. This model evaluates the conditional distribution function for BMI only at b = 30. Note that a change of the BMI cut-off point b leads to a different model, and thus different parameter estimates for all parameters αb, γ, and β. Such models have been reported for b = 25 or b = 3011,12.\n\nOrdered polytomous logistic regression This model is also known as proportional odds logistic regression for an ordered categorical outcome, such as the WHO categories3 underweight (BMI18.5 = I(BMI ≤ 18.5)), normal weight (BMI(18.5,25] = I(18.5 < BMI ≤ 25)), overweight (BMI(25,30] = I(25 < BMI ≤ 30)), and obese (BMI > 30). For these four categories, the model is defined by three category-specific regression functions\n\nr(18.5 | smk, sex, x) = α18.5 + γsmk:sex + x⊤β\n\nr(25 | smk, sex, x) = α(18.5,25] + γsmk:sex + x⊤β\n\nr(30 | smk, sex, x) = α(25,30] + γsmk:sex + x⊤β\n\nor, in more compact notation, by r(b | smk, sex, x) = α(b) + γsmk:sex + x⊤β with intercept function\n\n\n\nThe parameters γ and β are the same for all three regression functions and can be interpreted as category-independent log-odds ratios as a consequence of the proportional odds assumption on these parameters. The intercept function increases monotonically. Ordered polytomous logistic regression can be understood as a series of binary logistic regression models where only the intercept is allowed to change with increasing BMI values at cut-off points chosen ad hoc. Self-reported BMI values using the WHO criteria have been analyzed by such a model in 7. The BMI distribution of children categorized at marginal percentiles has been analyzed by a proportional odds model in 13.\n\nAn extension of ordered polytomous regression to continuous responses, treating the intercept function α as a step-function at the observations with subsequent non-parametric maximum likelihood estimation, was recently suggested by 21. Unlike the model and estimation procedure discussed here, their method does not allow for the different likelihood contributions presented in the next section.\n\nUnordered polytomous logistic regression Multinomial logistic regression is equivalent to polytomous logistic regression for an unordered outcome and is a generalization of the proportional odds model as it allows for category-specific parameters γ(b) and β(b) in the regression function\n\nr(b | smk, sex, x) = α(b) + γ(b)smk:sex + x⊤β(b)\n\nfor b ∈ {18.5, 25, 30}. The model can be used to test the proportional odds assumption, i.e., γ ≡ γ(b) and β ≡ β(b) for all b ∈ {18.5, 25, 30}. Typically, the model is introduced as a model of the conditional density by the relationship between density and distribution function for discrete variables (as in (2)). This model is very popular for the analysis of BMI-related outcomes8–10.\n\nThe novel continuous outcome logistic regression model can be viewed as a generalization of the above-introduced models from discrete to continuous outcomes. Like these discrete models, the continuous BMI logistic regression model does not require strong parametric assumptions for the conditional BMI distribution, yet it allows to model the conceptually continuous BMI variable by a continuous distribution, regardless of the scale of the actual BMI measurements.\n\nThe most important aspect here is a smooth and monotonically increasing intercept function α(b). In an unconditional model for the marginal BMI distribution\n\nlogit(ℙ(BMI ≤ b)) = r(b) = α(b),\n\nsuch an intercept function can model arbitrary BMI distribution functions by the term expit(α(b)) (technical details of the specification and estimation of such an intercept function are given in the Appendix). This essentially removes the need to specify a strict parametric distribution, such as the normal, for BMI. Because of a potential impact of both smoking and sex of the individual on the entire distribution, we stratify this intercept function with respect to these two variables, i.e., one specific intercept function is dedicated to each combination of smoking and sex:\n\nlogit(ℙ(BMI ≤ b | smk, sex)) = r(b | smk, sex) = α(b)smk:sex.\n\nThis model is also assumption free, because arbitrary BMI distribution functions can be assigned to each combination of sex and smoking.\n\nTo facilitate model interpretation, we assume that regression coefficients β of the remaining covariates are constant across the entire BMI distribution in our final model\n\nlogit(ℙ(BMI ≤ b | smk, sex, x)) = r(b | smk, sex) (4)\n\n= α(b)smk:sex + x⊤β.\n\nThe regression coefficients β are log-odds ratios of all possible events BMI ≤ b, b > 0. The interpretation of the parameters β is the same in logistic regression, proportional odds regression, and the novel continuous BMI logistic regression (4). Of course, these constant regression coefficients might be incorrectly specified. Residual analysis, for example using the residual U = ℙ(BMI ≤ b | smk, sex, x) for a subject with BMI b, can help to detect such misspecifications. Similar to Cox-Snell residuals, the residual U is uniform when the model is correct.\n\nOur model (4) can be understood as a joint model of all possible binary logistic regression models for the outcomes BMI ≤ b with b > 0 under two constraints: (1) the sex- and smoking-level-specific intercept is not allowed to jump abruptly, thus less parameters are required in this joint model, and increases for increasing cut-off points b; (2) the regression coefficients β are held constant as b increases. Instead of restricting our attention to specific binary logistic regression models defined by some cut-off points chosen ad hoc, we can answer questions about the odds ratios for all or specific events BMI ≤ b post hoc based on this model.\n\nThe interpretation of the sex- and smoking-specific intercept functions, and thus the associations of smoking and sex with BMI, however, is fundamentally different from the interpretation of the regression coefficients β. Because we allow the entire BMI distribution to change with these two variables in more complex ways, there is no simple interaction term γ that captures these parameters in model (4). However, model (4) allows computation of the log-odds ratios for some event BMI ≤ b between, for example, female former smokers and females who never smoked for all x as\n\nr(b | former smoker, female, x) – r(b | never smoked, female, x) = α(b)former smoker:female – α(b)never smoked:female\n\nIn this way, the parameters and contrasts we are interested in are not directly parameterized in model (4) but nevertheless can be obtained from this model by relatively simple contrasts. The events BMI ≤ b are not restricted to those of a specific categorization of the BMI measurements (such as the WHO categories). Due to the smoothness of the underlying intercept functions, log-odds ratios can be computed for arbitrary BMI values b > 0.\n\nLikelihoods for BMI models. Because the regression function r is defined for all possible BMI values b in model (4), the likelihood (2) can be evaluated for all types of intervals (b, b] and also for “exact” BMI values computed as the ratio of weight and squared height. We distinguished between four different likelihood contributions corresponding to four different BMI measurement scales.\n\nWHO categories (WHO) The BMI for each individual was reported in one of the four WHO categories corresponding to the intervals ≤ 18.5 (under-weight), (18.5, 25] (normal weight), (25, 30] (over-weight), > 30 (obese). The likelihood contribution of a normal-weight individual is thus\n\nexpit(r(25 | smk, sex, x)) – expit(r(18.5 | smk, sex, x)).\n\nOther categories (Int 1) Other studies might have used a different categorization scheme, e.g., the 21 categories defined by BMI intervals for length two:\n\n≤ 17, (17, 19], (19, 21], . . . , (35, 37], > 37.\n\nAn individual with a BMI value between 19 and 21 thus contributes\n\nexpit(r(21 | smk, sex, x))–expit(r(19 | smk, sex, x))\n\nto the likelihood.\n\nNumeric intervals (Int 2) With weight measured in kilogram and height in meters, the BMI is calculated according to its definition as BMI = weight/height2. However, for an individual 1.75m tall weighting 76kg, all BMI values between 75.5/1.7552 = 24.51 and 76.5/1.7452 = 25.12 are consistent with this individual due to rounding error. Thus, this individual contributes\n\nexpit(r(25.12 | smk, sex, x)) − expit(r(24.51 | smk, sex, x))\n\nto the likelihood, which automatically takes the measurement error into account. These intervals can be expected to be much larger in studies that rely on self-reported weights and heights.\n\nExact measurements (Exact) If extreme precision was used to measure weight and height, BMI = weight/height2 can be considered an “exact” observation. Because the interval around this value is very narrow, one can approximate the likelihood contribution by the density of the conditional BMI distribution\n\n\n\nevaluated at the “exact” BMI value.\n\nIt is important to note that it is possible to evaluate the likelihood when a mixture of these different BMI measurement scales is applied to subsets of the individuals. In subject-level meta analyses, for example, it would be possible to estimate a joint model based on studies using different BMI categorizations or no categorization at all. From a purely theoretical point of view, the application of numeric intervals that take rounding error into account (Int 2) is most appropriate. The remaining three procedures must be considered approximate.\n\n\nEmpirical results\n\nComparison of estimated probabilities obtained from the four different likelihoods for model (4) showed that these probabilities were practically identical. For females and males of all smoking categories with baseline covariates, the estimated conditional BMI distribution evaluated at the WHO categories b ∈ {18.5, 25, 30} obtained from model (4) are given in Table 1. The model was fitted to BMI observations categorized according to the WHO and to a different categorization with intervals of two BMI units (Int 1). Furthermore, numeric intervals taking rounding error into account (Int 2) and “exact” BMI values were used to estimate model (4). The approximation of the likelihood by the density was very accurate, as the estimated probabilities obtained from models estimated from numeric intervals taking rounding error into account (Int 2) and “exact” BMI values were very close. Differences occurred in the third decimal place if at all. Slightly larger differences were observed between numeric intervals (Int 2) and intervals obtained by categorization Int 1. The more extreme WHO categorization led to the largest differences in these estimated probabilities, but the results were still practically identical.\n\nFor baseline characteristics x, the probabilities obtained from model (4) for BMI ≤ 18.5, BMI ≤ 25, and BMI ≤ 30 are given for each combination of smoking and sex of the individual. The model was fitted using the likelihood (Lik) defined by BMI measurements categorized according to the WHO and according to a different categorization with intervals of two BMI units (Int 1). Numeric intervals taking rounding error into account (Int 2) and “exact” BMI values were used to estimate the model parameters. The differences between these four ways of evaluating the likelihood with respect to the estimated probabilities were marginal.\n\nIn addition to a comparison of the estimated probabilities, we also compared the proportional log-odds ratios β among the four BMI likelihoods (Table 2) and did not find relevant differences. The approximation of the likelihood based on the density resulted in odds ratios numerically almost identical to those obtained from numeric intervals that take the rounding error into account (Int 2). The odds ratios obtained with intervals of Int 1 differed more, but were still negligible. This also applied to the marginally less accurate odds ratios obtained from models fitted to BMI values categorized according to WHO criteria. It should be noted that the lengths of the confidence intervals between the four different BMI likelihoods were in line, which indicated that not only the estimated parameters β^ but also their estimated standard errors are comparable among the four approaches. The large sample size led to almost all odds ratios being significant. Age was associated with a shift towards larger BMI values, while higher alcohol intake was associated with marginally reduced BMI. Lower intake of fruits and vegetables as well as less physical activity also indicated a shift to higher BMI values. The BMI distributions of people with a higher education were shifted to the left compared to those of less well-educated people.\n\nThe odds ratios exp(β^) along with 95% confidence intervals for the covariates age (centered at 40 years), education, alcohol intake, fruit and vegetable consumption, physical activity, education, nationality, and region are given for the four ways of evaluating the likelihood of model (4), i.e.,, using BMI measurements categorized according to the WHO and according to a different categorization with intervals of two BMI units (Int 2), numeric intervals taking rounding error into account (Int 2), and “exact” BMI values.\n\nThe BMI values of people of the German-speaking part of Switzerland were higher than those of the French- and Italian-speaking regions.\n\nThe estimated conditional BMI distribution for all combinations of smoking and sex were clearly non-symmetric, and the impacts of smoking and sex of the individual related to changes in the mean and higher moments (distribution functions in Figure 1 and density functions in Figure 2). The BMI distribution shifted towards larger BMI values from males who never smoked to male former smokers. In this case, only the mean was affected; the shape of the distribution was constant. The BMI distribution of females who never smoked and female former smokers was similar to those of males. The difference between the two sexes could not be described by a simple shift because the shapes of the two distributions clearly differed. In general, the association of smoking and BMI was less pronounced for females than for males. Compared to the associations of sex (Figure 1 and Figure 2), the smoking associations were much smaller. We quantified the odds ratios of the smoking association for both sexes for the BMI categories. Table 3 presents the same information as the distribution functions evaluated with the BMI categories (gray vertical lines in Figure 1) on the odds ratio scale in a condensed form. The odds of lower BMI evaluated at BMI ∈ {25, 30} for male former smokers were smaller than for males who never smoked. The odds ratios for underweight and normal weight (BMI ≤ 25) and for non-obesity (BMI ≤ 30) increased for both males and females.\n\nFor each combination of smoking and sex, the conditional distribution function of BMI ℙ(BMI ≤ b | smk, sex, x) corresponding to model (4) was evaluated for baseline covariates x at all possible BMI values b. Red, female BMI distributions; blue, male BMI distributions; solid lines, BMI distributions of active smokers; dashed lines, never smoked; gray vertical lines, WHO categories 18.5, 25, 30. The model was fitted using “exact” BMI values.\n\nFor each combination of smoking and sex, the conditional density of BMI corresponding to model (4) was evaluated for baseline covariates x at all possible BMI values b. Red, female BMI distributions; blue, male BMI distributions; solid lines, BMI distributions of active smokers; dashed lines, never smoked; gray vertical lines, WHO categories 18.5, 25, 30. The model was fitted using “exact” BMI values.\n\nOdds ratios comparing all levels of smoking to the level never smoked for the events BMI ≤ 18.5, BMI ≤ 25, and BMI ≤ 30 obtained from model (4) were fitted to “exact” BMI measurements; 95% confidence intervals are given.\n\nFor current smokers, the odds ratio patterns that depended on BMI differed between males and females. All smoking levels were associated with larger odds of being underweight for females and had a U-shaped pattern. For males, this association was reversed and had an inverted U-shaped pattern. In the center of the BMI distribution (BMI ≤ 25), the odds ratios were much closer to 1 for both sexes. The odds ratios for non-obesity (BMI ≤ 30) for females indicated a trend towards smaller BMI values for current smokers. Except for heavy smokers, this effect was also found for males.\n\n\nDiscussion\n\nOur study showed that it was possible to analyze and compare BMI distributions in terms of standard parameters without the need of ad hoc categorization. Continuous BMI logistic regression, which avoided ad hoc categorization of BMI values, led to deeper insights into the impact of sex of the individuals and smoking status on the continuous BMI distribution. The model results were insensitive to BMI measurement scales or categorization schemes and matched previously reported findings on the impact of smoking and sex of the individuals on BMI. It was obvious from the conditional BMI densities (Figure 2) that more restrictive models, e.g., a conditional normal distribution with or without sex- and smoking-specific variance22, would describe the BMI distributions less accurately. The corresponding BMI-dependent odds ratios derived from continuous BMI logistic regression (Table 3) also indicated that a model that assumed proportional and thus BMI-independent odds would not be appropriate because odds ratios varied substantially as BMI cut-off points increased.\n\nWe used a parsimonious approach in defining covariate parameters and described the impact of the covariates on the BMI distribution as being linear on the log-odds scale. We therefore assumed that the covariate parameters would be the same in all binary or polytomous logistic regression models regardless of the ad hoc categorization applied. This corresponds to the proportional odds assumption in polytomous logistic regression models. In principle, this assumption could be relaxed by allowing BMI-dependent regression coefficients β (b), as in multinomial regression. Similar outcome-varying parameters are called time-varying parameters in survival analysis and distribution regression in econometrics23,24 and are a special case of conditional transformation models14. Ongoing research25 suggests that the assumption of a constant and sex-independent age effect for BMI is oversimplistic and conditional transformation models14,15, allowing BMI distributions to vary smoothly with age, might provide additional insights.\n\nFrom a practical point of view, one advantage of continuous outcome logistic regression is the possibility of evaluating the likelihood of BMI values obtained at different measurement scales or using different categorization schemes. This aspect allows the same model to be fitted to data obtained at different scales, and thus allows models from studies using different BMI measurement scales to be compared. The narrower the interval representing the BMI value for a particular individual, the more information is contributed by this individual to the likelihood. In contrast to the common procedure of downscaling all analyses by ad hoc categorization of BMI measurements to the ubiquitous WHO categories4,5, we propose to fit the same or even joint continuous BMI model to all studies by maximizing a likelihood with measurement-scale specific contributions. In subject-level meta analyses, these likelihood contributions are a mixture of exact, interval, or category-based BMI measurement scales. The likelihood can also be extended to incorporate study-specific left and right truncation when only individuals with BMI values in a pre-defined range are enrolled.\n\nOur findings on the association between smoking and BMI are consistent with the results of previous studies. It has been shown that former smoking is associated with being overweight as well as obesity, especially for males8,9,11,12,26. Other studies have also observed a positive association of male heavy smokers with obesity, although the association was non-significant when male heavy smokers were compared with males who never smoked8,9. By contrast, light and moderate smoking was associated with lower BMI values8,9. In general, current smoking is associated with lower BMI values12,27,28. These findings are consistent with previous findings on the effect of smoking on body weight29,30.\n\nWaiving the need for ad hoc categorization and thus also for agreement on standard categories that define the parameters in models for BMI distributions makes reported scientific results less dependent on these standard categories, and most importantly, less dependent on the WHO criteria. Considering that BMI distributions are subject to change at the population level over time2, insistence on the application of standards defined decades ago leads to an increasing discrepancy between models and data. Continuous BMI logistic regression is an attempt to narrow this gap.\n\n\nAppendix: Computational details\n\nThe intercept functions α(b)smk:sex for each combination of smoking and sex were estimated as smooth and monotonically increasing functions of b. The constraints expit(r(∞ | smk, sex, x)) = 1 and expit(r(0 | smk, sex, x)) = 0 restrict the BMI distribution on the positive numbers. For each of the ten strata given by the five smoking categories and two categories of sex, an intercept function was defined by six increasing parameters of a Bernstein polynomial31 of order five. This choice ensures smoothness and monotonicity and allows flexible intercept functions and thus regression functions r and conditional BMI distributions to be described by model (4). The monotonicity constrained on the intercept functions renders the addition of smoothing penalty terms to the likelihood unnecessary, because the effective number of parameters is less than the order of the Bernstein polynomial [see 15,32, for numerical experiments with varying numbers of parameters]. Simple maximum-likelihood estimation was performed for all model parameters simultaneously. When the likelihood was evaluated for BMI values in WHO categories, the sex- and smoking-specific intercept function was parameterized in terms of the step-function α(b) (see Formula (3)) defined for the proportional odds model. All computations were performed using R version 3.4.233. The mlt package32,34 was used to estimate continuous outcome logistic regression models. The underlying statistical theory is described in 15.\n\nA blueprint for the estimation of conditional BMI logistic regression using the mlt package in R, assuming the data are available in a data frame sgb with variables bmi (the numeric BMI values), smoking, and sex (smoking and sex as factors), as well as age and alcohol (numeric age and alcohol intake) with optional sampling weights weights, is\n\n\n\nContinuous outcome logistic regression, as a model for a continuous conditional distribution implemented in mlt, has a very strong connection to the Cox proportional hazards model, which describes the conditional continuous distribution of a survival time outcome with fully parameterized log-cumulative hazard function15,32. A Cox model for the conditional BMI distribution could be written as (see 35)\n\ncloglog(ℙ(BMI ≤ b | smk, sex, x)) = r(b | smk, sex, x).\n\nIn this case, the logistic link in (1) was replaced by the complementary log-log link. In the absence of covariates x, the results obtained from our continuous BMI logistic regression model and a Cox model stratified by sex and smoking would not be affected by this change, because for each combination of sex and smoking, a corresponding equivalent intercept function α(b)smk:sex (the sex- and smoking-specific log-cumulative hazard in the stratified Cox model) can be found on both the logit and cloglog scales. However, the interpretation of β changes from proportional log-odds ratios to proportional log-hazard ratios. In contrast to the partial likelihood of Cox models that treat the intercept functions as nuisance parameters, the likelihood for continuous outcome logistic regression is evaluated for fully parameterized intercept functions and all model parameters are estimated by maximum likelihood [similar to 36]. The corresponding monotonicity constraint allows smooth conditional distribution functions to be estimated without adding smoothing parameters to the likelihood15,32.\n\n\nData availability\n\nData from the Swiss Health Survey 2012 can be obtained from the Swiss Federal Statistics Office (Email: sgb12@bfs.admin.ch). Data is available for scientific research projects, and a data protection application form must be submitted. More information can be found here http://www.bfs.admin.ch/bfs/de/home/statistiken/gesundheit/erhebungenSupplementary",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nTL, SR and DF were supported by the Swiss Cancer Research foundation (grant no. KFS-3048-08-2012).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: BMI code.\n\nClick here to access the data.\n\n\nReferences\n\nWells JC, Fewtrell MS: Measuring body composition. Arch Dis Child. 2006; 91(7): 612–617. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNg M, Fleming T, Robinson M, et al.: Global, regional, and national prevalence of overweight and obesity in children and adults during 1980–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2014; 384(9945): 766–781. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: Obesity: Preventing and Managing the Global Epidemic. 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R Foundation for Statistical Computing, Vienna, Austria, 2015. Reference Source\n\nHothorn T: mlt: Most Likely Transformations. R package version 0.2-1, 2017. Reference Source\n\nDoksum KA, Gasko M: On a correspondence between models in binary regression analysis and in survival analysis. Int Stat Rev. 1990; 58(3): 243–252. Publisher Full Text\n\nMcLain AC, Ghosh SK: Efficient sieve maximum likelihood estimation of time-transformation models. J Stat Theory Pract. 2013; 7(2): 285–303. Publisher Full Text"
}
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[
{
"id": "27963",
"date": "29 Nov 2017",
"name": "Noora Kanerva",
"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 “Continuous outcome logistic regression for analyzing body mass index distributions” is a very well written paper. The issue is interesting and provides strong suggestion for future studies to consider continuous logistic regression instead of traditional logistic regression. I have only some minor questions/suggestions for the authors:\n\n1. The main research question was defined clearly in the end of Introduction section (association between smoking and BMI). However, it was less clearly explained why association between smoking and BMI was presented at different levels of sex in such detail in the results, but not the other covariates (e.g., consumption of fruits and vegetables)?\n\n2. In the discussion (page 8), the authors state “The corresponding BMI-dependent odds ratios from continuous BMI logistic regression (Table 3) indicated that a model that assumed proportional and thus BMI-independent odds ….”. Now, looking at the heading of the Table 3, it says “Estimated Non-proportional odds ratios for smoking”. Could you please clarify, whether the Table 3 presents results for proportional or non-proportional odds ratios?\n\n3. To demonstrate the difference between “traditional” and this “new” logistic regression approach, I would like to see also estimated odds ratios for traditional logistic regression with ad hoc categorization for this cohort. Would it be possible to add these results as supplemental material and discuss the differences between these results shortly? I understand that comparing these models was not the aim of this study, but still I think it would be interesting for the readers who are not familiar with this new approach to see this difference.\n\n4. In the discussion on page 9, the authors compare their results to previous studies. It would be also interesting to compare the magnitude of the association between smoking and BMI in their study and in the previous studies. If there are earlier studies from Switzerland or neighboring countries, are the results more similar with these compared to studies conducted in other, geographically more distant populations (e.g. USA, Asian countries)? Furthermore, it is not clear to me how the similarity of the results obtained using continuous logistic regression and traditional logistic regression should be interpreted. Is it a sign of validity of the new approach or does it imply that the traditional method is as good as the new one?\n\n5. Please add a paragraph about the possible limitations of the proposed new logistic regression to the Discussion section.\n\n6. What are the practical implications / future steps based on your findings? Should further studies move from traditional regression model to using this new approach or should the method still be tested in other data?\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": "27859",
"date": "07 Dec 2017",
"name": "Nikolaus Umlauf",
"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 \"Continuous outcome logistic regression for analyzing body mass index distributions\" presents a new approach for estimating conditional BMI probabilities, which avoids the necessity to specify ad hoc BMI categories/cutoff points, e.g., as defined by the World Health Organization. More precisely, the authors use recently developed conditional transformation models that directly model the distribution function of BMI outcomes and are therefore capable to estimate all moments of the response distribution simultaneously. Although computationally the model is quite complex, the authors show in numerous examples that probability statements can be easily obtained in a simple fashion, similar to commonly used models, e.g., when using ordered logistic regression models for BMI categories. The paper is excellently written and clearly shows how information loss can be avoided by the presented model in contrast to applying models onto ad hoc categorized BMI measurements. I only have minor points:\n* In section \"Likelihoods for BMI models\" the authors show how likelihood contributions are obtained using different measurement scales. This was immediately clear to me, however, in the next section the sentence:\n\n\"The model was fitted to BMI observations categorized according to the WHO and to a different categorization with intervals of two BMI units (Int 1).\"\n\nis confusing. What did the authors do exactly, please clarify.\n* In section \"Discussion\" the authors say that a conditional normal distribution with covariate specific variance would describe the BMI distributions less accurately. I agree, however, using distributions other than the normal might lead to equivalent results. It would be nice to add some more discussion on that.\n* In Table 2 there is a little typo, two commas \"i.e.,,\".\n\nRegarding the Data Availability, I think to ensure full reproducibility readers should also be able to download the data from F1000Research, that is why I answered \"NO\". However, this point is not too critical, since it is possible to obtain the data on request.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "27514",
"date": "22 Dec 2017",
"name": "Martyn Plummer",
"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 illustrates the methodology developed by Hothorn et al (20141, 20172) and implemented in the CRAN package mlt (most likely transformation). The methods are applied to a common epidemiological problem: estimating the effect risk factors for overweight and obesity measured in terms of body mass index (BMI). BMI is typically coded in categories determined by the World Health Organization (WHO), but ad hoc categories may also be used, and other encodings of BMI are possible (e.g. BMI recorded to a given numerical precision, or calculated exactly from the original height and weight data). The proposed continuous outcome logistic regression model is capable of handling all of these different data formats for BMI. This is illustrated using data from the Swiss Health Survey 2012. The BMI data from this survey are encoded in various ways and the results show consistency of results (table 1 and 2). With the exact encoding of BMI, the conditional distribution of BMI given baseline covariates is estimated from a flexible parametric model using Bernstein polynomials (Figure 1, 2) and odds ratios that depend on the cutpoints (referred to as \"non-proportional\" odds ratios) are also estimated.\nI have only minor comments on the manuscript\n1) Presentation of the non-proportional odds ratios is obviously more complex, but I wonder why a comparison across different encodings was not made for these odds ratios as it was for the risk factors assumed to have proportional odds ratios in Table 2.\n2) In Table 2, the results for the WHO encoding are somewhat different from the other encodings, which tend to be more consistent with each other. Why?\n3) In Table 2 , I would recommend different units for the continuous variables, as the per-unit odds ratios are attenuated towards 1 by the choice of scale, e.g. odds ratios for a 10-year age difference and a single unit of drink (12g in Switzerland, although 10g is more internationally comparable).\n4) The final paragraph of the discussion seems to imply that the WHO cutpoints will changes in the future as prevalence of overweight and obesity increases. This seems unlikely to me as these cutpoints are normative. For example, despite doubts raised about the utility of the current cutpoints in Asian popoulations, WHO recommends to continue to use them for international comparisons. Having said that, it is possible that the categories will become more detailed in the future as epidemiologists use the three classes of obesity (30-35, 35-59, 39+) and/or break down the \"normal\" category into two (18.5-23, 23-25).\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1933
|
https://f1000research.com/articles/6-1932/v1
|
01 Nov 17
|
{
"type": "Systematic Review",
"title": "The protective effectiveness of control interventions for malaria prevention: a systematic review of the literature",
"authors": [
"Thomas Kesteman",
"Milijaona Randrianarivelojosia",
"Christophe Rogier",
"Milijaona Randrianarivelojosia",
"Christophe Rogier"
],
"abstract": "Background: Thanks to a considerable increase in funding, malaria control interventions (MCI) whose efficacy had been demonstrated by controlled trials have been largely scaled up during the last decade. Nevertheless, it was not systematically investigated whether this efficacy had been preserved once deployed on the field. Therefore, we sought the literature to assess the disparities between efficacy and effectiveness and the effort to measure the protective effectiveness (PE) of MCI. Methods: The PubMed database was searched for references with keywords related to malaria, to control interventions for prevention and to study designs that allow for the measure of the PE against parasitemia or against clinical outcomes. Results: Our search retrieved 1423 references, and 162 articles were included in the review. Publications were scarce before the year 2000 but dramatically increased afterwards. Bed nets was the MCI most studied (82.1%). The study design most used was a cross-sectional study (65.4%). Two thirds (67.3%) were conducted at the district level or below, and the majority (56.8%) included only children even if the MCI didn’t target only children. Not all studies demonstrated a significant PE from exposure to MCI: 60.6% of studies evaluating bed nets, 50.0% of those evaluating indoor residual spraying, and 4/8 showed an added PE of using both interventions as compared with one only; this proportion was 62.5% for intermittent preventive treatment of pregnant women, and 20.0% for domestic use of insecticides. Conclusions: This review identified numerous local findings of low, non-significant PE –or even the absence of a protective effect provided by these MCIs. The identification of such failures in the effectiveness of MCIs advocates for the investigation of the causes of the problem found. Ideal evaluations of the PE of MCIs should incorporate both a large representativeness and an evaluation of the PE stratified by subpopulations.",
"keywords": [
"malaria",
"protective effectiveness",
"prevention and control",
"case-control studies",
"health surveys",
"insecticide-treated bed nets",
"indoor residual spraying",
"intermittent preventive treatment"
],
"content": "Introduction\n\nDuring the 2000s, several malaria control interventions have been largely adopted and scaled up in endemic countries. These interventions mainly include long lasting insecticidal nets (LLIN), indoor residual spraying (IRS), use of rapid diagnostic tests (RDT) to improve malaria diagnosis and artemisinin-based combination therapy (ACT) as first-line for the treatment of uncomplicated malaria, intermittent preventive treatment of pregnant women (IPTp), intermittent preventive treatment for infants (IPTi), and seasonal malaria chemoprevention (SMC). This considerable deployment of malaria control interventions has largely benefitted from the increase in international funding for the fight against malaria1.\n\nBefore being scaled up, the efficacy of control interventions needs to be demonstrated in controlled trials (phase III). These trials consist generally in randomizing individuals or clusters, half receiving the intervention tested, and the other half receiving a control, i.e. a placebo or the best intervention available. Both the intervention and the control are strictly delivered under monitored conditions, in order to preclude biases in the estimate of the efficacy. For any given intervention, especially life-saving interventions, a small number of trials are conducted, because once the efficacy or the superiority of the intervention has been demonstrated it would be unethical to keep providing a less effective intervention to the population, and also because of the important resources needed to conduct such trials. These studies are indispensable to ensure that people receive best interventions available but they poorly depict what will be the effectiveness of the intervention once deployed in real life, for two reasons: (i) phase III trials are conducted in a limited number of settings that don’t encompass all possible field conditions, and (ii) special attention is paid in delivering the intervention during controlled trials, thus rendering ‘ideal’ conditions.\n\nNational malaria control programs and donors should nevertheless make sure that the effectiveness of the interventions is confirmed once they have been deployed on the field. As a matter of fact, detecting suboptimal effectiveness of control interventions is critical for policy guidance. This is becoming particularly important since the global budget of the fight against malaria ceased growing1 and thus funding tends to be allocated for most effective interventions. The word “effectiveness” actually encompasses three concepts2,3: the coverage (does the intervention reach the population?), the individual protective effectiveness (does the intervention protect against or treat the disease?), and the community protective effectiveness (does the intervention benefit for others than the very ones who received the intervention?). Surveillance data and ecological studies can measure the overall impact of the control policy but they won’t be helpful in determining whether each intervention taken separately yielded the expected impact since all interventions are usually implemented concomitantly, and because they are influenced by environmental and social factors. Cross-sectional indicator surveys and social sciences studies will provide useful data regarding coverage of interventions and their determinants, but some data may be missing regarding the protective effectiveness (PE) of the interventions.\n\nAs mentioned above, it would be unethical - and laborious - to conduct controlled trials in all existing conditions and areas in order to verify the PE of malaria control interventions. Thus, alternative study designs must be applied. The PE can be evaluated (i) by biological studies measuring the bio-efficacy of drugs or insecticides (indirect measurement of the PE) or (ii) by epidemiological surveys yielding a direct measure of the PE of the intervention. The second approach encompasses three major study designs for phase IV assessment of the effectiveness of malaria control interventions, using historical non-compliant controls2: case-control surveys (CCS), cross-sectional surveys (CSS), and cohorts. The stepped-wedge design also allows for the evaluation of the PE, although it sometimes relates to the efficacy (phase III) when the implementation of the intervention is strictly overseen, and sometimes to the effectiveness (phase IV) when the intervention is implemented under field conditions. All these study designs share as a common drawback the possibility of biases: non-compliant controls are not strictly comparable to people having received and using the intervention. Adjusting for socio-demographic variables can reduce but not eliminate biases at the individual level, and adjustment variables can be challenging to identify and to quantify when it comes to the evaluation of the community PE (ecological bias).\n\nIn order to assess the disparities between efficacy and effectiveness of malaria control interventions, and the range of PE observed on the field, we conducted a systematic review of the literature on epidemiological studies providing data about the protective effectiveness of control interventions for malaria prevention (CIMP) under field conditions. This study also has as secondary objectives (i) to appreciate what were the study designs most used, (ii) which populations were most surveyed, and (iii) what was the representativeness of these studies.\n\n\nMethods\n\nThe PubMed database was searched for references using an algorithm (provided in Supplementary File 1) looking for (i) keywords related to malaria and (ii) keywords related to control interventions for prevention and (iii) keywords related to study designs that allow for the measure of the effectiveness. Bibliographies of the articles identified were also examined to find additional reports. The search was run for the last time on June 23, 2015.\n\nWe intentionally excluded efficacy studies such as controlled trials in the context of phase III assessment, and studies that aimed at measuring indicators such as coverage or factors associated with the uptake of interventions. Studies aiming at measuring the bio-efficacy of interventions using other methods than epidemiological, were also excluded from the database. The present study focuses on intervention for malaria prevention: LLINs, IRS, IPTp, SMC, IPTi, larval source management, and information, education and communication (IEC) campaigns. Given that the use of other insecticides than IRS, such as repellents or mosquito coils, have recently demonstrated an interest in preventing malaria4–6, their PE were also recorded. Articles presenting the effectiveness of IEC regarding prevention behaviours were included. Only articles in English, French, Dutch, or Spanish were considered.\n\nWe focused on two outcomes: (i) the measure of the PE against peripheral parasitemia, as measured by RDT and/or blood smears and/or PCR, and (ii) the measure of the PE against occurrence of acute clinical malaria. In studies having investigated other biological or clinical outcomes simultaneously, we recorded preferentially the two outcomes mentioned above. Whenever several PE results were available from a single study (e.g. for different subpopulations), all PE results were retrieved.\n\nOn the basis of the objectives of the study disclosed in the title, the abstract and the article, we determined whether the study was aimed at measuring the effectiveness of a CIMP or if this measure was done “accidentally”, e.g. for the purpose of controlling for other associations. The PE was defined as one minus odds ratio (OR) or one minus relative risk (RR), depending on the study design.\n\n\nResults\n\nOf the 1423 references retrieved, 523 were discarded on the basis of the title; 893 abstracts were checked and 683 of these didn't address the effectiveness of CIMP; seven abstracts could not be accessed (see flow diagram provided in Supplementary File 2). We thus identified 203 papers related to studies that aimed at measuring the effectiveness of CIMP or in which the effectiveness of CIMP was measured but 10 of them could not be accessed. One study was excluded because it focused on travellers and not resident populations of endemic areas; one reference was excluded because of the language; one study was excluded because it evaluated an intervention that was not particularly targeting malaria or mosquito control (cotrimoxazole in HIV positive pregnant women); two studies were excluded because it evaluated a CIMP out-dated (chloroquine chemoprophylaxis in pregnancy); 14 studies were excluded because no OR or RR value was presented and the data disclosed in the article didn’t allow for calculation of the PE of CIMP. Among the remaining 175 references, 13 presented methodological problems incompatible with the inclusion in the present review, such as absence of definition of cases or definition of the exposure to CIMP.\n\nThe final review included thus 162 studies. This included 133 (82.1%) studies on bed nets, 37 (22.8%) studies on IPTp, 25 (15.4%) studies on IRS, and 22 (13.6%) studies on other interventions (Figure 1). One third of the studies (52/162, 32.1%) addressed more than one CIMP. Regarding studies' design, 106 (65.4%) were CSS, 29 (17.9%) were CCS, 24 (14.8%) were cohorts, and 3 (1.9%) were stepped wedge (Figure 1).\n\nThe number of publications related to the PE of CIMP increased considerably during the years 2000’s and stagnated since 2010 (Figure 2). Two fifths (41.4%) of these studies were not directly aimed at measuring the PE of CIMP.\n\nThe last year is truncated since the search was performed in June 2015.\n\nRegarding the representativeness of the studies, two thirds (109/162, 67.3%) were conducted at the district level or below. Only one third of the studies that didn’t investigate the effectiveness of IPTp (43/125, 34.4%) included the whole population while the majority (71/125, 56.8%) included only children. Only 15 of these studies (12.0%) were conducted in the whole population and at a regional level (≥2 districts, province/region, or island) or above (national or multi-country).\n\nIn 18.1% (50/276) of the evaluations of PE retrieved or recalculated from the 162 studies included in the review, the association between the malaria outcome and the exposure to the CIMP was not adjusted on other variables (univariate logistic regression, two-by-two tables, etc). Since the adjustment on age is particularly important, we calculated the proportion of studies conducted in a population where the age of the oldest participants was ≥10 years older than the youngest, but for which no adjustment on age was done in the measure of the PE. We found that 38.9% of the studies conducted in such a population with heterogeneous age groups didn’t adjust the calculation of the PE for age.\n\nThe search retrieved 169 measures of the PE of bed nets in 133 studies (Supplementary File 3). Most of the time (82.2%), the exposure to bed nets was measured at the individual level, but in 23 cases (13.6%) it was done at household level (ownership or proportion of users) and in seven cases (4.1%) at cluster level. The majority of PE measurements involved insecticide-treated nets (ITN) or LLINs (42.0 and 12.4%, respectively) but in an important proportion of cases the definition of bed nets didn’t include impregnation or not (42.6%). In some instances (2.9%), the measurement was specifically done for non-impregnated bed nets (NIBN). Most PE evaluations used the Plasmodium infection as outcome (56.8%), especially CSS that accounted for 62.7% of study designs (Figure 3), or clinical malaria (31.9%), especially CCS that accounted for 20.1% of study designs; some used an obstetrical outcome (7.1%), and a few ones used the mortality as outcome (4.1%). Cohorts represented 15.4% of study designs and there were only three stepped-wedge designs (1.8%). More than a half of PE results (58.0%) were obtained from paediatric populations and 27.8% considered the whole population; the other studies (14.2%) were conducted on women of childbearing age.\n\nMost of the results (60.7%) demonstrated a significant PE from bed nets use. However, 38.2% of results were not significant (Figure 4, Figure 5 and Supplementary File 3). In 14.2% of cases, the PE value was negative, i.e. a trend towards a risk increased, and 1.2% of results showed a risk significantly increased. The median PE was 36.0% (interquartile range [IQR] 14.0–54.0%), and this differed only marginally according to CIMP definition: median PE was 39.8% (IQR 20.2–50.3%) for LLINs, 30.0% (IQR 16.5–52.0%) for ITNs, 51.0% (IQR 35.0–51.0%) for NIBNs, and 36.0 (IQR 13.8–54.6%) for bed nets without further precision of their impregnation.\n\nResults without CI are not displayed. Box size is proportional to the sample size.\n\nResults without CI are not displayed. Box size is proportional to the sample size.\n\nThe search retrieved 32 measures of the PE of IRS in 25 studies (Supplementary File 4). CSS survey design was largely predominant (90.6%) and three PE evaluations from CCS (9.4%) were observed. A third of studies (34.4%) considered the whole population while 59.4% were obtained from paediatric populations and 6.3% from women of childbearing age. Most of the time (78.1%), the exposure to bed nets was measured at the household level, but in seven cases (21.9%) it was measured at cluster level. Only 21.9% of PE measurements of IRS considered recent spraying (≤6 months before the survey or delay since last IRS round in months), and the rest considered IRS ‘last round’, ‘last year’, or even ‘ever’. Most PE evaluations used the Plasmodium infection as outcome (87.5%) and the rest considered clinical malaria (12.5%).\n\nHalf of results demonstrated a significant PE of IRS (median 28.5%, IQR 8.8–47.3%), but 43.8% of results were not significant and 6.2% of results showed a risk significantly increased (Figure 6 and Supplementary File 4). The PE value was positive in more than three results out of four (78.1%). Median PEs were comparable when considering recent (20.0%, IQR -2.5–41.0%) or older spraying (32.0%, IQR 9.0–46.0%).\n\nBox size is proportional to the sample size. Recent spraying: ≤6 months before the survey or delay since last IRS round in months.\n\nOur systematic search allowed us identifying only five studies and eight results about the PE of concurrent exposure to ITN and IRS (Table 1). Two results compared the exposure to both interventions versus IRS only, and the six other compared the exposure to both interventions versus no intervention. All study designs were CSS and all evaluated the effectiveness of ITN or LLIN against infection in children.\n\n*: Indicates significant result as compared with exposure to one CIMP only. MC: Multi-country study stratified by transmission (low-medium-high), the number between brackets indicates the number of countries. †: PE versus IRS only. ‡: PE versus no ITN and no IRS.\n\nFour out of eight results demonstrated a significant added PE of using both interventions as compared with one of these two CIMP only; in these studies (or sub-studies) ITN and IRS alone had both demonstrated significant PE –the PE of IRS in the study of Rehman et al. is borderline. In the other four (sub-)studies, one of the two CIMP had failed to demonstrate a significant PE and the exposure to both interventions either showed an added protection but non-significant as compared with one CIMP only or provided a PE inferior to the PE of IRS only.\n\nOur search retrieved 40 measures of the PE of IPTp using sulfadoxine-pyrimethamine (SP) in 37 studies (Supplementary File 5). Among these 40 results, 16 (40.0%) compared any regimen versus no SP dose, 13 (32.5%) compared the standard regimen versus no IPTp, and the remaining 11 (27.5%) compared the standard regimen versus substandard regimen. Most PE evaluations used an obstetrical or neonatal (e.g. low birth weight) outcome only (45.0%) or an outcome considering an obstetrical event or a maternal peripheral parasitemia (7.5%). The detection of Plasmodium in the mother’s blood was used as the outcome in 37.5%; three results (7.5%) had evaluated clinical malaria, and one used the mortality as outcome (2.5%). CSS represented 85.0% of study designs, cohorts 10.0% and there were only two CCS (5.0%). Most results (90.0%) were obtained from mothers, usually pregnant women at antenatal consultation and/or women at delivery units, but 10.0% considered paediatric populations (neonates or infants). The vast majority of studies were conducted at the district level or below (85.0%).\n\nMost results demonstrated a significant PE of IPTp (median PE 49.0%, IQR 23.0–67.3%), but 32.5% of results were not significant and 5.0% of results showed a risk significantly increased (Figure 7 and Supplementary File 5). Median PE was 24.7% (IQR 4.0–70.0%) in studies evaluating standard IPTp regimen versus no IPTp, 50.5% (IQR 30.0–65.0%) in studies comparing any IPTp regimen versus no IPTp, and 50.0% (IQR 35.8–65.4%) in studies evaluating standard IPTp regimen versus substandard IPTp regimen. These values were comparable between studies evaluating the PE of IPTp against infection (median PE 43.0, IQR 7.0–78.5%) and studies evaluating the PE of IPTp against obstetrical outcomes (median PE 53.5, IQR 33.3–66.2%).\n\nBox size is proportional to the sample size.\n\nOur systematic search identified 20 evaluations, from 16 studies (Supplementary File 6), of the PE of the use of other insecticides than IRS, including coils (45.0%), sprays (30.0%), and repellents (10.0%). In the remaining 15.0% of cases, the PE of two or three of these formulations together was evaluated. The vast majority of these results came from CCS evaluating clinical outcomes (15/20, 80.0%), and the four other results came from CSS evaluating clinical (1/20) infection (1/20), or obstetrical (2/20) outcomes. The majority of these studies were conducted in paediatric populations (65.0%), some in the whole population (20.0%) and the remaining 15.0% among adult women.\n\nOverall the PE of these insecticides was demonstrated in only four studies, whatever the formulation in coils, sprays, or repellents, and most (70.0%) results were non-significant (Figure 8 and Supplementary File 6). The median PE was 19.1% (IQR -21.0–38.5%).\n\nBox size is proportional to the sample size.\n\nOur search retrieved only one study aiming to evaluate the PE of IPTi. It was a CCS conducted among infants in Tanzania and its main result was that the PE against occurrence of clinical malaria cases was 18% and not significant (95% CI -129–71%165).\n\nWe identified two studies evaluating the PE of larviciding programs by comparing clusters receiving the intervention and clusters that were not treated, either in a CSS design applied in under-fives38 or in a stepped-wedge design encompassing all age groups43. Both studies showed a significant PE of larviciding against infection by Plasmodium of 72% (20–90%) and 21% (7–34%) respectively.\n\nIn this review, no study assessing the PE of IEC interventions on malaria indicators has been found, but we found two countrywide CSS evaluating the effectiveness of the exposure to IEC programs on bed net (or ITN) use. One was conducted in adult population of Cameroon166 and the other one in adult women of Zambia167. These two studies showed that being exposed to IEC interventions was associated with an increase in bed net use (OR 1.48, 95% CI 1.18–1.86, and OR 1.62, 95% CI 1.28–2.04, respectively) by logistic regression with propensity score matching.\n\nFinally, we found one study having evaluated the PE of availability of a village health worker trained for malaria management against Plasmodium infection through a CSS conducted among all age groups in a province of the Philippines27. They found a significant PE of 74% (16–92%).\n\n\nDiscussion\n\nThis review showed that the efforts made for the evaluation of the effectiveness is increasing with time, in parallel with the global funding available for malaria control. Nevertheless, the number of published studies about the effectiveness of CIMPs seems to be stagnating since 2010. This could hinder the progress towards more cost-effective control policies, as the strategy should be locally adapted depending on data about the effectiveness of CIMP.\n\nOverall, there is a sense of a low representativeness of the studies. Only one third of the studies were conducted at a large scale, and only one third included all ages and genders; only one out of eight had both features. Several CIMPs target the whole population of a region or a country, e.g. IRS or universal distributions of LLINs. When evaluating the PE of such CIMP, it’s crucial not to leave aside a part of the population since the effectiveness of CIMP may vary depending on transmission or between age groups for example67,168,169. On the contrary, nearly 40% of studies conducted across age groups didn’t include the age in regression models while age influences both malaria outcomes (e.g. probability to be infected) and CIMP coverage170. In order to yield unbiased evaluation of the PE of a CIMP, it is critical to adjust the measure of the association of malaria outcome and exposure to CIMP for age, as well as for other variables known to influence the outcome (e.g. socio-economic status, parity, rural or urban area) and to take into account the intra-cluster correlation in multi-stage sample designs. Moreover, several studies conducted at a large scale didn’t stratify the analysis. Since local features of malaria transmission or cultural behaviours may affect (or enhance) the PE of CIMPs, omitting stratified analysis precludes the identification of clusters where the effectiveness of CIMPs was suboptimal.\n\nOn the other hand, the multiplicity of local evaluations of the PE of CIMP offers an appreciation of the diversity of local conditions. Certain studies revealed that the PE was largely above what had been demonstrated in efficacy trials; this can result from biases inherent to observational studies, but it’s also possible that local conditions favour the effectiveness of CIMP, e.g. the PE of LLINs is expected to be especially high where vector populations exclusively bite indoor and late at night. Conversely, many studies failed to demonstrate the PE of CIMP studied or showed that it was lower than expected. This is where the interest of these surveys stands, for it urges policy makers and their research partners to investigate the causes of this failure and to propose alternative control interventions. This is also why we didn’t conduct a meta-analysis on the data retrieved in this review. Besides this, various meta-analyses of CIMP already exist, either reviewing efficacy studies only169,168,171, or mixed both efficacy and effectiveness studies172.\n\nThis review presents several limitations, including the search in one database only, the limits in languages considered (although one reference only was discarded for this reason), and the incomplete access to articles. This review is thus probably not exhaustive but it intends to be largely representative of effectiveness studies. On purpose, we didn’t include meta-analyses of the studies included as the overall objective was to get a sense of what kind of studies had been done, and to be strictly descriptive on the results obtained. Our take-home message is not so much that MCI are effective on average, but that their effectiveness might be locally lower -or higher- than what is expected by efficacy studies.\n\nOverall, the measures of the PE of bed nets demonstrated a fair effectiveness of this CIMP, even often above the protective efficacy measured in controlled trials. This phenomenon can be attributed to local features of malaria transmission (e.g. intensity of transmission, vector biting behaviour, vector sensitivity to insecticides, or human behaviour), or to differences in outcomes used in efficacy trials (often clinical outcomes) versus those used in effectiveness studies (often the infection by Plasmodium parasites), or to differences in the definition of the exposure to bed nets. For example, it has been shown that in low transmission areas LLIN perform better and/or parasitemia is a better indicator of LLIN performance37,168. Controlled trials performed in areas of high transmission (e.g. two meta-analyses of studies conducted in such areas showed protective efficacies of 13%168 and 17%173, respectively) have shown lower protective efficacy than those conducted in low transmission areas, e.g. in Kenyan highlands (protective efficacy 63%,174) or in Pakistan (protective efficacy 43%,175).\n\nVarious definitions of bed net exposure have been used throughout the studies included in the present review: type of bed net (LLIN, ITN, bed net without further definition of impregnation but sometimes in areas where most bed nets are actually impregnated, or NIBN), intensity of exposure (ownership, bed net/person ratio, use the previous night, or regular use), and level of measure of exposure (individual, household, cluster). Surprisingly, in our review, it seems that the definition of exposure to bed nets does not impacts importantly the measure of the PE. Therefore, it is possible that evaluations of the PE of LLINs or ITNs yield an estimation of the effectiveness provided by the physical barrier against vectors’ bites and underestimate the community effect offered by insecticides impregnation.\n\nEffectiveness studies generally verified the PE of IRS, whether at community or household level. It’s complicated to compare those results with efficacy studies since those are relatively scarce. Indeed, IRS has been deployed before the requirement of a demonstration of efficacy of MCI through randomized controlled trials. A meta-analysis from 13 efficacy and effectiveness studies conducted in 11 countries measured a pooled household-level and community-level protective efficacy of 62%172, but other controlled trials showed more limited protective efficacy at the community level, e.g. in India where it was 28%169,176 and in Nigeria during the wet season where it was 26%169,177. Controlled trials even sometimes showed very limited efficacy like in Nigeria during the dry season or in Tanzania (protective efficacy 6%169,177,178).\n\nAs for bed nets, the vectors’ biting behaviour, their sensitivity to insecticides, and the endemicity of malaria are expected to influence most the PE of IRS. These factors should be investigated in areas where IRS fails to demonstrate its effectiveness in order to guide local malaria control policies.\n\nOverall effectiveness studies plea for the combination of these two vector control interventions since it seems that all studies finding significant PE of the two CIMP separately also found a significant added PE in people benefitting from both CIMP simultaneously. On the contrary, in studies where at least one CIMP failed to demonstrate a significant PE, the added value of using LLIN in a household having received IRS also failed to be proven. Results from randomized controlled trials are more balanced: some did show an additional protection offered by IRS over LLIN only179,180, some did not181–183. Overall, evidence of additional protection of the combination against malaria remains inconclusive184.\n\nMost studies aiming to evaluate the effectiveness of IPTp were conducted at small scale, usually in one or two hospitals. Nevertheless, some studies were conducted at a larger scale and even stratified by regions, e.g. a study conducted in 3 regions of the Democratic Republic of Congo showed that, in one regions, the effectiveness of IPTp against low birth weight was affected while it was preserved in the 2 other regions161. Geographical stratification can thus detect an inhomogeneity in the PE that can reflect, for example, local parasitological resistance to SP. This resistance is the major cause to be investigated for policy guidance.\n\nAn important limitation of the present review is that IPTp aims at reducing malaria burden in terms of maternal and neonatal morbidity and mortality; obstetrical outcomes are therefore more adapted for the evaluation of the PE of IPTp than maternal peripheral parasitemia or acute clinical episodes of malaria –that we prioritized in our review. Similarly these outcomes were often considered as secondary in efficacy controlled trials. Nevertheless one meta-analysis of three studies conducted in two countries measured a pooled protective efficacy of IPTp against maternal peripheral parasitemia of 55%185 and another trial demonstrated a protective efficacy of 64%186. Besides this, the efficacy of IPTp has been evaluated against several obstetrical outcomes, including low birth weight (significant protective efficacy of 29%185), placental parasitemia (significant protective efficacy of 52%185), maternal anaemia (significant protective efficacy of 10%185), perinatal mortality (non-significant protective efficacy of 22%187), or stillbirth (non-significant protective efficacy of 4%187).\n\nThe PE of the use of insecticides was seldom demonstrated, despite the possibility of a socioeconomic bias that would be expected to increase their PE. Further studies will have to be conducted in order to verify that their efficacy translate into effectiveness under field conditions if they are adopted by policy makers.\n\nOur review identified no study having tried to evaluate the effectiveness of IEC interventions against clinical or biological malaria indicators, and only two that demonstrated the effectiveness of IEC programs on bed net coverage. Unfortunately, the uniqueness of media messages and cultural features in these studies preclude the extrapolation of their results. Generally few studies have evaluated the effectiveness of IEC intervention, not only for malaria188. This reflects the rarity of phase III studies aiming at demonstrating the efficacy of IEC interventions on epidemiological indices. This paucity of information is surprising given the popularity of IEC programs in public health.\n\nIn 2013, IPTi had been adopted by one country only1, which explains that we found only one study evaluating its PE. More surprisingly, SMC has been adopted by six countries1 but the PE of this CIMP has not been evaluated yet. The small number of studies regarding SMC, IEC or larviciding hinders the interpretation of these results.\n\n\nConclusions\n\nThis review shows that there is an increasing interest in measuring the PE of CIMPs. Most studies confirmed the PE of the CIMPs that they were evaluating, but an important part yielded a ‘negative’ PE and/or non-significant confidence interval. In this case, complementary investigations are needed in order to confirm the existence of a problem in the effectiveness of the CIMP and to propose alternative control measures if necessary.\n\nA frequent feature of the studies included in this review was the low geographical representativeness and/or the low representativeness in the population studied. Conversely the analyses of large samples were not systematically stratified by subpopulations. We believe that such investigations need to zoom out (encompass a large population) and to zoom in (stratify by subpopulations) to get a complete picture evaluating the effectiveness of CIMPs.\n\nTo evaluate properly the PE of a CIMP we recommend to pay attention to the following points: (i) encompass all age groups and genders, except for targeted interventions such as IPTp or IPTi, (ii) sample all geographical and/or cultural patterns, (iii) stratify the evaluation of the PE by subgroups, (iv) adjust for socio-demographic variables that are associated with the outcomes and at least adjust for age and gender if the population sampled is not homogeneous in this regard.\n\n\nAbbreviations\n\nACT: artemisinin-based combination therapy, CCS: case-control survey, CSS: cross-sectional survey, CIMP: control intervention for malaria prevention, IEC: information, education and communication, IPTi: intermittent preventive treatment for infants, IPTp: intermittent preventive treatment of pregnant women, IQR: interquartile range, ITN: insecticide-treated nets, IRS: indoor residual spraying, LLIN: long lasting insecticidal nets, NIBN: non-impregnated bed nets, RDT: rapid diagnostic tests, SMC: seasonal malaria chemoprevention; SP: sulfadoxine-pyrimethamine\n\n\nData availability\n\nDataset 1: Data underlying the results presented in this systematic review. DOI, 10.5256/f1000research.12952.d182415189.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by the Institut Pasteur de Madagascar.\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 Institut Pasteur de Madagascar, and in particular the Malaria Research Unit, for their helpfulness and their sympathy.\n\n\nSupplementary material\n\nSupplementary File 1: Algorithm for the systematic review of the literature.\n\nClick here to access the data.\n\nSupplementary File 2: PRISMA flow diagram.\n\nClick here to access the data.\n\nSupplementary File 3: PE of bed nets.\n\nClick here to access the data.\n\nSupplementary File 4: PE of IRS.\n\nClick here to access the data.\n\nSupplementary File 5: PE of IPTp.\n\nClick here to access the data.\n\nSupplementary File 6: PE of domestic use of insecticides.\n\nClick here to access the data.\n\nSupplementary File 7: PRISMA checklist.\n\nClick here to access the data.\n\n\nReferences\n\nWHO Global Malaria Programme: World Malaria Report 2014. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nDickinson KL, Randell HF, Kramer RA, et al.: Socio-economic status and malaria-related outcomes in Mvomero District, Tanzania. Glob Public Health. 2012; 7(4): 384–399. PubMed Abstract | Publisher Full Text\n\nAbdella YM, Deribew A, Kassahun W: Does Insecticide Treated Mosquito Nets (ITNs) prevent clinical malaria in children aged between 6 and 59 months under program setting? J Community Health. 2009; 34(2): 102–12. PubMed Abstract | Publisher Full Text\n\nMacedo De Oliveira A, Mutemba R, Morgan J, et al.: Prevalence of malaria among patients attending public health facilities in Maputo City, Mozambique. Am J Trop Med Hyg. 2011; 85(6): 1002–1007. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYamamoto SS, Louis VR, Sié A, et al.: The effects of zooprophylaxis and other mosquito control measures against malaria in Nouna, Burkina Faso. Malar J. 2009; 8: 283. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoosihermiatie B, Nishiyama M, Nakae K: The human behavioral and socioeconomic determinants of malaria in Bacan Island, North Maluku, Indonesia. J Epidemiol. 2000; 10(4): 280–289. PubMed Abstract | Publisher Full Text\n\nSharma PK, Ramanchandran R, Hutin YJ, et al.: A malaria outbreak in Naxalbari, Darjeeling district, West Bengal, India, 2005: weaknesses in disease control, important risk factors. Malar J. 2009; 8: 288. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeressa W, Ali A, Berhane Y: Household and socioeconomic factors associated with childhood febrile illnesses and treatment seeking behaviour in an area of epidemic malaria in rural Ethiopia. Trans R Soc Trop Med Hyg. 2007; 101(9): 939–947. PubMed Abstract | Publisher Full Text\n\nHaque U, Glass GE, Bomblies A, et al.: Risk factors associated with clinical malaria episodes in Bangladesh: a longitudinal study. Am J Trop Med Hyg. 2013; 88(4): 727–732. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nKurth F, Bélard S, Mombo-Ngoma G, et al.: Adolescence as risk factor for adverse pregnancy outcome in Central Africa--a cross-sectional study. PLoS One. 2010; 5(12): e14367. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSintasath DM, Ghebremeskel T, Lynch M, et al.: Malaria prevalence and associated risk factors in Eritrea. Am J Trop Med Hyg. 2005; 72(6): 682–687. PubMed Abstract\n\nMauny F, Viel JF, Handschumacher P, et al.: Multilevel modelling and malaria: a new method for an old disease. Int J Epidemiol. 2004; 33(6): 1337–1344. PubMed Abstract | Publisher Full Text\n\nGies S, Coulibaly SO, Ouattara FT, et al.: Individual efficacy of intermittent preventive treatment with sulfadoxine-pyrimethamine in primi- and secundigravidae in rural Burkina Faso: impact on parasitaemia, anaemia and birth weight. Trop Med Int Health. 2009; 14(2): 174–82. 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}
|
[
{
"id": "27508",
"date": "13 Nov 2017",
"name": "Immo Kleinschmidt",
"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 piece of work that examines the effectiveness of malaria control interventions in programmatic, non-research settings. Whilst such studies do not deliver the same quality of evidence as randomised trials, they provide important insights, and their sheer volume provides an understanding that we do not get from efficacy trials. This is particularly important for interventions like IRS, which have a wealth of programmatic evidence of effectiveness, but a dearth of randomised trials demonstrating impact. To see this body of work brought together and systematically synthesised in this paper is a great achievement indeed.\nStudies such as the ones that are included in this review are by their very nature somewhat messy and subject to bias and confounding. They therefore need to be interpreted with caution, and in relation to the available trial evidence, where this exists. This has largely been done in this review.\nPerhaps a bit more could have been said about adjustment for confounding other than by age, and the discussion could have mentioned the strong possibility of publication bias against non-significant findings. One group of studies that have not been included here are studies that compare post intervention outcomes with pre-intervention outcome in the same population. These studies are of course also methodologically flawed, but given the nature of this review I would have thought that some should have been eligible. This is particularly true for IRS studies where it is difficult to obtain measures of effect from cross-sectional surveys because the intervention acts at a community level, and because non-randomised contemporaneous control communities are as problematic as the before versus after design. Some studies have recorded such large changes in outcomes after the introduction of IRS, that it is unlikely that this is not associated with the intervention. Studies which represent an interrupted time series (for example Sharp et al, 2007) are particularly convincing in this regard. To me this review seems somewhat incomplete without their consideration. However, I fully accept that the authors may have had good reason for the exclusion of such studies from this review.\n\nA point to add to the discussion is the possibility of the impact of insecticide resistance on effectiveness, particularly in the case of IRS. A minor point is to use the term ‘susceptibility’ (of vectors to insecticide), rather than ‘sensitivity’ (presumably a translation issue).\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": "27509",
"date": "24 Nov 2017",
"name": "Michel Cot",
"expertise": [
"Reviewer Expertise Epidemiology",
"tropical diseases",
"mother and child health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis review reports on the effectiveness of malaria prevention interventions, worldwide. Whereas the subject of the efficacy of malaria control measures has been extensively assessed, the originality of this paper is to deliberately exclude efficacy studies (eg, randomized trials) and to focus on the protective effectiveness (PE) provided by control interventions under field conditions, such as case-control, cross-sectional surveys or cohort studies that are usually left aside. The context and the objectives are clearly laid out, and the methods are described in detail. Also, this considerable work addresses two important difficulties which have to be overcome, the high frequency of biases in non controlled designs, and the representativeness of such highly heterogeneous studies. Indeed, they relate to very different interventions or populations (especially age groups which are adequately adjusted for, although they should not be the only ones), rendering the overall effect on PE difficult to interpret, so the cautious discussion on the final results is particularly precious.\n\nMy major concern is that the search does not include references published after June 2015. In particular, the statement (Results, 3rd paragraph) that the number of publications related to the PE of interventions decreased since 2010, considering that some major studies were published in the last two years, is not true when performing a quick search in the databases. Some important results have been published in the period 2015 - 2017, such as a study on malaria prevention measures on six Sub-Saharan African countries by Drakeley et al.1; a complete issue of the American Journal of Tropical Medicine and Hygiene (2017, 97, issue 3 Suppl) with reports from several East African countries; a study on IRS in Northern Uganda2. All these studies should be included in the review, as they may change the results (or at least, significantly contribute). Besides, the final paragraph of the discussion claims that the PE of SMC (seasonal malaria control) implemented in six African countries has not been evaluated. It was probably true in 2015, but the evaluation has been done since, by Diawara et al. 3 in Mali, Cissé et al.4 in Senegal, Tagbor et al.5 in Ghana. These important results must definitely be included in the review, which would be incomplete otherwise.\n\nOn the nature of the interventions that have been included, I agree that IPTp is a major intervention in the prevention of malaria and that IPTp studies in pregnant women should not be excluded from the review. However, the main outcome is generally different from parasitological or clinical indicators found in other studies, as it mainly consists in the measurement of the baby's birthweight (and the proportions of LBW babies). This particular outcome appears in the supplementary file 5, it should be more clearly specified in the text (results, and particularly in the methods where only two outcomes are mentioned).\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": "27512",
"date": "07 Dec 2017",
"name": "Christian Lengeler",
"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 systematic review by Kesterman et al. is both timely and important. Measuring effectiveness of health interventions is important and increasingly done. Malaria control is fortunate to have many such studies done, especially with the strong development of secondary data analysis since the year 2000. But as shown very well by this review, the studies differ widely in their study designs, settings, age groups, and a myriad of other parameters. When reading publications on such studies it is often difficult to see the significance or generalizability of an individual study. With the present review, Kesterman et al. contribute greatly to show the range of studies and their results, and put some order in that vast body of information. By and large this is an excellent paper, well designed, researched and written up. The figures in particular are excellent.\nI have only one major issue, which is that their literature search stopped in 2015. For a paper submitted in November 2017, this is not really acceptable. Given that the analysis of the data is limited, an additional search and addition of the newest studies to the paper should not represent an enormous investment in time, and I would strongly suggest that this be done.\nFurther, I have a number of small points, relating largely to the interpretation of the data and they are listed below:\n1. One more general reference on the difference between efficacy and effectiveness would be useful in the Introduction, at the end of para 2.This would help to clarify the difference for readers less familiar with these concepts. I have listed a few possible references below.\nJP Habicht et al. 1999. Evaluation designs for adequacy, plausibility and probability of public health programme performance and impact. International Journal of epidemiology 28; 10-18.\nCG Victora et al. 2004. Evidence-based public health: moving beyond randomized trials. American Journal of Public Health 94 (3), 400-405.\nTanner M, Lengeler C, Lorenz N. Case studies from the biomedical and health systems research activities of the Swiss Tropical Institute in Africa. Trans R Soc Trop Med Hyg. 1993 Sep-Oct;87(5):518-23.\n2. Discussion para 1: The number of studies on effectiveness is stable since 2010, but at a high level. Malaria control interventions are really very well documented in comparison to many other health interventions. So the rather negative formulation that this could “hinder progress” is not really justified.\n3. Discussion para 3: a little more discussion on the risk of bias, which is substantial in the reviewed studies, would be useful.Certainly, the fact that some studies show a significantly increased risk (for which it is hard to find plausibility) is a good indication of how strong the bias can be.\n4. Discussion para 3: I agree in principle that these studies should guide local control activities. But given the risk of bias mentioned above, this should be really careful and guided by circumstances.\n5. Finally, in the discussion it would be good to re-visit briefly the point made in the introduction (3rd para) that surveillance and ecological studies also provide data on the effect of malaria control. While it is to some extent correct that these tools look at the overall effect of different interventions and attribution is difficult, in practice in many settings only one preventive tool is applied with a high coverage - usually LLINs or IRS. IPTp, case management and other interventions contribute probably little to impacting transmission, so surveillance can actually be a useful way to triangulate the results from specific studies.\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/6-1932
|
https://f1000research.com/articles/6-1929/v1
|
01 Nov 17
|
{
"type": "Study Protocol",
"title": "Development of a deep neural network derived from contours defined by consensus-based guidelines for automatic target segmentation in hepatocellular carcinoma radiotherapy: A study protocol",
"authors": [
"Jiandong Zhao",
"Jiazhou Wang",
"Mingxia Cheng",
"Jiandong Zhao",
"Jiazhou Wang"
],
"abstract": "Hepatocellular carcinoma (HCC) is a leading cause of cancer death in China and around the world. Tumoricidal doses of modern radiation therapy (RT) can now be safely delivered with excellent local control and minimal toxicity. Delivering adequate doses of radiation to the primary tumor, while preserving adjacent healthy organs, depends on accurate target identification. In recent years, different novel machine learning techniques, including artificial intelligence technology, have been exploited in RT with impressive results in automatic image segmentation. If the machine learning algorithms are trained on delineated contours, according to consensus contouring guidelines, it promises greatly reduced interobserver and intraobserver variability in target delineation, thus substantially improving the quality and efficiency of HCC radiotherapy. This study protocol proposes to develop a fully-automated target structure contouring system, which is based on deep neural networks trained on contours delineated according to consensus contouring guidelines in HCC radiotherapy. In addition, the study will evaluate the contouring system’s feasibility and performance during application in normal clinical operations. The study is ongoing (data analysis).",
"keywords": [
"hepatocellular carcinoma",
"radiation therapy",
"artificial intelligence",
"automatic target segmentation",
"deep neural network"
],
"content": "Introduction\n\nHepatocellular carcinoma (HCC) is the third leading cause of cancer death in China (estimated 422,100 deaths in 2015) (Chen et al., 2016), and the second leading cause of cancer-related death worldwide (approximately 745,000 deaths per year) (Siegel et al., 2015), with China's deaths accounting for over 50% of cases. The incidence of HCC is on the rise due to viral hepatitis (hepatitis B and C virus), alcohol use, and nonalcoholic fatty liver disease (Hemming et al., 2016).\n\nOnly a small group of HCC patients are eligible for liver transplant or resection, for the majority of HCC patients ineligible for surgery, radiation therapy (RT) has been playing an increasingly important role in achieving acceptable local control (Krishnan et al., 2008; Ren et al., 2011; Zhao et al., 2008), especially with the recent advances in external beam RT technique, including 3-dimensional conformal RT, intensity modulated RT, stereotactic body RT, proton beam RT, and/or image-guided RT with respiratory motion management (Bujold et al., 2013; Yeung et al., 2017). Tumoricidal doses of modern RT can now be safely delivered with excellent local control and minimal toxicity. RT has thus become a recommended option in some international consensuses and current clinical practice guidelines (Park et al., 2016; Rim & Seong, 2016).\n\nAccurate target delineation in HCC RT is essential to deliver adequate doses of radiation to the primary tumor, while preserving adjacent healthy organs. Target delineation is a complex part of RT planning, and manual segmentation is the most common way in clinical practice and research, but manual segmentation is tedious and time-consuming. Besides that, the interobserver variability in target delineation of HCC gross target volume (GTV) is noteworthy (Kim et al., 2016).\n\nRecently, a clear consensus on how to delineate GTV in radiotherapy for HCC was recommended by the Radiation Therapy Oncology Group (RTOG) (Hong et al., 2014). For organs at risk (OARs) in the upper abdominal region, a contouring consensus was also proposed by RTOG, as well to improve the quality and consistency of contouring uniformity in radiation oncology (Jabbour et al., 2014).\n\nIt is believed that, other than the use of guidelines, provision of autocontours shows promise to reduce the interobserver and intraobserver variability in target delineation, thus improving the quality of RT (Vinod et al., 2016). Moreover, auto-segmentation also has the potential advantage to greatly reduce contouring time.\n\nIn recent years, different novel machine learning techniques, including artificial intelligence technology, have been exploited in RT with impressive results in image segmentation. Machine learning may mimic labor intensive tasks, for example, target delineation via complex computational algorithms trained using the input from an expert radiation oncologist (Chu et al., 2016; Trebeschi et al., 2017).\n\nMany algorithms have been proposed and explored for automatic normal organ segmentation in liver cancer RT, such as atlas-based method for liver auto-segmentation (Li et al., 2017), a random walker based framework segment for liver CT images (Moghbel et al., 2016), and a deep fully convolutional neural networks for the segmentation of four organs (liver, spleen and both kidneys) (Hu et al., 2017). As for liver tumors, a multi-channel fully convolutional network was developed to segment GTV from CT images (Sun et al., 2017). However, those studies applied in a radiation oncology field need to be carefully implemented and validated before application in daily clinical practice. A set of recommendations regarding the ontology definition, performance evaluation tools and benchmark evaluation methods should be complied to meet the clinical standard (Valentini et al., 2014).\n\nA fully-automated target structure contouring system based on deep neural networks could automatically localize and segment GTV and OARs in RT. This study protocol proposes a study that aims to develop a contouring system derived from contours defined by consensus-based guidelines for automatic target segmentation in hepatocellular carcinoma radiotherapy.\n\n\nProtocol\n\nTo develop a fully-automated target structure contouring system trained on contours delineated according to consensus contouring guidelines. In addition, to evaluate the contouring system’s feasibility and performance during application in normal clinical operations.\n\nUpper abdominal CT scans with or without contrast enhancement acquired from HCC patients who requested medical second opinion services from Shangfang Health Inc. between 1st October 2014 and 1st September 2017 will be retrospectively selected in this study. Patients in the supine position with arms extended overhead, and tumor stage meeting the liver-directed radiotherapy criteria (from the 5th Asia-Pacific primary liver cancer expert meeting) (Park et al., 2016) will be eligible for inclusion.\n\nCT datasets will be stored according to the digital imaging and communications in medicine (DICOM) standards of practice. The number of slices and the slice thickness in each CT series varies from 23 to 191 and 1.0 to 8.0 mm, respectively.\n\nCT image sets acquired from each patient will be loaded into a radiation treatment planning system (Pinnacle v9.0, Philips Healthcare, Madison, WI) or MIM Maestro software v6.7.5 (MIM Software Inc., OH) for manual delineation of targets. Following nomenclature for structure naming conventions in RT (Santanam et al., 2012), liver tumors will be delineated as GTV. Thirteen abdominal OARs (liver, stomach, duodenum, spleen, left kidney, right kidney, small bowel, colon, gallbladder, esophagus, left lung, right lung and spinal cord) will be named liver, stomach, duodenum, spleen, kidney_L, kidney_R, smallbowel, colon, gallbladder, esophagus, lung_L, lung_R and spinalcord, respectively.\n\nTo ensure that a standardized approach to delineate OARs is used across radiation oncologists involved in the study, each participant will be provided with “upper abdominal normal organ contouring guidelines and atlas: A RTOG consensus” (Jabbour et al., 2014). As for the GTV delineation, it will be contoured according to the recommendation previously reported by RTOG - using the union of GTVs across all available phases of multiphasic imaging (Hong et al., 2014).\n\nDelineating OARs using the standard abdominal soft tissue window setting: window width (WW) of 350-400 Hounsfield unit (HU), window level (WL) of 35-50 HU; for GTV, using liver window setting: WW of 150 HU, WL of 50-100 HU.\n\nTwo well-trained residents in radiation oncology will be trained by the expert physician (JZ), making sure that they understand all the requirements. Subsequently, they will manually delineate all the targets and check each other’s targets and revise them when necessary. All contours will be reviewed by an attending physician independently to detect any other gross errors (e.g., missing slices) and revise any violations or deviations from the contouring guidelines, any gross errors and violations or deviations detected during the review process will be corrected. After the attending physician’s review and revise, the contours will be reviewed and finally approved by the expert physician. These contours are to be saved as DICOM-RT structure format and transferred to an in-house web based RT-PACS platform for the fully-automated target structure contouring system (deep neural network) development.\n\nIn order to develop the auto-contouring system, the following machine learning networks will be used, including supervised and semi-supervised convolutional neural networks, recurrent neural networks, unsupervised clustering, reinforcement learning, etc. The whole technique workflow is shown in Figure 1.\n\nTo consider the 3D structure of the CT image, 5 CT image slices were simultaneously input into the network. To increase model training efficiency, a two phases training process was implemented. In phase 1, we only identify whether this slice has an organ or GTV, which is the classification task. Then a segmentation task follows with adapted weight in last phase. Both phases were trained 200 epochs. A recurrent neural structure, such as LSTM, will be tested in research if available. After initial training, the results will be review by physician, the correction information will be recorded and feed back to automatic contouring network as the input of a reinforcement learning.\n\nApproximately 400 retrospective CT data sets from patient cases will be used to establish the automatic contouring system for OARs, and an additional 200 CT data sets will be needed for GTV delineation. Another 50 CT data sets will be used as an external validation data set. The sample size is informed by the existing literature (Chu et al., 2016; Hu et al., 2017; Sun et al., 2017) and by our previous work on knowledge-based radiotherapy treatment planning prediction (Fan et al., 2017).\n\nFor external validation patients, all the contours will be manually delineated by expert teams following the same process mentioned previously (in Targets delineation) and used as ground truth. In order to evaluate the performance of auto-segmentation of the deep neural network, segmentation results will be compared with the ground truth for those patients. Analysis and comparison with four methods (HD, MDA, Dice and Jaccard) will be utilized to detect the common areas of disagreement. The explanation of these indies is as follows: HD = Hausdorff Distance, the maximum distance across all points on a surface and their closest point on another surface; MDA = Mean Distance to Agreement, which is similar but using the mean; Dice = 2 * (volume of intersection of A and B) / (volume of A + volume of B); Jaccard = (volume of intersection of A and B) / (volume of union of A and B).\n\nThis research is not a clinical trial, and doesn’t involve any direct patient contact. The CT images were acquired with permission from Shangfang Health Inc., who own the data. Shangfang Health Inc. has already obtained written informed consent from the patients, who allowed their anonymized CT data to be used for scientific research purposes. Since the data were acquired during standard procedures, with patient consent for use in research, and were anonymized, this study doesn’t need any approval from a Human Research Ethics Committee.\n\nThe results of our research will be disseminated through presentations at conferences, such as radiation oncologist and medical physicist education and radiation oncology conferences, regionally and nationally, and through articles published in peer-reviewed journals related to the treatment of cancer using radiation.\n\n\nConclusion\n\nWe propose a machine learning proof of concept system to develop a fully-automated target structure contouring system aiming to utilize in HCC radiotherapy. The study is ongoing (data analysis). It will be of help to improve the quality and efficiency of RT. The progress made here can be subsequently applied to other tumor sites in clinical settings.",
"appendix": "Competing interests\n\n\n\nAndreas Hartmann has received consultant's honoraria from Lundbeck and Noema Pharma. Natalia Szejko: None. Nanette Mol Debes: None. Andrea E. Cavanna: None.\n\n\nGrant information\n\nThis study is funded by Shangfang Health Inc.\n\n\nAcknowledgments\n\nThe authors thank the residents and the attending physician for their works on target delineation. Ying Zhao is responsible for the data protection and security in this research protocol.\n\n\nReferences\n\nBujold A, Massey CA, Kim JJ, et al.: Sequential phase I and II trials of stereotactic body radiotherapy for locally advanced hepatocellular carcinoma. J Clin Oncol. 2013; 31(13): 1631–1639. PubMed Abstract | Publisher Full Text\n\nChen W, Zheng R, Baade PD, et al.: Cancer statistics in China, 2015. CA Cancer J Clin. 2016; 66(2): 115–132. PubMed Abstract | Publisher Full Text\n\nChu C, De Fauw J, Tomasev N, et al.: Applying machine learning to automated segmentation of head and neck tumour volumes and organs at risk on radiotherapy planning CT and MRI scans [version 1; referees: 1 approved with reservations]. F1000Res. 2016; 5: 2104. Publisher Full Text\n\nFan J, Wang J, Zhang Z, et al.: Iterative dataset optimization in automated planning: Implementation for breast and rectal cancer radiotherapy. Med Phys. 2017; 44(6): 2515–2531. PubMed Abstract | Publisher Full Text\n\nHemming AW, Berumen J, Mekeel K, et al.: Hepatitis B and Hepatocellular Carcinoma. Clin Liver Dis. 2016; 20(4): 703–720. PubMed Abstract | Publisher Full Text\n\nHong TS, Bosch WR, Krishnan S, et al.: Interobserver variability in target definition for hepatocellular carcinoma with and without portal vein thrombus: radiation therapy oncology group consensus guidelines. Int J Radiat Oncol Biol Phys. 2014; 89(4): 804–813. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu P, Wu F, Peng J, et al.: Automatic abdominal multi-organ segmentation using deep convolutional neural network and time-implicit level sets. Int J Comput Assist Radiol Surg. 2017; 12(3): 399–411. PubMed Abstract | Publisher Full Text\n\nJabbour SK, Hashem SA, Bosch W, et al.: Upper abdominal normal organ contouring guidelines and atlas: a Radiation Therapy Oncology Group consensus. Pract Radiat Oncol. 2014; 4(2): 82–89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim YS, Kim JW, Yoon WS, et al.: Interobserver variability in gross tumor volume delineation for hepatocellular carcinoma : Results of Korean Radiation Oncology Group 1207 study. Strahlenther Onkol. 2016; 192(10): 714–721. PubMed Abstract | Publisher Full Text\n\nKrishnan S, Dawson LA, Seong J, et al.: Radiotherapy for hepatocellular carcinoma: an overview. Ann Surg Oncol. 2008; 15(4): 1015–1024. PubMed Abstract | Publisher Full Text\n\nLi D, Liu L, Chen J, et al.: Augmenting atlas-based liver segmentation for radiotherapy treatment planning by incorporating image features proximal to the atlas contours. Phys Med Biol. 2017; 62(1): 272–288. PubMed Abstract | Publisher Full Text\n\nMoghbel M, Mashohor S, Mahmud R, et al.: Automatic liver segmentation on Computed Tomography using random walkers for treatment planning. EXCLI J. 2016; 15: 500–517. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPark HC, Yu JI, Cheng JC, et al.: Consensus for Radiotherapy in Hepatocellular Carcinoma from The 5th Asia-Pacific Primary Liver Cancer Expert Meeting (APPLE 2014): Current Practice and Future Clinical Trials. Liver Cancer. 2016; 5(3): 162–174. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRen ZG, Zhao JD, Gu K, et al.: Three-dimensional conformal radiation therapy and intensity-modulated radiation therapy combined with transcatheter arterial chemoembolization for locally advanced hepatocellular carcinoma: an irradiation dose escalation study. Int J Radiat Oncol Biol Phys. 2011; 79(2): 496–502. PubMed Abstract | Publisher Full Text\n\nRim CH, Seong J: Application of radiotherapy for hepatocellular carcinoma in current clinical practice guidelines. Radiat Oncol J. 2016; 34(3): 160–167. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSantanam L, Hurkmans C, Mutic S, et al.: Standardizing naming conventions in radiation oncology. Int J Radiat Oncol Biol Phys. 2012; 83(4): 1344–1349. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSiegel RL, Miller KD, Jemal A: Cancer statistics, 2015. CA Cancer J Clin. 2015; 65(1): 5–29. PubMed Abstract | Publisher Full Text\n\nSun C, Guo S, Zhang H, et al.: Automatic segmentation of liver tumors from multiphase contrast-enhanced CT images based on FCNs. Artif Intell Med. 2017; pii: S0933-3657(16)30593-0. PubMed Abstract | Publisher Full Text\n\nTrebeschi S, van Griethuysen JJM, Lambregts DMJ, et al.: Deep Learning for Fully-Automated Localization and Segmentation of Rectal Cancer on Multiparametric MR. Sci Rep. 2017; 7(1): 5301. PubMed Abstract | Publisher Full Text | Free Full Text\n\nValentini V, Boldrini L, Damiani A, et al.: Recommendations on how to establish evidence from auto-segmentation software in radiotherapy. Radiother Oncol. 2014; 112(3): 317–320. PubMed Abstract | Publisher Full Text\n\nVinod SK, Min M, Jameson MG, et al.: A review of interventions to reduce inter-observer variability in volume delineation in radiation oncology. J Med Imaging Radiat Oncol. 2016; 60(3): 393–406. PubMed Abstract | Publisher Full Text\n\nYeung RH, Chapman TR, Bowen SR, et al.: Proton beam therapy for hepatocellular carcinoma. Expert Rev Anticancer Ther. 2017; 17(10): 911–24. PubMed Abstract | Publisher Full Text\n\nZhao JD, Xu ZY, Zhu J, et al.: Application of active breathing control in 3-dimensional conformal radiation therapy for hepatocellular carcinoma: the feasibility and benefit. Radiother Oncol. 2008; 87(3): 439–444. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "28784",
"date": "07 Dec 2017",
"name": "Laura A. Dawson",
"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 is rationale for investigating how to standardize contouring. Hepatocellular carcinoma is an excellent site to investigate neural networks, since there is variability in contouring, and sometimes, the full extent of HCC is challenging to delineate. However, normal tissues and OAR atlases already exist, so there is less rationale to include some OARs where automated segmentation is already in the clinic.\n\nDesign of the research is reasonable, with one exception. Training two residents to contour is likely to be associated with inaccuracies in contours. It would be better to have experienced faculty radiation oncologists contour, and a radiologist to provide input, especially for challenging cases\n\nDatasets are described, but more details are needed. E.g. what exact cases are to be used? Early stage HCC, or HCC with vascular invasion. Snapshots of the exact cases in an appendix may be helpful.\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? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Partly",
"responses": []
},
{
"id": "36679",
"date": "09 Aug 2018",
"name": "Sergio Casciaro",
"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 study protocol to develop a fully-automated contouring system based on deep learning neural network to identify target delineation of Hepatocellular carcinoma (HCC) gross target volume (GTV) in liver CT images.\nThe protocol of the study clearly describes the data selection and the manual contouring procedure. However, the level of details provided to develop the auto-contouring system based on neural network should be improved. For example, the authors should provide more details about the architecture of the chosen neural network and additional information about the neural network input data that they are expecting to use.\nThe content of the paper is an acceptable scientific standard and therefore it is suitable for publication. Authors should provide more details about the automatic data analysis workflow.\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? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1929
|
https://f1000research.com/articles/6-1919/v1
|
31 Oct 17
|
{
"type": "Research Article",
"title": "Cancer complaints: The profile of patients from the emergency department of a Brazilian oncology teaching hospital",
"authors": [
"Felipe Batalini",
"Millena Gomes",
"Fábio I",
"Flávio Kuwae",
"Giselle Macanhan",
"Julio L.B. Pereira",
"Millena Gomes",
"Fábio I",
"Flávio Kuwae",
"Giselle Macanhan",
"Julio L.B. Pereira"
],
"abstract": "Background: With the increase in prevalence of cancer in our society, we aim to clarify through primary data use what drives emergency department (ED) utilization among patients with cancer. Methods: This is a cross-sectional study. A direct survey was applied to cancer patients over 277 visits in 2015. Variables including chief complaint for current and last visit, frequency of visits, primary tumor site, and demographics were collected. Results: Pain was the most common complaint, responsible for 40% of visits, followed by constitutional symptoms (17%), and gastrointestinal complaints (11%). Abdominal pain was the single most noted pain type, with 18.4%, and had the highest rate of recurrence. It was followed by back pain, dyspnea, asthenia and fever, accounting for 8.5%, 8.5%, 8.1% and 7%, respectively. Cervical cancer represented 14.8% of patients, followed by breast (11.6%) and lung (7.6%) cancers. The majority of patients visited the emergency department less than once a month. Conclusion: The drivers of emergency department utilization among patients with cancer found through primary use data mostly confirm findings from larger studies with secondary use data. Our research underscores the burden of pain to patients with cancer, as it is the most common complaint leading to ED visits, and generally requires multiple visits. Abdominal pain was more likely to recur than other complaints. Patients could benefit from focused outpatient pain management, and from more research and education targeting cancer-related pain.",
"keywords": [
"cancer",
"complaints",
"neoplasms",
"pain",
"oncology",
"hospital"
],
"content": "Introduction\n\nThe progressive increase in life expectancy of cancer patients, which is associated with the development and availability of newer more effective therapies has raised the prevalence of cancer in our society1,2. Even though recent therapies, such as immunotherapy, tend to have an improved side-effect profile3, patients still suffer from stigma and progression of the disease, especially those with incurable conditions. Good outpatient care is a crucial component of the treatment of the oncologic patient, and emergency department (ED) visits are a strong indicator of low quality of life among cancer patients4. Other authors have studied the profile of cancer patients in general hospitals and through registries5–9. The use of secondary data can sometimes lead to information bias10. We aim to understand what drives ED utilization among cancer patients in an oncology teaching hospital using primary source data.\n\n\nMethods\n\nThis is a cross-sectional study. We analyzed survey data from 277 patients from Araújo Jorge Hospital, a major oncology-only teaching hospital located in the city of Goiânia, Goiás, Brazil. After approval by the institutional review board (IRB approval number: CAAE: 43909215.7.0000.0031), written informed consent was obtained from patients, or from caregivers for very debilitated patients, for participation in the study. The only inclusion criterion was arrival at the ED, and the only exclusion criterion was the refusal to participate in the study. Data was collected upon arrival at the ED for 12 consecutive days, 24 hours a day, in May 2015. The questionnaire is provided as Supplementary File 1. Medical records were used to obtain specific demographical information only when necessary. There was potential of recall bias for information regarding prior visits. In order to minimize information bias, all the authors reviewed all the data and whenever there was doubt in categorization of chief complaints, consensus was achieved before final categorization.\n\nIt is important to remark that some patients presented at the ED multiple times; therefore, the variable unit is the patient visit and not the patient itself, so frequencies and proportions will reflect those. The primary study variable is the chief complaint, but we also collected other variables such as gender, age, main complaint, primary tumor site, city of origin, age at diagnosis, insurance type, frequency of visit to the ED, time and reason for the previous visit.\n\nDescriptive analysis of data was performed through SPSS version 24, from IBM.\n\n\nResults\n\nPatient demographics are shown in Table 1.\n\nPain was the most common complaint in their presentation to the emergency department, accounting for 40.4% (n=112) of all visits (Figure 1). Constitutional symptoms were second with 17.3% (n=48) of visits. Gastrointestinal-related complaints were third with 10.5% (n=29). Respiratory symptoms were the fourth most common complaints with 9.4% (n=26) of all visits. Altogether, these four most common complaints formed 77.6% (n=215) of all visits. Less common were complaints related to wounds (3.6%, n=10), malfunctioning of tubes and catheters (3.6%, n=10), genitourinary complaints (2.9%, n=8), visit for procedures (1.8%, n=5) and others not specified (8.7%, n=24).\n\nGI, gastrointestinal; GU, genitourinary.\n\nAmong all pain-related complaints, abdominal pain was the most common corresponding to 44.6% of those cases, followed by back pain with 20.5%. Constitutional symptoms accounted for 17.3% of all visits; divided into asthenia, fever, anorexia and malaise, responsible respectively for 46%, 40% and 12.5% of the complaints in this category. From the gastrointestinal complaints, nausea and vomit were responsible for 48% of cases, followed by diarrhea (17%) and constipation (14%). Within the respiratory category, 88% of complaints were dyspnea, more common than cough and hemoptysis.\n\nIn individual complaint analysis (Table 2), as opposed to analysis by categories (Figure 1), the most common finding was abdominal pain with 18.4% (n=50); following was back pain (8.5%, n=23), dyspnea (8.5%, n=23), asthenia (8.1%, n=22), fever with (7.0%, n=19) and vomiting and nausea (4.8%, n=13).\n\n*Missing data on chief complaint in 5 of 277 interviewed patients.\n\nFor patients who reported a previous ED visit, the chief complaint at the last visit was recorded and from the 192 visits amenable for analysis, 55.2% (n=106) patients were returning to the ED with the same complaint as the last visit. From those presenting with pain, 79.7% (59/74) had a prior visit for the same reason. Those presenting with abdominal pain described abdominal pain as the chief complaint at their last visit in 85.7% (24/28) of times.\n\nTable 3 shows the most common neoplasias. Cervical cancer was the most frequent primary tumor, accounting for 14.8% (n=41) of all visits during the study period. Breast cancer was second (11.6%, n=32), followed by lung (7.6%, n=21) and colorectal cancers (7.6%, n=21). Prostate and esophageal cancer formed 5.4% (n=15) each. Gastric cancer: 3.2% (n=9), followed by liver and pancreatic cancers, each one of them with 1.4% (n=4) of cases.\n\nIn analysis by biological systems (Table 4), genitourinary tract cancers were the most common primary neoplasms (26.3%, n=73), driven mostly by cervical cancer with 56.1% (n=41) within this category. Gastrointestinal neoplasias constituted 20.6% (n=57) of all visits. Head and neck cancers were 15.5% (n=43), where laryngeal cancer corresponded to 3.2% (n=9), followed by tongue cancer with 1.8% (n=5). Hematologic malignancies accounted for 5.8% (n=16) cases, within these: 4% (n=11) were lymphomas. Primary skin cancers accounted for 2.9% (n=8) of the visits, among which the most common tumor was melanoma, representing 2.2% (n=6) of total cases. Central nervous system cancers constituted 2.2% (n=6), from which only 1.4% (n=4) were primary.\n\nAccording to patients, 19.3% (n=52) had visited the ED for the first time, and 13.0% (n=35) had come before in the prior month. 10.0% (n=27) stated that their last visit was the day before. This matches the finding of 8.2% (n=22) reporting visiting the ED daily. Overall, the most reported frequency was “less than once a month” (50.2%, n=139).\n\nThe 277 patients were seen over 288 consecutive hours, average 0.96 patients per hour. Most of them (71.1%, n=197) arrived during day shifts, defined from 7 am to 6:59 pm, and the minority (28.9%, n=80) came at night shifts, from 7 pm to 6:59 am. The busiest time was between 10 am and 10 pm, with average of 1.52 patient per hour, when 79.1% (n=219) arrived. In contrast, the period from 10 pm to 10 am had an average of 0.48 patients per hour.\n\n\nDiscussion\n\nThis study registered 277 consecutive ED visits to Araújo Jorge Hospital, an oncology-only teaching hospital. The main limitations of the study are the absence of staging information and the fact it is based in a single institution. Its strength resides in the quality of data - collected for primary use - and despite the limited number of visits, it shows that pain is the main driver of patients with cancer to the ED, corroborating findings of previous studies with larger numbers from secondary use data6–9. Demographic analysis showed that the hospital is a strong regional reference for oncologic care, serving patients not only from the mid-west but also from the north and northeastern regions. There were no patients from the south or southeastern regions of the country.\n\nAbdominal pain was the most common single complaint with 18.4% of all visits; patients with these complaints were more likely to return with the same complaint than others, leading to more frequent visits than those with different complaints. One possible explanation is that abdominal pain unites complications from many different organs and systems, including tumors from the most common sites (see Table 4). Also, its higher frequency can be at least in part explained by the higher frequency of cervical cancer in our population, which commonly complicates with intra-abdominal and pelvic metastasis.\n\nComplaints at the current visit was the same as the last visit in 55.2% of times, revealing the opportunity to predict the chief complaint of future visits, especially in the case of abdominal pain. In practice, nausea and vomit are major complaints of this population, but it ranked only sixth in this study, this is possibly explained by either easier good outpatient control or not enough severity to bring patients to the hospital.\n\nIn our study, cervical cancer had the highest frequency in the ED, with 14.8% (n=41) of all cases, followed by breast, lung and colorectal cancers, with 11.6% (n=32), 7.6% (n=21) and 7.6% (n=21), respectively. Another Brazilian study, Borges et al.6 also found cervical cancer as the most common at the ED, and almost two-thirds of the patients had one of the following primary tumors: urological, breast, gastrointestinal tract and lung cancer. In contrast, most studies found lung cancer as the major driver of visits, followed by breast and colorectal tumors5,7,11,12. This inequality is associated with low-resource settings with suboptimal programs such as screening and vaccination13. According to The Brazilian Cancer National Institute (INCA), cervical cancer has high incidence and prevalence in Brazil, and it is estimated to be responsible for 70% of the total of uterine cancer. Furthermore, although it’s ranked third in incidence in the country, it is the second in the mid-west region of the country, where this study was performed, responsible for 11.4% of all female malignancies. In parallel, in the US, only 17% of uterine cancers are expected to originate from the cervix, according to public domain reports from the National Cancer Institute Surveillance, Epidemiology, and End Results Program. This disparity should encourage more focus on preventive measures, such as better vaccination rates against HPV and Pap smear coverage in Brazil13. Despite not being the most incident, melanoma was the most common skin cancer leading to ED usage, likely due to its higher aggressiveness and invasion potential than other skin cancers.\n\nWe found a high rate of patients with multiple visits, including 10% of patients reporting daily visits. More than half of patients (63.8%, n=172) visited the ED in the previous month. These are findings that differ from Leak et al.12, who showed that 71% of the patients had visited the ED only once before their death. The absolute majority (95%) of patients were insured by Sistema Único de Saúde (SUS), the Brazilian public health system, therefore suggesting low-income population, with limited access to costly pain medications; requiring some of our patients to come to the ED on a daily basis for analgesia. In addition, cultural behavior limits goals of care discussions, causing a significant barrier to adequate end-of-life care. In this scenario, the expansion of the role of pain clinic and palliative care initiatives could immensely benefit patients by easing the dying process. Good outpatient symptom control could lead to decrease of ED utilization. Furthermore, this study once again highlights the importance of pain management in oncology, as a major topic in the field, and as such, it should be given extra emphasis in oncology training. More research is needed for the development of new therapies for pain in cancer patients.\n\n\nEthical statement\n\nThe research meets all applicable standards with regard to the ethics of experimentation and research integrity, and the following is being declared true. As an expert scientist and along with co-authors of concerned field, the paper has been submitted with full responsibility, following due ethical procedure, and there is no duplicate publication, fraud, plagiarism, or concerns about animal or human experimentation.\n\nIRB approval number: CAAE: 43909215.7.0000.0031\n\n\nData availability\n\nDataset 1: Cancer patients presenting at the emergency department in Brazil. Doi, 10.5256/f1000research.12632.d18227714",
"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 in Portuguese and English.\n\nClick here to access the data.\n\n\nReferences\n\nFerlay J, Soerjomataram I, Dikshit R, et al.: Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015; 136(5): E359–86. PubMed Abstract | Publisher Full Text\n\nDeSantis CE, Lin CC, Mariotto AB, et al.: Cancer treatment and survivorship statistics, 2014. CA Cancer J Clin. 2014; 64(4): 252–71. PubMed Abstract | Publisher Full Text\n\nWeber JS, Yang JC, Atkins MB, et al.: Toxicities of Immunotherapy for the Practitioner. J Clin Oncol. 2015; 33(18): 2092–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEarle CC, Park ER, Lai B, et al.: Identifying Potential Indicators of the Quality of End-of-Life Cancer Care From Administrative Data. J Clin Oncol. 2003; 21(6): 1133–8. PubMed Abstract | Publisher Full Text\n\nSadik M, Ozlem K, Huseyin M, et al.: Attributes of cancer patients admitted to the emergency department in one year. World J Emerg Med. 2014; 5(2): 85–90. PubMed Abstract | Free Full Text\n\nBorges G, Rovere RK, de Maman K: Perfil dos pacientes oncológicos que procuraram o departamento de emergência de um hospital de blumenau no período de 01 abril de 2011 a 31 de outubro. Revista Brasileira De. 2013.\n\nMayer DK, Travers D, Wyss A, et al.: Why Do Patients With Cancer Visit Emergency Departments? Results of a 2008 Population Study in North Carolina. J Clin Oncol. 2011; 29(19): 2683–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBozdemir N, Eray O, Eken C, et al.: Demographics, Clinical Presentations and Outcomes of Cancer Patients Admitted to the Emergency Department. Turk J Med Sci. 2009; 39(2): 235–240. Publisher Full Text\n\nBarbera L, Taylor C, Dudgeon D: Why do patients with cancer visit the emergency department near the end of life? CMAJ. 2010; 182(6): 563–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGardiner RC: Quality considerations in medical records abstracting systems. J Med Syst. 1978; 2(1): 31–43. PubMed Abstract | Publisher Full Text\n\nTanriverdi O, Beydilli H, Yildirim B, et al.: Single center experience on causes of cancer patients visiting the emergency department in southwest Turkey. Asian Pac J Cancer Prev. 2014; 15(2): 687–90. PubMed Abstract | Publisher Full Text\n\nLeak A, Mayer DK, Wyss A, et al.: Why do cancer patients die in the emergency department?: an analysis of 283 deaths in NC EDs. Am J Hosp Palliat Care. 2013; 30(2): 178–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVaccarella S, Laversanne M, Ferlay J, et al.: Cervical cancer in Africa, Latin America and the Caribbean and Asia: Regional inequalities and changing trends. Int J Cancer. 2017; 141(10): 1997–2001. PubMed Abstract | Publisher Full Text\n\nBatalini F, Gomes M, I F, et al.: Dataset 1 in: Cancer complaints: The profile of patients from the emergency department of a Brazilian oncology teaching hospital. F1000Research. 2017. Data Source"
}
|
[
{
"id": "36629",
"date": "02 Aug 2018",
"name": "Evandro Dantas Bezerra",
"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\nVery interesting topic and study design.\nVery well written.\nI approve this article without changes.\nI have the following minor comments:\n\"Constitutional symptoms accounted for 17.3% of all visits; divided into asthenia, fever, anorexia and malaise, responsible respectively for 46%, 40% and 12.5% of the complaints in this category.\" - Not clear what was the prevalence of malaise, if you would like to report.\n\nInteresting that hematologic malignancies were only 5.8%. Is this cancer center focused in solid tumors? We know that hematologic cancers are less prevalent, but usually the patients are more ill.\n\nWhat was the prevalence of patients on chemotherapy? Recent surgery? Radiation?\n\nWhat was the rate of admission? If possible would be interesting to know if any specific complain or cancer were more likely to be admitted. I know this was not the focus of this paper, but this might interesting for second project.\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": "37018",
"date": "19 Oct 2018",
"name": "Hugo Akabane",
"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 strengths of this research project relies on:\n\nThe paper is well written and there is good fluidity in the text. The study was well designed and performed in a simple format, focusing in descriptive analysis, which was appropriate to its goals. It was a short study but its execution clearly required significant effort from the research team to interview patients in their arrival to the emergency department 24 hours a day and 7 days a week. This is not an ambitious project but rather important in the sense that the primary data acquired mostly validates findings from larger studies from billing codes (secondary data use). The project was performed in an academic hospital from the middle west of Brazil with a huge coverage area, involving patients from many different regions of the country, and therefore represents a large patient population. This project also has a clear message that patients with cancer suffer enormously from their disease and pain control should be a major topic in oncology. It underscores that patients need great support, especially at the end-of-life care. The references include the main papers within the scope of the article, and include studies from higher and lower income countries. The author does a good job reviewing and comparing findings.\n\nThe weaknesses are:\nThe economics of implementing more intensive and patient-centered approaches for end-of-life care are not discussed in depth, and if possible it would be interesting to explore this more, perhaps in a different paper. Economics do play a major role on how resources are allocated, and one wonders what are the specific implications in a low-resource setting. The data was collected in a single institution, limiting generalizability. There is a challenge when a single symptom attempts to represent the reason for a patient’s visit. Not uncommonly, some patients visit the ED for more than one complaint, and some symptoms clearly overlap. A longitudinal follow-up was not performed in order to assess complaints that pose a higher risk of admission or poor outcome.\n\nIn summary, this is a meaningful article that explores the particularities of an Academic Oncology Hospital in Brazil with high volume ED visits for emergencies. It is a simple, easily reproducible study. Its main strengths relies on the fact that the authors acquired primary data from patients as opposed to much larger studies from secondary data use, and it validates pain a the most important burden for cancer patients. The high rate of multiple visits stress the importance of this type of service and perhaps indicate room for improvement in the care of patients in their end of life with more intense palliative care. Hopefully, this work aids hospital from low-income settings to allocate the limited resources available in the health system. It would be interesting if the author could explore the combined symptoms at presentation but this may not be able to be recovered from when the data was collected.\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/6-1919
|
https://f1000research.com/articles/6-517/v1
|
20 Apr 17
|
{
"type": "Research Article",
"title": "Results of early warning indicators for HIV/AIDS in 42 outpatient clinics in 25 northern provinces of Vietnam",
"authors": [
"Huong Le Thi",
"Linh Thuy Thi Doan",
"Dung Kim Pham",
"Huyen Phuc Do",
"Huong Lan Thi Nguyen",
"Huong Le Thi",
"Linh Thuy Thi Doan",
"Dung Kim Pham",
"Huyen Phuc Do"
],
"abstract": "Background: The emergence of HIV drug resistance (HIVDR) is an unavoidable consequence of antiretroviral therapy (ART), and HIVDR early warning indicators (EWIs) could specifically assess factors at individual clinics associated with HIVDR. Thus, the present study aimed to collect data on EWIs for HIV/AIDS at 42 outpatient clinics (OPCs) in 25 northern provinces and cities of Vietnam in 2012. Methods: A retrospective cohort study was conducted. Data was collected from 42 OPCs in 25 northern provinces between October and December 2012. The information was collected retrospectively from outpatient records from 2008 to 2011. Results: In total, 99.8% ART patients were prescribed the correct regimen when starting ART treatment. All facilities met the target of under 20% patients lost to follow-up at 12 months. A total of 33/42 facilities reached the goal for on-time appointment keeping and 37/42 facilities achieved the target of first-line retention after 12-month ARV treatment. Conclusions: EWIs should be performed routinely in HIV/AIDS facilities. The data collected will contribute to monitoring, supervision, periodic assessment, and future plans for HIV/AIDS care and treatment programs in the area.",
"keywords": [
"ARV",
"HIV drug resistance",
"outpatient clinics",
"early warning indicators"
],
"content": "Introduction\n\nAccording to UNAIDS, the number of people living with HIV/AIDS globally has continuously increased and reached to approximately 33 million at end of 2009. The number of new infections in 2009 was 2.6 million, and the number of people living with HIV (PLHIV) rose from 8 million in 1990 to 33 million in 20091. Fortunately, the number of new infections and deaths caused by AIDS has significantly reduced thanks to antiretroviral treatment (ART). According to the World Health Organization, at the end of 2008, an estimated 4 million PLHIV were receiving ART in low-income and middle-income countries. Worldwide, ART coverage has increased from 7% in 2003 to 42% in 20092.\n\nAlthough Vietnam is among the most-at-risk countries for HIV1, the government has responded to HIV epidemics. As of December 2011, there was a total of 318 antiretroviral (ARV) treatment facilities nationwide, including 287 outpatient clinics (OPCs) for adults and 118 ARV treatment facilities for children. The total number of HIV infections treated with ARV was increased to 600, an increase of 25 times compared to the end of 20053,4.\n\nAccording to the national plan regarding preventing and monitoring HIV drug resistance (HIVDR) in 2008–2012, Vietnam collects early warning indicators (EWIs) for HIVDR annually. This important activity not only contributes to the prevention and monitoring of HIVDR, but also supports the usage of available data to monitor and evaluate HIV/AIDS care and treatment programs, in order to improve the quality of service delivery. Within the framework of the Global Fund supported project on HIV/AIDS, we conducted this study to interpret results after collecting HIVDR EWIs at 42 HIV/AIDS treatment facilities in 25 northern provinces of Vietnam in 2012. The results of this study evaluate HIV/AIDS treatment effectiveness based on care and treatment national indicators.\n\n\nMethods\n\nA cross-sectional study was conducted between October and December 2012 in 25 northern provinces, including: Bac Giang, Bac Kan, Bac Ninh, Cao Bang, Dien Bien, Ha Giang, Ha Nam, Ha Noi, Hai Duong, Hai Phong, Hoa Binh, Hung Yen, Lang Son, Nam Dinh, Ninh Binh, Nghe An, Phu Tho, Quang Ninh, Son La, Thai Binh, Thai Nguyen, Thanh Hoa, Tuyen Quang, Vinh Phuc and Yen Bai. OPCs met the following inclusion criteria would be included in the study:\n\n- Regarding administrative units: included urban and rural facilities.\n\n- Regarding patients at the facilities: included adults and children.\n\n- Regarding care and treatment supported agencies: included institutions under the National target programs; Global Fund and President’s Emergency Plan for AIDS Relief (PEPFAR) funding.\n\nWe involved all OPCs that met the inclusion criteria. A total of 42 OPCs in these provinces were selected.\n\nEWI systems has been implemented according to the guidelines of the Vietnam Authority of HIV/AIDS Control (VAAC), Ministry of Health5. EWI data was extracted by the research team using a data collection form that was developed by VAAC (Supplementary File 1). HIV drug resistance EWIs collected in the present study, from the OPCs, included: percentage of patients that were prescribed the correct regimen when starting ART treatment; percentage of ART patients that were lost to follow-up after 12 months; percentage of patients that arrived on time for a doctor's appointment; and percentage of patients that retained first-line ART after 12 months of treatment.\n\nExcel 2010 software (Microsoft Corp.) was used to clean and analyze the data. Descriptive statistical analysis, including frequency and percentage, was used to analyze the data.\n\nThe study received ethical approval from VAAC, Ministry of Health. Data collection procedures and the use of data for analysis were also approved by the directors of the OPCs. No personal patient data was collected in this study.\n\n\nResults\n\nTable 1 describes EWIs in 42 HIV/AIDs facilities in 2012. Regarding the availability of antiretroviral drugs, EWIs for HIVDR showed that 7/42 facilities did not reach the target of patients receiving prescriptions for ART, congruent with national guidelines5. Regarding patients that abandoned ART treatment after 12 months, only Cao Bang Hospital had a relatively high proportion (15.79%). All facilities (100%) reached the target of <20% of patients lost to follow-up after 12 months of treatment.\n\na: Number of patient received correct ART regimen when starting treatment; b: Number of patients starting treatment; c: Number of patients lost to follow-up after 12 months of treatment; d: Number of patients registered in the cohort\n\nSome facilities had very low rate of patients arriving on time for appointments, such as Pho Yen, Thai Nguyen, Cao Loc - Lang Son (Table 2). Data regarding patient appointments could not be collected at Quang Yen Hospital, Quang Ninh, because this hospital did not make appointments with patients. Regarding the rate of patients retaining on a first line ARV regimen after 12 months of treatment, only a few facilities did not achieve its objectives, including Dai Tu, Thai Nguyen, Hung Yen provincial AIDS center, Thanh Hoa hospital.\n\na: Number of patients arriving on time for appointments in last quarter of 2011; b: Number of patients having appointments in last quarter of 2011; c: Number of patients retaining ART after 12 months of treatment; d: Number of patients registered in the cohort\n\n\nDiscussion\n\nThis study aimed to interpret results after collecting HIVDR EWIs at 42 HIV/AIDS treatment facilities in 25 northern provinces of Vietnam in 2012. The findings indicate that 35/42 (83.3%) of facilities reached the goal for patients receiving prescriptions for ART congruent with national guidelines, and 100% of facilities reached the target of <20% of patients lost to follow-up after 12 months. In addition, 33/42 (78.6%) facilities reached the goal for patients arriving on time for appointments and 37/42 (88.1%) facilities achieved the target for first line retention after a 12-month ARV treatment. These figures are higher in comparison with other Asian countries, found in a multi-country survey by the WHO - among 1048 clinics, 64–80% reached the goals of the EWIs6.\n\nSeveral implications are drawn from this study. First, since the data about EWIs could be used to optimize the ART program6, based on the result of this study, clinics that have a low performance should identify their weaknesses and find corresponding solutions to improve their services. Second, several achievements that were reported in this study should be maintained by the OPCS, including maintaining that all patients receive correct ARV regimens on admission, and continuing a patient roll-out rate of ≤20%. Third, health care providers in ARV facilities should ensure that there are no ARV drug shortages in order that patients are sufficiently provided. It is important to ensure a minimum of 80% of patients receive ARV drugs as scheduled4. This could be performed by enhancing outpatient records to track medication dispensing history. Finally, all facilities should regularly review treatment-related data regarding treatment monitoring and EWIs of HIVDR, and issuing data collection activity of HIVDR EWIs should become a routine activity in clinics. Health care providers should address the reasons patients dropped out of treatment regimens, and why patients did not come to follow-up appointments. This could help to improve the quality of service delivery and optimize the benefits of ARV treatment.\n\n\nConclusions\n\nOur study suggests that all outcomes measured in the OPCs reached standard goals, according to national guidelines. These remarkable achievements should be maintained. In addition, other considerations should be addressed, including the roll-out rate, the follow-up rate, and the rate of first line retention after 12-month ARV treatment.\n\n\nData availability\n\nDataset 1: Raw data used in the construction of Table 1 and Table 2. Datasets from early warning indicator systems detailing number of patients starting ART; number of patients in cohort; number of patients lost to follow-up; number of patients retaining ART treatment; number of patients receiving correct regime; number of patients having appointments in last quarter of 2011; and number of patients keeping appointments on-time. doi, 10.5256/f1000research.11010.d1575087",
"appendix": "Author contributions\n\n\n\nHTL, LTTD, DKP, HPD, HLN conceived of the study, and participated in its design and implementation and wrote the manuscript. HTL, LTTD analyzed the data. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to express our deepest gratitude for the great contribution of authorship to complete this study.\n\n\nSupplementary material\n\nSupplementary File 1: Data collection form developed by Vietnam Authority of HIV/AIDS Control, used to collect data about early warning indicators from outpatient clinics.\n\nClick here to access the data.\n\n\nReferences\n\nJoint United Nations Programme on HIV/AIDS: AIDS epidemic update: December 2009. Geneva UNAIDS; 2010. Reference Source\n\nWorld Health Organization: World health statistics 2010. Reference Source\n\nBureau of HIV/AIDS: Pilot of treatment accessibility 2.0 in Vietnam Hanoi: Bureau of HIV/AIDS. 2012.\n\nNational Committee for Prevention and Control of AIDS DaP: Outcomes of the implementation of the National Strategy on HIV/AIDS Prevention and Control until 2010 with a vision to 2020. Hanoi: 2010.\n\nThe Ministry of Health: Decision 4139/QD-BYT on amending and supplementing several contents in the \"Guidelines for Diagnosis and Treatment of HIV/AIDS\", issued together with Decision No. 3003/QD-BYT dated 19/08/2009 of Health Minister. 2011.\n\nBennett DE, Jordan MR, Bertagnolio S, et al.: HIV drug resistance early warning indicators in cohorts of individuals starting antiretroviral therapy between 2004 and 2009: World Health Organization global report from 50 countries. Clin Infect Dis. 2012; 54(Suppl 4): S280–S9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThi HL, Doan LT, Pham DK, et al.: Dataset 1 in: Results of early warning indicators for HIV/AIDS in 42 outpatient clinics in 25 northern provinces of Vietnam. F1000Research. 2017. Data Source"
}
|
[
{
"id": "22968",
"date": "13 Jun 2017",
"name": "Nghia Van Khuu",
"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\nStrengths: The paper has described some early warning indicators (EWI) recorded at 42 outpatient clinics (OPC) in northern Vietnam, assisting on having better directions for minimizing risks for HIV drug resistance given the growing widespread of antiretro therapy (ART).\nWeak points and suggestions for revision: the paper has some points in need of being re-considered:\nIntroduction: paragraph 2, the authors wrote, “As of December 2011, (…….). The total number of HIV infections treated with ARV was increased to 600, an increase of 25 times…” The number 600 seems not to be correct because, as of 2011, more than 60,000 HIV cases were reported to get ART according to a VAAC report. The authors should check information and revise the number accordingly.\n\nMethods: There were inconsistencies between assessing indicators and outcomes. In the paragraph “Variables and measurements”, the paper listed only four indicators, including (1) percentage of patients that were prescribed the correct regimen when starting ART treatment; (2) percentage of ART patients that were lost to follow-up after 12 months; (3) percentage of patients that arrived on time for a doctor’s appointment; and (4) percentage of patients that retained first-line ART after 12 months of treatment whereas in RESULTS section, there is one additional indicator entitled “the availability of antiretroviral drugs”. However, the latter indicator has not been described and discussed thereafter. The authors should provide additional information for this indicator in the method, results and discussion sections.\n\nResults: Information described in the results section are not compatible with those in the data table. In paragraph 2 of Results section, the paper has the OPC name as “Quang Yen Hospital, Quang Ninh” whereas Table 2 reported the information as “Yen Hung, Quang Ninh”. Information in descriptive words and in the table should be consistent.\n\nDiscussion: The authors wrote, “33/42 (78.6%) facilities reached the goal for patients arriving on time for appointments” but data in Table 2 revealed that only 31/42 OPC reached the goal for patients arriving on time for appointments and 1/42 OPC had no data (N/A). We suggest the authors review and revise information to correct this.\n\nIn short, the paper is not well-cohesive and information insufficient. The outcomes described in results section are quite simple, but few indicators unstated in the former sections have been discussed in DISCUSSION section. Moreover, the limitation section has been intertwined into the results section, making it more or less difficult for readers to follow through. The authors should re-structure information pieces to appropriate order to make the paper more cohesive.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-517
|
https://f1000research.com/articles/6-1649/v1
|
06 Sep 17
|
{
"type": "Opinion Article",
"title": "The future of metabolomics in ELIXIR",
"authors": [
"Merlijn van Rijswijk",
"Charlie Beirnaert",
"Christophe Caron",
"Marta Cascante",
"Victoria Dominguez",
"Warwick B. Dunn",
"Timothy M. D. Ebbels",
"Franck Giacomoni",
"Alejandra Gonzalez-Beltran",
"Thomas Hankemeier",
"Kenneth Haug",
"Jose L. Izquierdo-Garcia",
"Rafael C. Jimenez",
"Fabien Jourdan",
"Namrata Kale",
"Maria I. Klapa",
"Oliver Kohlbacher",
"Kairi Koort",
"Kim Kultima",
"Gildas Le Corguillé",
"Nicholas K. Moschonas",
"Steffen Neumann",
"Claire O’Donovan",
"Martin Reczko",
"Philippe Rocca-Serra",
"Antonio Rosato",
"Reza M. Salek",
"Susanna-Assunta Sansone",
"Venkata Satagopam",
"Daniel Schober",
"Ruth Shimmo",
"Rachel A. Spicer",
"Ola Spjuth",
"Etienne A. Thévenot",
"Mark R. Viant",
"Ralf J. M. Weber",
"Egon L. Willighagen",
"Gianluigi Zanetti",
"Christoph Steinbeck",
"Merlijn van Rijswijk",
"Charlie Beirnaert",
"Christophe Caron",
"Marta Cascante",
"Victoria Dominguez",
"Warwick B. Dunn",
"Timothy M. D. Ebbels",
"Franck Giacomoni",
"Alejandra Gonzalez-Beltran",
"Thomas Hankemeier",
"Kenneth Haug",
"Jose L. Izquierdo-Garcia",
"Rafael C. Jimenez",
"Fabien Jourdan",
"Namrata Kale",
"Maria I. Klapa",
"Oliver Kohlbacher",
"Kairi Koort",
"Kim Kultima",
"Gildas Le Corguillé",
"Nicholas K. Moschonas",
"Steffen Neumann",
"Claire O’Donovan",
"Martin Reczko",
"Philippe Rocca-Serra",
"Antonio Rosato",
"Reza M. Salek",
"Susanna-Assunta Sansone",
"Venkata Satagopam",
"Daniel Schober",
"Ruth Shimmo",
"Rachel A. Spicer",
"Ola Spjuth",
"Etienne A. Thévenot",
"Mark R. Viant",
"Ralf J. M. Weber",
"Egon L. Willighagen",
"Gianluigi Zanetti"
],
"abstract": "Metabolomics, the youngest of the major omics technologies, is supported by an active community of researchers and infrastructure developers across Europe. To coordinate and focus efforts around infrastructure building for metabolomics within Europe, a workshop on the “Future of metabolomics in ELIXIR” was organised at Frankfurt Airport in Germany. This one-day strategic workshop involved representatives of ELIXIR Nodes, members of the PhenoMeNal consortium developing an e-infrastructure that supports workflow-based metabolomics analysis pipelines, and experts from the international metabolomics community. The workshop established metabolite identification as the critical area, where a maximal impact of computational metabolomics and data management on other fields could be achieved. In particular, the existing four ELIXIR Use Cases, where the metabolomics community - both industry and academia - would benefit most, and which could be exhaustively mapped onto the current five ELIXIR Platforms were discussed. This opinion article is a call for support for a new ELIXIR metabolomics Use Case, which aligns with and complements the existing and planned ELIXIR Platforms and Use Cases.",
"keywords": [
"metabolomics",
"databases",
"bioinformatics infrastructure",
"data standards",
"computational workflows",
"cloud computing",
"training",
"multi-omics approaches"
],
"content": "Introduction\n\nMetabolomics aims to provide novel insights into the biochemistry of organisms by characterising the presence and concentrations of low molecular weight compounds from biological samples. It measures both endogenous (produced within an organism) and exogenous (those introduced from the environment including food components and drugs) metabolites. The primary analytical tools for such high-throughput data collection are mass spectrometry (MS), often preceded by chromatographic or electrophoretic separation technologies, and nuclear magnetic resonance spectroscopy (NMR). These technologies produce relatively large and complex data sets that require bioinformaticians, cheminformaticians, biostatisticians and computer scientists to develop and apply a wide range of algorithms, software tools, repositories and computational resources to process, analyse, report and store the data and metadata.\n\nThe field celebrated its coming of age in 20161 and progressed primarily through developments in analytical and computational tools, from which biomedical discoveries followed. As shown in Figure 1, the term ‘metabolomics’ is still gaining momentum and the global market for metabolomics was valued at $5.9 billion in 2014 and was expected to reach $12.5 billion by 2020, with a compound annual growth rate (CAGR) of 13.0% (https://goo.gl/yXTiJD). The future is bright for the application of metabolomics in academic and industrial laboratories, scientific instrument companies, government laboratories and contract research organisations. Yet, several challenges remain. Discussions amongst both independent metabolomics experts, and those within ELIXIR (http://elixir-europe.org/), culminated at the recent workshop the “Future of metabolomics in ELIXIR”. This opinion article summarises the interactions in the workshop and its outcomes.\n\nSource: Data from Google Scholar.\n\nELIXIR coordinates bioinformatics resources across its member states and help researchers to find, analyse, and exchange biological data. It is a distributed infrastructure with a single Hub based in Hinxton, United Kingdom, and an increasing number of Nodes located throughout Europe. As of July 2017, ELIXIR has 20 national Nodes, with European Bioinformatics Institute (EMBL-EBI; co-located with the Hub), working as a separate Node.\n\nThe workshop was organised at the Airport Conference Center on April 25th 2017 in Frankfurt (Germany). The invitation to the workshop was widely advertised through the newsletter of the Metabolomics Society (http://metabolomicssociety.org), the PhenoMeNal (Phenome and Metabolome aNalysis) project website (http://phenomenal-h2020.eu) and ELIXIR dissemination channels, including ELIXIR Technical Coordinators, Heads of Node mailing lists and the ELIXIR newsletter (https://goo.gl/KVUb8y). The invitation was also extended to the partners of the MetaStar consortium (http://meta-star.eu, the H2020 Metabolomics Starting Community), and interested members of the metabolomics community.\n\nThe workshop included 35 participants from across 10 countries, representing the ELIXIR Nodes including Belgium, EMBL-EBI, Estonia, France, Germany, Greece, Italy, Luxembourg, The Netherlands, Spain, The United Kingdom, and the ELIXIR hub. The objective of the meeting was to identify the principal challenges within this field and prioritise actions, in particular those within the scope and mission of ELIXIR. The workshop showcased flash presentations on the national metabolomics activities in the participating Nodes and ideas and requirements for future developments of ELIXIR metabolomics activities. An overview of ELIXIR Use Cases and Platforms was also presented by Rafael Jiménez (ELIXIR Chief Technology Officer), representing the ELIXIR Hub. The presentations were followed by discussions on the needs and challenges present in the metabolomics community. The following challenges were identified:\n\n1. Minimum information standards and early data capture\n\n2. Global spectral databases\n\n3. Tools and standards registries\n\n4. Compound identifier mapping\n\n5. Omics data integration\n\n6. Metabolite identification\n\nThese challenges are described in more detail in the following paragraphs.\n\n\nIdentification of challenges\n\nIn 2007 the Metabolomics Standards Initiative (MSI)2,3 published a set of seminal papers with recommendations on minimal reporting standards for a number of aspects in metabolomics, summarized in 4. This work built on earlier efforts by the Standard Metabolic Reporting Structure initiative5 and the Architecture for Metabolomics consortium (ArMet)6. Although critical, the MSI work served only as a starting point. Since then, several initiatives have been set up to address elements of standardisation, particularly on data standards, building on the work of the MSI. The COSMOS project (COordination Of Standards In MetabOlomicS, http://www.cosmos-fp7.eu)7, coordinated data standards efforts amongst database providers, ontologists, software engineers and instrument vendors working towards open access data standardization and agreements. A Metabolomics Society Data Standards Task Group was subsequently established to foster and coordinate efforts in enabling efficient storage, compression, terminological annotation, exchange and verification of information within metabolomics datasets. However, no significant and coordinated effort has since been attempted on minimum reporting itself: there is now widespread agreement that the early work of the MSI needs to be taken up in a new initiative, linked with related activities such as the HUPO-PSI (http://www.psidev.info), and be ‘completed’. Indeed, the lack of standardised and validated reporting in the field of metabolomics has now become a significant hindrance for data reuse and the translation of this technology into regulated areas of science. For example, one conclusion from a European workshop on regulatory toxicology stated that “there are a number of R&D needs, including a database to support metabolomics, standardisation, validation and reporting formats” (https://goo.gl/EiuTfM), and progress is now underway8 (https://goo.gl/HapKhz). The multiple task groups of the international Metabolomics Society have objectives to educate and reform reporting standards including the Metabolite identification and QA/QC task groups.\n\nIn contrast to genomics, there is a lack of metabolite databases with sufficient depth and breadth covering entire metabolomes. There are no databases capable of providing a near comprehensive snapshot of the molecular diversity available.\n\nWhile a number of databases around the globe store and serve spectral data, there is no open and comprehensive resource targeted at the needs of metabolomics. The public repository MassBank (http://www.massbank.jp/en/database.html), which stores mass spectral data, is actively developed and used by members of the metabolomics community. However, it is designed to hold only reference spectra and not experimental raw data. For NMR, a few and sparsely populated repositories provide raw data for individual metabolites, such as the metabolomics collection in BioMagResBank (http://www.bmrb.wisc.edu/www/resources.shtml)9.\n\nEfficient metabolite identification requires several fundamental resources and developments, which mandate concerted efforts across research groups and countries in Europe and worldwide. In the field, two main types of resources exist:\n\n1. Databases of measurements: Resources such as the Human Metabolome Database (HMDB, http://www.hmdb.ca)10, and the Yeast Metabolome Database (YMDB, http://www.ymdb.ca)10,11 store MS and NMR measurements of known compounds but also collect and curate published results with the aim to establish specific organism reference metabolomes.\n\n2. Genome-based metabolic reconstruction databases: These databases are built based on genome annotation, mostly of enzymes and their association to known reactions, and thus may not cover the entire metabolome since some genes that are enzymes have no enzymatic function associated and some functions may not yet be known. Normally they do not include spectral data, but they are valuable in associating expected metabolites to defined species. Examples include KEGG12, BioCyc13 and Recon14, which is a human genome-scale metabolic reconstruction.\n\nOpen-access metabolite databases such as the commercial Reaxys database (https://www.reaxys.com), Chemical Entities of Biological Interest (ChEBI, https://www.ebi.ac.uk/chebi/15), and KNApSAcK16 contain data on species-metabolite associations, besides those mentioned in the Genome-based metabolic reconstruction databases category.\n\nOne important consideration in this arena is that different confidence levels can be associated with structural identification of metabolites17, and standardized annotation schemes for such evidence descriptions are currently emerging18. The highest confidence level can only be observed when instrumental data for an authentic chemical standard is matched to the data for the biological samples. However, authentic chemical standards are not available for many metabolites and therefore bioinformatics and chemoinformatics, including through ELIXIR supported resources, are essential to solve this community-wide hurdle.\n\nRecently, as one strategy to accelerate the identification of metabolites in biological systems, a task group within the International Metabolomics Society has promoted the idea of characterising model organism metabolomes19,20. The philosophy of this task group is to leverage upon the critical mass of knowledge and activity that already exists for model organisms, linking to these other efforts and exploiting resources such as sequenced genomes to predict metabolism (as targets for experimental investigation) using genome-wide metabolic reconstructions or metabolic pathway databases KEGG12, BioCyc13, and WikiPathways21. The task group has set a grand challenge for the community, to identify and map all metabolites onto metabolic pathways, to develop quantitative metabolic models for model organisms, and to relate organism metabolic pathways within the context of evolutionary metabolomics22. To assist the experimental community in generating and gathering high-quality metabolomics data about biological models, an Implementation Study proposal called “MetabolHomes” has been submitted to ELIXIR as part of the present Metabolomics Use Case, which aims at providing users with a generic data model and the associated software tools for data management, visualisation, and annotation.\n\nSince its inception, the Metabolomics community has developed a plethora of computational tools for data analysis (https://goo.gl/Crf2Ye), as well as data and minimum information standards (https://goo.gl/gouSQY). There is a general feeling that it is difficult for the average researcher to navigate both areas.\n\nThe suggestion here was to contribute to and improve long-term supported registries of tools and standards that help researchers decide which mature, well-tested tools and standards to use for which purpose. There is a need for more metabolomics tools to be appropriately included in ELIXIR's Tools and Data Service Registry (currently, there are 56 tools/DBs tagged with 'metabolomics' in the registry).\n\nIn order to interpret the biological relevance of measured metabolites, their structures must be mapped to existing knowledge i.e., to pathways using a multi-omics data integration approach. Many metabolomics experiments measure a metabolite and characterise the structure with a retention time, one or more m/z values, or an NMR spectrum. Sometimes this characterisation can be linked to an identity of a specific chemical compound but often it can be only linked to a compound class. However, the tagged identity is commonly different from what is found in metabolism knowledge bases. In fact, different knowledge bases may have different focuses and representations of compounds. For example, one knowledge base may focus on the biological role of the metabolite, while another contains precise representations of the metabolites chemical structure and properties. Furthermore, knowledge bases are typically either too broad or too narrow in scope, and frequently not interoperable. Chemical structure mapping is therefore an important aspect to ensure interoperability between experimental and biochemical resources.\n\nNo generic solution currently exists, and people use either mapping based on expert knowledge23,24 or on equivalence based on the chemical structure, e.g. with the InChI string or key25,26. However, neither approach is well-suited for solving the issues around ambiguities in the characterisations of both the experimental side and the knowledge side. Theoretical solutions exist for linking these facts, such as scientific lenses27, but these need to be extended to service the metabolomics research field.\n\nIn addition, pattern recognition analysis, such as Pavlidis template matching (Pavlidis and Noble, 2001) could further assist in identifying the biological role of the metabolite in the metabolic network and add to its chemical identification. Pavlidis template matching clusters a metabolite based on its concentration pattern with other metabolites of known identity or class in an experiment (or multiple experiments that are combined in a meta-analysis).\n\nMetabolites function as both reactants and products of metabolic reactions. However, they also serve as regulatory molecules of proteins, affecting the structure and control of protein interaction and gene regulatory networks. This dual role of metabolites ensures that metabolomics is an integral aspect of systems biology research.\n\nThere is a great need for standardised integrated multi-omic analyses for the comprehensive understanding of the cellular physiology with significant applications in biomedicine and all the spectrum of biotechnology. Thus, establishing standardized protocols of multi-omic (i.e. metabolomic, transcriptomic, proteomic, and interactomic) data representation, integration, visualization and interpretation is of great importance. Currently, metabolomics data can be integratively visualised with transcriptomics data. However, there is a lack of integrated omic databases for most model systems and it is not self-explanatory how a genomicist/proteomicist could integrate his/her data with metabolomics data that refers to the same biological system and vice versa. Additionally, there is a lack of harmonization of the experimental design, sample collection, handling and quenching protocols of metabolomics monitoring, which further increases the challenge of integrated omics analysis. The ELIXIR group proposed that these issues of integrated omic analysis and the standardization of metabolomic data interpretation in this context, could be tested and explored via comparison and analysis of a controllable reference biological system, such as a well-characterized human cell line.\n\nGenome scale metabolic networks and metabolic pathway databases contain information both on metabolites and their reactions with corresponding genes and proteins. Thus, these networks provide valuable context for simultaneous interpretation of metabolomics data and other omics data. However, mapping metabolites in these databases is a heavy workload (see above), since most of databases use specific identifiers for small molecules, where ideally chemical structure mapping should be applied. This is a particularly striking issue with genome-scale metabolic models, as these were initially built for constraint-based computational studies (flux balance analysis and related), where the chemical structure of small molecules plays no role. Because of this, no effort was put into those models to use proper small molecules identifiers, and instead only short and ambiguous names were used for small molecules, making mappings very difficult. Hence, omics data integration was not considered at all in their design and most of these databases (available in SBML format) do not provide standard metabolite identifiers. The MSI recommends a number of identifiers e.g. HDMB ID, ChEBI ID, CAS ID, and IUPAC Name, but only InChI and InChIKey encode the chemical structure themselves.\n\nRecently, the Recon human genome-scale metabolic reconstruction network was enriched to incorporate InChIs, but more comprehensive chemical structure mapping is needed to capture all of the biological details. However, still most of the existing genome-scale metabolic reconstructions available for other organisms have not been enriched at this level. There is thus a strong need to coordinate with this community in order to facilitate the integration of metabolomics data in the context of these networks.\n\nThe problem of integrating metabolomics data into genome-scale metabolic models does not end in the community being able to map small molecules available there to proper identifiers, it only begins there. Classically, most established analysis methods used to simulate those models (Constraint-based analysis methods) are not prepared to use metabolite concentrations/abundances, as they only aim to balance incoming and outgoing reaction fluxes on each metabolite. So the use of metabolomics data in the context of these networks will present new challenges to the modelling community as well.\n\nUnlike genomics, the analytical Platforms used in metabolomics and lipidomics will not per se deliver a molecular identity, i.e. a specific chemical compound, but only the spectral characteristics. In untargeted metabolomics, metabolite identification remains the main bottleneck in data analysis and interpretation28. The typical output is a spectrum containing (a large number of) features, which are characterised in NMR by the location and intensity of signals on a frequency axis, and in MS by m/z values (and possible additional information like retention time if coupled to a chromatographic system or drift times if coupled to ion mobility). In targeted metabolomics, data acquisition instrument parameters are tuned to detect (a list of) target compounds, thus making it possible to deliver tables of metabolite abundances, ideally with absolute quantification, for downstream biochemical interpretation. In recent years, both approaches have been improved towards each other, resulting in widely targeted metabolomics, covering hundreds of compounds29.\n\nFurthermore, computational tools for untargeted metabolomics methods have improved their ability to deliver metabolite annotation, albeit with varying levels of certainty30. Concerted efforts in ELIXIR and the community can facilitate to improve the current situation, by removing the burden on developers to manually connect different tools into pipelines, and on experimentalists, by providing the tools and resources that give access to the knowledge required for biochemical interpretation of the data.\n\n\nMetabolomics Use Case in ELIXIR\n\nAfter extensive discussions during the workshop, metabolite identification was identified by popular vote as the one area where:\n\nA. A maximal impact of computational metabolomics and metabolomics data management could be aligned with other fields, in particular with the existing four ELIXIR Use Cases (https://www.elixir-europe.org/use-cases).\n\nB. Metabolomics community would benefit most\n\nC. Can be exhaustively mapped onto the existing five ELIXIR Platforms.\n\n\nELIXIR technical activities\n\nELIXIR technical activities are performed by ELIXIR Nodes and supported by the Hub. The Nodes run bioinformatics resources and services focusing on national priorities and contributing to a harmonised strategy across Europe. At the national level, an ELIXIR Node consists of research institutes within a member country, building on their national strengths. At the European level, ELIXIR’s activities are structured around Platforms and Use Cases, which bring resources and expertise together from both the ELIXIR Nodes and the basic unit of operation within ELIXIR. The ELIXIR Platforms are responsible for the implementation of the ELIXIR Scientific Programme, which is organised into five key areas: Data, Tools, Compute, Interoperability and Training.\n\nThe four Use Cases that currently represent four scientific communities are: Human Data, Rare Diseases, Marine Metagenomics and Plant Sciences. The Use Cases drive the work of the ELIXIR Platforms by describing their bioinformatics requirements. A close collaboration between the ELIXIR Use Cases and Platforms safeguards services developed by the ELIXIR Platforms would be fit for purpose.\n\nMetabolomics activities are well represented within Europe and ELIXIR nodes. Following the establishment of national efforts such as the French MetaboHUB31 and the Netherlands Metabolomic Centre, transnational efforts such as the FP7 coordination action COSMOS and the H2020 e-infrastructure PhenoMeNal followed. ELIXIR Greece has included a computational metabolomics and protein interactomics Use Case in the strategic planning for the national infrastructure. An ELIXIR Use Case on Metabolomics can have a positive impact on the community, strengthening collaboration and delivering a more harmonised strategy amongst service providers. Metabolomics aligns with the ELIXIR Platforms and Use Cases as well as other Omics themes represented and proposed in ELIXIR like Genomics and Proteomics. Though “metabolite identification” is not the only activity of interest within the ELIXIR Nodes, we proposed this Use Case as a starting point of common interest to catalyze the collaboration amongst ELIXIR Nodes and ELIXIR Platforms.\n\n\nAlignment with ELIXIR Platforms\n\nThe Metabolomics community in ELIXIR has vast expertise in the five areas represented by ELIXIR Platforms (Data, Tools, Interoperability, Compute and Training). Next, we summarise the current Platforms’ priorities, how the selected metabolomics Use Case aligns with the Platforms and a general alignment of metabolomics activities within Europe.\n\nThe Data Platform focuses on sustaining long term Europe’s life science data infrastructure by working on guidelines and indicators to improve data resources impact and long-term sustainability. Additionally, this platform aims to improve links between curated and non curated data resources and literature.\n\nOn the data side, metabolite identification requires a) the availability of high-quality curated resources for compound de-replication (the process of finding known chemical compounds in databases based on their spectroscopic and chromatographic fingerprints) as well as b) the establishment of workflows to push data on newly identified metabolites into the existing repositories. For a), the reference layer of the MetaboLights32 database plays a crucial role and needs to be extended. The reference layer holds information about individual metabolites, their chemistry, their spectral data (MS, NMR), as well as their role in pathways and biological systems. New metabolites identified in studies deposited into MetaboLights (http://www.ebi.ac.uk/metabolights/) are being curated by the MetaboLights team and added to the reference layer. In particular, characterization of the metabolome of biological models (e.g. organisms, tissues, biofluids, cell lines) is of major importance for the understanding of biochemical mechanisms, for the exploration of phenotype diversity and for the identification of new biomarkers. Due to the variety and the complexity of each biological system, gathering and curating knowledge about metabolomes can be best achieved by expert communities. In genomics, the GMOD project (http://gmod.org/wiki/Main_Page) provides biological research communities with open-source software components for annotating and managing data about model organisms. Developing such data models and software tools in metabolomics to gather, analyse, and curate data will therefore be critical to produce high-quality knowledge about model metabolomes. The curated data (spectra, compounds, networks) and workflows will be of high value as input for the corresponding reference repositories and e-infrastructures (MetaboLights, ChEBI, MetExplore, Workflow4Metabolomics, PhenoMeNal).\n\nEurope is a major provider of massive and high-quality metabolomics data. Large endeavors such as the MRC-NIHR National Phenome Centre (http://www.imperial.ac.uk/phenome-centre), Phenome Centre Birmingham (http://www.birmingham.ac.uk/research/activity/phenome-centre/index.aspx), the Netherlands Metabolomics Center (http://www.metabolomicscentre.nl), and the French MetaboHUB (http://www.metabohub.fr/home.html31) infrastructure are producing data in key scientific and socio-economic areas, including the ELIXIR Use Cases (https://www.elixir-europe.org/use-cases). Valorisation of this wealth of data requires annotation practices to be refined and new software tools to be developed to assist chemists in formatting, validating, referencing, and curating their annotations. All parties mentioned above have been engaging in the European GO-FAIR (https://www.dtls.nl/go-fair/) initiative through their participation in PhenoMeNal, which has recently been co-organising the launch of a hub for FAIR metabolomics data in goFAIR. The FAIR data movement has gained considerable momentum in Europe, where FAIR33 stands for data being Findable, Accessible, Interoperable and Reusable.\n\nThe Tools Platform drives access and exploitation of bioinformatics research software by working closely with services and connectors. Additionally, this Platform aims to facilitate the discovery, benchmarking and interoperability of bioinformatics software by focusing on software development, best practices and on strategy for workflows and software containers.\n\nWorkflow management e-infrastructures such as Workflow4Metabolomics (http://workflow4metabolomics.org/)34,35, PhenoMeNal, and Galaxy-M36 are key European resources built on the Galaxy environment37 that simultaneously address the two challenges of 1) high-performance, user-friendly, modular, and reproducible data analysis (needed by the experimental community), and 2) collaborative contributions from the bioinformatics community. Comprehensive workflows for preprocessing, statistical analysis, and annotation of data from liquid chromatography - MS (LC-MS), direct infusion MS (DIMS), gas chromatography - MS (GC-MS), and NMR technologies can be created, tailored, run, saved, shared, and publicly referenced with digital object identifiers (http://workflow4metabolomics.org/referenced_W4M_histories). A recent questionnaire has shown a need to further develop such tools and workflows, as part Galaxy, that are well supported through community-based training, to further improve the standardisation and automation of data processing and analysis38.\n\nStandardization of compound annotation is critical for such Platforms i) to enable individual modules to communicate between each other and with external resources (e.g., repositories for raw data, mass spectra, compounds and metabolic networks) and ii) to deliver useful and FAIR data to the end-user. Conversely, new modules can be developed to integrate and harmonize annotations from complementary resources.\n\nThe Interoperability Platform provides support to the discovery, integration and analysis of biological data organised in projects, centred around persistent identifiers, metadata and data standards for exchange and storage formats in addition to controlled vocabularies and linked data. This Platform facilitates work on the description of interoperability services and organises specialised BYOD (Bring Your Own Data) workshops with the aim to improve the FAIRness of data resources.\n\nExperimental metabolomics data must be interoperable in order to facilitate integration with existing knowledge bases and other omics data. Interoperability can be realized by community accepted data standards and ontological molecule representations. Persistent Identifiers for metabolites are a central need here, as is the more general chemical structure mapping problem (see above). The latter is a need that this metabolomics Use Case has in common with the Human Data, Rare Diseases, Marine Metagenomics and Plant Science Use Cases. The interoperability needs for metabolomics, however, extends beyond chemical structures: more standardized interoperability of experimental data, such as NMR spectra, is also required. Introduction of the SPLASH39 for NMR would benefit the other Use Cases too.\n\nThe Compute Platform is devoted to the compute, transfer, storage, authentication and authorization related to biological data relying on services provided by ELIXIR Nodes and other e-infrastructures.\n\nWith PhenoMeNal, Europe now has at least one major initiative to support computing with large-scale metabolomics data. The PhenoMeNal e-infrastructure enables researchers to deploy and test metabolomics workflows in public clouds (Amazon EC2, Google Compute Platform) or local, in-house OpenStack environments in cases where sensitive data cannot leave the institution. It also provides a number of commonly used workflows for metabolomics that include the eventual identification of metabolites in metabolomics experiments and the mapping onto biological pathways. PhenoMeNal unites major metabolomics laboratories across Europe and forms an essential component for our next steps to launch a European infrastructure for metabolomics service laboratories.\n\nThis Platform aims to increase the professional skills for managing and exploiting data. The training activities focus on researchers, trainers and service providers, but also include e-learning, the discovery of training materials and measuring the impact of training.\n\nThe need for further development of training programmes in metabolomics across Europe is well recognised, including training in metabolite identification40. Over the last few years multiple training courses have been established, somewhat ad hoc, both in relatively large training centres as well as individual laboratories that specialise in a particular aspect of metabolomics. Currently, there is a critical need to improve the coordination between these training courses and initiatives, and to ensure that all stakeholders across Europe and beyond (e.g. NIH training centres in USA and Metabolomics Australia) are able to readily access courses, from introductory to advanced, including online and face to face. This is one of the objectives of the newly formed European Metabolomics Training Coordination Group (EmTraG, http://www.emtrag.eu), led initially by a team in ELIXIR-UK with support from several other ELIXIR Nodes and the ELIXIR Training Platform.\n\nSpecifically in the context of metabolite identification, several introductory training courses teach the basics of metabolite annotation and identification. In addition the Birmingham Metabolomics Training Centre (BMTC), an ELIXIR-UK training resource, runs a course “Metabolite identification with the Q Exactive and LTQ Orbitrap” in partnership with Thermo Scientific. Formalising a Metabolomics Use Case within ELIXIR could enable the expansion of the delivery of such courses, for example through the EmTraG network. Training partnerships with instrument vendors can be extremely valuable, increasing the quality of the training material and facilities, as also has been achieved by Waters Corporation partnering with the Imperial International Phenome Training Centre and the BMTC.\n\nTraining in metabolite identification requires materials and case studies related to both data acquisition and bioinformatic analysis of the acquired data and a multi-disciplinary training team of analytical chemists and bioinformaticians to deliver courses. The provision of courses currently focuses on hands-on training at training centres, as described above at the BMTC, and which typically can train 6–12 scientists per course. However, in the growing discipline of metabolomics, there is a requirement to provide training to larger numbers that is only achievable through online training resources. The matching of trainee learning objectives to the type of course provided is key and recent examples of online courses have demonstrated their power in delivering Massive Open Online Courses (MOOCs) or more specialised Small Private Online Courses (SPOCs). At BMTC, the introductory course on metabolomics MOOC has been used by greater than 3000 active learners and the first SPOC focussed on data processing and analysis in metabolomics was completed by more than 50 people. However, courses for greater levels of hands-on training in the laboratory are focused on training in the laboratory; through the use of video media we can envisage some courses operating via online resources.\n\nFor training purposes, the Galaxy framework has also shown that it could be an efficient Platform to explain tools, parameters and workflows to life scientists without any skills in scripting (R, Bash, Python). The trainees can focus on their scientific questions regardless the technical aspects and programming language barrier. Since 2014, the Workflow4Metabolomics group (ELIXIR-FR, MetaboHUB) have conducted three sessions of one week based on their Galaxy instance. For those who wish to use the command line after the training session, the bridge is easy since the parameters within Galaxy are mapped exactly on the native software.\n\nELIXIR training modules can be classified into three types of trainers:\n\n1) Life Scientist (TrR): “Bring Your Own Data” training addressed to the experimentental community optimally promote good practices for using software and critically interpreting the results, and provide feedback about specific training needs. As an example, during the Workflow4Experimenters (ELIXIR-FR, MetaboHUB) one-week courses (W4E) (http://workflow4metabolomics.org/events), participants learn to analyze their own MS or NMR datasets by using the Workflow4Metabolomics Platform. Morning sessions are dedicated to methodology and tools and afternoon sessions are devoted to tutoring. Such training offers unique opportunities to discuss the designs, methods, and tools for comprehensive and rigorous data preprocessing, statistical analysis, and annotation.\n\n2) Communities of Developers (TrD): to enrich the tools and compute Platforms based on good practices guidance. As an example, the ELIXIR-EXCELERATE and IFB (ELIXIR-FR) European Galaxy Developer Workshop (EGDW, https://www.elixir-europe.org/events/elixir-excelerate-and-ifb-european-galaxy-developer-workshop), hosted in Strasbourg, aimed to teach the best-practices about the tool integration and advanced features like the Galaxy API, visualisation and administration (high performance computing (HPC), Docker).\n\n3) Trainers expert (TrT): Train the trainers program with the best strategies in Learning principles and didactic strategies for delivering bioinformatics in the most effective way\n\n\nAlignment with ELIXIR Use Cases\n\nThe ELIXIR Platforms are currently complemented by four Use Cases across four scientific communities:\n\n1. The Human Data Use Case for long-term strategies for managing and accessing sensitive human data.\n\n2. The Rare Disease Use Case for development of new therapies for rare diseases.\n\n3. The Marine Metagenomics Use Case works towards a sustainable metagenomics infrastructure to support research and innovation in the marine domain.\n\n4. Plant Science Use Case for infrastructure development for genotype-phenotype analysis of crop and tree species.\n\nAs one of the core omics technologies, metabolomics forms an important component in all of the science-driven Use Cases established so far in ELIXIR. In particular, for the human data Use Case, the PhenoMeNal e-infrastructure is very relevant as is working to develop cloud-based resources for computing with big clinical metabolomics data. Significant synergies between the privacy and ethics work package in PhenoMeNal and the parties working on the human data Use Case are obvious.\n\nAs one of the best established molecular phenotypes, metabolomics is already widely used for the genotype-phenotype analysis of crops and in tree species, providing an immediate interplay with the Plant Science Use Case. A rich corpus of work exists on the metabolomics of marine organisms where communities of, for example, marine sponges and marine microorganisms produce pharmacologically active polyketides with diverse chemical structures, which are investigated based on genomic and macroeconomic data of these communities.\n\nIn all those application scenarios, the mapping of spectral features in metabolomics data to identified chemical compounds (metabolites) and thereby to molecular pathways is crucial for the understanding of the biochemistry underlying the Use Case in question.\n\nFollowing the formation of national infrastructures for metabolomics, for example with the French infrastructure in Metabolomics and Fluxomics MetaboHUB and the Netherlands Metabolomics Centre Foundation (NMC), the COSMOS initiative7 for the coordination of standards in metabolomics provided the first coordination action between all relevant efforts in metabolomics in Europe. Leading to the establishment of the worldwide metabolomeXchange network, COSMOS paved the way for PhenoMeNal. As a core organiser of the launch of the node for FAIR data33 in metabolomics as part of the goFAIR initiative, the PhenoMeNal consortium made another necessary step to establish itself as an authority for metabolomics data ELIXIR Greece has included a computational metabolomics and protein interactomics Use Case, including the formation of standardized integrated metabolomic and proteomic databases and the evolvement of tools for combined metabolic and protein network analysis, in the strategic planning of its national infrastructure management and processing for the European Open Science Cloud (EOSC).\n\nSignificant synergies can been leveraged with the suggested sister Use Case for proteomics41. The metabolomics and proteomics communities have been extensively interacting on the data standards and formats side, where metabolomics has been able to adapt and adopt work that has been started by the proteomics community. Based on these preparatory steps, our proposal to establish metabolomics as a Use Case in ELIXIR is a logical progression.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe meeting was funded by PhenoMeNal, European Commission's Horizon2020 programme, grant agreement number 654241.\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 the proteomics community for sharing their experiences from their meeting “The future of proteomics in ELIXIR” on March 1–2 2017 in Tübingen, allowing us to build on this and organise our workshop as a one-day event.\n\n\nReferences\n\nKell DB, Oliver SG: The metabolome 18 years on: a concept comes of age. Metabolomics. 2016; 12(9): 148. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFiehn O, Robertson D, Griffin J, et al.: The metabolomics standards initiative (MSI). Metabolomics. 2007; 3(3): 175–8. Publisher Full Text\n\nSansone SA, Schober D, Atherton HJ, et al.: Metabolomics standards initiative: ontology working group work in progress. Metabolomics. 2007; 3(3): 249–56. Publisher Full Text\n\nGoodacre R: Water, water, every where, but rarely any drop to drink. Metabolomics. 2014; 10(1): 5–7. Publisher Full Text\n\nLindon JC, Nicholson JK, Holmes E, et al.: Summary recommendations for standardization and reporting of metabolic analyses. Nat Biotechnol. 2005; 23(7): 833–8. PubMed Abstract | Publisher Full Text\n\nJenkins H, Hardy N, Beckmann M, et al.: A proposed framework for the description of plant metabolomics experiments and their results. Nat Biotechnol. 2004; 22(12): 1601–6. PubMed Abstract | Publisher Full Text\n\nSalek RM, Neumann S, Schober D, et al.: COordination of Standards in MetabOlomicS (COSMOS): facilitating integrated metabolomics data access. Metabolomics. 2015; 11(6): 1587–97. 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PubMed Abstract | Publisher Full Text\n\nSanchon-Lopez B, Everett JR: New Methodology for Known Metabolite Identification in Metabonomics/Metabolomics: Topological Metabolite Identification Carbon Efficiency (tMICE). J Proteome Res. 2016; 15(9): 3405–19. PubMed Abstract | Publisher Full Text\n\nSchober D, Salek RM, Neumann S: Towards standardized evidence descriptors for metabolite annotations. In: Loebe F, Boeker M. Herre H, Jansen L, Schober D, editor. Proceedings of ODLS. 2016; 1–5. Reference Source\n\nEdison AS, Hall RD, Junot C, et al.: The Time Is Right to Focus on Model Organism Metabolomes. Metabolites. 2016; 6(1): pii: E8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nViant MR, Kurland IJ, Jones MR, et al.: How close are we to complete annotation of metabolomes? Curr Opin Chem Biol. 2017; 36: 64–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKutmon M, Riutta A, Nunes N, et al.: WikiPathways: capturing the full diversity of pathway knowledge. Nucleic Acids Res. 2016; 44(D1): D488–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdison AS, Hall RD, Junot C, et al.: The Time Is Right to Focus on Model Organism Metabolomes. Metabolites. 2016; 6(1): pii: E8.PubMed Abstract | Publisher Full Text | Free Full Text\n\nWohlgemuth G, Haldiya PK, Willighagen E, et al.: The Chemical Translation Service--a web-based tool to improve standardization of metabolomic reports. Bioinformatics. 2010; 26(20): 2647–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Iersel MP, Pico AR, Kelder T, et al.: The BridgeDb framework: standardized access to gene, protein and metabolite identifier mapping services. BMC Bioinformatics. 2010; 11: 5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChambers J, Davies M, Gaulton A, et al.: UniChem: a unified chemical structure cross-referencing and identifier tracking system. J Cheminform. 2013; 5(1): 3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMerlet B, Paulhe N, Vinson F, et al.: A Computational Solution to Automatically Map Metabolite Libraries in the Context of Genome Scale Metabolic Networks. Front Mol Biosci. 2016; 3: 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrenninkmeijer C, Evelo C, Goble C, et al.: Scientific lenses over linked data: an approach to support task specific views of the data. A vision. In: Proceedings of 2nd international workshop on linked science. 2012. Reference Source\n\nDias DA, Jones OA, Beale DJ, et al.: Current and Future Perspectives on the Structural Identification of Small Molecules in Biological Systems. Metabolites. 2016; 6(4): pii: E46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSawada Y, Akiyama K, Sakata A, et al.: Widely targeted metabolomics based on large-scale MS/MS data for elucidating metabolite accumulation patterns in plants. Plant Cell Physiol. 2009; 50(1): 37–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchymanski EL, Ruttkies C, Krauss M, et al.: Critical Assessment of Small Molecule Identification 2016: automated methods. J Cheminform. 2017; 9(1): 22. Publisher Full Text\n\nRolin D, Agasse A, Bertrand-Michel J, et al.: MetaboHUB: a national infrastructure dedicated to metabolomics and fluxomics. In: 7 Journées scientifiques du Réseau Français de Métabolomique et de Fluxomique (RFMF). 2013. Reference Source\n\nHaug K, Salek RM, Conesa P, et al.: MetaboLights--an open-access general-purpose repository for metabolomics studies and associated meta-data. Nucleic Acids Res. 2013; 41(Database issue): D781–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGiacomoni F, Le Corguillé G, Monsoor M, et al.: Workflow4Metabolomics: a collaborative research infrastructure for computational metabolomics. Bioinformatics. 2015; 31(9): 1493–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuitton Y, Tremblay-Franco M, Le Corguillé G, et al.: Create, run, share, publish, and reference your LC-MS, FIA-MS, GC-MS, and NMR data analysis workflows with the Workflow4Metabolomics 3.0 Galaxy online infrastructure for metabolomics. Int J Biochem Cell Biol. 2017; pii: S1357-2725(17)30157-7.PubMed Abstract | Publisher Full Text\n\nDavidson RL, Weber RJ, Liu H, et al.: Galaxy-M: a Galaxy workflow for processing and analyzing direct infusion and liquid chromatography mass spectrometry-based metabolomics data. Gigascience. 2016; 5: 10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoecks J, Nekrutenko A, Taylor J: 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\nWeber RJ, Lawson TN, Salek RM, et al.: Computational tools and workflows in metabolomics: An international survey highlights the opportunity for harmonisation through Galaxy. Metabolomics. 2017; 13(2): 12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWohlgemuth G, Mehta SS, Mejia RF, et al.: SPLASH, a hashed identifier for mass spectra. Nat Biotechnol. 2016; 34(11): 1099–101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeber RJ, Winder CL, Larcombe LD, et al.: Training needs in metabolomics. Metabolomics. 2015; 11(4): 784–786. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVizcaíno JA, Walzer M, Jiménez RC, et al.: A community proposal to integrate proteomics activities in ELIXIR. F1000Res. 2017; 6: pii: ELIXIR-875. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "25758",
"date": "15 Sep 2017",
"name": "Charles F. Burant",
"expertise": [
"Reviewer Expertise Integrative biology",
"metabolomics",
"metabolism",
"endocrinology",
"obesity"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 nice summary of a meeting of European scientists who have a range of interests in metabolomics-based science. The review provides a summary of the main areas that the investigators considered critical to move move both the technological aspects of the work forward, with securing the identification of 'unknown' metabolites found in biological samples, as the primary need. The group also identified various aspects of data science, data science and bioinformatics as other high priority needs.\nI found the review to be detailed, informative and well referenced. The review is unabashedly Eurocentric, appropriately so given that it was generated with the goal of highlighing European efforts. Thus, it sometimes reads as an advert for ELIXIR, but that does not distract measurably from the fine recitation of the opinions of the participants.\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": "25762",
"date": "18 Sep 2017",
"name": "Carl Brunius",
"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: I have answered “partly” to some of the questions above, since this article constitutes a condensation of a workshop/meeting and as such contains opinions, not necessarily easily backed with either results or literature. However, it should be noted that the authors have done a remarkable job of justifying opinions and claims as far as possible.\n\nThe authors have submitted a manuscript which to a large degree gives an accurate representation of challenges and bottlenecks in metabolomics along the pipeline from data capture down to biological interpretation and data integration. The identification of metabolite identification as the single major challenge reflects well the discussions in the metabolomics community. The article places emphasis on the maturity of metabolomics and expertly points out the need for integration of platforms and infrastructure, with an emphasis on European research.\n\nThe manuscript promotes a phenome-oriented view on metabolomics, with emphasis on bioinformatics of known/identified compounds and permeating a systems biology view at the expense of the more explorative side of untargeted approaches. This is understandable from the perspective of Elixir and PhenoMeNal, but somewhat misrepresentative of the larger metabolomics community, which obviously hasn’t yet matured to the stages of other (older) omics disciplines due to e.g. technical heterogeneity in platforms and difficulties in identification. On the other hand, these potentially different perspectives converge in the emphasis on metabolite identification.\n\nThe manuscript is furthermore likely difficult to penetrate for those readers that may not be fully up to date on European and international efforts for streamlining bioinformatics infrastructures. A more thorough introduction would help lower that threshold.\n\nMore detailed comments: The article pre-supposes knowledge from the reader on organisations, infrastuctures and current research political agendas on European level, as well as what similar trajectories can be observed outside of Europe. A presentation of past efforts in streamlining standards and needs for international organisations, as well as a more thorough introduction on Elixir, PhenoMeNal and other initiatives would have been in order to help the less initiated reader to follow the article. These background issues should be more thoroughly explained, broadened and put into relevant context to reduce the feeling of this being an Elixir-internal lobbying paper. E.g.\nConsider in relation to this aspect that: “The objective of the meeting was to identify the principal challenges within this field and prioritise actions, in particular those within the scope and mission of ELIXIR.” AND the article title: “The future of metabolomics in ELIXIR” The authors state that: “ELIXIR coordinates bioinformatics resources across its member states and help researchers to find, analyse, and exchange biological data. It is a distributed infrastructure with a single Hub based in Hinxton, United Kingdom, and an increasing number of Nodes located throughout Europe. As of July 2017, ELIXIR has 20 national Nodes, with European Bioinformatics Institute (EMBL-EBI; co-located with the Hub), working as a separate Node.” This description, however, hardly describes Elixir from a functional point of view. Moreover, considering that: “This opinion article is a call for support for a new ELIXIR metabolomics Use Case” there is surprisingly little information on the Elixir use cases in the background section. What are the aims, objectives and functionalities of the use cases and how do they relate to the core challenges in the metabolomics community? Overlaps between use cases and challenges are addressed later in the article, but for something this important, this should have been addressed in the introduction.\n\nThe challenges/bottlenecks in metabolomics have been expertly identified. However, these challenges are not exclusive to Elixir, but represents challenges to the entire metabolomics community.\n\nCorrection: 2. “Genome-based metabolic reconstruction databases: These databases are built based on genome annotation, mostly of enzymes and their association to known reactions, and thus may not cover the entire metabolome since some genes that are enzymes have no enzymatic function associated”\n\nThe section on current standards and techniques for multiOMICs integration is sketchy: “Currently, metabolomics data can be integratively visualised with transcriptomics data.”. There are several interesting and promising initiatives for data integration extending well beyond visual integration with transcriptomics as the authors are well aware. There is, however, a lack of validated tools. Especially for supervised data analysis of integrated data (data integration isn’t limited to systems biology through pathway analysis, but is also highly relevant from a biostatistical point of view). But I also agree that there is “a lack of integrated omic databases”, although I believe that there are several crucial aspects of data integration that need to be addressed well before we go into database issues. The systems biology approach will furthermore not be advisable in an untargeted metabolomics setting: Where complementarity in biological information from the different omics layers may be utilised for predictive modelling of e.g. disease pathophysiology of risk modelling before compound identification (and may, in fact even be used as a strategy for compound identification through e.g. Bayesian network modelling).\n\nRegarding metabolite identification, the authors state: “Concerted efforts in ELIXIR and the community…” The order should probably be the inverse.\n\nCorrection: “The Data Platform focuses on sustaining long term Europe’s life science data infrastructure”\n\nA reflection on the “Data Platform” section is that many researchers seem to have perceived reporting formats for the MetaboLights service as somewhat of a hurdle (as are most “general” issue added value tasks not corresponding to the core task at hand for most funded research (e.g. identifying predictive biomarkers of a specific disease). Whereas I have seen several BYOD initiatives for actual analysis of data, I still haven’t seen BYOD approaches would metadata handling. Maybe these exist – From a user-friendliness perspective it would be interesting to see how this hurdle is being tackled. It would also be interesting to have survey results on perceived bottlenecks in metabolights (and similar repositories) if such exist, which could potentially focus efforts into FAIR reporting.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": []
},
{
"id": "25890",
"date": "29 Sep 2017",
"name": "Emmanuel Mikros",
"expertise": [
"Reviewer Expertise NMR based Metabolomics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript presents an opinion paper concerning the outcomes of the workshop that took place within the context of the strategic planning of ELIXIR and PhenoMeNal consortia. The article describes the actual level and the diversity of efforts within the European Metabolomics community. It represents main bottlenecks in reporting QA/QC, the metabolite identification, and the lack of comprehensive and coordinated metabolite databases. The authors describe their plans how to address challenges in metabolomics mainly within the ELIXIR platforms. The paper is undoubtedly, informative for the major part of common efforts of the European Metabolomics Community. It is mainly focusing on ELIXIR plans yet the intentions of the authors are to inform the community on this issue and this is clearly reflected in the title.\nThis reviewer feels that the complexity of the non human metabolome (plant, microbe etc) is not appropriately addressed and the relative databases of secondary metabolites existing worldwide should be more seriously considered.\nComments:\nSome corrections should be considered before indexing.\nIn page 5 the statement “For NMR, a few and sparsely populated repositories provide raw data for individual metabolites, such as the metabolomics collection in BioMagResBank” is somehow contradictory with the fact that HMDB is mentioned just few sentences later and MetaboLights is also mentioned in a completely different context in another paragraph. The sentence “ELIXIR Greece has included a computational metabolomics and protein interactomics Use Case” is repeated twice in page 7 and page 10 In page 6 the reference Pavlidis and Noble 2001 appears without any relevant number and is not mentioned in the references section\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1649
|
https://f1000research.com/articles/6-1712/v1
|
20 Sep 17
|
{
"type": "Research Article",
"title": "Atopic dermatitis patients show increases in serum C-reactive protein levels, correlating with skin disease activity",
"authors": [
"Anjali S. Vekaria",
"Patrick M. Brunner",
"Ahmad I. Aleisa",
"Lauren Bonomo",
"Mark G. Lebwohl",
"Ariel Israel",
"Emma Guttman-Yassky",
"Anjali S. Vekaria",
"Patrick M. Brunner",
"Ahmad I. Aleisa",
"Lauren Bonomo",
"Mark G. Lebwohl",
"Ariel Israel"
],
"abstract": "Background: Atopic dermatitis (AD), the most common chronic inflammatory skin disease, is evolving as a systemic disease, and associated systemic inflammation is possibly linked to increases in cardiovascular disease. Methods: We assessed levels of the inflammatory marker CRP in 59 patients with moderate-to-severe AD compared to matched healthy controls, and to determine correlation with skin disease severity. Clinical severity was measured using SCORing of Atopic Dermatitis (SCORAD) and body surface area (BSA). Control subjects (n=118), matched by age, gender, smoking status and ethnicity, were obtained from the National Health and Nutrition Survey (NHANES). Results: AD patients had significantly increased serum CRP levels compared to controls (0.7±1.0 vs. 0.4±0.7mg/dl; p=0.001). CRP levels were significantly correlated with both SCORAD (r=0.427, p=0.0008) and BSA (r=0.407, p=0.0015). IgE levels in AD were highly elevated (median 2903U/ml, IQR [234,10655]), but only weakly correlated with SCORAD (r=0.282, p=0.0427) and BSA (r=0.382, p=0.0052), but not with CRP levels. AD patients also showed increased LDH levels, but without significant correlations with disease severity (SCORAD, BSA) or CRP. Conclusions: Our study strongly supports CRP as a marker for disease severity in moderate-to-severe AD patients, further demonstrating its chronic systemic nature.",
"keywords": [
"Atopic dermatitis",
"C-reactive protein",
"systemic inflammation",
"disease biomarker"
],
"content": "Introduction\n\nAtopic dermatitis (AD), the most common chronic inflammatory skin disease, frequently starts during infancy, and in adults it has usually been present for several decades1. Similar to moderate-to-severe psoriasis, there is now evolving evidence that AD also has a systemic component beyond the classic atopic/allergic comorbidities, with increases in cardiovascular risk factors such as obesity2–4, and associations with cardiovascular diseases in population-based studies2,5. A comparison of AD and psoriasis patients with healthy individuals, using cardiac computed tomography angiography, showed higher rates of coronary artery disease in both psoriasis and AD, compared to controls6. Systemic immune activation in adult moderate-to-severe AD patients is reflected by highly activated circulatory T-cells as measured using T-cell activation markers (ICOS and HLA-DR), at even higher frequencies than in psoriasis7. Also, several inflammatory blood biomarkers (e.g. Thymus and Activation Regulated Chemokine /TARC or CCL17) were consistently shown to correlate with AD clinical severity8. The important contribution of chronic inflammation to the development of atherosclerosis and cardiovascular disease events is now well established9. Therefore, C-reactive protein/CRP, an acute phase reactant reflecting systemic inflammation, has been suggested as potential biomarker for cardiovascular disease9. In patients with a history of myocardial infarction, the anti-inflammatory monoclonal antibody canakinumab (IL-1β blocker) led to a significant decrease in cardiovascular events10. Patients also showed reductions in serum CRP levels, without changes in their lipid profile10, demonstrating that anti-inflammatory treatment can indeed have an impact on cardiovascular disease. In psoriasis, it has been demonstrated that CRP is significantly elevated and associated with disease severity11. One recent study suggests that CRP levels are also increased in adult chronic AD patients vs. matched controls12, but it remains to be determined whether CRP could serve as a marker for disease severity. In contrast to adults, studies in children and adolescents with active AD did not show increases in overall CRP levels compared to controls13, and elevated CRP levels early in life were claimed to have a protective role against the development of AD14 and allergic sensitization15, suggesting that chronic low-grade inflammation in infants might provide some protection from allergen sensitization. In order to better clarify the potential role of CRP as disease biomarker, we sought to investigate CRP serum levels in moderate-to-severe adult AD patients in relation to skin disease severity.\n\n\nMethods\n\nWe retrospectively assessed CRP levels in serum from 59 adult AD patients (>18yo), with active AD and a Body Surface Area/BSA>10% (mean 59.6±27.9%, range 11–99%), that had presented to the outpatient clinic of the Department of Dermatology at Mount Sinai Hospital, New York, NY. All patients reported chronic AD since early infancy, and were off systemic anti-inflammatory AD treatment. Clinical severity was measured using SCORing of Atopic Dermatitis (SCORAD), and the vast majority of patients were in the moderate-to-severe category16 (mean SCORAD 62.2±20.86, range 15–97.5). Other demographic data was also collected, including age (mean 39.5±15.2, range 18–67 years), gender (49.2/51.8% F:M), BMI (mean 27.5±5.6kg/m2, range 18.99–41.62), blood pressure (mean 123.5/77.1mmHg, range systolic 80–154, diastolic 58–109), smoking status (11.9% smokers), total serum IgE (median 2903U/ml, IQR [234,10655]), ethnicity, comorbid conditions, concomitant medications and lipid profiles (Dataset 117). We also evaluated serum lactate dehydrogenase/LDH (mean 293.8U/L±115.3, range 117–597U/L), previously reported as a possible serum biomarker of AD severity18. None of the patients showed clinical signs of active skin infection.\n\nMatched control subjects were obtained with a ratio of 2-to-1 (n=118) from the National Health and Nutrition Survey/NHANES (https://www.cdc.gov/nchs/nhanes). They were matched to AD patients for age, gender, smoking status and ethnicity, using the R procedure MatchIt, method ‘nearest’, with a ratio of 2 control subjects for each case subject. We used individuals from the SPRINT survey nationwide between the years 2005 and 2010, for which CRP laboratory data were available. There were no changes (from the previous 2 years of NHANES) to equipment, laboratory methods or lab site.\n\nSerum CRP levels in AD patients were assessed using an immunoturbidimetric test (Abbott Laboratories, Lake Bluff, Illinois). For NHANES, CRP levels were assessed using a Siemens/Behring Nephelometer (Siemens HealthcareDiagnostics, Deerfield, IL), as described at https://wwwn.cdc.gov/Nchs/Nhanes/2009-2010/CRP_F.htm. Both assays had a lower limit of detection of 0.02mg/dl. While different assays were used to measure CRP levels in patients and controls, both methods have the same lower level of detection (0.02mg/dL) and were shown to be comparable19.\n\nFor comparisons between AD and the control group, we used the two sample t-test for age; Fisher exact test for gender, ethnicity and smoking status; and the two sample Wilcoxon test for biomarkers. When variables were missing for some of the individuals, comparison was performed only for the individuals for which the variable was available.\n\nPearson correlation coefficients were used to calculate the association between the logarithm of the biomarkers (CRP, LDH, total serum IgE) and disease activity measures SCORAD and BSA. We used a univariate linear regression formula to draw the regression line for these correlations. Each correlation was performed only for the individuals for which relevant biomarker data was available. All analyses were performed using R statistical software (Version 3.3).\n\n\nResults\n\nThere were no significant differences between demographic data of AD patients and controls (age, gender, ethnicity), blood lipids (triglycerides, LDL, HDL), body mass index (BMI), or smoking status (Table 1).\n\nComparisons of AD patients with matched healthy controls. Two samples t-test (age), Fisher exact test (gender, ethnicity, smoking), Wilcoxon test (CRP, LDH, triglycerides, LDL, HDL, BMI).\n\nAD patients had significantly increased serum CRP levels (0.7±1.0mg/dl) when compared to controls (0.4±0.7mg/dl; p=0.001; Table 1 and Figure 1a). CRP levels in AD ranged from undetectable in one patient (<0.02mg/dl) to a maximum value of 6.2mg/dl in a patient with very severe AD and a SCORAD of 95 (Dataset 117). 23 out of 59 patients (39%) showed CRP levels outside the reference range of 0-0.5mg/dl. Furthermore, CRP levels were significantly correlated with both SCORAD (Figure 1b) and BSA (Figure 1c). As 14 patients reported a history of asthma, a disease that has been shown to be associated with increased CRP blood levels20, we performed a sensitivity analysis to assess the non-asthma AD patients (Supplementary Table 1). However, differences between CRP levels in AD patients and controls remained highly significant after exclusion of all the patients with a history of asthma (Figure 2, Supplementary Table 1).\n\nComparison of CRP levels (mg/dL) in AD patients and healthy control subjects; Wilcoxon-test: p=0.001 (a); Pearson correlation and linear regression of log2 CRP levels with SCORAD (b) and body surface area/BSA (c).\n\nCRP levels (mg/dL) in AD patients excluding those with a history of asthma, compared to matched healthy control subjects; Wilcoxon-test: p<0.001.\n\nConsistent with previous publications18, the AD patients also showed increased LDH levels, but without significant correlations with disease severity measures (SCORAD, BSA) or CRP (Figure 3a–c). While IgE levels in AD were highly elevated (median 2903U/ml, IQR [234,10655]) and correlated with SCORAD and BSA, they were not correlated with CRP (Figure 3d–f).\n\nLDH and total serum IgE levels correlated with SCORAD, body surface area/BSA and log2 CRP levels (a–f); Pearson correlation and linear regression.\n\n\nDiscussion\n\nThis study is the first to demonstrate a correlation of AD disease severity with CRP levels in moderate-to-severe adult AD patients with decades of chronic disease activity, independent of co-existence of asthma. This finding is in line with the evolving concept that chronic AD has a considerable systemic inflammatory component12 that is directly linked with the overall inflammatory burden in the skin. This increase in systemic inflammation might not only be a biomarker for skin disease severity, but one might speculate that it could also contribute to AD comorbid conditions, such as the evolvement of cardiovascular disease2. This concept is strongly supported by the fact that canakinumab led to a significant reduction in serum CRP levels and cardiovascular events in a recent clinical trial10. According to the joint guidelines of the Centers for Disease Control and Prevention and the American Heart Association on CRP levels and cardiovascular risk11, 20 of our AD patients (33.9%) showed CRP levels in the range of ≥0.1mg/dl and ≤0.3mg/dl, predicting intermediate risk, and 31 patients (52.5%) showed CRP levels >0.3mg/dl, which is within the high risk range. While CRP is predominantly produced by hepatocytes, it has also been detected in tape stripping experiments from AD skin, and its expression responded to emollients21.\n\nFuture large and prospective studies in chronic severe AD patients should determine whether the up-regulated CRP levels observed in our AD cohort are indeed linked to increased cardiovascular risk, beyond its role as a marker of systemic inflammation. Nevertheless, there is some circumstantial evidence that even these small increases might be clinically relevant, as CRP above 0.42mg/dL showed differences in statin treatment outcomes for cardiovascular events in a clinical trial22, and CRP levels in the canakinumab trial were in the same order of magnitude10.\n\nFuture clinical trials investigating new therapeutic agents might follow changes in CRP levels during treatment as a potential serum biomarker of disease severity and systemic inflammation, and these may clarify whether correcting CRP can serve as a surrogate for decreasing cardiovascular risk in AD patients. However, increases in CRP levels can be a result of various conditions such as infections and malignancies, which needs to be taken into account.\n\nOur study harbors a few limitations. Besides being a retrospective study, healthy controls were not available at our site and were based on published historic controls matched for age, gender, and ethnicity. Also, it focused on a moderate-to-severe AD patient population (all but two patients had moderate-to-severe AD, i.e. a SCORAD >2516) in a large tertiary academic center in New York, while controls were obtained across the United States, which might introduce some bias. To ensure that our results are applicable to the general AD population across ethnicities, larger international studies across different ethnic backgrounds that will also evaluate for existence of “silent” cardiovascular disease in chronic AD patients are needed. However, our data supports the role that persistent skin disease has in the systemic burden of inflammation in AD patients, mandating further investigation.\n\n\nEthical statement\n\nThis study has been approved by the IRB of the Icahn School of Medicine at Mount Sinai, New York, NY (approval number, 16-00717), according to the Declaration of Helsinki.\n\n\nData availability\n\nDataset 1: Individual demographics, biomarkers and comorbid conditions of the AD study patients. doi, 10.5256/f1000research.12422.d17778417",
"appendix": "Competing interests\n\n\n\nPMB has received personal fees from LEO Pharma and Sanofi. EGY is a board member for Sanofi Aventis, Regeneron, Stiefel/GlaxoSmithKline, MedImmune, Celgene, Anacor, AnaptysBio, Celsus, Dermira, Galderma, Glenmark, Novartis, Pfizer, Vitae, Leo Pharma, Abbvie and Asana Biosciences; has received consultancy fees from Regeneron, Sanofi, MedImmune, Celgene, Stiefel/GlaxoSmithKline, Celsus, BMS, Amgen, Drais, AbbVie, Anacor, AnaptysBio, Dermira, Galderma, Glenmark, LEO Pharma, Novartis, Pfizer, Vitae, Mitsubishi Tanabe, Eli Lilly, Abbvie, and Asana Biosciences; and has received research support from Janssen, Regeneron, Celgene, BMS, Novartis, Merck, LEO Pharma, Dermira, Glenmark, Innovaderm, and UCB. The rest of the authors declare that they have no relevant conflicts to disclose.\n\n\nGrant information\n\nPMB was supported in part by grant # UL1 TR0001866 from the National Center for Advancing Translational Sciences and National Institutes of Health, Clinical and Translational Science Award program.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary Table 1. Baseline characteristics and blood biomarker levels of AD subset without asthma. AD patients excluding those with a history of asthma, compared with matched healthy controls. Two samples t-test (age), Fisher exact test (gender, ethnicity, smoking), Wilcoxon test (CRP, LDH, triglycerides, LDL, HDL, BMI).\n\nClick here to access the data.\n\n\nReferences\n\nWeidinger S, Novak N: Atopic dermatitis. Lancet. 2016; 387(10023): 1109–22. PubMed Abstract | Publisher Full Text\n\nBrunner PM, Silverberg JI, Guttman-Yassky E, et al.: Increasing Comorbidities Suggest that Atopic Dermatitis Is a Systemic Disorder. J Invest Dermatol. 2017; 137(1): 18–25. PubMed Abstract | Publisher Full Text\n\nSilverberg JI, Greenland P: Eczema and cardiovascular risk factors in 2 US adult population studies. J Allergy Clin Immunol. 2015; 135(3): 721–8.e6. PubMed Abstract | Publisher Full Text\n\nZhang A, Silverberg JI: Association of atopic dermatitis with being overweight and obese: a systematic review and metaanalysis. J Am Acad Dermatol. 2015; 72(4): 606–16.e4. PubMed Abstract | Publisher Full Text\n\nSilverberg JI: Association between adult atopic dermatitis, cardiovascular disease, and increased heart attacks in three population-based studies. Allergy. 2015; 70(10): 1300–8. PubMed Abstract | Publisher Full Text\n\nHjuler KF, Böttcher M, Vestergaard C, et al.: Increased Prevalence of Coronary Artery Disease in Severe Psoriasis and Severe Atopic Dermatitis. Am J Med. 2015; 128(12): 1325–34.e2. PubMed Abstract | Publisher Full Text\n\nCzarnowicki T, Malajian D, Shemer A, et al.: Skin-homing and systemic T-cell subsets show higher activation in atopic dermatitis versus psoriasis. J Allergy Clin Immunol. 2015; 136(1): 208–11. PubMed Abstract | Publisher Full Text\n\nThijs J, Krastev T, Weidinger S, et al.: Biomarkers for atopic dermatitis: a systematic review and meta-analysis. Curr Opin Allergy Clin Immunol. 2015; 15(5): 453–60. PubMed Abstract | Publisher Full Text\n\nYousuf O, Mohanty BD, Martin SS, et al.: High-sensitivity C-reactive protein and cardiovascular disease: a resolute belief or an elusive link? J Am Coll Cardiol. 2013; 62(5): 397–408. PubMed Abstract | Publisher Full Text\n\nRidker PM, Everett BM, Thuren T, et al.: Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease. N Engl J Med. 2017; (in press). PubMed Abstract | Publisher Full Text\n\nStrober B, Teller C, Yamauchi P, et al.: Effects of etanercept on C-reactive protein levels in psoriasis and psoriatic arthritis. Br J Dermatol. 2008; 159(2): 322–30. PubMed Abstract | Publisher Full Text\n\nWang J, Suárez-Fariñas M, Estrada Y, et al.: Identification of unique proteomic signatures in allergic and non-allergic skin disease. Clin Exp Allergy. 2017. PubMed Abstract | Publisher Full Text\n\nSilverberg JI: Association between childhood atopic dermatitis, malnutrition, and low bone mineral density: a US population-based study. Pediatr Allergy Immunol. 2015; 26(1): 54–61. PubMed Abstract | Publisher Full Text\n\nMarschan E, Kuitunen M, Kukkonen K, et al.: Probiotics in infancy induce protective immune profiles that are characteristic for chronic low-grade inflammation. Clin Exp Allergy. 2008; 38(4): 611–8. PubMed Abstract | Publisher Full Text\n\nMustonen K, Orivuori L, Keski-Nisula L, et al.: Inflammatory response and IgE sensitization at early age. Pediatr Allergy Immunol. 2013; 24(4): 395–401. PubMed Abstract | Publisher Full Text\n\nPucci N, Lombardi E, Novembre E, et al.: Urinary eosinophil protein X and serum eosinophil cationic protein in infants and young children with atopic dermatitis: correlation with disease activity. J Allergy Clin Immunol. 2000; 105(2 Pt 1): 353–7. PubMed Abstract | Publisher Full Text\n\nVekaria AS, Brunner PM, Aleisa AI, et al.: Dataset 1 in: Atopic dermatitis patients show increases in serum C-reactive protein levels, correlating with skin disease activity. F1000Research. 2017. Data Source\n\nMorishima Y, Kawashima H, Takekuma K, et al.: Changes in serum lactate dehydrogenase activity in children with atopic dermatitis. Pediatr Int. 2010; 52(2): 171–4. PubMed Abstract | Publisher Full Text\n\nMali B, Armbruster D, Serediak E, et al.: Comparison of immunoturbidimetric and immunonephelometric assays for specific proteins. Clin Biochem. 2009; 42(15): 1568–71. PubMed Abstract | Publisher Full Text\n\nAgassandian M, Shurin GV, Ma Y, et al.: C-reactive protein and lung diseases. Int J Biochem Cell Biol. 2014; 53: 77–88. PubMed Abstract | Publisher Full Text\n\nKoppes SA, Brans R, Ljubojevic Hadzavdic S, et al.: Stratum Corneum Tape Stripping: Monitoring of Inflammatory Mediators in Atopic Dermatitis Patients Using Topical Therapy. Int Arch Allergy Immunol. 2016; 170(3): 187–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaul S, Morrissey RP, Diamond GA: By Jove! What is a clinician to make of JUPITER? Arch Intern Med. 2010; 170(12): 1073–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "26213",
"date": "11 Oct 2017",
"name": "Alexander A. Navarini",
"expertise": [
"Reviewer Expertise Inflammatory skin disease"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nVekaria, Brunner et al. present a nice and clear clinical Investigation into AD severity and CRP levels. The title should be adapted to \"Moderate-to-severe AD patients ...\" as you have really investigated just this population.\n\nIn the correlations, the patients with high CRP Levels are omitted for some (graphical?) reason. Please state why and whether the Pearson r is calculated with or without them. I don't think this changes the conclusion of the paper but IMHO should be shown. If you have access to the raw SCORAD data, you might be able to check whether subcomponents of the SCORAD have a closer connection to the Serum CRP than others: - eczema involvement of some body regions - crusting, oozing - excoriations (scratch marks)\nI think it may be worth adding to the abstract that >50% of the moderate-to-severe AD patients were in the range of cardiovascular high-risk CRP levels. Also, you should probably discuss all ways to lower the high CRP. The best of them may be anti-IL6R, which also works in atopic dermatitis according to a case series.\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? No\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3121",
"date": "27 Oct 2017",
"name": "Emma Guttman-Yassky",
"role": "Author Response",
"response": "We thank the reviewer for his positive and encouraging comments. We have changed the title accordingly. All patients have been included in the graphs. We have now corrected the axis labeling for a more clear display of CRP levels in the correlation graphs Figures 1b and 1c, and Figures 3c and 3d. Due to the retrospective nature of the study, we do not have access to the sub-components of SCORAD. Therefore, we cannot calculate these correlations. We have now modified the abstract and the discussion section accordingly."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1712
|
https://f1000research.com/articles/6-1896/v1
|
27 Oct 17
|
{
"type": "Research Article",
"title": "Prenatal maternal plasma DNA screening for cystic fibrosis: A computer modelling study of screening performance",
"authors": [
"Robert W. Old",
"Jonathan P. Bestwick",
"Nicholas J. Wald",
"Robert W. Old",
"Jonathan P. Bestwick"
],
"abstract": "Background: Prenatal cystic fibrosis (CF) screening is currently based on determining the carrier status of both parents. We propose a new method based only on the analysis of DNA in maternal plasma. Methods: The method relies on the quantitative amplification of the CF gene to determine the percentage of DNA fragments in maternal plasma at targeted CF mutation sites that carry a CF mutation. Computer modelling was carried out to estimate the distributions of these percentages in pregnancies with and without a fetus affected with CF. This was done according to the number of DNA fragments counted and fetal fraction, using the 23 CF mutations recommended by the American College of Medical Genetics for parental carrier testing. Results: The estimated detection rate (sensitivity) is 70% (100% of those detected using the 23 mutations), the false-positive rate 0.002%, and the odds of being affected given a positive screening result 14:1, compared with 70%, 0.12%, and 1:3, respectively, with current prenatal screening based on parental carrier testing. Conclusions: Compared with current screening practice based on parental carrier testing, the proposed method would substantially reduce the number of invasive diagnostic procedures (amniocentesis or chorionic villus sampling) without reducing the CF detection rate. The expected advantages of the proposed method justify carrying out the necessary test development for use in a clinical validation study.",
"keywords": [
"cystic fibrosis",
"prenatal screening",
"DNA sequencing"
],
"content": "Introduction\n\nCystic fibrosis (CF) is a severe monogenic autosomal recessive inherited disorder. Over 1,000 mutations have been documented1. In Europe, CF prevalence is about 1 in 2500 live births2,3, with about 1 in 25 people being a carrier4. In current prenatal screening, parental CF carrier testing identifies couples who are both carriers and offers an invasive diagnostic procedure (amniocentesis or chorionic villus sampling [CVS]) to expectant mothers, one in four of whom will have an affected pregnancy5–8.\n\nDetecting paternal CF mutations in DNA from maternal plasma as a possible alternative screening method fails because in about 50% of cases the paternal and maternal CF mutations are the same9. The screening method described here overcomes this and does not require parental CF carrier testing. It relies on sequencing and counting DNA fragments, as currently carried out in prenatal DNA screening for Down syndrome10–12. Unlike carrier testing, which aims simply to detect the presence or absence of mutations in each parent, our method depends on determining the proportion of mutant and non-mutant DNA fragments in maternal plasma. We describe how this proportion can be determined with sufficient statistical precision to distinguish affected from unaffected pregnancies.\n\nThe DNA analysis pathway is summarized in Box 1. Plasma DNA comprises short fragments that are typically 100–200bp. Primer pairs are used to hybridise with target sites within about 150bp in the CFTR gene for polymerase chain reaction (PCR) amplification of DNA regions that include specified CF mutations. Plasma DNA fragments are tagged with barcodes that provide unique molecular identifiers of each DNA fragment. Such tagging adjusts for and minimises variation in the ratio of mutant to non-mutant DNA sequences that arises from the PCR13–15. The number of DNA fragments with and without a CF mutation are counted after massively parallel DNA sequencing of the amplified products.\n\nThis concept paper uses results from computer modelling to estimate the number of DNA fragments to be counted at each CF mutation site, and the number of CF mutation sites to be analysed to achieve good expected discrimination between affected and unaffected pregnancies. We then estimate the expected population screening performance of the method.\n\n\nMethods\n\nAn affected pregnancy is one in which a fetus has a CF mutation on each of the pair of chromosomes 7; other pregnancies, including those with fetuses that are CF carriers, are designated unaffected. For any CF mutation site, the expected (mean) percentage of DNA fragments with a CF mutation in affected and unaffected pregnancies was determined for a given fetal fraction (the proportion of plasma DNA from the placenta). These expected percentages depend on whether the DNA analysis detects two different CF mutations. If not, there are four possible situations, illustrated in Figure 1. In case A in the figure (an affected pregnancy with a 10% fetal fraction), the mother’s plasma will, in expectation, contribute 45% of DNA fragments with a CF mutation and the fetus 10%, i.e. a total of 55%. We estimated the distribution of the percentages of DNA fragments with a CF mutation in affected and unaffected pregnancies for specific fetal fractions using Gaussian distributions with a mean m and a standard deviation m×(100−m)/n, where n is the number of sequenced DNA fragments in the mutation site. There is one distribution in affected pregnancies with an expected mean of (100-ff) / 2 (maternal contribution to all CF fragments) + ff (the fetal contribution), where ff is the fetal fraction. There are four distributions in unaffected pregnancies; (i) if neither parent is a carrier the mean and the standard deviation are zero, (ii) if the mother but not the fetus is a carrier the expected mean is (100-ff)/ 2, (iii) if the fetus is a carrier, with the mutation inherited from the mother the expected mean is (100-ff)/ 2 +ff/2, (iv) if the fetus is a carrier with the mutation inherited from the father the mean is ff/2.\n\nOne or no CF mutation found.\n\nWhere two different CF mutations are found in the maternal plasma, only the predominant CF mutation (i.e. the more abundant mutation, which is always the one inherited from the mother) is informative. For example, in an affected pregnancy with a 10% fetal fraction, the mother’s plasma will, in expectation, contribute 45% of DNA fragments with a CF mutation and the fetus 10%, half of which is from the father and can be disregarded, i.e. 50% of DNA fragments at the relevant site have a CF mutation. As above, we estimated the distribution of percentages in affected and unaffected pregnancies for specified fetal fractions. The expected mean of the distribution in affected pregnancies is (100-ff)/2 + ff/2 and is (100-ff)/2 if the fetus is a carrier.\n\nThe distributions were derived for increasing numbers of DNA fragments counted (the more counted, the larger n, and the smaller the standard error) and for different fetal fractions (the larger the fetal fraction, the more separated the distributions) to determine the minimum counts needed to obtain complete or near complete separation of the distribution in affected and unaffected pregnancies. A positive result was defined as one in which the percentage of DNA fragments with a CF mutation was equal to or greater than a specified cut-off. A screen negative result was one with values below the cut-off.\n\nThe above analyses were applied to the 23 CF mutations selected by the American College of Medical Genetics and the American College of Obstetrics and Gynecology16 for parental carrier testing, taking account of their separate prevalence in the pan-ethnic standard population, accounting for an estimated 83.4% of CF carriers.\n\nThe detection rate (DR, sensitivity: proportion of affected pregnancies with a positive result) was estimated from the proportion of the total area under the distribution of percentage of DNA fragments with a given CF mutation in affected pregnancies equal to or greater than specified cut-off levels, multiplied by the proportion of all CF mutations in the population attributable to the CF mutations in the panel.\n\nThe false-positive rate (FPR: proportion of unaffected pregnancies with a positive result), was estimated from the proportion of the total area under the distributions of the percentage of DNA fragments with a given CF mutation in unaffected pregnancies equal to or greater than the specified cut-off levels, multiplied by the proportion of all CF mutations in the population attributable to the CF mutations in the panel. We adjusted for confined placental mosaicism involving trisomy 7 by taking account of its prevalence and the 50% chance that the extra chromosome has the CF mutation17–19.\n\nThe population odds of being affected given a positive result (OAPR) was estimated from the DR divided by the FPR times the prevalence of CF expressed as an odds. The pregnancy prevalence of CF was taken to be 1 in 2500, or 1:2499 as an odds.\n\n\nResults\n\nFigure 1 shows the percentage of DNA fragments with a CF mutation in maternal plasma from an affected pregnancy when the two CF mutations are the same (XX) (i.e. only one CF mutation found) according to parental CF carrier status (OX = carrier, OO = unaffected non-carrier). The figure is based on a 10% fetal fraction. If the fetus is affected, the percentage of DNA fragments with the CF mutation in the maternal plasma is, in expectation, 55%. If the fetus is unaffected and is not a carrier, it is 45% or 0% (depending on the parental carrier status). If the fetus is a CF carrier, it is 50% or 5% (again depending on the parental carrier status). In this way, affected pregnancies are distinguished and a result of 55%, which can be statistically separated from the expected 50% or less, defines a positive screening result.\n\nFigure 2 shows the percentage of DNA fragments with a CF mutation in maternal plasma in an affected pregnancy when the two CF mutations are different (X1 and X2) (i.e. two CF mutations found) according to parental CF carrier status. The fetal fraction is taken as 10%. In maternal plasma the predominant mutation is necessarily from the mother. The expected percentage of CF mutations at the predominant CF mutation site in an affected pregnancy is 50% and 45% in an unaffected pregnancy.\n\nTwo different CF mutations found.\n\nFigure 3 shows the estimated relative distributions of DNA fragments with one or no CF mutation found according to fetal fractions (10%, a typical value, and 4%, a lower limit typically used in prenatal DNA Down syndrome screening)20–22, the number of DNA fragments sequenced that include the mutation site, and whether the pregnancy is affected or unaffected. With a 10% fetal fraction, counting 8,000 sequenced DNA fragments gives almost complete separation of the relative distributions for the three possible fetal genotypes, with complete (or near complete) discrimination, and consequently a very low FPR. With a 4% fetal fraction, counting 32,000 targeted fragments still gives good discrimination between affected and unaffected pregnancies.\n\nOne or no CF mutation found.\n\nFigure 4 shows the relative distributions of the percentage of DNA fragments with a CF mutation according to fetal fraction if 32,000 DNA fragments are counted. Figure 4A applies if one or no CF mutations are found, and Figure 4B if two different CF mutations are found. Figure 4A shows that with a 3% or greater fetal fraction, there is good discrimination between affected and unaffected pregnancies, but less so with a 2% fetal fraction.\n\n(A) One or no CF mutation found; (B) Two different cystic fibrosis (CF) mutations found.\n\nFigure 4B shows that when two different mutations are found the mean for the predominant CF mutation in affected pregnancies is always 50% regardless of the fetal fraction. This is also shown in Figure 2 with a 10% fetal fraction. If the fetal fraction were 4%, the contributions from the mother and the fetus would still sum to 50% i.e. 2% + 48% instead of 5% + 45% as in Figure 2. In an unaffected pregnancy in which the fetus is a carrier, the mean increases towards 50% with decreasing fetal fraction and consequently the cut-off to determine a positive test result is dependent on the fetal fraction. As in Figure 4A, with a 3% or greater fetal fraction, there is good discrimination, but not with a 2% fetal fraction.\n\nConfined placental mosaicism involving trisomy 7, with an estimated prevalence of 0.2%17,18, has a small influence on the FPR. This effect arises from pregnancies in which the fetus is a CF carrier and has inherited the CF mutation from the mother, and the placental mosaicism is 0XX. This results in more than the expected 50% of DNA fragments with the CF mutation in the maternal plasma. The increase depends on the fetal fraction. Instead of the (unaffected) carrier fetus contributing half fetal fraction to the proportion of mutant fragments in the maternal plasma, it will be two-thirds fetal fraction, i.e. an increase of one-sixth fetal fraction. Given that the mother and fetus are both carriers, and in the estimated 0.1% of pregnancies with confined placental mosaicism of the OXX type, this shift in the distribution of mutant DNA fragments increases the FPR. For example, with a cut-off of 51%, a fetal fraction of 10% and 32,000 DNA fragments counted, the effect of confined placental mosaicism contributes about 0. 002% to the FPR (prevalence of OXX type of confined placental mosaicism (0.1%), times the prevalence of mother and fetus carrier status (2%), times the proportion of CF mutations in the population included in the ACMG panel (0.834) , and making the conservative assumption that nearly all of these false positives would be shifted across the 51% cut-off). Our estimates of screening performance take account of this correction to the FPR. The prevalence of confined placental mosaicism of either the OXX or OOX type (0.1%) is sufficiently low to have a negligible effect on the DR.\n\nThe 23 CF mutations in the selected panel account for an estimated 83.4% of people with a CF mutation, so the maximum CF DR (proportion of CF pregnancies detected) is 70% (83.4% × 83.4%), because for a fetus to be affected it must have two CF mutations, one from each parent, assuming random mating.\n\nTable 1 shows the estimated screening performance according to the screening cut-off (expressed as the percentage of targeted DNA fragments with a CF mutation), and fetal fraction using the 23 CF mutation panel. The cut-off of choice is 51% when one or no CF mutation is found in the maternal plasma sample. When two CF mutations are found in the maternal plasma, the cut-off will vary according to fetal fraction (eg. 46% with a 10% fetal fraction or 49% with a 4% fetal fraction. Provided the fetal fraction is 3% or more, a DR of 67–70% (limited mainly because of the number of mutations used in the test, not by the DNA analysis) can be achieved with a very low FPR (≤ 0.002%). Counter-intuitively, the OAPR increases with decreasing fetal fraction. This arises as a result of two competing effects; an underlying decrease in the OAPR with decreasing fetal fraction and a diminishing false-positive rate due to the placental mosaicism, the latter dominating. Even in the presence of placental mosaicism a low fetal fraction is a disadvantage because of the reduced detection rate. Table 1 also shows that the DR is reduced to 35% with a 2% fetal fraction, setting a practical lower limit of 3%; <1% of pregnancies have a fetal fraction <3%20. The 70% DR shown in Table 1 requires the use of 23 CF mutations in the test and an estimated 736,000 targeted DNA fragments need to be counted (23 mutations × 32,000 fragments per mutation).\n\n*Cut-off to yield same screening performance as when only 1 mutation is found.\n\nDR=detection rate; FPR=false-positive rate; OAPR=odds of being affected given a positive result\n\nIf FPR < 0.001% OAPR is precise unless over 1000:1.\n\n\nDiscussion\n\nPrenatal DNA screening for CF has a higher predicted screening performance than conventional screening based on parental carrier testing. The improved screening performance is based on maintaining the DR achieved using a given parental carrier testing CF mutation panel but with a 60 times lower FPR, 0.002% compared with 0.12% (prevalence of carrier couples is 4% × 4% = 0.16%, assuming random mating and, among these, 75% are false-positive (0.16% × 0.75 = 0.12%)).\n\nFigure 5 compares prenatal CF screening based on parental carrier testing with plasma DNA screening. The figure shows its clinical advantage in achieving a much lower FPR and hence a much higher OAPR, 42 times higher (14:1/ 1:3) than parental carrier testing. An estimated 98% of invasive diagnostic tests in unaffected pregnancies are avoided, without loss of detection (82/84 in Figure 5).\n\nIllustration of prenatal screening for cystic fibrosis (CF) based on A parental CF carrier testing using the 23 common mutation panel recommended by the American College of Medical Genetics16 and B screening using the maternal plasma DNA with the same 23 CF mutations (fetal fraction 10%, 51% cut-off if one CF mutation found, 46% if 2 found [see Table 1]).\n\nThe selection of a screening cut-off of targeted DNA fragments with a CF mutation should balance maximizing the DR, minimizing the FPR, and achieving an acceptably high OAPR. A cut-off of 51% is reasonable (Table 1). This cut off, with a 10% fetal fraction, achieves an expected 70% DR with a FPR of 0.002%. A higher cut-off of 52% results in a loss of detection with low fetal fractions even though the FPR is decreased. A 50% cut-off retains detection but at the cost of a much increased FPR.\n\nPlasma DNA screening is simpler than parental carrier testing because it only requires a maternal plasma sample. It also avoids a problem with parental carrier based screening that arises when the biological father (but not the assumed father) is a carrier, and the pregnancy is affected. The rate of non-paternity varies among populations; in one study it was 2%23.\n\nPlasma DNA screening treats each pregnancy as a fresh screening opportunity, which is similar to prenatal screening for neural tube defects or Down syndrome. The screening aims to identify an affected pregnancy. There is no intention to identify carriers, which is a benefit from a screening perspective, because almost all carriers will never have an affected pregnancy. Being a carrier is of minor or no medical consequence. The fact that the disorder being screened for is inherited is, from the screening perspective, irrelevant. This approach has the advantage of screening women who have a pregnancy with a different partner without the need to retrieve a report on her previously determined carrier status, and if a carrier, determining the carrier status of her new partner.\n\nUnique molecular identifiers are needed to overcome random error in DNA fragment amplification. With the use of unique molecular identifiers amplification, sequencing, and counting can be corrected for under- and over-amplification13–15. Plasma DNA sequencing as described here, using unique molecular identifiers, overcomes the limitation associated with digital PCR24 that does not quantify mutant and non-mutant DNA sequences sufficiently accurately to reliably distinguish affected from unaffected pregnancies. Whole-genome sequencing could overcome the digital PCR limitation, but is impractical because of the cost of unnecessary sequencing of most of the genome25. Our paper provides a potentially practical cost-effective solution, the screening algorithms needed, and computer modelled estimates of population screening performance.\n\nTo obtain the 32,000 DNA fragments containing a CF mutation site needed for the test if the fetal fraction is as low as 2% requires about 13ml of plasma (i.e. about 30ml blood), because 1ml typically contains about 2400 haploid (single duplex DNA strand) whole genomic equivalents, each of which has one copy of each CF mutation site26,27 (32,000/ 2,400). Figure 6 illustrates how 13.3ml of plasma provides enough target sites to distinguish an affected fetus from a maternally derived fetal carrier fetus (one CF mutation found). There would be enough blood for a DNA screening test for trisomy 21, 18, and 13, as well as CF. The plasma DNA test should cost little more than about half the cost of a Down syndrome DNA screening test because sequencing accounts for about half the test cost, and the sequencing cost of the CF test is reduced to about a tenth or less of that required for Down syndrome screening\n\nAs the number of CF mutations used in the test increases, the number of DNA fragments to be counted also increases without, however, requiring a larger plasma sample because many CF mutation sites can be amplified simultaneously14. Using 23 CF mutations, 736,000 (23 × 32,000) DNA fragments need to be counted. Adding prenatal CF screening to DNA-based screening for Down syndrome is feasible and involves only an extra step to amplify the selected CF DNA sites prior to sequencing.\n\nThe number of CF mutations tested limits the overall DR. Some current parental carrier testing uses more CF mutations than the 23 used here; one programme uses a 106 mutation panel, accounting for an estimated 91% of people with a CF mutation in the population specified. This yields an 81% DR (91% × 91%). A panel of about 100 mutations should be feasible with maternal plasma DNA screening; however, the incremental increase in detection with increasing number of CF mutations is very small. Any panel used could be modified according to the ethnic distribution of the population screened. Maternal plasma DNA screening using full exon sequencing (which would include all CF mutations) may be a future option, but there may be a limit to the number of DNA regions that can be amplified.\n\nWhile plasma DNA screening does not rely on parental CF carrier detection, it still has a useful role to play where CF carrier testing has already been established. The relevant CF mutations would be known from parental testing and, provided the parental CF mutations were included in the method, all affected pregnancies would be identified and amniocentesis or chorionic villus sampling would be avoided in nearly all unaffected pregnancies. Therefore, about 3/4 women would avoid an invasive diagnostic procedure.\n\nThe proposed method could be used in a two-step screening procedure in which the CF carrier status of the mother is first identified and then, if she is a carrier, the described method adopted. This may have cost savings as, if the mother is not a carrier, there is no need to proceed with further testing. Also, it would lend itself to other autosomal recessive disorders. The method described here need not, therefore, be limited to CF.\n\nAlthough de novo CF mutations are rare, the proposed method detects them, provided they are included in the specified mutation set. Interpretation of test results if the fetus has a de novo mutation applies as set out in this paper, with three exceptions: (i) mother is not a carrier but the father is a carrier - in this situation the percentage DNA CF fragments is on average 10% if the mutation is the same as the father’s mutation or 5% if it is different (assuming a 10% fetal fraction); (ii) mother and father are carriers, the father carries a different CF mutation, the de novo mutation is the same as the father’s mutation and the fetus inherits the father’s mutation - in this situation the affected pregnancy would be missed; (iii) mother and father are carriers, the father carries with a different mutation, the fetus has a mutation that is different from both and the fetus inherits the father’s mutation - in this situation the affected pregnancy would be detected by the presence of three different CF mutations. While in one of these situations an affected pregnancy would be missed, the method achieves a higher level of detection than screening based on parental carrier testing, which misses all cases with a de novo mutation except for those where both parents are carriers.\n\n\nConclusion\n\nPrenatal maternal plasma DNA screening for CF has an estimated screening performance substantially higher than current screening based on parental carrier testing. While amniocentesis is still required for the diagnosis of CF, the proposed method means that nearly all amniocenteses would be performed in affected pregnancies, without reducing the DR. The overall expected advantages are sufficiently large to merit developing the test for use in routine screening practice and evaluation in a clinical validation study.\n\n\nData availability\n\nAll data analysed in this study are cited through the article.",
"appendix": "Competing interests\n\n\n\nRobert Old has applied for a UK patent (application nos. 1619812.9 and 1702924.0 priority date 23 November 2016) for the method described in this paper and assigned the invention to Logical Medical Systems, of which Nicholas Wald is a Director.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBobadilla JL, Macek M Jr, Fine JP, et al.: Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening. Hum Mutat. 2002; 19(6): 575–606. PubMed Abstract | Publisher Full Text\n\nFarrell PM: The prevalence of cystic fibrosis in the European Union. J Cystic Fibrosis. 2008; 7(5): 450–453. PubMed Abstract | Publisher Full Text\n\nSouthern KW, Munck A, Pollitt R, et al.: A survey of newborn screening for cystic fibrosis in Europe. J Cystic Fibrosis. 2007; 6(1): 57–65. PubMed Abstract | Publisher Full Text\n\nRomeo G, Devoto M, Galietta LJ: Why is the cystic fibrosis gene so frequent? Hum Genet. 1989; 84(1): 1–5. PubMed Abstract | Publisher Full Text\n\nWald NJ: Couple screening for cystic fibrosis. Lancet. 1991; 338(8778): 1318–9. PubMed Abstract | Publisher Full Text\n\nWald NJ, George L, Wald N, et al.: Further observations in connection with couple screening for cystic fibrosis. Prenat Diagn. 1995; 15(6): 589–90. PubMed Abstract | Publisher Full Text\n\nHaddow JE, Bradley LA, Palomaki GE, et al.: Issues in implementing prenatal screening for cystic fibrosis: results of a working conference. Genet Med. 1999; 1(4): 129–35. PubMed Abstract | Publisher Full Text\n\nWald NJ, Morris JK, Rodeck CH, et al.: Cystic fibrosis: selecting the prenatal screening strategy of choice. Prenat Diagn. 2003; 23(6): 474–483. PubMed Abstract | Publisher Full Text\n\nDebrand E, Lykoudi A, Bradshaw E, et al.: A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity. PLoS One. 2015; 10(11): e0142729. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChiu RW, Chan KC, Gao Y, et al.: Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natnl Acad Sci USA. 2008; 105(51): 20458–20463. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFan HC, Blumenfeld YJ, Chitkara U, et al.: Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing dNA from maternal blood. Proc Natl Acad Sci USA. 2008; 105(42): 16266–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLo YM, Chiu RW: Genomic analysis of fetal nucleic acids in maternal blood. Annu Rev Genomics Hum Genet. 2012; 13: 285–306. PubMed Abstract | Publisher Full Text\n\nKivioja T, Vähärautio A, Karlsson K, et al.: Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods. 2012; 9(1): 72–74. PubMed Abstract | Publisher Full Text\n\nStåhlberg A, Krzyzanowski PM, Jackson JB, et al.: Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing. Nucl Acids Res. 2016; 44(11): e105. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKinde I, Wu J, Papadopoulos N, et al.: Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci USA. 2011; 108(23): 9530–9535. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWatson MS, Cutting GR, Desnick RJ, et al.: Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Genet Med. 2004; 6(5): 387–391. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKalousek DK, Vekemans M: Confined placental mosaicism. J Med Genet. 1996; 33(7): 529–533. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrati FR, Grimi B, Frascoli G, et al.: Confirmation of mosaicism and uniparental disomy in amniocytes, after detection of mosaic chromosome abnormalities in chorionic villi. Eur J Hum Genet. 2006; 14(3): 282–288. PubMed Abstract | Publisher Full Text\n\nHochstenbach R, Nikkels PG, Elferink MG, et al.: Cell-free fetal DNA in the maternal circulation originates from the cytotrophoblast: proof from an unique case. Clin Case Rep. 2015; 3(6): 489–491. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCanick JA, Palomaki GE, Kloza EM, et al.: The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies. Prenat Diagn. 2013; 33(7): 667–674. PubMed Abstract | Publisher Full Text\n\nHudecova I, Sahota D, Heung MM, et al.: Maternal plasma fetal DNA fractions in pregnancies with low and high risks for fetal chromosomal aneuploidies. PLoS One. 2014; 9(2): e88484. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPalomaki GE, Kloza EM, Lambert-Messerlian GM, et al.: DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med. 2011; 13(11): 913–920. PubMed Abstract | Publisher Full Text\n\nKing TE, Jobling MA: Founders, drift, and infidelity: the relationship between Y chromosome diversity and patrilineal surnames. Mol Biol Evol. 2009; 26(5): 1093–1102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLun FM, Tsui NB, Chan KC, et al.: Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma. Proc Natl Acad Sci USA. 2008; 105(50): 19920–19925. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChan KC, Jiang P, Sun K, et al.: Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends. Proc Natl Acad Sci USA. 2016; 113(50): E8159–E8168. PubMed Abstract | Publisher Full Text | Free Full Text\n\nManokhina I, Singh TK, Peñaherrera MS, et al.: Quantification of cell-free DNA in normal and complicated pregnancies: overcoming biological and technical issues. PLoS One. 2014; 9(7): e101500. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBischoff FZ, Lewis DE, Simpson JL: Cell-free fetal DNA in maternal blood: kinetics, source and structure. Hum Reprod Update. 2005; 11(1): 59–67. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "27399",
"date": "02 Nov 2017",
"name": "James E Haddow",
"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 concept paper describes a methodology in maternal plasma, aimed at detecting homozygous CF mutations in free fetal DNA. The philosophy underlying this proposal places emphasis on screening directly for fetal CF, thereby avoiding first level screening that focuses on screening to identify CF carriers when both parents are found to be heterozygous for individual mutations. In this latter circumstance, the fetus would have a 1 in 4 risk of having CF. Invasive testing (amniocentesis or chorion villus sampling) would provide the definitive answer. The methodology described here, which detects the fetus directly, also requires invasive testing for confirmation but avoids 3 out of 4 invasive procedures required by parental carrier screening.\n\nThe present concept is a product of expertise involving several disciplines, including molecular biology, population screening (with emphasis in pregnancy), and biostatistics. Description of the methodology is plausible, including limitations resulting from low percentage of free fetal DNA in a given sample, and volume of maternal blood sample necessary for adequate numbers of DNA fragments. This concept has been extensively thought out, including potential applications in various screening scenarios. Although not explicitly stated, the methodology appears ready for testing as a proof of concept. Assuming success, this could then serve as a prelude to determining how most appropriately to introduce implementation.\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": "27536",
"date": "08 Nov 2017",
"name": "Ioannis Prassas",
"expertise": [
"Reviewer Expertise Biomarker discovery",
"proteomics",
"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\nA novel study which introduces a novel method for prenatal detection of CF. It is based on the direct quantitative amplification of the CF gene to determine the percentage of targeted DNA fragments (CF mutation sites) in maternal plasma. Avoiding the need for standard parental carrier testing, the proposed procedure is positioned to reduce the number of invasive diagnostic procedures (amniocentesis or chorionic villus sampling) without compromising the total CF detection rate.\n\nThe study is well-designed and the results are carefully interpreted. The conclusions drawn are adequately supported by the results. The few associated limitations of the proposed technology are properly highlighted by the authors.\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/6-1896
|
https://f1000research.com/articles/6-1889/v1
|
26 Oct 17
|
{
"type": "Research Article",
"title": "Risk of solid cancer in patients with mast cell activation syndrome: Results from Germany and USA",
"authors": [
"Gerhard J. Molderings",
"Thomas Zienkiewicz",
"Jürgen Homann",
"Markus Menzen",
"Lawrence B. Afrin",
"Thomas Zienkiewicz",
"Jürgen Homann",
"Markus Menzen",
"Lawrence B. Afrin"
],
"abstract": "Background: It has been shown repeatedly that mast cells can promote or prevent cancer development and growth. If development and/or progression of a solid cancer is substantially influenced by mast cell activity, the frequencies of occurrence of solid cancers in patients with primary mast cells disorders would be expected to differ from the corresponding prevalence data in the general population. In fact, a recent study demonstrated that patients with systemic mastocytosis (i.e., a rare neoplastic variant of the primary mast cell activation disease) have increased risk for solid cancers, in particular melanoma and non-melanoma skin cancers. The aim of the present study is to examine whether the risk of solid cancer is increased in systemic mast cell activation syndrome (MCAS), the common systemic variant of mast cell activation disease. Methods: In the present descriptive study, we have analysed a large (n=828) patient group with MCAS, consisting of cohorts from Germany and the USA, for occurrence of solid forms of cancer and compared the frequencies of the different cancers with corresponding prevalence data for German and U.S. general populations. Results: Sixty-eight of the 828 MCAS patients (46 female, 22 male) had developed a solid tumor before the diagnosis of MCAS was made. Comparison of the frequencies of the malignancies in the MCAS patients with their prevalence in the general population revealed a significantly increased prevalence for melanoma and cancers of the breast, cervix uteri, ovary, lung, and thyroid in MCAS patients. Conclusions: Our data support the view that mast cells may promote development of certain malignant tumors. These findings indicate a need for increased surveillance of certain types of cancer in MCAS patients irrespective of its individual clinical presentation.",
"keywords": [
"mast cell",
"systemic mast cell activation disease",
"systemic mast cell activation syndrome",
"systemic mastocytosis",
"cancer",
"melanoma",
"breast cancer",
"cervical carcinoma"
],
"content": "Introduction\n\nSystemic mast cell activation disease (MCAD) comprises a heterogeneous group of multifactorial polygenic disorders characterized by aberrant release of variable subsets of mast cell (MC) mediators together with accumulation of either morphologically altered and immunohistochemically identifiable mutated MCs due to MC proliferation (systemic mastocytosis [SM] and mast cell leukemia [MCL]) or morphologically ordinary MCs due to decreased apoptosis (MC activation syndrome [MCAS] or well-differentiated systemic mastocytosis; Table 1; for details, see 1). The various MCAD classes and clinical subtypes represent varying manifestations of a common process of MC dysfunction2–5 (for details, see Supplementary File 1). While the prevalence of SM in Europeans ranges between 0.3 and 13 per 100,0006–8, the prevalence for MCAS may be in the single-digit percentage range (at least in Germany9).\n\nMCs are best known for their effector functions in IgE-associated allergic reactions, but they are also involved in a variety of processes maintaining homeostasis or contributing to disease. Studies have shown that MCs accumulate in tumors and their microenvironment, inferring potential for influencing tumor development, tumor-induced angiogenesis, tissue remodeling, and shaping of adaptive immune responses to tumors by release of certain subsets of mediators (e.g., EGF, NGF, PDGF, SCF, angiopoetin, heparin, IL-8, VEGF). Increased accumulation of MCs within tumor environments has been correlated with poor prognosis, increased metastasis, and reduced survival in several types of human cancer, including melanoma10–12, prostate carcinoma13, pancreatic adenocarcinoma14, squamous cell carcinomas of the esophagus15, mouth16, and lip17, and Merkel cell carcinoma18. Conflicting findings have been reported for lung adenocarcinoma and breast carcinomas: increased numbers of MCs have been shown to correlate with either a good19–21 or poor22,23 prognosis in non-small cell lung cancer. In breast carcinomas, most24–27 but not all28,29 studies have linked increased MC density to a good prognosis. For prostate cancer and colorectal cancer, MC densities within the tumors seem to be an independent favorable prognostic factor30–35, whereas high numbers of peritumoral MCs were associated with a poor prognosis36,37.\n\nTumorigenic effects of MCs could be pronounced in MCAD, which is characterized by an increased systemic density and activity of MCs. Knowledge of increased risk for cancer in patients with MCAD would first have important implications for counseling and care of these patients and second would support the idea of an involvement of MCs in tumorigenesis. In fact, a recent study demonstrated that SM patients bear increased risk for solid cancers, in particular melanoma and non-melanoma skin cancers38. However, studies of occurrence of solid cancer in the proportionally prevalent MCAD patient group with MCAS patients have not yet been performed. Therefore, the aim of the present study was to analyze a large MCAS patient group, consisting of two roughly equally sized cohorts from Germany and the USA retrospectively, for the occurrence of solid cancers and to compare their frequencies with corresponding prevalences in German and U.S. general populations.\n\n\nMethods\n\nMCAS was diagnosed per current provisional criteria (for detailed discussion of the criteria, see Supplementary File 1: Table S2). For differential diagnosis, other diseases presenting similar symptoms were ruled out by appropriate assessments, including laboratory testing, imaging, and/or endoscopy. Occurrence of solid cancer was determined from the medical history obtained at the time MCAS was diagnosed. Due to the retrospective character of the study, pathological material of the solid malignancy could not be specifically stained for MCs. Since in MCAD the profile of MC mediator elevations is highly dependent on individual conditions, correlation analysis of mediator levels determined as part of MCAS diagnosis and the occurrence of specific tumor types was not performed.\n\nGerman MCAS patients. 417 Caucasians presented consecutively to the Bonn Interdisciplinary Research Group for Systemic Mast Cell Diseases between May 2005 and May 2016 for diagnostic evaluation, and were assigned a diagnosis of MCAS diagnosed per current criteria (39, Table S2). These patients were included in this retrospective study. The presence of MCAS was the only inclusion criterion; there were no exclusion criteria. From the patients´ clinical files, data concerning the occurrence of solid tumors and hematologic neoplasms up to the time of the presentation at our research group were extracted. All data in this study were collected during routine clinical evaluations of MCAS patients who provided informed consent for use of such data in research. Patient information was anonymized prior to analysis. As such, the Ethics Committee of the Medical Faculty of the University of Bonn classified this study as exempt from requiring specific patient consent. This committee also approved the protocol for this study.\n\nThe frequencies of solid cancers in the German MCAS patient cohort were compared with the 10-year prevalences of these cancers in the general German population40.\n\nU.S. MCAS patients. The full population of 411 U.S. patients included in this study was comprised of a 296 patient population examined retrospectively (protocol Pro00015852, diagnoses made between November 2008 and September 2012 per current criteria (Table S2)) and a 115 patient population examined prospectively (protocol Pro00015857, diagnoses made between April 2012 and October 2013 per current criteria (Table S2)) at a single center (the Medical University of South Carolina). Protocols were approved by the center’s institutional review board (IRB); the retrospective protocol was deemed IRB-exempt, and all prospective subjects provided written informed consent. Eligible patients for the retrospective protocol were all living, and deceased adult (18 years or older) patients who were, or had been, diagnosed with MCAS from the first such diagnosis at MUSC to the opening of the protocol; the accrual goal for the prospective protocol (targeting adult patients clinically suspected of having MCAS and undergoing diagnostic testing for such, and primarily designed to assess differences in monocyte growth factors between MCAS patients with monocytosis vs. healthy control subjects) was set based on expectations (derived from preliminary data) of finding monocytosis in 70% of MCAS patients.\n\nRetrospective patients were identified through interrogation of the MUSC enterprise data warehouse for patients diagnosed with MCAS; the subjects’ medical records at MUSC served as the sole source of data for the study, and no patient was contacted to obtain additional information. Prospective patients were identified in the course of the principal investigator’s (LBA’s) clinical work and provided written informed consent. All diagnoses met published criteria39 and were made at age 16 or older. Data items abstracted from eligible patients’ medical records included gender, race, age, symptoms, comorbidities, date of MCAS diagnosis, and all results on file of routine complete blood counts, routine chemistry panels, and diagnostic testing for MCAS per published criteria.\n\nThe frequencies of solid cancers in the U.S. MCAS patient cohort were compared with the 32-year prevalences of these cancers in the general U.S. population (from Cancer Statistics Review 1975–2013; all races)41.\n\nOur MCAS patient cohorts were not corrected for cancer risk factors, such as smoking, alcohol intake and body mass index, since the data for the German and U.S. population that were used for comparison were also not corrected in that way. Due to the retrospective nature of the study, we could only calculate the prevalence of a given tumor from the medical histories of the patients, not its incidence. Therefore, standardized incidence ratios could not be calculated and differences between the frequencies of tumor occurrence in our patient groups and the corresponding prevalences in the German and U.S. general population were analyzed by means of two-sided χ2 test using GraphPad InStat V3.05. Here, a significance level of α=0.05 was set.\n\n\nResults\n\nThe 828 MCAS patients included in the study showed male:female ratios of about 1:2.5 in both population groups, which did not differ significantly in age distribution (Table 2). Forty-four MCAS patients had additional associated hematologic neoplasms, most frequently multiple myeloma, JAK2-positive essential thrombocytosis, and chronic lymphocytic leukemia (Table 2). In these patients there was no comorbidity of a solid cancer.\n\nMCAS, systemic mast cell activation syndrome; SD, standard deviation.\n\nGerman patient group. Eighteen of 417 MCAS patients (15 female, 3 male) had developed a solid tumor before the diagnosis of MCAS was made (Table 3). The most frequent tumor was breast cancer in eight patients (Table 3). The comparison of the frequencies of the malignancies in the MCAS patients with their 10-year prevalence in the German general population revealed in subsets of the MCAS patients a significantly increased prevalence for melanoma (P<0.001), lung cancer (P<0.0001), breast cancer (P<0.003), cervical carcinoma (P<0.0001), cancer of the urinary bladder (P<0.03) and testis (P=0.05) (Table 3).\n\nParentheses, number of patients in the respective age group; bold print, number of affected patients; f, female; m, male. P, two-sided P value in the Chi-square-test; ns, not significant\n\nUS patient group. Sixty-three of the 411 MCAS patients had developed a solid tumor before the diagnosis of MCAS was made (Table 4), 47 female and 16 male patients. The difference to the numbers of solid tumors listed in Table 4 is due to the fact that some patients had more than one solid tumor. The most frequent tumors were non-melanoma skin cancer and breast cancer (Table 4). The comparison of the frequencies of the malignancies in the MCAS patients with their 32-year prevalence in the U.S. general population revealed in subsets of the MCAS patients a significantly increased prevalence for lung cancer (P<0.0001), cervical uterine carcinoma (P<0.0001), ovarian cancer (P<0.02), cancer of the urinary bladder (P<0.04) and thyroid cancer (P<0.01) (Table 4).\n\nParentheses, number of patients in the respective age group; bold print, number of affected patients; f, female; m, male, ys, years; BCC, basal cell carcinoma. P, two-sided P value in the Chi-square-test; ns, not significant\n\n1Statistics of basal and squamous cell skin cancers are not reported to and tracked by cancer registries. Data based on mathematical modeling are taken from 48.\n\n\nDiscussion\n\nIt has been shown that MCs can promote tumor development and growth (for references, see Introduction). These tumorigenic effects of MCs could be pronounced in MCAD (Table 1), which is characterized by an increased systemic density and activity of MCs. An increased risk for solid cancer has been demonstrated for SM patients38, but MCAS patients have not been investigated in this respect so far.\n\nThe present analysis of the frequencies of solid malignancies in the MCAS patients revealed a significantly increased prevalence of melanoma in a subset (male, <50 years) of the German patient group. The more heterogeneous ethnic makeup of the U.S. patient group might be a factor in that group’s lower rate of melanoma compared to the Caucasian-only German group, as different ethnic groups might have different risk for melanoma. An increased risk for melanoma has been observed in previous studies with Caucasian SM patients38,42–45 with a prevalence of 5% in 81 Swedish SM patients43. It has been speculated that, as in SM, mutations in tyrosine kinase KIT, which have also been reported in melanoma46,47, may predispose MCAD patients to melanoma. In addition, the cytokines produced by MCs may recruit melanocytes and stimulate proliferation12,43.\n\nFurthermore, an increased risk for cervical carcinoma, lung and bladder cancer was found in the present study in both the German and U.S. cohorts, and increased risk for breast carcinoma and cancer of the testis was found in the German cohort. The marginal discordances in the type of solid cancers observed in the two groups could be explained by the more heterogeneous ethnic makeup of the U.S. patient group and the limited number of patients included in the study.\n\nIt is striking that it is the skin and respiratory and genitourinary tracts – i.e., environmental interfaces – where the increased frequencies were (mostly) seen. Given that (1) MCs preferentially site themselves at the environmental interfaces, (2) MCs have ample capacity to promote local and systemic inflammatory states, and (3) risk for many forms of cancer appears correlated with chronic inflammation, one wonders if the increased risk for these skin and genitourinary tract cancers bears any relationship to the relatively increased density of MCs under normal circumstances in these sites, and thus also potential for greater chronic inflammatory stimulus in these sites.\n\nIn the combined study population, intestinal cancer and prostate cancer were as frequent as in the German and U.S. general population. Thus, the reported protective effect of an increased systemic MC activity and density on the development of these two cancer forms is not observed in our study population who have increased systemic MC activity and density. Our findings are in contrast to data from epidemiological studies suggesting that allergic disease, which is also characterized by increased MC activity, is associated with decreased risk for colorectal cancer (48,49; for review, see 50). It is conceivable that in the present study the ethnic heterogeneity of the combined study population might have masked the expected protective effect51. In addition, the number of patients included in the present study might still be too low to clearly reveal the purported protective effect.\n\nIt is a limitation of the present study that, due to our limited numbers of MCAS patients, we had to partition the patients into only two age groups for each type of cancer for statistical analysis, whereas data from the German and U.S. general populations were partitioned into at least five age groups. However, according to the distribution of the prevalence data within those five groups, it was possible to break them down into two age groups for comparison with our frequency data. Moreover, the reliability of our frequency data is supported by recent similar findings in a Danish MCAD cohort consisting of 687 SM patients38. Unfortunately, it was not possible to compare the frequencies of the solid malignancies determined in our MCAS patient cohorts with those in SM because either the respective frequencies were not reported38 or the MCAD variant of the included patients were not defined exactly (i.e., SM or MCAS)52. Moreover, neither of these publications provided the age of the patient at which the cancer occurred. Finally, our German and U.S. cohorts appear to differ in their frequencies of hematologic neoplasms. At present, we could only speculate about reasons for this difference.\n\nIn conclusion, our data support the view that MCs may promote development of certain malignancies. These findings indicate a need for increased surveillance of cancers more frequently seen in MCAS patients. Given the influence of inflammation on neoplasia and the chronic multisystem inflammatory state that is the essence of MCAS, it is conceivable that treatment of MCAS (presuming recognition of MCAS in the first place) may reduce risk for neoplasia. It also is possible that treatment of MCAS in the setting of cancer (regardless of whether the MCAS or the cancer is discovered first) may favorably influence the course of the cancer (e.g. 53), similar to the favorable effects often seen when SM is treated in the setting of associated hematologic neoplasia54.\n\n\nData availability\n\nDataset 1: Raw data supporting the findings in this study. Sheet 1, Data for the German patient cohort; Sheet 2, Data for the U.S. patient cohort. doi, 10.5256/f1000research.12730.d18145055",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nPublication was supported by the Förderclub Mastzellforschung e.V., Germany. Collection of the U.S. data was supported by The Mastocytosis Society.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: Classification of and diagnostic criteria for mast cell activation disease.\n\nClick here to access the data.\n\nTable S1. WHO criteria defining Systemic Mastocytosis\n\nTable S2. Criteria defining Mast Cell Activation Syndrome\n\nTable S3. Criteria alternatively proposed to define mast cell activation syndrome\n\n\nReferences\n\nAfrin LB, Butterfield JH, Raithel M, et al.: Often seen, rarely recognized: mast cell activation disease--a guide to diagnosis and therapeutic options. Ann Med. 2016; 48(3): 190–201. PubMed Abstract | Publisher Full Text\n\nMolderings GJ, Kolck UW, Scheurlen C, et al.: Multiple novel alterations in Kit tyrosine kinase in patients with gastrointestinally pronounced systemic mast cell activation disorder. Scand J Gastroenterol. 2007; 42(9): 1045–1053. PubMed Abstract | Publisher Full Text\n\nMolderings GJ, Meis K, Kolck UW, et al.: Comparative analysis of mutation of tyrosine kinase kit in mast cells from patients with systemic mast cell activation syndrome and healthy subjects. Immunogenetics. 2010; 62(11–12): 721–727. PubMed Abstract | Publisher Full Text\n\nHermine O, Lortholary O, Leventhal PS, et al.: Case-control cohort study of patients' perceptions of disability in mastocytosis. PLoS One. 2008; 3(5): e2266. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAkin C, Valent P, Metcalfe DD: Mast cell activation syndrome: proposed diagnostic criteria. J Allergy Clin Immunol. 2010; 126(6): 1099–104.e4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaenisch B, Nöthen MM, Molderings GJ: Systemic mast cell activation disease: the role of molecular genetic alterations in pathogenesis, heritability and diagnostics. Immunology. 2012; 137(3): 197–205. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCohen SS, Skovbo S, Vestergaard H, et al.: Epidemiology of systemic mastocytosis in Denmark. Br J Haematol. 2014; 166(4): 521–528. PubMed Abstract | Publisher Full Text\n\nvan Doormaal JJ, Arends S, Brunekreeft KL, et al.: Prevalence of indolent systemic mastocytosis in a Dutch region. J Allergy Clin Immunol. 2013; 131(5): 1429–31.e1. PubMed Abstract | Publisher Full Text\n\nMolderings GJ, Haenisch B, Bogdanow M, et al.: Familial occurrence of systemic mast cell activation disease. PLoS One. 2013; 8(9): e76241. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRibatti D, Ennas MG, Vacca A, et al.: Tumor vascularity and tryptase-positive mast cells correlate with a poor prognosis in melanoma. Eur J Clin Invest. 2003; 33(5): 420–425. PubMed Abstract | Publisher Full Text\n\nRibatti D, Vacca A, Ria R, et al.: Neovascularisation, expression of fibroblast growth factor-2, and mast cells with tryptase activity increase simultaneously with pathological progression in human malignant melanoma. Eur J Cancer. 2003; 39(5): 666–674. PubMed Abstract | Publisher Full Text\n\nTóth-Jakatics R, Jimi S, Takebayashi S, et al.: Cutaneous malignant melanoma: correlation between neovascularization and peritumor accumulation of mast cells overexpressing vascular endothelial growth factor. Hum Pathol. 2000; 31(8): 955–960. PubMed Abstract | Publisher Full Text\n\nNonomura N, Takayama H, Nishimura K, et al.: Decreased number of mast cells infiltrating into needle biopsy specimens leads to a better prognosis of prostate cancer. Br J Cancer. 2007; 97(7): 952–956. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStrouch MJ, Cheon EC, Salabat MR, et al.: Crosstalk between mast cells and pancreatic cancer cells contributes to pancreatic tumor progression. Clin Cancer Res. 2010; 16(8): 2257–2265. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElpek GO, Gelen T, Aksoy NH, et al.: The prognostic relevance of angiogenesis and mast cells in squamous cell carcinoma of the oesophagus. J Clin Pathol. 2001; 54(12): 940–944. PubMed Abstract | Free Full Text\n\nIamaroon A, Pongsiriwet S, Jittidecharaks S, et al.: Increase of mast cells and tumor angiogenesis in oral squamous cell carcinoma. J Oral Pathol Med. 2003; 32(4): 195–199. PubMed Abstract | Publisher Full Text\n\nRojas IG, Spencer ML, Martinez A, et al.: Characterization of mast cell subpopulations in lip cancer. J Oral Pathol Med. 2005; 34(5): 268–273. PubMed Abstract | Publisher Full Text\n\nBeer TW, Ng LB, Murray K: Mast cells have prognostic value in Merkel cell carcinoma. Am J Dermatopathol. 2008; 30(1): 27–30. PubMed Abstract | Publisher Full Text\n\nTomita M, Matsuzaki Y, Onitsuka T: Correlation between mast cells and survival rates in patients with pulmonary adenocarcinoma. Lung Cancer. 1999; 26(2): 103–108. PubMed Abstract | Publisher Full Text\n\nWelsh TJ, Green RH, Richardson D, et al.: Macrophage and mast-cell invasion of tumor cell islets confers a marked survival advantage in non-small-cell lung cancer. J Clin Oncol. 2005; 23(35): 8959–8967. PubMed Abstract | Publisher Full Text\n\nCarlini MJ, Dalurzo MC, Lastiri JM, et al.: Mast cell phenotypes and microvessels in non-small cell lung cancer and its prognostic significance. Hum Pathol. 2010; 41(5): 697–705. PubMed Abstract | Publisher Full Text\n\nImada A, Shijubo N, Kojima H, et al.: Mast cells correlate with angiogenesis and poor outcome in stage I lung adenocarcinoma. Eur Respir J. 2000; 15(6): 1087–1093. PubMed Abstract | Publisher Full Text\n\nTakanami I, Takeuchi K, Naruke M: Mast cell density is associated with angiogenesis and poor prognosis in pulmonary adenocarcinoma. Cancer. 2000; 88(12): 2686–2692. PubMed Abstract | Publisher Full Text\n\nAaltomaa S, Lipponen P, Papinaho S, et al.: Mast cells in breast cancer. Anticancer Res. 1993; 13(3): 785–788. PubMed Abstract\n\nDabiri S, Huntsman D, Makretsov N, et al.: The presence of stromal mast cells identifies a subset of invasive breast cancers with a favorable prognosis. Mod Pathol. 2004; 17(6): 690–695. PubMed Abstract | Publisher Full Text\n\nRajput AB, Turbin DA, Cheang MC, et al.: Stromal mast cells in invasive breast cancer are a marker of favourable prognosis: a study of 4,444 cases. Breast Cancer Res Treat. 2008; 107(2): 249–257. PubMed Abstract | Publisher Full Text | Free Full Text\n\ndella Rovere F, Granata A, Familiari D, et al.: Mast cells in invasive ductal breast cancer: different behavior in high and minimum hormone-receptive cancers. Anticancer Res. 2007; 27(4B): 2465–2471. PubMed Abstract\n\nXiang M, Gu Y, Zhao F, et al.: Mast cell tryptase promotes breast cancer migration and invasion. Oncol Rep. 2010; 23(3): 615–619. PubMed Abstract | Publisher Full Text\n\nRibatti D, Finato N, Crivellato E, et al.: Angiogenesis and mast cells in human breast cancer sentinel lymph nodes with and without micrometastases. Histopathology. 2007; 51(6): 837–842. PubMed Abstract | Publisher Full Text\n\nNielsen HJ, Hansen U, Christensen IJ, et al.: Independent prognostic value of eosinophil and mast cell infiltration in colorectal cancer tissue. J Pathol. 1999; 189(4): 487–495. PubMed Abstract | Publisher Full Text\n\nTan SY, Fan Y, Luo HS, et al.: Prognostic significance of cell infiltrations of immunosurveillance in colorectal cancer. World J Gastroenterol. 2005; 11(8): 1210–1214. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYodavudh S, Tangjitgamol S, Puangsa-art S: Prognostic significance of microvessel density and mast cell density for the survival of Thai patients with primary colorectal cancer. J Med Assoc Thai. 2008; 91(5): 723–732. PubMed Abstract\n\nElezoğlu B, Tolunay S: The relationship between the stromal mast cell number, microvessel density, c-erbB-2 staining and survival and prognostic factors in colorectal carcinoma. Turk Patoloji Derg. 2012; 28(2): 110–118. PubMed Abstract | Publisher Full Text\n\nAcikalin MF, Oner U, Topcu I, et al.: Tumour angiogenesis and mast cell density in the prognostic assessment of colorectal carcinomas. Dig Liver Dis. 2005; 37(3): 162–169. PubMed Abstract | Publisher Full Text\n\nGulubova M, Vlaykova T: Prognostic significance of mast cell number and microvascular density for the survival of patients with primary colorectal cancer. J Gastroenterol Hepatol. 2009; 24(7): 1265–1275. PubMed Abstract | Publisher Full Text\n\nFisher ER, Paik SM, Rockette H, et al.: Prognostic significance of eosinophils and mast cells in rectal cancer: findings from the National Surgical Adjuvant Breast and Bowel Project (protocol R-01). Hum Pathol. 1989; 20(2): 159–163. PubMed Abstract | Publisher Full Text\n\nJohansson A, Rudolfsson S, Hammarsten P, et al.: Mast cells are novel independent prognostic markers in prostate cancer and represent a target for therapy. Am J Pathol. 2010; 177(2): 1031–1041. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBroesby-Olsen S, Farkas DK, Vestergaard H, et al.: Risk of solid cancer, cardiovascular disease, anaphylaxis, osteoporosis and fractures in patients with systemic mastocytosis: A nationwide population-based study. Am J Hematol. 2016; 91(11): 1069–1075. PubMed Abstract | Publisher Full Text\n\nMolderings GJ, Brettner S, Homann J, et al.: Mast cell activation disease: a concise practical guide for diagnostic workup and therapeutic options. J Hematol Oncol. 2011; 4: 10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobert Koch-Institut (eds): Verbreitung von Krebserkrankungen in Deutschland. Entwicklung der Prävalenzen zwischen 1990 und 2010. Beiträge zur Gesundheitsberichterstattung des Bundes. RKI, Berlin, [ISBN 978-3-89606-208-6], 2010. Reference Source\n\nHowlader N, Noone AM, Krapcho M, et al. (eds): SEER Cancer Statistics Review, 1975–2013. National Cancer Institute. Bethesda, MD, based on November 2015 SEER data submission, posted to the SEER web site, 2016. Reference Source\n\nStern RS: Prevalence of a history of skin cancer in 2007: results of an incidence-based model. Arch Dermatol. 2010; 146(3): 279–282. PubMed Abstract | Publisher Full Text\n\nHägglund H, Sander B, Gülen T, et al.: Increased risk of malignant melanoma in patients with systemic mastocytosis? Acta Derm Venereol. 2014; 94(5): 583–584. PubMed Abstract | Publisher Full Text\n\nTodd P, Garioch J, Seywright M, et al.: Malignant melanoma and systemic mastocytosis--a possible association? Clin Exp Dermatol. 1991; 16(6): 455–457. PubMed Abstract | Publisher Full Text\n\nPaolino G, Belmonte M, Trasarti S, et al.: Mast Cell Disorders, Melanoma and Pancreatic Carcinoma: From a Clinical Observation to a Brief Review of the Literature. Acta Dermatovenerol Croat. 2017; 25(2): 112–119. PubMed Abstract\n\nSlipicevic A, Herlyn M: KIT in melanoma: many shades of gray. J Invest Dermatol. 2015; 135(2): 337–338. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPhung B, Kazo JU, Lundby A, et al.: KIT/D816V induces SRC-mediated tyrosine phosphorylation of MITF and altered transcription program in melanoma. Proceedings of the AACR 107th Meeting.2016; 76(14 Suppl): Abstract 1127. Publisher Full Text\n\nSherman PW, Holland E, Sherman JS: Allergies: their role in cancer prevention. Q Rev Biol. 2008; 83(4): 339–362. PubMed Abstract | Publisher Full Text\n\nNegri E, Bosetti C, La Vecchia C, et al.: Allergy and other selected diseases and risk of colorectal cancer. Eur J Cancer. 1999; 35(13): 1838–1841. PubMed Abstract | Publisher Full Text\n\nMarech I, Ammendola M, Gadaleta C, et al.: Possible biological and translational significance of mast cells density in colorectal cancer. World J Gastroenterol. 2014; 20(27): 8910–8920. PubMed Abstract | Free Full Text\n\nHempel HA, Kulac I, Cuka NS, et al.: A relationship between mast cells and the racial disparity of prostate cancer. Cancer Epidemiol Biomarkers Prev. 2016; 25(Suppl 3): Abstract C73. Publisher Full Text\n\nTravis WD, Li CY, Bergstralh EJ: Solid and hematologic malignancies in 60 patients with systemic mast cell disease. Arch Pathol Lab Med. 1989; 113(4): 365–368. PubMed Abstract\n\nAfrin LB, Spruill LS, Schabel SI, et al.: Improved metastatic uterine papillary serous cancer outcome with treatment of mast cell activation syndrome. Oncology (Williston Park). 2014; 28(2): 129–131. PubMed Abstract\n\nValent P, Sperr WR, Akin C: How I treat patients with advanced systemic mastocytosis. Blood. 2010; 116(26): 5812–5817. PubMed Abstract | Publisher Full Text\n\nMolderings GJ, Zienkiewicz T, Homann J, et al.: Dataset 1 in: Risk of solid cancer in patients with mast cell activation syndrome: Results from Germany and USA. F1000Research. 2017. Data Source"
}
|
[
{
"id": "27327",
"date": "21 Nov 2017",
"name": "Bastian Walz",
"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\nThey use very large cohorts for the study.\nThe authors found an accumulation of hematological neoplasia in the US cohort. This circumstance is probably due to a selection bias, as the attending physician is a haemato-oncologist.\nThe accumulation of malignant skin tumours could also be caused by a photosensitisation due to mast cell activation disease, as many patients suffer from a rapid skin reaction to UV light.\nThe present work would be proposed for publication, not least because there are still only a few working groups in this area and there is a strong unmet scientific need for further investigation and information in mast cell activation disease.\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": "28307",
"date": "30 Nov 2017",
"name": "Leonard Weinstock",
"expertise": [
"Reviewer Expertise MCAS",
"RLS",
"SIBO",
"IBS",
"GI"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 mast cell (MC) was discovered by Paul Ehrlich in 1876. The MC is a unique bone marrow-derived cells since mast cells (MCs) are not found in the blood stream. They are located in tissues of the body where they can reside for many months or years.\nThe first historical MC path led to the understanding of its role in health. The next path of MC research led to the discovery of the rare, yet well accepted disease mastocytosis - a primary malignant process that had systemic MC-mediator induced symptoms. The next remarkable discovery was that mutations could lead to active MCs and be responsible for over 48 symptoms throughout the body. This confounding presentation of mast cell activation syndrome (MCAS) usually causes confusion and frustration to the physician as well as to the patient. Once MCAS is diagnosed, treatment is often helpful and allows most patients to regain a better quality of life.\nThe next query posed by Molderings et al is whether or not the MC plays a role in cancer in humans. There are four animal models to suggest that there was a modulating effect in cancer development. Other studies show potential suppression of cancer by MCs. The human connection has been explored in this journal in a large population of MCAS patients. In this study, 828 MCAS were investigated. A history of solid malignant lesions was queried prior to establishing the diagnosis of MCAS.\nThe authors suggest that this data should be compelling evidence for physicians to perform more intense screening for malignancies in those who have MCAS. The ultimate challenge is to see if modulation, suppression or stabilization of MCs and/or MCAS can decrease the risk and/or progression of cancer.\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/6-1889
|
https://f1000research.com/articles/6-1888/v1
|
25 Oct 17
|
{
"type": "Research Article",
"title": "A putative role for genome-wide epigenetic regulatory mechanisms in Huntington’s disease: A computational assessment",
"authors": [
"Eleni Mina",
"Willeke van Roon-Mom",
"Pernette Verschure",
"Peter A.C. 't Hoen",
"Mark Thompson",
"Rajaram Kaliyaperumal",
"Kristina Hettne",
"Erik Schultes",
"Barend Mons",
"Marco Roos",
"Willeke van Roon-Mom",
"Pernette Verschure",
"Peter A.C. 't Hoen",
"Mark Thompson",
"Rajaram Kaliyaperumal",
"Kristina Hettne",
"Erik Schultes",
"Barend Mons"
],
"abstract": "Background: Huntington's Disease (HD) is an incurable disease of the adult brain. Massive changes in gene expression are a prominent feature. Epigenetic effects have been reported to be implicated in HD, but the role of chromatin is not well understood. We tested if the chromatin state of dysregulated genes in HD is affected at a genome-wide scale and examined how epigenetic processes are associated with CpG-island-mediated gene expression. Methods: Our general approach incorporates computational and functional analysis of public data before embarking on expensive wet-lab experiments. We compared the location in the genome of the genes that were deregulated in HD human brain, obtained from public gene expression data, to the location of particular chromatin marks in reference tissues using data from the ENCODE project. Results: We found that differentially expressed genes were enriched in the active chromatin state, but not enriched in the silent state. In the caudate nucleus, the most highly affected brain region in HD, genes in the active state were associated with transcription, cell cycle, protein transport and modification, RNA splicing, histone post-translational modifications and RNA processing. Genes in the repressed state were linked with developmental processes and responses related to zinc and cadmium stimulus. We confirmed that genes within CpG-islands are enriched among HD dysregulated genes in human and mouse in HD. Epigenetic processes were associated more with genes that overlap with CpG-islands than genes that do not. Conclusion: Our results suggest that massive transcriptional dysregulation in HD is not matched by large-scale relocation of gene activity, i.e. inactive chromatin regions are altered into actively expressed chromatin regions and vice versa. We expect that changes in epigenetic chromatin state might occur at the level of single genes (e.g. promoters, gene body) and scattered genomic sites (e.g. CTCF sites, enhancer regions) instead of large-scale genomic regions.",
"keywords": [
"Huntington's disease",
"gene expression",
"CpG-islands",
"caudate nucleus",
"chromatin regions"
],
"content": "Introduction\n\nHuntington’s Disease (HD) is a complex disease of the brain associated with massive changes in gene expression. The genetic cause was identified in 19931, but no successful treatment has been found yet. HD is a dominantly inherited neurodegenerative disease that affects 1 – 10/100.000 individuals, making it the most common heritable neurodegenerative disorder2.\n\nAlthough HD is considered a monogenic disease3, extensive research since 1993 into the underlying pathology suggests that the disease mechanisms are more complex than originally considered. Transcriptional dysregulation is a widespread phenomenon in HD that can be observed well before the first clinical symptoms appear4. It suggests that mutant huntingtin causes a broad and complex cascade of downstream effects. There are several ways in which mutant huntingtin can interfere with the transcriptional machinery and alter gene expression5,6. Given the extent of the transcriptional changes, the question arises if epigenetic mechanisms that operate at a genome-wide scale are also involved in HD. There is increasing evidence for epigenetic mechanisms playing a role in HD in human and model systems. For instance, earlier computational analysis of HD gene expression data showed that expression is deregulated in large genomic regions, indicative of a coordinated genome-wide mechanism7. These studies did not determine whether genome-wide alterations in gene expression are associated with changes in the composition of histone modifications.\n\nMore recently, H3K4me3 was shown to be enriched in 136 loci in an HD case/control study, which included genes that may affect the neuronal epigenome at large8. In HD mice, down-regulated genes were found to be associated with a selective decrease of H3K27ac and RNA Polymerase II9. Inhibitors of HDAC1, broad-range regulators of chromatin structure, have indeed been shown to be effective in HD mice10. The interplay of enzymes involved in post-translational histone modifications like histone deacetylaces (HDACs), may make chromatin less accessible in many places and therefore alter gene expression patterns. In Drosophila, the homolog of htt was found to suppress position effect variegation (PEV), possibly by influencing PEV modifier genes11.\n\nA role for epigenetic mechanisms in HD is further corroborated by numerous neurodevelopmental and neurodegenerative disorders that have been associated with an altered chromatin structure12–14. Neuroepigenetics has therefore become a prime topic of interest15, with researchers seeking to identify epigenomic signatures and how they can contribute to brain health or brain disease16.\n\nConsidering the growing body of evidence that epigenetic regulation is involved in neurological pathology, we asked if the massive transcriptional dysregulation in HD coincides with large scale changes in chromatin state across the genome. Under that assumption, we hypothesize that significant numbers of differentially expressed genes in HD will be found in regions that are not normally associated with active chromatin states.\n\nHere we report on a computational test of our hypothesis before laboratory experiments using only publicly available data sets: HD gene expression from the Gene Expression Omnibus (GEO)17 and chromatin state from ENCODE18. We assessed the overlap between differentially expressed genes and chromatin state, and we applied literature-based concept profile analysis (CPA) to interpret our findings19,20.\n\nOur results indicate that the massive transcriptional dysregulation in HD is not matched by a significant largescale change of activity of genes that are part of inactive chromatin states in reference tissue. Our report includes a functional characterization of differentially expressed genes in HD in relation to huntingtin, chromatin state and CpG islands, based on a literature-based semantic analysis.\n\nThe analysis we performed to test our hypothesis is part of an interdisciplinary research approach where computational analysis is used to help steer laboratory experiments to increase the overall efficiency of a research laboratory21.\n\n\nMethods\n\nIn Concept Profile Analysis (CPA), the vector space model is used to associate two concepts mined from the literature with each other. Advantages of this model include efficient and transparent comparisons, and the possibility of attaching a weight to the association20. The CPA algorithms have previously been used for a range of different gene expression data analysis purposes such as functional annotation22, comparison of studies23, prediction of novel interactions24, generation of gene sets19,25, and association with chemical structures26). The methodology has been described previously27. In short: In our database, every concept is associated with PubMed records using the indexing engine Peregrine (https://trac.nbic.nl/data-mining/) which is equipped with an in-house thesaurus of biomedical and chemical concepts that have been prepared for text mining28,29. For all concepts except genes and Gene Ontology (GO) terms the PubMed records are comprised of the texts in which the concept is mentioned. For genes, only a subset of PubMed records are used in order to limit the impact of ambiguous terms and distant homologs. GO terms are sometimes given as words or phrases that are infrequently found in the normal texts. To still provide broad coverage of GO terms, the PubMed records that were used as evidence for annotating genes with the GO term are added. For every concept in the thesaurus that is associated to at least five PubMed records, a vector containing all concepts related to the main concept (direct co-occurrence), weighted by the symmetric uncertainty coefficient is created. We call this a \"Concept Profile\". Concept profiles are matched to identify similarities via their shared concepts (indirect relations). Any distance measure can be used for this matching such as the mutual information, inner product, cosine angle, Euclidean distance or Pearson’s correlation. The CPA Web Services that we used for our analysis use an inner product measure30. These web services can be found in the BioCatalogue web service registry https://www.biocatalogue.org/services/3559.\n\nFor data analysis and interpretation we implemented a series of workflows using the Taverna workbench (Taverna workbench version 2.4.0)31,32. Taverna is an open source software for the development and execution of workflows. Our workflows are deposited online on the Zenodo repository (https://doi.org/10.5281/zenodo.16420133 and https://doi.org/10.5281/zenodo.16419834) and the myExperiment platform (http://www.myexperiment.org/packs/553).\n\nThe first Taverna workflow load_data_identify_DE_genes_Array_A, was implemented to examine differential gene expression between control and HD samples. Required workflow inputs were two data files with gene expression values and the phenotype information that describes the samples from the microarray experiment. Differential expression was computed using moderated t statistics with the package limma35 (version 3.14.1), which is provided by the bioconductor project36, R version https://www.bioconductor.org/. We analyzed each brain region separately, because previous analysis37 revealed regional patterns in gene expression. The workflow maps the expression data from probes to entrez gene ids using the Affymetrix Human Genome U133 set annotation data, (packages hgu133a and hgu133b, version 2.8.0; https://bioconductor.org/packages/hgu133a.db; https://bioconductor.org/packages/hgu133b.db/). When multiple probe names map to the same gene id, the ones exhibiting the most significant changes were used for further analysis. Final outcome of this workflow is a report. Each row is composed of a gene id, a fold change and its corresponding P-value indicating the significance of every change in gene expression, between HD and controls for each brain region. Adjusted P-values, generated by Benjamini and Hochberg’s method for multiple testing correction, are also included38. This workflow can be adjusted to compute differential gene expression between other variables such as male/female or the grade of disease pathology by editing the nested workflow “compute_DE_limma” within the R workflow component. An additional workflow create_exprs_obj_download_files is included in the myExperiment pack that was used to download data from the ArrayExpress repository. The workflow saves the gene expression data and the corresponding phenotype file in the directory indicated in the workflow input.\n\nWe note that this particular microarray experiment was composed of two microarrays, Human Genome U133A and U133B. For convenience we added the workflow: Get differentially expressed genes for Array B one brain region, but in principle the first workflow could be reused by adjusting the “libraries” component.\n\nThe second workflow map_genes_on_chromosome uses the output from the first workflow in order to map genes to their corresponding genomic location. The workflow uses the Biomart39 service within R, to obtain information regarding the position of each gene at the chromosome, HGNC gene symbols40, transcription start and end site and the transcription strand. The database that was used was the Ensembl genes 68 from Sanger institute and the Homo Sapiens dataset GRCh37.p8. The mouse assembly that was used to map genes to their chromosomal location was Dec 2011, GRCm38mm10.\n\nThe last workflow get_promoter_region_calculate_overlaps, first computes a promoter region for each gene and then operates on genomic intervals to compute gene promoters that overlap with a genomic region. The promoter region is computed for each gene, according to prespecified values, indicating the number of base pairs (bp) upstream and downstream of the transcriptional start site (TSS); for the CpG island analysis 5000bp upstream and 2000bp downstream and for the chromatin states analysis 50bp upstream and 50bp downstream was used. The decision for the promoter size in each case was taken after discussing with the domain experts and from knowledge acquired from previous experiments using the ENCODE data from Ernst et al.41. Using those data we performed multiple runs with different input values for the “upstream” and “downstream” variables, and the overlap parameter (Supplementary File 1).\n\nWhen genes had multiple transcription start sites, we computed a promoter region for every TSS. Next part of this workflow is to compute overlapping regions between the input datasets. It includes a two sample Kolmogorov – Smirnov test to compare the empirical cumulative distribution functions (ecdf) of the P-values between the gene promoters that overlap with a specific genomic region and the ones that do not. The null hypothesis tested here was that there is no difference between the two groups. The workflow returns two lists of genes, one for the genes that overlap with a particular genomic region and another that does not. Furthermore, the results of the statistical test are reported: the ks test statistic (maximum distance D between the ecdf of the two samples) and the P-value of the test. If P-value < 0.05 we reject the null hypothesis.\n\nThe workflows that we implemented for gene interpretation and gene prioritization are based on the workflow pack at Zenodo, https://doi.org/10.5281/zenodo.164198 (see also, at myExperiment: http://www.myexperiment.org/packs/368), and are implemented using CPA web services30.\n\nThe CPA workflow Annotate gene list with top ranking concepts annotates a gene list with top ranking concepts by matching concept profiles of genes with for example in our case the concept set of Biological Processes. The web services part of this workflow query the Anni database27 that stores the concept profiles for each concept of interest. The first web service mapDatabaseIDListToConceptIDs maps a list of concepts, in our case Entrez gene identifiers, to their corresponding concept profile ids. Necessary inputs are a concept list (gene list) in a comma separated file, and the database identifier of the gene list necessary for the mapping (EG for Entrez Gene, see here: https://www.biocatalogue.org/soap_operations/41197 for more details on database identifiers). The next web service getSimilarConceptProfilesPredefined matches our gene list with the predefined concept set “Biological Process” (ID = “5”), and gives the top scoring biological processes that describe our gene list. For a complete list of predefined concept sets, the workflow List Concept Sets (provided in the current pack) can be run to choose the ID of the predefined concept set of interest. The web service getConceptName, gives the complete (human readable) names of the top matching biological processes. Lastly, the workflow Explain score between two concepts can be included to the analysis to provide evidence for the association between each gene and the annotations of the biological processes. The evidence reported is a list of concepts that link one concept with another and the contribution to the overall strength of the association. In addition, the corresponding concept ids are reported.\n\nThe workflow Prioritize gene list can prioritize a set of genes with respect to their association with particular concepts, in our case the HTT concept and epigenetics (concept profile: “epigene”). In order to obtain the concept profile identifiers the workflow getConceptSuggestionsFromTerm needs to run first.\n\nHuman brain data. The HD human brain data that was used in this analysis was originally produced and analyzed by A. Hodges and co-workers37. This experiment contains 44 HD positive cases and 36 age and sex matched controls. The processed data are available from the public repository NCBI Gene Expression Omnibus, entry GSE3790. Three brain regions were included and analyzed; the caudate nucleus, frontal cortex and cerebellum, with an Affymetrix Microarray GeneChip (Human Genome U133A and U133B). Furthermore, the HD positive cases were further classified based on whether symptoms were present or absent and according to Vonsattel grade of disease pathology (scale = 0 – 4). In our analysis we used the processed data and performed our own differential gene expression analysis (Dataset 142) with the workflow that performs differential gene expression analysis that was described previously in Methods.\n\nCpG island data. CpG island information in the human genome was obtained from UCSC genome browser43, hg19 assembly FEB 2009 1 (Dataset 244). Here, CpG islands are marked as the DNA regions where the following conditions hold:\n\nGC content of 50% or greater\n\nlength greater than 200 bp\n\nratio greater than 0.6 of observed number of CG dinucleotides to the expected number on the basis of the number of Gs and Cs in the segment\n\nThe ratio observed/expected (Obs/Exp) CpG was calculated as follows:\n\n\n\nwhere N is the total amount of nucleotides in the sequence that is being analyzed. For CpG island information of the mouse genome the assembly dec 2011 (GRCm38/mm10) was used.\n\nChromatin states data. The chromatin marks were obtained from the encode project45. The chromatin states were part of an integrative analysis of 111 reference human epigenomes profiled for histone modification patterns based on DNA accessibility, DNA methylation and RNA expression. We used the two cell types that were more suitable for our analysis; the anterior caudate and dorsolateral prefrontal cortex (Dataset 346 and Dataset 447, respectively). The chromatin states we used for the current analysis were: active transcription start site proximal promoter (TssA), bivalent regulatory region (TssBiv), heterochromatin (Het) and repressed Polycomb (ReprPC).\n\nMouse data. The mouse brain data was taken from a published study10. There, total RNA was extracted from cortex, striatum, and cerebellum from WT and R62 transgenic mice. This study examines the effect of the HDACi 4b inhibitor on the disease phenotype. However only the data from animals treated with vehicle was used (Dataset 142). This study used Illumina Mouse Mouseref-8 Expression Beadchips v1. The raw data were analyzed using the Bioconductor packages and contrast analysis of differential expression was performed by using the LIMMA package. The differential expression values are available in the supplemental material of that publication.\n\n\nResults\n\nTo test if and how differential gene expression in HD is associated with particular chromatin states we used publicly available datasets from the GEO and ENCODE public repositories. We selected HD gene expression data from three regions of the brain made available by Hodges and coworkers37, and data from the ENCODE consortium carrying information about four chromatin states. These were: active TSS proximal promoter (TssA), bivalent regulatory region (TssBiv), heterochromatin (Het) and repressed Polycomb (ReprPC)45. Briefly, the first state pertains to active genes, the second to repressed genes that are ready to be activated, and the latter two represent repressed genes. The chromatin states were part of an integrative analysis of 111 reference human epigenomes profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. The different chromatin states were defined by a computational model that is based on a multivariate Hidden Markov Model48.\n\nFirst we confirmed the results reported by the previous study37: large and numerous changes in transcriptional activity in the caudate nucleus brain region and notably smaller changes in the frontal cortex and cerebellum. More specifically, the number of differentially expressed genes was 5219, 127 and 96 for each brain region respectively with a FDR of 0.05. These results confirm previous observations illustrating that specific HD-affected brain regions exhibit defined changes in gene expression, in line with observable physiological effects37.\n\nSecondly, we paired the data describing gene expression changes in the caudate nucleus and frontal cortex from the Hodges study to chromatin state data from the anterior caudate and dorsolateral prefrontal cortex from ENCODE. We considered these brain regions as the most comparable between the two studies. The cerebellum was excluded from this part of our analysis due to the absence of chromatin state data at ENCODE for the cerebellum. We determined the reference chromatin state of the genes from the gene expression study by determining the overlap of their promoter regions with the start and end positions of the chromatin state from the aforementioned brain regions used by ENCODE. The effect of a chromatin state on differential gene expression in HD was assessed by comparing the distribution of expression levels of genes overlapping with a particular chromatin state with the distribution of expression of non-overlapping genes. If chromatin state has substantial functional impact on the genes that are differentially expressed in HD, then these are expected to be significantly different. More details on the promoter region calculation and the overlap between the genomic regions can be found in the Methods section.\n\nSpecifically, we compared the distribution of the P-values for differential expression between these two groups of genes and assessed the difference by a KolmogorovSmirnov test, in the two brain regions and for each chromatin state (Figure 1). In the caudate region, we found an enrichment of genes overlapping with the active TSS and a depletion of genes overlapping with ReprPC (Figure 1). Heterochromatic or bivalent chromatin were not significantly associated with a genomic location of differentially expressed genes. A similar, but less pronounced pattern was observed for the frontal cortex for both the active TSS and ReprPC. In addition, in the frontal cortex the number of genes overlapping with the bivalent state was reduced. The magnitude of the association of a brain region with each chromatin state is in line with the HD neurodegeneration pattern, where the caudate nucleus exhibits the largest gene expression changes while the frontal cortex exhibits an intermediate to low pathology. In summary, we found enrichment of differentially expressed genes in the active chromatin state of reference tissue, but no strong evidence for significant enrichment in chromatin regions that are associated with repressed gene expression activity.\n\nFor each brain region, the P-value distribution for differential expression in HD patients compared to controls was compared between genes overlapping with a specific chromatin state and all other genes, in the anterior caudate and dorsolateral prefrontal cortex cells. The plot displays the maximum absolute distance D between the cumulative distribution function of the P-values for each group of genes (overlapping and non overlapping), as reflected by the KS test statistic. Positive values correspond to an enrichment of differentially expressed genes in a chromatin state, negative values with a depletion. Stars indicate a significant enrichment/depletion cf the KS test (P-value < 0.05). TssA= active TSS, TssBiv= bivalent Tss, Heterochr= Heterochromatin, ReprPC= repressed Polycomb state. For caudate: TssA D= 0.31, TssBiv D=0.04, Heterochr D=0.17, ReprPC D= 0.27, for frontal cortex TssA D=0.168, TssBiv D=0.097, Heterochr D=0.095, ReprPC D=0.147. The corresponding p-values were for the caudate : TssA pval < 2.2e-16, TssBiv pval= 0.59, Heterochr pval= 0.10, ReprPC pval=4.5167e-12, for frontal cortex TssA pval < 2.2e-16, TssBiv pval= 0.0002, Heterochr pval= 0.872, ReprPC pval= 1.4596e-05.\n\nTo further interpret the results from the chromatin state analysis, we used literature information (CPA19,27) to assess the biological processes within the lists of genes per chromatin state that were associated with gene deregulation (Dataset 549).\n\nUsing CPA, we found that for the caudate nucleus, genes in the active TSS state are mainly associated with processes related to transcription, cell cycle, protein transport and modification, RNA splicing, chromatin modifications and RNA processing. Genes in the repressed polycomb chromatin state are mainly associated with (brain) developmental processes and responses related to zinc and cadmium stimulus.\n\nFor the frontal cortex, genes in the active TSS state are associated with protein transport and modification, cell cycle, RNA splicing and signal transduction pathways (MAPK, notch, smoothened). Genes in the bivalent TSS are associated with brain development, neurogenesis, synapse and responses related to zinc and cadmium stimulus. We found that the genes that are part of the polycomb repressed group to be associated with similar functions as the genes in the bivalent state: (brain) developmental processes, responses related to zinc, cadmium and copper stimuli.\n\nWe note that at this point of our analysis, we filtered out genes of which the promoters were labelled with more than one state, using the criterion of at least 50 bps overlap in a promoter region of 100 bps. Interpretation of this set of genes would be ambivalent.\n\nBecause CpG island methylation is a known epigenetic regulatory mechanism that is ubiquitous in the human genome and may be a target for genome-wide regulation50,51, we also applied our approach to test if genes within CpG islands are overrepresented among differentially expressed genes in HD. We measured this by similar KS test statistics as in the previous section. We found that genes overlapping with CpG-islands in their promoter region, were significantly enriched in the group of HD-deregulated genes in all three brain regions (Figure 2A; P-value < 0.05), which supports taking this mechanism into account to formulate hypotheses when studying gene deregulation in HD.\n\nMaximum distance between the cumulative distribution function of the P-values between genes containing a CpG island in their promoter and genes that do not. The maximum distance (D) for each brain region was plotted for both datasets (human (A) and mouse (B) datasets). The gradient adjacent to each plot indicates the extent of neurodegeneration in each brain region. Black represents severe and white mild neurodegeneration. A: The analysis performed in the human data. Caudate nucleus is exhibiting the largest differences with a distance D between the two distributions of 0.2808, frontal cortex follows with D = 0.1482 and cerebellum with D = 0.147. B: The analysis performed in the mouse data. The results are analogous to the human data with striatum showing the largest differences with D= 0.118, cortex following with D=0.0739 and cerebellum with an insignificant difference (P-value = 0.596 >> 0.05) of D = 0.0383. * : depicts significance.\n\nTo further analyze and verify the association of gene deregulation in HD with the presence of CpG islands, we also analysed overrepresentation in data obtained from a HD transgenic mouse model10. Our results based on the mouse data corroborated those from the human data (Figure 2B). Comparable to the results for caudate nucleus in human data, the most highly affected mouse brain region, the striatum, showed the biggest overrepresentation of CpG islands among deregulated genes.\n\nWe next examined the biological processes that are associated with the differentially expressed genes that do or do not overlap with CpG islands by CPA. We inspected the top 50 annotations of each group. We identified many similarities between these two groups, but also annotations that were specific to each group (Dataset 652). For example, concepts related to chromatin alterations, such as chromatin remodelling and chromatin (dis)assembly, and histone post-translational modifications, such as (de)acetylation, and (de)methylation, were only found with a high rank in the list of genes containing CpG islands in their promoters.\n\nConversely, lymphocyte activation, angiogenesis, antigen presentation and neurogenesis were only high ranking associations for the non-CpG containing genes.\n\nSome annotations were found in both groups but in a different ranking order. For example, the rankings of gene silencing, RNA splicing and phosphorylation were increased for genes within CpG islands while transcriptional activation and mitotic cell cycle rankings were higher for non-CpG containing genes (Dataset 652).\n\nCPA can also be used to prioritize findings by a specific biological interest. In our case, we aimed to prioritise the list of genes overlapping with CpG islands that were also differentially expressed in HD caudate nucleus by their association with HTT and epigenetics. Here, we prioritised 100 proteins based on their association with HTT and epigenetics and absence of a direct relation with HTT by CPA (Dataset 753). Such relations are typically novel, or lost in tables or figures. If a novel relation is found (i.e. the relation is not found in our database of relations in MedLine abstracts), then CPA also provides intermediate concepts that link the two concepts.\n\nIn Table 1, we present the top 5 novel proteins that have the strongest association, and the intermediate concepts that link each prioritized protein to HTT and epigenetics. The intermediate concepts are grouped under the semantic categories “General”, “Biological Processes”, “Disease or Syndrome”, “Homo Sapiens proteins” and “Molecular Functions”.\n\nGrouping the evidence provides a more complete insight into the processes involved in gene deregulation in HD mediated by CpG islands. For example, for one of our candidates, the amyloid beta (A4) precursor protein-binding, family A, member 1 (APBA1), we found nerve tissue (General), endocytosis (Biological Process), Alzheimer’s Disease (disease or syndrome), GRIN2B (H. sapiens Genes) and kinesin activity (Molecular Function) as intermediate links with HTT. This suggests that mechanisms involving APBA1 in HD share common components with the mechanisms involved in endocytosis and Alzheimer’s Disease and those involving GRIN2B and kinesin activity. Intermediate links with epigenetics were respectively: epigene (General), methylation (Biological Process), hypomyelination and congenital cataract (disease or syndrome), CDKN2A (H. sapiens Genes) and MGMT - O6-alkylguanine DNA alkyltransferase - (Molecular Function). Accordingly, these concepts provide suggestions about the epigenetic role of APBA1, which can be taken into account when further studying the role of APBA1 in HD.\n\nWe next investigated whether the prioritized genes reflect valid biological knowledge. Figure 3 shows that CPA is able to prioritize true associations with huntingtin as measured by a gene expression experiment, but that combining experimental (differential expression) measurements and literature evidence enables to select even more specific HD signatures. We used our concept profile technology to match all genes in our database that have a concept profile (12,391 genes) to the “huntingtin” concept profile (black line). We then compared the distribution of those CPA scores to the CPA scores of genes that were found to be differentially expressed in the caudate nucleus (p value < 0.05). We included two gene lists in our analysis: the top 100 most differentially expressed genes (red line) and the top 1000 (green line). The shift in the distribution of CPA match scores between the differentially expressed genes (top 100 and top 1000) and the scores of all genes reflects the added value of CPA (CPA scores of top 100 and top 1000 can be found in Dataset 854). We found a significant shift in the scores when comparing all CPA scores from our database with the top 100 differentially expressed genes: p = 2.67e −08 and the top 1000 p < 2.2e− 16.\n\nCumulative distribution of match scores of the concept profiles (CP) between differentially expressed genes in HD with the concept profile of htt: match scores of all genes with a concept profile (black), match scores of the top 1000 differentially expressed genes (green), and the match scores of the top 100 of differentially expressed genes (red).\n\nAlso the top 100 and top 1000 differ significantly (p = 0.03184), showing that it is useful to narrow down on the top ranks for follow-up research. In principle, more extreme p-values are associated with higher CPA scores. In addition, to show that our list of 100 prioritized gene-HTT CPA match scores would not have been found by chance, we assessed the percentile score of our list when compared to the frequency distribution of 100 match scores of randomly sampled gene-HTT concept pairs out of the 12,391 genes in our concept profile database (Dataset 955). All genes were in the top 95 percentile, except NTRK3 (55 percentile).\n\n\nDiscussion\n\nThe computational analysis with public data that we present in this paper shows that there is no strong evidence for genome-wide relocalization of gene activity to repressed chromatin states, at least not at a scale that could explain the massive transcriptional deregulation that we observe in HD. Most of the deregulated genes mapped to the active chromatin state of our reference tissue and were underrepresented in silenced states of chromatin (Figure 1). Previous reports supported the implication of large scale chromatin alterations in gene deregulation in HD. For instance, Anderson et al. reported that gene expression is deregulated in large genomic regions in blood and post mortem tissue of HD patients7. The authors inferred a relation with repressed and active chromatin, but did not use chromatin annotation data directly. Our results suggest that the association with genome clusters is mostly within the active state and does not extend to disruption of chromatin states at large scales.\n\nActive chromatin is normally more prone to regulation and deregulation. Our results suggest that the epigenetic mechanisms that have been observed in HD are mainly bound to this fraction of chromatin. We speculate that the effects are more closely associated with transcription regulation of individual genes, than with large scale higher-order rearrangements of chromatin structure56. Our CPA analysis of deregulated genes with CpG-islands corroborates this suggestion: chromatin-related concepts from our CPA ranked high in this set of genes, while CpGislands are mostly known for their direct role in transcription initiation of the proximal gene. Nevertheless, CpG-mediated changes in gene expression are a common mechanism for many genes and deregulation of this mechanism is thus likely to have a genome-wide effect. It was unexpected that the differentially expressed genes are significantly depleted in the bivalent chromatin state in the prefrontal cortex. The bivalent state is expected to be associated with genes that are prone to become active. Our expectation was that deregulated genes in this brain region would be weakly enriched in this state, as we did find for the caudate nucleus. We currently lack a good explanation for this result.\n\nWe incorporated the computational analysis on public data in our research strategy to advise on next experiments. However, working with public data has its limitations. Here, we had to rely on reference tissue for the chromatin state in order to compare it with gene expression. In addition, the reference tissue chromatin state was measured in healthy individuals. Therefore, we cannot rule out that large scale chromatin effects will be observed when chromatin state and differential expression are measured together in new HD specific experiments. This could reveal new evidence for chromatin state deregulation in HD and give insight in the relationship with transcriptional deregulation, possibly at higher resolution than we could achieve in our current study. However, our results suggest that this is not very likely, leading us to advise fellow HD researchers to prioritise experiments that assess the role of epigenetic mechanisms in HD at the scale of individual genes or small clusters of genes, and within the active fraction of chromatin.\n\nOur results can be used to refine hypotheses about molecular mechanisms involved in HD. For instance, we surmise that the reported association of DNA methylation and chromatin organisation, and the effects of HDAC on the HD phenotype in mice57 are bound to the active fraction of chromatin. Furthermore, it appears that CpG islands located within the promoter region of a gene increase the probability that genes in such genomic regions are deregulated in HD. This is in accordance with a study where changes in DNA methylation were observed in cells expressing mutant huntingtin58. This in turn suggests that DNA methylation in promoters is implicated in alterations in the brain, which is in accordance with a study that noted changes in DNA methylation in cells expressing mutant huntingtin58. Based on our semantic analysis chromatin remodelling, chromatin (dis)assembly, and histone modification were associated with altered gene expression profiles. In contrast, non-CpG containing genes are more likely involved in immune response and neurogenesis, which represents functionally linked processes59.\n\nFurthermore, we used CPA as a means to prioritise genes for our hypotheses. For instance, we prioritised genes overlapping with CpG islands by their association with HTT, assuming that CPA rank scores are higher for genes that are higher-up in the cascade of events caused by the mutant protein. We recently showed that this is a fair assumption for CPA, although literature bias cannot be completely mitigated60. Similarly, we further prioritized candidate genes in terms of their association with epigenetics. Genes such as APBA1, KAT2A, CARM1, SLIT2 and BNIP3 came forward as the most likely candidates to play a role in HD in the context of downstream effects of HTT involving epigenetic mechanisms. These candidates were filtered on potential novelty: only those genes were reported for which there was no direct association found in our database of PubMed abstracts. This does not exclude associations that were reported in tables and supplemental material that are much harder to mine for technical and legal reasons. Our study also retrieved several well established associations consistent with earlier studies.\n\nIn summary, our results show how literature information in combination with data analysis present useful tools for exploration of hypotheses for possible future experiments.\n\n\nConclusions\n\nOur methodology offers support for hypothesis generation to elucidate missing links in mechanisms involved in a complex disease such as HD. We have shown how the analysis of microarray data and the integration of publicly available datasets and literature information enables prioritization of associations, such as proteins and mechanisms, that are likely to be involved in HD. In addition, we were able to focus on mechanisms that are associated with epigenetic regulation that may regulate changes that are part of the disease pathology. We argue that such a methodology can be of great value to the scientific community for narrowing down the amount of possible associations but also providing evidence to support a particular hypothesis.\n\n\nData availability\n\nAll workflows are deposited in Zenodo (https://doi.org/10.5281/zenodo.16420133 and https://doi.org/10.5281/zenodo.16419834), and the myExperiment platform (http://www.myexperiment.org/packs/553).\n\nDataset 1: Gene expression data. This folder contains the gene expression data for the three human brain regions and the three mouse brain regions. doi, 10.5256/f1000research.9703.d17946842\n\nDataset 2: CpG islands. This folder contains the CpG island information both for human and the mouse data. doi, 10.5256/f1000research.9703.d17946944\n\nDataset 3: Chromatin states for the anterior caudate. This folder contains the four chromatin state data for the anterior caudate. Active TSS proximal promoter: TssA, bivalent regulatory region: TssBiv, heterochromatin: Het, repressed Polycomb: ReprPC. doi, 10.5256/f1000research.9703.d17947046\n\nDataset 4: Chromatin states for the prefrontal cortex. This folder contains the four chromatin state data for the prefrontal cortex. Active TSS proximal promoter: TssA, bivalent regulatory region: TssBiv, heterochromatin: Het, repressed Polycomb: ReprPC. doi, 10.5256/f1000research.9703.d17947147\n\nDataset 5: Unique annotations of genes in each chromatin state with Biological Processes. This file contains the results from the semantic analysis with Biological Processes of the genes that were overlapping with active TSS, bivalent TSS, heterochromatin and repressed polycomb chromatin state. The annotations that we present here are uniquely characterizing each gene list (as resulted from the CPA out of the top 50 annotations). doi, 10.5256/f1000research.9703.d17947249\n\nDataset 6: Annotations of CpG containing genes and non CpG containing genes. The top 50 annotations characterising each group of genes with CpG islands and without, resulted from the semantic analysis of those two groups of genes with Biological Processes. doi, 10.5256/f1000research.9703.d17947352\n\nDataset 7: Gene prioritization. Top 100 novel proteins, resulted from the semantic analysis associated with HTT and epigenetics. doi, 10.5256/f1000research.9703.d17947453\n\nDataset 8: Concept profile analysis (CPA) scores for the two gene lists. This folder contains the CPA scores per gene list (top 100 differentially expressed genes in the caudate nucleus and top 1000 differentially expressed from the same brain region) that were obtained by matching the gene lists against the HTT concept profile. doi, 10.5256/f1000research.9703.d17947554\n\nDataset 9: Percentile scores. This document presents the percentile score of the prioritized gene list when compared to the frequency distribution of 100 match scores of randomly sampled gene-HTT concept pairs out of the 12,391 genes in our concept profile database. doi, 10.5256/f1000research.9703.d17947655",
"appendix": "Author contributions\n\n\n\nEM designed and executed the experiments, analyzed the data and wrote the manuscript; WvRM helped design and interpret the experiments as the Huntington’s Disease expert and reviewed the manuscript; PH helped design and interpret the experiments as bioinformatics expert and reviewed the manuscript; PJV reviewed the manuscript; RK, MT provided technical support for the web services; KMH helped with the CPA and reviewed the manuscript; EAS reviewed the manuscript; MR helped design and interpret the experiments, reviewed the manuscript, general supervision; BM: senior advice.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe research leading to these results is supported by grants received from the Netherlands Bioinformatics Centre (NBIC) under the BioAssist program, the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreements No. 305444 (RD-Connect; HEALTH.2012.2.1.1-1-C) and No. 270129 (Wf4Ever; ICT-2009.4.1), and theInnovative Medicines Initiative Joint Undertaking project Open PHACTS (grant agreement No. 115191).\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 Herman van Haagen for methodological input, Jelle J. Goeman and Maarten van Iterson for statistical advice and Silvere van der Maarel for advice on the role of epigenetics in disease.\n\n\nSupplementary material\n\nSupplementary File 1: Decision about the promoter region of the chromatin states analysis and CpG islands. This document is included as a reference to describe the decisions that were made concerning the promoter size using an older version of epigenetic data from ENCODE. In this file we present the additional runs of the workflow compute_overlaps that were performed with different parameters in order to test and decide for the best promoter region and overlap parameters.\n\nClick here to access the data\n\n\nReferences\n\nMacDonald ME, Ambrose CM, Duyao MP, et al.: A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Group. Cell. 1993; 72(6): 971–983. PubMed Abstract | Publisher Full Text\n\nLandles C, Bates GP: Huntingtin and the molecular pathogenesis of Huntington’s disease. Fourth in molecular medicine review series. EMBO Rep. 2004; 5(10): 958–963. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArning L: The search for modifier genes in huntington disease - multifactorial aspects of a monogenic disorder. Mol Cell Probes. 2016; 30(6): 404–409. PubMed Abstract | Publisher Full Text\n\nCha JH: Transcriptional dysregulation in Huntington’s disease. Trends Neurosci. 2000; 23(9): 387–392. PubMed Abstract | Publisher Full Text\n\nLuthi-Carter R, Cha JH: Mechanisms of transcriptional dysregulation in huntington’s disease. Clin Neurosci Res. 2003; 3(3): 165–177. Publisher Full Text\n\nValor LM: Transcription, epigenetics and ameliorative strategies in huntington’s disease: a genome-wide perspective. Mol Neurobiol. 2015; 51(1): 406–423. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nDietz KN, Di Stefano L, Maher RC, et al.: The Drosophila Huntington's disease gene ortholog dhtt influences chromatin regulation during development. Hum Mol Genet. 2015; 24(2): 330–345. PubMed Abstract | Publisher Full Text\n\nUrdinguio RG, Sanchez-Mut JV, Esteller M: Epigenetic mechanisms in neurological diseases: genes, syndromes, and therapies. Lancet Neurol. 2009; 8(11): 1056–1072. PubMed Abstract | Publisher Full Text\n\nJakovcevski M, Akbarian S: Epigenetic mechanisms in neurological disease. Nat Med. 2012; 18(8): 1194–1204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHe F, Todd PK: Epigenetics in nucleotide repeat expansion disorders. Semin Neurol. 2011; 31(5): 470–483. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShin J, Ming GL, Song H: Seeking a roadmap toward neuroepigenetics. Neuron. 2015; 86(1): 12–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMo A, Mukamel EA, Davis FP, et al.: Epigenomic Signatures of Neuronal Diversity in the Mammalian Brain. Neuron. 2015; 86(6): 1369–1384. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdgar R, Domrachev M, Lash AE: Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002; 30(1): 207–210. PubMed Abstract | Publisher Full Text | Free Full Text\n\nENCODE Project Consortium: An integrated encyclopedia of DNA elements in the human genome. Nature. 2012; 489(7414): 57–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJelier R, Goeman JJ, Hettne KM, et al.: Literature-aided interpretation of gene expression data with the weighted global test. Brief Bioinform. 2011; 12(5): 518–529. PubMed Abstract | Publisher Full Text\n\nJelier R, Schuemie MJ, Roes PJ, et al.: Literature-based concept profiles for gene annotation: the issue of weighting. Int J Med Inform. 2008; 77(5): 354–362. PubMed Abstract | Publisher Full Text\n\nMina E, Thompson M, Hettne KM, et al.: Multidisciplinary collaboration to facilitate hypotheses generation in huntington’s disease. In: IEEE 11th International Conference on e-Science (e-Science). 2015; 118–125. Publisher Full Text\n\nJelier R, Jenster G, Dorssers LC, et al.: Text-derived concept profiles support assessment of DNA microarray data for acute myeloid leukemia and for androgen receptor stimulation. BMC Bioinformatics. 2007; 8: 14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJelier R, ’t Hoen PA, Sterrenburg E, et al.: Literature-aided meta-analysis of microarray data: a compendium study on muscle development and disease. BMC Bioinformatics. 2008; 9: 291. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Haagen HH, ’t Hoen PA, de Morrée A, et al.: In silico discovery and experimental validation of new protein-protein interactions. Proteomics. 2011; 11(5): 843–853. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nMina E: HD data analysis workflows for paper: A putative role for genome-wide epigenetic regulatory mechanisms in Huntington’s disease: A computational assessment_v2. Zenodo. 2016. Data Source\n\nMina E: HD data interpretation workflows for paper: A putative role for genome-wide epigenetic regulatory mechanisms in Huntington’s disease: A computational assessment_v2. Zenodo. 2016. Data Source\n\nSmyth DK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004; 3: Article3. PubMed Abstract | Publisher Full Text\n\nBioconductor - home. Reference Source\n\nHodges A, Strand AD, Aragaki AK, et al.: Regional and cellular gene expression changes in human Huntington’s disease brain. Hum Mol Genet. 2006; 15(6): 965–977. PubMed Abstract | 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; 57(1): 289–300. Reference Source\n\nKasprzyk A: BioMart: driving a paradigm change in biological data management. Database (Oxford). 2011; 2011: bar049. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHUGO gene nomenclature committee home page | HUGO gene nomenclature committee. Reference Source\n\nErnst J, Kheradpour P, Mikkelsen TS, et al.: Mapping and analysis of chromatin state dynamics in nine human cell types. Nature. 2011; 473(7345): 43–49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMina E, van Roon-Mom W, Verschure P, et al.: Dataset 1 in: A putative role for genome-wide epigenetic regulatory mechanisms in Huntington’s disease: A computational assessment. F1000Research. 2017. Data Source\n\nUCSC genome browser home. Reference Source\n\nMina E, van Roon-Mom W, Verschure P, et al.: Dataset 2 in: A putative role for genome-wide epigenetic regulatory mechanisms in Huntington’s disease: A computational assessment. F1000Research. 2017. Data Source\n\nRoadmap Epigenomics Consortium, Kundaje A, Meuleman W, et al.: Integrative analysis of 111 reference human epigenomes. Nature. 2015; 518(7539): 317–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMina E, van Roon-Mom W, Verschure P, et al.: Dataset 3 in: A putative role for genome-wide epigenetic regulatory mechanisms in Huntington’s disease: A computational assessment. F1000Research. 2017. Data Source\n\nMina E, van Roon-Mom W, Verschure P, et al.: Dataset 4 in: A putative role for genome-wide epigenetic regulatory mechanisms in Huntington’s disease: A computational assessment. F1000Research. 2017. Data Source\n\nErnst J, Kellis M: ChromHMM: automating chromatin-state discovery and characterization. Nat Methods. 2012; 9(3): 215–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMina E, van Roon-Mom W, Verschure P, et al.: Dataset 5 in: A putative role for genome-wide epigenetic regulatory mechanisms in Huntington’s disease: A computational assessment. F1000Research. 2017. Data Source\n\nBlackledge NP, Klose R: CpG island chromatin: a platform for gene regulation. Epigenetics. 2011; 6(2): 147–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTeodoridis JM, Strathdee G, Brown R: Epigenetic silencing mediated by CpG island methylation: potential as a therapeutic target and as a biomarker. Drug Resist Updat. 2004; 7(4–5): 267–78. PubMed Abstract | Publisher Full Text\n\nMina E, van Roon-Mom W, Verschure P, et al.: Dataset 6 in: A putative role for genome-wide epigenetic regulatory mechanisms in Huntington’s disease: A computational assessment. F1000Research. 2017. 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PubMed Abstract | Publisher Full Text\n\nJia H, Morris CD, Williams RM, et al.: HDAC inhibition imparts beneficial transgenerational effects in Huntington's disease mice via altered DNA and histone methylation. Proc Natl Acad Sci U S A. 2015; 112(1): E56–E64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNg CW, Yildirim F, Yap YS, et al.: Extensive changes in DNA methylation are associated with expression of mutant huntingtin. Proc Natl Acad Sci U S A. 2013; 110(6): 2354–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKohman RA, Rhodes JS: Neurogenesis, inflammation and behavior. Brain Behav Immun. 2013; 27(1): 22–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHettne KM, Thompson M, van Haagen HH, et al.: The Implicitome: A Resource for Rationalizing Gene-Disease Associations. PLoS One. 2016; 11(2): e0149621. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "27321",
"date": "24 Nov 2017",
"name": "Núria Queralt-Rosinach",
"expertise": [
"Reviewer Expertise Data 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\nSummary: This is a computational study devoted to investigate the hypothesis that epigenetic mechanisms dysregulate transcription at genome-wide scale in Huntington's disease (HD). The authors designed their experiments to evaluate two regulatory mechanisms: 1) change of the chromatin state, and 2) CpG island methylation. By means of statistical analysis of experimental data they evaluated the association of dysregulated genes in HD with these two processes, and they provided results and a literature-based semantic analysis for functional interpretation. Furthermore, by application of a semantic analysis they provided a list of prioritized proteins based on their newly predicted association with HTT and epigenetics. Lastly, they evaluated their semantic analysis for gene prioritization. The main conlusions are that their findings do not support the hypothesis of a massive transcriptional dysregulation in HD is linked to large-scale relocation of gene activity, thus the authors speculate that epigenetic effects might be more closely related to dysregulation of individual genes. Finally, the authors claim that their methodology for hypothesis generation can be of great value for the scientific community as it helps in narrowing down the key associations and the evidence underlying them.\n\nReviewer notes:\nThis is an interesting work on the possible epigenetic mechanisms that contribute to transcription dysregulation in Huntington Disease (HD). It is very well written in a clear and accurate manner. They based their hypothesis on a very cited current literature. I have only detected one typo in the whole manuscript: limma vs LIMMA, the authors should be consistent in the format.\n\nIntroduction: I would like to highlight that this study is the first time that is done, so is novel and relevant. Good literature citation.\n\nMethods: The experiments are properly designed and the methods used are well established and based on state-of-the-art approaches. All is very FAIR: data, workflows and webservices used. The study is based on public and open resources (data and software). The methodology is well described but i still missed some information: lack of the statement about the R version, and lack of the specific parameters used in the statistical analyses that other scientists would need to replicate the experiments. Why the authors chose these statistical tests? Regarding CPA- co-occurrence can come from refuting evidence of association, is this taken into account in the concept profile? if does, it is that reflected in the evidence graph downweighting some edges? state the version of CPA database of relations used.\n\nDesign: They tested two regulatory mechanisms: 1) via changes in the chromatin state, 2) via methylation in CpG island content. Both using the promotor gene region to 1) overlap with the chromatin state, 2) to overlap with the CpG content of deregulated genes. They assessed the association via KS statistic test, why this test? As the authors said, there are more regulatory regions in the DNA that could be target of epigenetic regulation, could a cumulative dysregulation in all these regions derive to a large-scale?\n\nResults: All the resulted data is available for reproducibility check. Highlight they reproduce previous published results. I am wondering if they had issues accessing, pre-processing the data to adapt it for their analysis workflows. An explanation of these issues and if their workflows help the community on this regard facilitating to overcome these issues in a systematic, reproducible and traceable manner would be of importance.\n\nImportantly, their analyses integrate experimental data and knowledge and evidence from the literature. Regarding the text mined noisy/literature-biased knowledge that may come from their CPA approach, inclusion of ontologies could be benefitial by leveraging the intrinsic knowledge using automated logical reasoning. Have the authors any plans on this regard?\n\nDiscussion: I agree with the authors that the combination of experimental data analyses with Literature-based functional interpretation of the results (CPA) is relevant and add value to the results. In the second paragraph, can the authors suggest next steps to try to explain the unexpected results?\n\nConclusions: The conclusions are supported by their results. In the conclusions section I missed the conclusion about the title of the paper is investigating, which is the plausibility of a genome-wide scale epigenetic dysregulation of transcription in HD although are stated in the abstract and discussion.\n\nI would emphasize the relevance of their approach performing synergystic research work between computational and wet lab researchers. This interdisciplinary research approach seems to me the way to go in an innovative and efficient big data driven 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?\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": "30246",
"date": "08 Feb 2018",
"name": "Andreas R. Pfenning",
"expertise": [
"Reviewer Expertise Neurobiology",
"epigenetics",
"computational biology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: The purpose of this paper is to leverage publicly-available data to investigate the association between chromatin state and Huntington’s Disease (HD). The authors do this by identifying genes that are differentially expressed in individuals with HD relative to healthy individuals and identifying the locations of these genes in the genome and the biological processes associated with these genes. They find that many of these genes’ promoters are in the active chromatin state in healthy individuals and in CpG islands. They also find that many of these genes are related to biological processes related to HD and that some are in chromatin modification biological processes. Although this study suggests that there may be an association between chromatin state and HD, the nature of that association remains unclear.\n\nMajor Comments:\nI appreciate how the authors integrated existing literature with differential gene expression results to prioritize biological processes, diseases, genes, and molecular functions. In addition, defining the similarity between concepts based on the number of shared concepts is similar to approaches that have been used for community detection in social networks (Blondel et al., Journal of Statistical Mechanics: Theory and Experiment, 2008) and, more recently, for clustering cells based on protein expression (Levine et al., Cell, 2015) (I do not think that the authors need to cite these papers), so I am not surprised that it worked well. I hope that the author’s use of this approach will inspire others to use such methods for comparing biological concepts in literature and encourage future researchers to directly integrate literature with differential gene expression. I found many of the results difficult to interpret because the authors seem to have done all of the analyses on the set of all differentially expressed genes. My expectations for up-regulated genes are different from those for down-regulated genes. In the Minor Comments, I point out specific analyses for which I think that separating the genes based on the direction of the differential expression would be helpful. If the authors did use only down-regulated or only up-regulated genes, it would be great if they could make this clear in the Methods section and include what fold-change cutoff they used. I thought that some of the claims in the Discussion section were not well-supported by the results. I have pointed out what these are in the Minor Comments. Most concerns come from the lack of separation between down-regulated and up-regulated differentially expressed genes in the analyses in this paper. Although there is no chromatin state data from anywhere in the brain in HD individuals, there are H3K27ac and PolII datasets in the striatum of HD and control mice (Achour et al., Human Molecular Genetics, 2015). This paper would be more convincing if it included a comparison between the differentially-expressed genes in mouse HD individuals versus controls and the differential H3K27ac regions from this dataset. I found much of the Methods section difficult to follow. In the Minor Comments, I point out specific parts that I think should be re-ordered and specific details that I think should be added to make the Methods clearer. The authors should also include the exact version and settings that they used for every publicly available software package so that others can reproduce the results.\n\nMinor Comments: Introduction: Page 3: Although the authors clearly describe literature suggesting that epigenetic mechanisms may be involved in HD, there is also some evidence against the role of epigenetics in HD. For example, a recent study profiled methylation in the cortex of HD individuals and controls using the Illumina HumanMethylation450K BeadChip array and found that there are no significantly differentially-methylated regions between HD individuals and controls (De Souza et al., Human Molecular Genetics, 2016). The authors should cite this paper and explain why it does not demonstrate that epigenetics is not involved in HD (the assay used was not genome-wide, methylation is not the only component of transcriptional regulation, etc.). Page 3: It is not clear to me why transcriptional dysregulation in HD would be associated with differentially expressed genes in regions that are not normally associated with active chromatin states. My understanding from the literature cited in the introduction is that many of the genes that are differentially expressed in HD individuals have lower expression in HD individuals than they do in control individuals. I would therefore expect that these genes would fall in regions that are normally associated with active chromatin states but may not be associated with active chromatin states in individuals with HD. It would be great if the authors could clarify the motivation behind this hypothesis. Page 3: At the end of the introduction, the computational method is introduced as an approach to intelligently select which experiments to do. However, I was not sure from the introduction what types of experiments this method is designed to guide. It would be great if the authors could add a more detailed explanation of this earlier in the manuscript.\n\nMethods: Page 3: It would be easier to understand the advantages of the vector space model if they were listed after the description of the vector space model instead of before it. Page 3: It would be helpful if the authors could describe how the subset of PubMed records are selected for genes or cite a previous paper that uses the same method that they used. Page 3: It would be helpful if the authors could define “symmetric uncertainty coefficient.” Page 4: It would be helpful if the authors could list exactly what publicly available datasets were used for each differential expression test before describing the differential expression test. Page 4: It would be helpful if the authors could state what microarrays were used to generate the gene expression data before describing how the differential expression analysis was done. Page 4: It seems like the authors did not account for potential confounding factors that were available, such as sex, age, and brain tissue, in the differential expression analysis. I am concerned that these confounding factors may affect the results. Page 4: The authors state which human and mouse assemblies they used, but they had not previously stated that their analysis included data from mouse. It would be helpful if the authors could state exactly what species they are using for each part of their analysis earlier in the manuscript. Page 4: The authors state that they used a Kolmogorov-Smirnov test to compare p-value distributions. It was not clear to me where these p-values come from. Are they the p-values for differential expression of the genes corresponding to the promoters? It would be helpful if the authors could clarify this. Page 4: It was not clear how many Kolmogorov-Smirnov tests were done. The authors said that they rejected the null hypothesis if the p-value was < 0.05. If they did more than one test, then they should do multiple hypothesis correction. Page 4: It would be helpful if the authors could clarify the purpose of the concept ids. Page 4: It would be helpful if the authors could provide a more detailed explanation of how the concept linking is done. Page 5: It would be helpful if the authors could define the HTT concept and explain why they used it for prioritizing genes. Page 5: It would be helpful if the authors could explain why they decided to prioritize genes based on the “epigene” concept. It seems like the authors are interested in genes that affect epigenetics, such as demethylases or histone modifiers. It is not clear to me how this selection relates to the hypothesis that was described in the introduction. Page 5: It would be helpful if the authors could clarify exactly what differential expression tests were done with the human brain data and what the categories were for each test. Page 5: It would be helpful if the authors could clarify whether the human brain data described here was the only human data used for differential expression analysis and, if it was not, what other data was used. Page 5: It would be helpful if the authors could briefly describe how the CpG island data fits into the rest of the analysis. Page 5: It would be helpful if the authors could explain why they selected the two cell types and four chromatin states that they used in the Methods section. Page 5: I think that it might make sense to incorporate additional chromatin states, such as quiescent, weak repressed Polycomb, and enhancer, as strong repression is not always the cause of a promoter’s inactivity. Page 5: It would be helpful if the authors could clarify why they used only the mouse data from animals treated with the vehicle. My intuition is that it would make more sense to use the animals that did not receive the HDACi 4b inhibitor since the human subjects did not receive any kind of treatment. It is possible that I misunderstood the purpose of the mouse analysis.\n\nResults: Page 6: It is not clear to me why a difference in distribution of expression levels between genes overlapping a chromatin state and genes not overlapping that chromatin state implies that chromatin state has an effect on HD. I think that the authors mean that, if the difference in gene expression between individuals with and without HD is higher for genes overlapping a specific chromatin state than overlapping other chromatin states, then there is an association between the chromatin state and HD. Page 6: It would be helpful to split Figure 1 into two parts, one for genes that have higher expression in people with HD and another for genes that have lower expression for people with HD. My intuition is that most of the differences in p-value distribution are coming from the second category because, since the chromatin state data comes from people without HD, I would expect that genes in an active chromatin state would have higher expression in healthy individuals. Adding onto that, regions of closed chromatin cannot decrease because the genes are not expressed. Regions of open chromatin could either increase or decrease, potentially leading to more variability. Page 6: It would be helpful to have a supplemental figure with all chromatin states because it is not clear from Figure 1 if the differences occur for TSS’s in all active chromatin states (including inactive genes that are acting as enhancers for other genes) or only from genes that are transcribed. Page 6: It would be helpful if the authors could clarify if the overlaps in Figure 1 are done using the entire gene, only the TSS, or the gene’s promoter. Page 7: For the biological process analyses, I think that using a tool for differential enrichment between the two groups of genes would provide more interpretable results than comparing the top hits from CPA because such a tool looks for terms that are significantly enriched in one gene set relative to another. An example of such as tool is CompGO (Waardenberg et al., BMC Bioinformatics, 2015). Page 8: It would be helpful if the authors clarified what they mean by “top novel protein.” Does novel mean that the gene had not been associated with HD in a previous paper? Page 8: It was not clear why Figure 3 shows that CPA is able to prioritize true associations with huntington as measured by a gene expression experiment and why combining differential expression measurements and literature evidence enables the selection of even more specific HD signatures. It would be great if the authors could clarify this. Page 8: It would be helpful if the authors could include the direction of the CPA score shifts for the different groups of differentially expressed genes. Page 8: The authors say that “the top 100 and top 1000 differ significantly.” It would be helpful if they stated the way in which these gene sets differ. Page 11: It would be helpful if the authors could clarify what x is in Figure 3.\n\nDiscussion: Page 11: I am not sure if the paper provides a lack of evidence for genome-wide re-localization of gene activity to repressed chromatin states. The paper combined all of the up-regulated and down-regulated genes instead of separating them. If the paper had shown that the genes that are up-regulated in people with HD are not found in repressive chromatin states in healthy individuals, then I would be more convinced of this lack of re-localization. However, I would not be fully convinced because changes in chromatin state do not always cause changes in gene expression. For example, a previous study showed that most single nucleotide polymorphisms associated with histone modifications are not associated with transcription, suggesting that histone modification differences between individuals do not always correspond to gene expression differences (Grubert et al., Cell, 2015). Thus, it is possible that there are chromatin state differences between HD individuals and controls in parts of the genome where there are no differentially-expressed genes. Page 11: The authors suggest that HD is not associated with the disruption of chromatin states at a large scale. To investigate the association of HD with chromatin state using existing data, the authors would need determine if genes that are up-regulated in people with HD tend to fall in repressive chromatin states and if those that are down-regulated in people with HD tend to fall in active chromatin states. Because there do not seem to be separate evaluations of up-regulated and down-regulated genes, I do not think that the results in this paper can be used to evaluate the relationship between chromatin state disruptions and HD. Page 12: I think that CPA’s high ranking of chromatin-related concepts for differentially expressed genes suggests an association between chromatin reorganization and HD. If differentially expressed genes near CpG islands include genes involved in chromatin structure, that suggests that there is cis-regulatory change in the regulation of those genes, which could have a downstream effect on chromatin organization. Page 12: Although the paper shows that there are more differentially expressed genes in the active chromatin state in healthy individuals, I am not sure that there is sufficient evidence to conclude that most important changes in HD are occurring in the active chromatin state. For example, if the majority of differentially expressed genes are down-regulated in individuals with HD, then the findings in this paper would match my expectations, even if the most important differentially-expressed genes are up-regulated and are not found in the active chromatin state in healthy individuals.\n\nSupplemental Datasets Dataset 1: Some of the line breaks seem to be missing. Dataset 2: The column breaks seem to be missing. Dataset 8: The column breaks seem to be missing for the top 100 differentially expressed genes. Supplementary File 1: The first word in the first figure caption seems like it should be “Illustration.” Supplementary File 1: It would be great if the authors could clarify what they mean by “x2.” Supplementary File 1: It would be great if the authors could explain why they are using the HMEC and NHEK cell lines.\n\nIs the work clearly and accurately presented and does it cite currently literature? The authors seem to clearly describe what they do and cite most of the relevant literature. However, as I mentioned in the fifth major comment, I found parts of the Methods section difficult to follow.\n\nIs the study design appropriate and is the work technically sound? As I mentioned in the first major comment, I do not think that the authors can test their hypothesis with their study design because they combine the up-regulated and down-regulated genes.\n\nAre sufficient details of methods and analysis provided to allow replication by others? The authors provide publicly available workflows for almost everything they did. However, as I mentioned in my fifth major comment, the lack of clarity in parts of the Methods section might make reproducing some of the results difficult.\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Most of the statistical analysis seems appropriate, but most of the interpretation does not make sense because the up-regulated and down-regulated genes were combined.\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? As I mentioned in my second major comment, I think that many of the conclusions are not supported by the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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
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https://f1000research.com/articles/6-1888
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https://f1000research.com/articles/6-1875/v1
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24 Oct 17
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{
"type": "Research Article",
"title": "Identification of predictive cytokine biomarkers of scleroderma via local causal neighborhood methods",
"authors": [
"Ali Shojaee Bakhtiari",
"Galina S. Bogatkevich",
"Alexander V. Alekseyenko",
"Galina S. Bogatkevich",
"Alexander V. Alekseyenko"
],
"abstract": "Background: Scleroderma is an autoimmune disease with established relationship between immune cytokines and prognosis. Therefore, it is necessary to identify and investigate the causal relationship between cytokines and scleroderma diagnosis and to use this information to identify predictive biomarkers of scleroderma status. Methods: Forty scleroderma positive patients and twenty-four healthy controls have been included in this study. Twenty-nine cytokines implicated in scleroderma have been measured in the bronchoalveolar lavage fluid of these patients, and eight have been found to be univariately associated with scleroderma status. Results: Using local causal neighborhood learning methods, we have found two cytokines, Osteoprotegerin (OPG), also known as osteoclast genesis inhibitory factor, or tumor necrosis factor receptor superfamily member 11B and macrophage inflammatory protein-1 delta, to be causally related to the scleroderma status. Logistic regression predictor based on these cytokines achieves 73% AUC for the task of identifying the scleroderma status. Conclusions: Our results demonstrate the feasibility of developing predictive local causal neighborhood biomarkers of scleroderma status based on bronchoalveolar lavage fluid.",
"keywords": [
"Local causal neighborhood",
"Predictive modeling",
"Scleroderma",
"Cytokine biomarkers."
],
"content": "Introduction\n\nScleroderma, also known as systemic sclerosis (SSc), is an autoimmune disease of the connective tissues. The disease is characterized by accumulation of collagen and other connective tissue macromolecules in skin as well as internal organs. The disease is not considered heritable, although it is argued that genetic predisposition plays an important role in its development1.\n\nThe mechanisms that cause interstitial lung disease in scleroderma (SSc-ILD) remain poorly understood. It is well documented that fibrosis is associated with increased expression of the profibrotic cytokines and chemokines. Several cytokines such as transforming growth factor (TGF)2, connective tissue growth factor (CTGF)3, interleukins, tumor necrosis factor (TNF)4, oncostatin M (OSM)5 and others have been reported to be increased in bronchoalveolar lavage fluid (BALF) or serum from scleroderma patients when compared to healthy controls. It has been postulated that production of profibrotic cytokines and chemokines is likely to be key components in the pathology that leads to progressive pulmonary fibrosis in scleroderma6.\n\nIt has been suggested that T-helper (Th) cells and their associated cytokines may play a role in the pathophysiology of scleroderma7. Different subsets of Th cells play prominent roles in the progression of scleroderma7. Although heavily implicated in disease pathogenesis, immunoproteomic biomarkers do not currently provide an effective way to predict SSc-ILD8.\n\nIn this paper, we use computational local causal neighborhood (LCN) discovery techniques9,10 to identify the cytokines that have direct causal relationship to scleroderma status. Rather than reconstructing a detailed causal graph, LCN methods identify variables (cytokines) that are causally ‘close’ to a target variable (SSc-ILD). Based on theoretical results and empirical studies, this task can be achieved even from observational data under very common assumptions about the distribution of the data9,10. The causal nature of the identified variables is complemented by provably advantageous properties with respect to the predictive value of these variables. Specifically, the set of biomarkers in the LCN of a target node make all other biomarkers conditionally independent. This implies that LCN yield the most parsimonious yet maximally predictive diagnostic biomarkers. This has been demonstrated in a number of empirical studies with real biological data11,12. Therefore, we seek to develop a biomarker of SSc-ILD by discovering its LCN in cytokine data measured from the bronchoalveolar lavage fluid (BALF).\n\n\nMaterials and methods\n\nForty patients with SSc-ILD (20 African American: 5 males, 15 females, mean age 43.7 ± 11.0; 20 Caucasian: 10 males, 10 females, mean age 53.8 ± 11.6) and 24 healthy subjects (12 African American: 5 males, 7 females, mean age 32.0 ± 9.5; 12 Caucasian: 4 males, 8 females, mean age 29.6 ± 9.9), all nonsmokers, were examined.\n\nBALF Preparation. Bronchoalveolar lavage was performed as a part of standard care after informed consent was obtained under a protocol approved by the Institutional Review Board for Human Research of Medical University of South Carolina. Recovered BALF was centrifuged at 500 g for 10 minutes at 4°C. Pellet was removed and subjected to total and differential cell counts. Supernatant was dialyzed against sterile distilled H2O overnight at 4°C, lyophilized, and stored at -80°C until assayed.\n\nCytokine Array. Lyophilized BALF powder was recovered in 5 mM Tris, pH 7.4 to a protein concentration of 1 mg/ml, and analyzed using Human Cytokine Antibody Array V from RayBiotech, Inc. (Norcross, GA) according to the manufacturer’s instructions. Briefly, 500 µg of BALF samples were incubated with array support at room temperature for 2 h followed by the incubation with cocktail of biotin conjugated antibodies at 4°C overnight. The arrays were then incubated with horseradish peroxidase-conjugated streptavidin at room temperature for 2 h, and developed by using enhanced chemiluminescence-type solution. The images were scanned and analyzed with the NIH Image software. The net optical density (OD) was obtained by subtracting a background measurement of the same size negative area from the OD measurement of the signal area. Positive controls (six per membrane) were used to normalize the results from different membranes being compared. The variation between two identical cytokine spots ranged from 0 to 10% in duplicated experiments.\n\nThe final dataset consisted of 28 measured cytokines. Figure 1 shows the description of the cytokine array map.\n\nPos, positive; Neg, negative; ENA, epithelial cell-derived neutrophil-activating peptide; GCSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte/macrophage-colony stimulating factor; GRO, growth-regulated oncogene; IL, interleukin; IFN, interferon; MCP, monocyte chemotactic protein; MCSF, macrophage colony stimulating factor; MDC, macrophage-derived chemokine; MIG, monokine induced by gamma interferon; MIP, macrophage inflammatory protein; RANTES, regulated upon activation in normal T cells, expressed, and secreted; SCF, stem cell factor; SDF, stromal cell-derived factor; TARC, thymus and activation regulated chemokine; TNF, tumor necrosis factor; EGF, epidermal growth factor; IGF, insulin-like growth factor; Ang, angiogenin; OSM, oncostatin; TPO, thrombopoietin; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; BDNF, brain-derived neurotrophic factor; BLC, B-lymphocyte chemoattractant; IGFBP, insulin-like growth factor binding protein; IP-10, interferon-inducible protein 10; LIF, leukemia inhibitory factor; LIGHT, lymphotoxins, inducible expression, competes with HSV glycoprotein for HVEM, a receptor expressed on T-lymphocytes; MIF, microphage migration inhibitory factor; NAP, neutrophil activating peptide; NT, neurotrophin; PARC, pulmonary and activation regulatory chemokine; PIGF, placenta growth factor; TIMP, tissue inhibitors of metalloproteinases.\n\nThe raw cytokine measurements have been log transformed. We have imputed the missing cytokine values from their 10 nearest neighbors identified by K-nearest neighbors (KNN) algorithm using Bioconductor package impute, version 3.413. Adjustment for sex and race have been performed by fitting linear regression model to individual cytokine values and extracting the residuals for further analysis. All analyses have been performed in R, version 3.2.514.\n\nWe have performed Welch t-test15 to find cytokines univariately associated with scleroderma status. We have adjusted for multiple comparisons using False Discovery Rate correction (FDR)16 with significance threshold set at 0.05. Table 2.\n\nWe define LCN of a variable as a set of variables in its vicinity. LCNs can be constructed in such a way as to maximize their utility for biomarker development. For instance, Markov blanket (MB) of a variable is an LCN defined as the most compact set of variables (cytokines) rendering other variables independent of a target variable (SS)17. Intuitively, MB is the optimal solution of variable selection problem for predictive purposes. In practice, this means that the selected biomarkers contain all essential predictive information about the target node and are causally close to it (causal parents, children or spouses). Inference of MB requires inference of causal directionality, which is infeasible in most biomedical datasets due to their limited sample size and complexity. A closely related LCN that preserves much of the predictive utility of MB is the parent-child set (PC-set), which only consists of direct causal parents and children of the target node. As opposed to MB the PC-set does not include common confounders of the effects of the target node, and thus can be slightly less predictive. Nonetheless, PC-sets are much easier to discover computationally. Overall, the major advantage LCNs for biomarker discovery is that the size of the biomarker is typically small and has a causal interpretation.\n\nIn order to infer LCN of SS, we have used the HITON-PC algorithm18 from causal explorer toolbox19 in MATLAB Release 2016b20. This algorithm performs a series of conditional independence tests (Fisher’s Z test with 0.05 significance threshold) to infer the PC-set of the target variable (SS).\n\nWe use logistic regression model for predictive analysis. To ensure accurate and unbiased estimates of predictive power of the local causal neighborhood, we have performed 1,000 cross validation (CV) runs of the predictive analysis. First, we have randomly split the sample into training (70% of the data) and testing sets preserving case-control balance in each of the splits. Next, we have identified the PC-set of SSc-ILD using HITON-PC algorithm. The cytokines in the PC-set served as the predictors in building the logistic regression model of SSc-ILD from the training set data. Finally, we estimated the performance of the logistic regression model on the data in the test set. The performance estimates from each of the 1,000 CV runs have been used to determine the overall predictive power of the PC-set.\n\nOur main metric of predictive performance is area under receiver operating characteristic (ROC) curve (AUC). Curve specifies the relationship between the true positive rate (TPR) and the false positive rate (FPR) of the model under all possible model parameter thresholds. The value of AUC from 0.5 (no predictive signal) to 1 (perfect prediction). We have extracted the following additional performance metrics from the ROC: (i) mean classification success rate, defined as the average rate of correctly identifying the scleroderma status on the test set, over the entire simulation runs; (ii) mean optimal sensitivity, the average value of the optimal sensitivity of the predictive model over the entire simulation runs. (ii) mean optimal specificity, the average of the optimal specificity of the predictive model over the entire simulation runs.\n\n\nResults\n\nCytokine arrays were utilized to explore and compare the expression levels of multiple cytokines in BALF samples. The array revealed elevated expression of 28 cytokines in BALF from scleroderma patients when compared to controls, which were selected for further analysis. Figure 2.\n\nSelect cytokine expression locations are shown by arrows. In this instance Caucasian and African American controls are brought against Caucasian and African American with positive scleroderma status.\n\nFrom the 28 cytokines measured, 8 are significantly associated with scleroderma status at p-value of 0.05 (Table 1). After correction for multiple comparisons using FDR, 3 cytokines, MIP-1delta, VEGF and Osteoprotegerin,remain significantly associated. The same cytokines are significant after adjustment for subject sex and race.\n\n* Welch t-test, p-values significant at 0.05 are shown in bold.\n\nHITON-PC algorithm identifies two cytokines, OPG and MIP-1delta, as the members of the PC-set LCN of SS in both adjusted and unadjusted data. Figure 3 shows the inferred LCN for the scleroderma status.\n\nHITON-PC algorithm identified two cytokines, Osteoprot and MIP-1delta, to be in the PC-set of the scleroderma status (SS). 6 other cytokines are found to be univariately associated with SS. These additional cytokines as well as every other cytokine not in PC-set, however, are conditionally independent of the SS, given the PC-set. The causal connectivity of the remaining cytokines with SS is unknown.\n\nIn order to assess the stability of the PC-Set estimate, we performed a re-sampling analysis as described in the methods section. The histogram of the appearance of the different cytokines in the causal neighborhood of scleroderma status is shown in Figure 4. Osteoprot and MIP-1delta appeared most frequently in the PC-set LCN of SS. In the 1000 simulation runs, either one or both cytokines appeared in the PC-set of scleroderma status 847 times. The scatterplot of MIP1-delta against Osteoprot adjusted values (Figure 5) shows apparent concentration of scleroderma cases in the top right quadrant, indicating association of higher expression values of these cytokines with disease.\n\nWe performed 1,000 iterations of repeated cross validation to determine stability of the two cytokine biomarkers. The number of times each cytokine (adjusted for sex and race) appears in the PC-set LCN of SS is depicted on this plot. Osteoprot and MIP-1delta appear in the LCN of SS with high frequency, indicating high likelihood of their causal proximity to scleroderma status. These results are similar if no adjustments for sex and race are made (data not shown).\n\nThe scatterplot shows the distribution of the two cytokines against each other in data adjusted by sex and race. Red and black points indicate cases and controls, respectively. The concentration of the red points in the upper right quadrant indicates potential presence of predictive signal towards disease status in these cytokines.\n\nWe first fit the logistic regression model using all cytokines as predictors. The resulted model attains cross-validated predictivity indistinguishable from random predictor on the testing data and almost perfect predictivity in the training data. This indicates high levels of noise in the weakly predictive data, which requires feature selection before attempting to build predictive model in order to obtain accurate models and estimates of model predictivity. To estimate the predictive utility of PC-set LCN in an unbiased way, we have performed 1,000 iterations of repeated cross-validated predictive model building using logistic regression. In data adjusted for sex and race, the mean classification success rate is 68.46% (st. dev. 6.66%). The mean optimal sensitivity of the model is 65.49%( st. dev. 11.38%). The mean optimal specificity of the model is 73.44% (± 11.88% st. dev.). We estimate the AUC of the cross-validated model to be 73% (Figure 6), indicating non-negligible power of LCN biomarker for SSc prediction.\n\nThe ROC curve shows the estimated values of true positive rate against the false positive rate for the predictive model. The estimated value for the area under curve is 73%. The significant difference of the AUC value from 0.5 confirms the strength of the identified biomarkers in predicting the SS.\n\n\nDiscussion\n\nILD is the leading cause of morbidity and mortality in scleroderma patients. The mechanisms leading to SSc lung disease remain unknown. However, a variety of cytokines and growth factors have been reported to be increased in BALF from SSc patients. It is known fact that, Osteoprotegerin acts as a competitive receptor for the membrane-bound receptor activator of nuclear factor-kappaB21. It is also known that, osteoprotegerin-deficient mice exhibit osteoporosis at early stage of life span with the activation of RANK ligand (RANKL)22. Macrophage inflammatory protein (MIP) has been previously shown to be associated with scleroderma diagnosis23. OPG in primary myelofibrosis (PMF) expressed significantly higher (up to 71-fold) when compared with prefibrotic cellular PMF and control cases24.\n\nIn this paper, we present a model for inferring the cytokines predictive of SSc-ILD using local causal neighborhood framework. The model identified osteoprotegerin (OPG) and macrophage inflammatory protein-1 delta (MIP-1delta), to be closely related to the scleroderma status. We tested the hypothesis that the identified cytokines can be used as predictive biomarkers.\n\nWe compared the biomarkers selected by LCN with those resulting from LASSO regression25. In our data, the regression model selects the same features as LCN. This suggests that LCN approach can result in equivalent inference to LASSO. However, the major advantage of an LCN feature selection approach is in the theoretical causal guarantees it provides. LCN identifies features based on their causal proximity to the target variable, something that LASSO regression feature selection cannot match.\n\nIn this study, clinical disease characterization and demographic information, other than sex and race, have not been incorporated in the predictive model of SS. In practice, we maintain that combining molecular biomarkers with clinical and demographic data should improve the quality of the predictor. Such a biomarker, however, will require a larger cohort of patients to be developed.\n\nUnderstanding the role of BALF’s cytokines in pathogenesis of SSc-ILD may identify new targets for the development of diagnostic biomarkers predicting the biological behavior of the disease.\n\n\nData and code availability\n\nZENODO: SLE_cytokines - Identification of predictive cytokine biomarkers of scleroderma via local causal neighborhood methods data\n\nRaw and imputed cytokine array expressions, relevant code provided in Matlab and R format, and the results of applying LASSO regression on the BALF cytokine arrays are provided in the repository. https://zenodo.org/record/1001044; DOI: 10.5281/zenodo.100104426\n\n\nEthics and consent\n\nAll procedures were performed as a part of patient standard care after informed consent was obtained under a protocol approved by the Institutional Review Board for Human Research of Medical University of South Carolina.",
"appendix": "Competing interests\n\n\n\nThe authors declare no competing interests.\n\n\nGrant information\n\nThe project described was supported by the NIH National Center for Advancing Translational Sciences (NCATS) through Grant Number UL1 TR001450. AVA is funded by NIH/NCI R01 CA164964, NIH/NIDCR R34 DE025085, and NIH/NIAMS R21 AR067459. AVA and ASB are funded by MUSC College of Medicine Enhancing Team Science (COMETS) Pilot. GSB is funded by NIH/NIAMS P60 AR062755.\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\nAssassi S, Radstake TR, Mayes MD, et al.: Genetics of scleroderma: implications for personalized medicine? BMC Med. 2013; 11(1): 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoberts AB, Sporn MB, Assoian RK, et al.: Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci U S A. 1986; 83(12): 4167–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoussad EE, Brigstock DR: Connective tissue growth factor: what's in a name? Mol Genet Metab. 2000; 71(1–2): 276–92. PubMed Abstract | Publisher Full Text\n\nLeithäuser F, Dhein J, Mechtersheimer G, et al.: Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells. Lab Invest. 1993; 69(4): 415–29. PubMed Abstract\n\nGearing DP, Bruce AG: Oncostatin M binds the high-affinity leukemia inhibitory factor receptor. New Biol. 1992; 4(1): 61–5. PubMed Abstract\n\nAtamas SP, White B: Cytokine regulation of pulmonary fibrosis in scleroderma. Cytokine Growth Factor Rev. 2003; 14(6): 537–50. PubMed Abstract | Publisher Full Text\n\nKurzinski K, Torok KS: Cytokine profiles in localized scleroderma and relationship to clinical features. Cytokine. 2011; 55(2): 157–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCastro SV, Jimenez SA: Biomarkers in systemic sclerosis. Biomark Med. 2010; 4(1): 133–147. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAliferis CF, Statnikov A, Tsamardinos I, et al.: Local Causal and Markov Blanket Induction for Causal Discovery and Feature Selection for Classification Part I: Algorithms and Empirical Evaluation. J Mach Learn Res. 2010; 11: 171–234. Reference Source\n\nAliferis CF, Statnikov A, Tsamardinos I, et al.: Local Causal and Markov Blanket Induction for Causal Discovery and Feature Selection for Classification Part II: Analysis and Extensions. J Mach Learn Res. 2010; 11: 235–284. Reference Source\n\nStatnikov A, Alekseyenko AV, Li Z, et al.: Microbiomic signatures of psoriasis: feasibility and methodology comparison. Sci Rep. 2013; 3: 2620. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStatnikov A, Henaff M, Narendra V, et al.: A comprehensive evaluation of multicategory classification methods for microbiomic data. Microbiome. 2013; 1(1): 11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHastie T, Tibshirani R, Narasimhan B, et al.: Impute: Imputation for microarray data. R package version 1.48.0. 2016. Reference Source\n\nR Core Team: R: A Language and Environment for Statistical Computing. 2015. Reference Source\n\nWelch BL: The generalisation of student's problems when several different population variances are involved. Biometrika. 1947; 34(1–2): 28–35. PubMed Abstract | Publisher Full Text\n\nBenjamini Y, Hochberg Y: Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J R Stat Soc Series B (Methodol). 1995; 57(1): 289–300. Reference Source\n\nPearl J: Probabilistic reasoning in intelligent systems: networks of plausible inference. Morgan Kaufmann Publishers Inc. 1988; 552. Reference Source\n\nAliferis CF, Tsamardinos I, Statnikov A: HITON: a novel Markov Blanket algorithm for optimal variable selection. AMIA Annu Symp Proc. 2003; 2003: 21–25. PubMed Abstract | Free Full Text\n\nAliferis CF, Statnikov AR, Tsamardinos I, et al.: Causal explorer: A causal probabilistic network learning toolkit for biomedical discovery. In International Conference on Mathematics and Engineering Techniques in Medicine and Biological Sciences (METMBS’ 03). 2003. Reference Source\n\nMATLAB version 2016b, Natick, Massachusetts: The MathWorks Inc.\n\nCoetzee M, Kruger MC: Osteoprotegerin-receptor activator of nuclear factor-kappaB ligand ratio: a new approach to osteoporosis treatment? South Med J. 2004; 97(5): 506–11. PubMed Abstract\n\nBucay N, Sarosi I, Dunstan CR, et al.: osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998; 12(9): 1260–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScala E, Pallotta S, Frezzolini A, et al.: Cytokine and chemokine levels in systemic sclerosis: relationship with cutaneous and internal organ involvement. Clin Exp Immunol. 2004; 138(3): 540–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKreipe H, Büsche G, Bock O, et al.: Myelofibrosis: molecular and cell biological aspects. Fibrogenesis Tissue Repair. 2012; 5(Suppl 1): S21. PubMed Abstract | Free Full Text\n\nTibshirani R: Regression Shrinkage and Selection via the Lasso. J R Stat Soc Series B (Methodol). 1996; 58(1): 267–288. Reference Source\n\nBakhtiari AS, et al.: ashojaee/SLE_cytokines: Identification of predictive cytokine biomarkers of scleroderma via local causal neighborhood methods data. Zenodo. 2017. Publisher Full Text"
}
|
[
{
"id": "27316",
"date": "01 Dec 2017",
"name": "Mark M Wurfel",
"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 an interesting report that addresses an important clinical problem. However, the methods and presentation potentially obscure what the key findings from these data might be. The following should be addressed.\nThere is very little data presented describing the study subjects. They authors should present more demographic information (age) and disease characteristics (time from diagnosis, organs involved, lung function, current therapy).\n\nThe rationale for using lyophilized BALF for the analyses is not presented nor are the potential challenges in interpretation addressed. For instance, the absolute numbers will not be useful for future diagnostics as it is highly unlikely that alveolar fluid would be lyophilized/concentrated before measurement. It would be helpful to know what the concentration of albumin or other background proteins might be to appreciate the relative abundance of the mediators measured.\n\nThe text refers to Table 1 as having results of the cytokine analyses but this appears in Table 2. Table 2 has \"unadjusted\" and \"adjusted\" analyses without any statement of how the \"adjusted\" analyses were performed. Given its exploratory nature I would like to see the entirety of the dataset presented here as it has more utility to the community than a multivariate model that is not validated and is likely to be highly over-fit.\n\nThe rationale for using LCN for these analyses is unclear, particularly given that LASSO appears to perform similarly and is more straightforward to interpret. Notably, there does not appear to be any comparison of LCN and LASSO presented even though it is mentioned in the discussion.\n\nFigure 4 and Figure 5 are not very useful. I would like to see more details about the outputs from the LASSO model (i.e. what were the driving mediators and model performance measures like BIC or other measures of fit/overfit). It would also be helpful to readers to see some form of correlation matrix or cluster graphic to show how the different mediators track or don't track with each other. This will allow for more insights on pathways that are differentially up/down regulated.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "28122",
"date": "11 Dec 2017",
"name": "Kathryn Torok",
"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 an interesting paper regarding the study of cytokines derived from BALF which offers the possibility to study local disease mechanisms and potential biomarkers associated with disease propagation. Most of the paper and references were dedicated to the local casual neighborhood (LCN) biostatistical approach. My main comments to develop this paper further would be to 1) display the directionality and interaction of cytokines on a correlation matrix or cluster graphic to allow for more insight regarding the relationship of these cytokines/insight into pathways, and 2) expand the clinical information about the systemic sclerosis subjects to assist the reader in understanding the generalizability to the patient population they are treating.\n\nA few constructive comments to address these concerns:\n\nRegarding citations, this institution has published before regarding certain cellular subtypes and cytokines determined in SSc BALF. It would be a strength to include these and also potentially tie into the discussion if similar or different cytokines were determined in these current analyses. Particularly, MIP-1alpha (not MIP-1delta as in current paper), was found to associate with degree of alveolitis.\nCytokine concentrations in bronchoalveolar lavage fluid of patients with systemic sclerosis. Arthritis Rheum. 1997 Apr;40(4):743-51. Bolster MB1, Ludwicka A, Sutherland SE, Strange C, Silver RM.\n\nIn a similar framework, these prior publications have included more SSc patient clinical data, which would be helpful to understand the subset of patients analysed and the generalizability to clinical practice. I would recommend including featured such as disease subtype (dcSSc, lcSSc), disease duration, degree of lung fibrosis or CT changes if quantified, average FVC, DLCO etc if available. 2a. In the discussion it was mentioned these were not displayed due to lack of numbers for subanalyses, which is understandable, but the general summary of these characteristics would be helpful of added to Table 1.\n\nPresenting the general cell types derived from the BALF would be helpful between SSc and controls. This may give more insight in context with cytokines found more predominately in SSc BALF.\n\nFigure 4 displays the frequency of cytokines generated with repeated analyses, although MIP-1delta and OPG are the most frequent, PARC and VEGF are not close behind. A Figure showing the Causal modeling PC algorithm for cytokines in proximity and direction to the target variable (SSc disease status) would be very instructional here. One would think PARC and VEGF might be close enough to include in the ROC curve combining these 4 cytokines. Revising Figure 3 to display directionality from the casual modeling, which is the strength of utilizing this statistical approach, would be advisable to help understand the narrow focus of just the 2 cytokines.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1875
|
https://f1000research.com/articles/6-1676/v1
|
11 Sep 17
|
{
"type": "Data Note",
"title": "BeerDeCoded: the open beer metagenome project",
"authors": [
"Jonathan Sobel",
"Luc Henry",
"Nicolas Rotman",
"Gianpaolo Rando",
"Luc Henry",
"Nicolas Rotman"
],
"abstract": "Next generation sequencing has radically changed research in the life sciences, in both academic and corporate laboratories. The potential impact is tremendous, yet a majority of citizens have little or no understanding of the technological and ethical aspects of this widespread adoption. We designed BeerDeCoded as a pretext to discuss the societal issues related to genomic and metagenomic data with fellow citizens, while advancing scientific knowledge of the most popular beverage of all. In the spirit of citizen science, sample collection and DNA extraction were carried out with the participation of non-scientists in the community laboratory of Hackuarium, a not-for-profit organisation that supports unconventional research and promotes the public understanding of science. The dataset presented herein contains the targeted metagenomic profile of 39 bottled beers from 5 countries, based on internal transcribed spacer (ITS) sequencing of fungal species. A preliminary analysis reveals the presence of a large diversity of wild yeast species in commercial brews. With this project, we demonstrate that coupling simple laboratory procedures that can be carried out in a non-professional environment, with state-of-the-art sequencing technologies and targeted metagenomic analyses, can lead to the detection and identification of the microbial content in bottled beer.",
"keywords": [
"metagenomic",
"beer",
"citizen science",
"crowdfunding"
],
"content": "Introduction\n\nBeer is probably the world’s oldest and most widely consumed alcoholic beverage on the planet, with a worldwide production of nearly 2 billion hectolitres (2·10E11 litres) annually [The Barth Report, Hops 2015/2016], and, as DNA sequencing becomes increasingly cheap, whole genome sequencing and metagenomic analyses are being explored as tools to better understand brewing in particular, and food fermentation in general1. Complex microbial communities influence the wine- and cheesemaking process throughout2,3. Indeed, microbial communities contribute to nutritional and aromatic properties, as well as shelf life of the products. In the case of wine, microorganisms are present in the soil, on the grapes, and in the fermenter, being carried over from the vine to the must to the wine, and there is increasing evidence for the existence of an important microbial contribution to the notion of “terroir”4–7. One question that remains unanswered is whether there is such a thing as a “terroir” for beer.\n\nOf particular interest is sour beer, such as lambic and gueuze, a beverage produced without the controlled addition of known yeast cultivates. Instead, the wort is exposed to ambient air, allowing naturally occurring bacteria and yeasts to start the fermentation and leading to a production that is difficult to standardize. To our knowledge, three initiatives are currently exploring the role of the beer microbiome in the brewing process and how it shapes the characteristics of the final product. Using metagenomic analyses, Kevin Verstrepen and colleagues at KU Leuven, Belgium, study the production of lambic, a traditional Belgian beer produced by spontaneous fermentation [VIB project 35]. Similarly, Matthew Bochman and colleagues at Indiana University, USA, have recently published preliminary results showing how the microbial community evolved over the fermentation process, together with the relative abundance of the organic acids that give sour beer its characteristic taste8,9. Similarly, researchers at the University of Washington, USA, have studied open-fermentation beer using chromosome conformation capture and discovered a novel interspecific hybrid yeast10.\n\nTo investigate the microbial composition of a collection of commercial beers, we initiated BeerDeCoded in the context of Hackuarium, a Swiss not-for-profit organisation that supports unconventional research projects and promotes the public understanding of science. Members of the Hackuarium community are interested in participatory biology and want to promote interdisciplinary citizen research and innovation outside traditional institutions, using low-cost, simple and accessible technologies. The goal of the BeerDeCoded project is not only to broaden the scientific knowledge about beer, but also to improve the public understanding of issues related to personal genomics, food technology, and their role in society. With the release of this first data set, we built the proof of concept for a targeted metagenome analysis pipeline for beer samples that can be used in high schools, citizen science laboratories, craft breweries or industrial plants.\n\n\nMethods\n\nThe content of each beer sample was mixed to homogeneity by inversing the bottle several times. 50 mL were transferred into a conical tube and centrifuged (5000 rpm, 20 min, 4°C) to collect cells and other precipitable material. Pellets were resuspended with 1 mL TE buffer (Tris 10 mM, EDTA 1 mM, pH 8.0) and transferred into 1.5 mL tubes. The samples were centrifuged (10000 rpm, 10 min, 4°C), the supernatant was removed and the pellet stored frozen (- 20°C) until future analyses. For DNA extraction, the ZR Fecal DNA MiniPrep kit (Zymo Research) was used with minor modifications.\n\nTo ensure the DNA was free from proteins and other contaminants, the absorbance of DNA samples was measured at 230, 260 and 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).\n\nYeast genomic DNA was amplified using the fungal hypervariable region ITS1 (internal transcribed spacer 1) as previously described11 using the following primers: BITS (5’–CTACCTGCGGARGGATCA–3’) and B58S3 (5’– GAGATCCRTTGYTRAAAGTT–3’). Typical PCR reactions contained 5–100ng of DNA template. Amplicon size (500nt) was verified using gel electrophoresis and with a fragment analyser. ITS amplicons were purified using AMPure XP beads following the manufacturer‘s instructions (Beckman Coulter). Dual indices and Illumina sequencing adapters were attached using the Nextera XT Index Kit following manufacturer’s instructions (Illumina).\n\nMiSeq sequencing was performed using the MiSeq v3 reagent kit protocol (Illumina). Briefly, the amplified DNA was quantified using a fluorimetric method based on ds-DNA binding dyes (Qubit). Each DNA sample was diluted to 4 nM using 10 mM Tris pH 8.5 and 5 uL of diluted DNA from each library were pooled. In preparation for cluster generation and sequencing, 5 uL of the pooled final library was denatured with 5 uL of freshly diluted 0.2 N NaOH and combined with 30% PhiX control library to serve as an internal control for low-diversity libraries. After loading the samples on the MiSeq, paired 2x 300bp reads were generated and exported as FASTq files.\n\nThe curated set of ITS sequences from the Refseq database (targeted loci) was used to build an ITS index for the Burrows-Wheeler Aligner (BWA)12. The BWA was used to map the reads of each beer from the fastq files to our ITS index. The bam files were sorted and indexed using samtools13. Subsequently, the number of ITS per beer and per species were counted and only species with over 10 reads were taken into consideration.\n\n\nResults\n\nOver the month of June 2015, a total of 124 individuals contributed over 10,000 Euros to a crowdfunding campaign that provided financial resources for the first stage of the BeerDeCoded project. Reaching out to the public through this campaign also enabled crowdsourcing a collection of 120 beer samples from 20 countries. We have subsequently demonstrated that it is possible to extract DNA directly from bottled beer using low cost methodologies, typically available to citizen scientists (see Methods).\n\nThe internal transcribed spacer regions (ITS) of fungal species14 were then amplified and, after quality control, 39 samples were sent for DNA sequencing. These 39 commercial beers originated from 5 different countries: 30 were from Switzerland, five from Belgium, two from Italy, one from France and one from Austria. We obtained an average library size of 600K reads (min 350K, max 2400K) per sample and a total of 88 fungal species were identified, including 52 unique occurrences. This high variety of wild yeasts in commercial beers was unexpected (Figure 1A), with some brews containing traces of up to more than 30 different fungal species (Figure 1B). Using hierarchical clustering, we built a proximity tree of the different beers (Figure 2).\n\nBarplot graph representing (A) the number of beers containing the species (n=36) occurring in at least two samples. Species (n=52) present in only one sample were excluded for clarity. (B) represents the number of fungal species identified in each of the 39 bottled beers.\n\nConsistent with its widespread use for fermentation, brewer’s yeast (Saccharomyces cerevisiae) was detected in all beer samples, accounting for between 11% (Orval, an ale beer by Belgian Brasserie d’Orval) and 99% (Tempête, an ale from the Swiss brewery Docteur Gab’s) of all sequencing reads. In most samples, S. cerevisiae was present at very high levels (typically 90–97% of reads, Figure 3). More surprisingly, Saccharomyces mikatae, a species used in winemaking15 was also relatively abundant in all samples (0.5–5%). Interestingly, most brews were found to contain low to medium presence of multiple other yeast species, including Saccharomyces bayanus (used in winemaking and cider fermentation), Saccharomyces kudriavzevii and Saccharomyces pastorianus (used in lager manufacturing), Saccharomyces eubayanus (a probable parent of Saccharomyces pastorianus) and Brettanomyces bruxellensis (typically used for the production of the Belgian beer styles). B. bruxellensis represented 86% and 15% of the reads in the Orval and the “Chicha” experimental beer, respectively. Non-conventional, as well as wild yeast, such as Saccharomyces cariocanus and Saccharomyces paradoxus, two species closely related to Saccharomyces cerevisiae were also found. Another example is Kazachstania sp., a wild yeast of commonly found in brines. The presence of this species may be of interest, as it was previously reported that adding the parent Kazachstania servazzi to the brewing process 24 hours before the ale yeast contributed to the production of high level of esters, producing a strong fruity and floral aroma16.\n\nOnly ITS with more than 10 reads and present in at least two beers are shown.\n\nThe beer in which we measured the highest ITS diversity was Waldbier 2014 “Black Pine”, an Austrian beer brewed using pine cones collected in local forests, with 38 fungal species. Two other beers contained more than 20 fungal species: La Nébuleuse Cumbres Rijkrallpa (a sour/wild ale beer made with cranberries and the fermented corn “Chicha”) and Chimay Red Cap, a Belgian trappist beer. Fungal DNA from 52 species was unique for specific beers. For instance, Chimay Red Cap, was the only beer containing traces of Ganoderma sp., a fungus rich in terpenoids well known for its bitter taste17. A fungus naturally present in the kernels of cereals and used in organic agriculture as natural herbicide, Alternaria sp., was found in 15 of the 39 samples, suggesting that microorganisms, or their DNA, could be carried over from the ingredients to the final product.\n\n\nDiscussion and future perspectives\n\nWhile a continuous process of market consolidation has lead to 5 companies controlling more than half of global beer production, there has been an explosion of craft industries over the past years, especially in Europe and North America. In 1978 there were 89 large industrial breweries in the USA. In 2016, there were 5,301, among them 3,132 small, independent microbreweries (American Brewers Association). There is a parallel with Hackuarium, an independent “craft” science initiative that has branched out from large institutional research institutes and provides an environment that allows scientists to explore topics that are rarely found in academia or industry. What is truly unique is the participation of individuals with no formal science training, and therefore the strong focus on citizen science and communication. With the BeerDeCoded project, we explored the potential of crowdfunding and crowdsourcing in engaging members of the general public in the production of scientific knowledge. We demonstrated that it is possible to execute complex molecular analyses on everyday products using limited resources and technical support from research institutions, and no financial support from traditional funding sources. The resulting dataset contains the ITS profile of 39 bottled beers from five different countries, revealing the low abundance but widespread presence of wild fungal species. Further analyses could go as far as shedding light on the so-called biological “dark matter” of the beer ecosystem18,19.\n\nWith the costs of DNA sequencing falling dramatically, and with the emergence of portable and user-friendly instrumentation, we believe that it is a favorable time to expand the application of DNA analysis to novel fields, including food and beverage. This industry is starting to explore the potential of genome sequencing to understand the contribution of various species to product characteristics. The sequencing of the full genome of 157 brewing yeast strains was, for example, recently reported20. Metagenomic analyses could also have important implications for the optimization and batch-to-batch reproducibility of the various fermentation processes, as well as quality control, traceability and authentication of the products. One hypothesis that could be investigated further in the future is whether the presence of a specific fungal species can be diagnostic for a unique geographic area. In our data set, the non Saccharomyces yeast that contributes to wine aroma through the production of volatile compounds, Wickerhamomyces anomalus, was found exclusively in five of the brews manufactured in Switzerland. The limited sample size, however, does not allow us to draw a statistically significant conclusion, and it remains to be seen if W. anomalus is present in beers from other locations as well. Due to inherent limitations of DNA sequencing, it is difficult to anticipate whether the microbes identified are likely to be having an impact on the fermentation process. However, based on the identification of strains present in brews with desired characteristics, controlled experiments in which the microbial composition of the brew is altered could allow us to investigate if the presence of specific microorganisms affects flavour21. The origin of each yeast species could also be investigated; i.e. whether they come with the ingredients or from the environment at the production site. Techniques to sample airborne DNA exist22. Furthermore, other protocols could also be used to catalogue plant DNA23, such as malt and hop varieties, and to map the bacterial diversity.\n\nIn order to standardize and simplify our pipeline, and facilitate the contribution of new data and their further analysis by individuals not involved in this initial study, we are in the process of developing a BeerDeCoded repository and a Galaxy instance24. This tool will enable any citizen scientist to carry out beer metagenomics and reproduce our analysis. In the meantime, we encourage researchers from other laboratories, microbreweries and citizen laboratories to further explore our data set, and invite them to consider contributing additional data in the future.\n\n\nData availability\n\nThe dataset contains the metagenetic profiles for 39 beers. The data was obtained using a targeted approach based on the phylogenetic typing with internal transcribed spacers (ITS) of ribosomal sequences. All methods, quality control, processed tables, metadata and code are accessible at: https://github.com/beerdecoded/Beer_ITS_analysis. The raw data are stored in the SRA database in the bio project PRJNA388541",
"appendix": "Competing interests\n\n\n\nGR is the co-founder, CTO and a shareholder of SwissDeCode, a company selling point-of-need DNA tests to food manufacturers for food safety and compliance. JS, LH and NR declare no competing interests.\n\n\nGrant information\n\nThis project was crowdfunded thanks to the support of 124 contributors to the BeerDeCoded campaign that took place in June 2015. For a full list of backers, see the kickstarter project page. Some of these individuals played a role in data collection, as they provided the beer samples of their choice for analysis and participated in DNA extraction workshops.\n\n\nAcknowledgements\n\nThe authors would like to thank the following people for their invaluable contributions: Vanessa Lorenzo (Hackuarium) and Alex Hantson (nativs.ch) for their help with the crowdfunding campaign; Gabrielle Salanon for her help with sample extraction and analysis; Keith Harshman (Lausanne Genomic Technology Facility, University of Lausanne) and Stéphane Bernard (Debiopharm International) for providing access to the sequencing platform; Onecodex for providing access to its metagenomic analysis tool; Vital-it high-performance computing centre of the Swiss Institute of Bioinformatics (SIB) for providing access to their analysis cluster; UniverCite and InArTiS for hosting the Hackuarium laboratory; Rachel Aronoff (Hackuarium) for her critical revisions of the manuscript; Patrick Roelli (Bioinformatics Core Facility, University of Lausanne) for the review of the code; Bérénice Batut (University of Freiburg) for the development of the Galaxy instance; Yan Amstein and the backers of the crowdfunding campaign for providing beer samples; all members of the Hackuarium community, past and present, for their contribution to such an inspiring environment.\n\n\nReferences\n\nDe Filippis F, Parente E, Ercolini D: Metagenomics insights into food fermentations. Microb Biotechnol. 2017; 10(1): 91–102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu Y, Rousseaux S, Tourdot-Maréchal R, et al.: Wine microbiome: A dynamic world of microbial interactions. Crit Rev Food Sci Nutr. 2017; 57(4): 856–873. PubMed Abstract | Publisher Full Text\n\nDelcenserie V, Taminiau B, Delhalle L, et al.: Microbiota characterization of a Belgian protected designation of origin cheese, Herve cheese, using metagenomic analysis. J Dairy Sci. 2014; 97(10): 6046–56. PubMed Abstract | Publisher Full Text\n\nBokulich NA, Collins TS, Masarweh C, et al.: Associations among Wine Grape Microbiome, Metabolome, and Fermentation Behavior Suggest Microbial Contribution to Regional Wine Characteristics. mBio. 2016; 7(3): pii: e00631–16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBokulich NA, Thorngate JH, Richardson PM, et al.: Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. Proc Natl Acad Sci U S A. 2014; 111(1): E139–E148. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPinto C, Pinho D, Cardoso R, et al.: Wine fermentation microbiome: a landscape from different Portuguese wine appellations. Front Microbiol. 2015; 6: 905. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBelda I, Zarraonaindia I, Perisin M, et al.: From Vineyard Soil to Wine Fermentation: Microbiome Approximations to Explain the \"terroir\" Concept. Front Microbiol. 2017; 8: 821. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBochman ML, Young J: Mapping the sour beer microbiome (Experiment.com). Technical report, Indiana University; Wild Pitch Yeast, LLC and Blue Owl Brewing, 2017. Reference Source\n\nOsburn K, Amaral J, Metcalf SR, et al.: Primary souring: a novel bacteria-free method for sour beer production. 2017. Publisher Full Text\n\nHeil CS, Burton JN, Liachko I, et al.: Identification of a novel interspecific hybrid yeast from a metagenomic open fermentation sample using Hi-C. 2017. Publisher Full Text\n\nBokulich NA, Bergsveinson J, Ziola B, et al.: Mapping microbial ecosystems and spoilage-gene flow in breweries highlights patterns of contamination and resistance. eLife. 2015; 4: e04634. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14): 1754–1760. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi 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\nSchoch CL, Seifert KA, Huhndorf S, et al.: Nuclear ribosomal internal transcribed spacer (its) region as a universal dna barcode marker for fungi. Proc Natl Acad Sci U S A. 2012; 109(16): 6241–6246. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBellon JR, Schmid F, Capone DL, et al.: Introducing a new breed of wine yeast: interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast and Saccharomyces mikatae. PLoS One. 2013; 8(4): e62053. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGibson B, Krogerus K, Ekberg J, et al.: Non-conventional yeast as a new tool for beer flavour modification. Technical report, VTT Technical Research Centre of Finland. 2015. Reference Source\n\nLeskošek-Ĉukalović I, Despotović S, Nedović V, et al.: New Type of Beer – Beer with Improved Functionality and Defined Pharmacodynamic Properties. Food Technol Biotechnol. 2010. Reference Source\n\nRobbins RJ, Krishtalka L, Wooley JC: Advances in biodiversity: metagenomics and the unveiling of biological dark matter. Stand Genomic Sci. 2016; 11(1): 69. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRinke C, Schwientek P, Sczyrba A, et al.: Insights into the phylogeny and coding potential of microbial dark matter. Nature. 2013; 499(7459): 431–7. PubMed Abstract | Publisher Full Text\n\nGallone B, Steensels J, Prahl T, et al.: Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts. Cell. 2016; 166(6): 1397–1410.e16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHong X, Chen J, Liu L, et al.: Metagenomic sequencing reveals the relationship between microbiota composition and quality of Chinese Rice Wine. Sci Rep. 2016; 6(1): 26621. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJiang W, Liang P, Wang B, et al.: Optimized DNA extraction and metagenomic sequencing of airborne microbial communities. Nat Protoc. 2015; 10(5): 768–779. PubMed Abstract | Publisher Full Text\n\nNakamura S, Tsushima R, Ohtsubo K: A novel method for the preparation of template DNA for PCR from beer to detect materials and to develop DNA markers to evaluate the quality of beer. Biosci Biotechnol Biochem. 2013; 77(4): 820–831. PubMed Abstract | Publisher Full Text\n\nAfgan E, Baker D, van den Beek M, et al.: The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 2016; 44(W1): W3–W10. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "25904",
"date": "13 Sep 2017",
"name": "Bastian Greshake",
"expertise": [
"Reviewer Expertise Fungal metagenomics & 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 article describes how innovative, participant-driven research projects can create an interesting data set outside the traditional Academy. This is an extremely laudable goal and the resulting data will be of interest to a broad audience. In its current state the article is a hybrid between data note, methods article and research article that delivers preliminary results. Regardless of the form of the article, the methods section would benefit a lot from a more detailed description of the methodology (see details below). Similarly, the analyses and results are a bit lacking at this stage, especially with respect to the basic metrics of the data sets (again, see below).\n\nMajor comments\nMethods\n\nthe methods section should be significantly improved/extended for a better understanding. I'm aware that most/all the things are on GitHub, but having to crosslink these makes it hard to follow. Specifically the following things should be improved upon:\n\nWhat modifications were done to the protocol of the ZR Fecal DNA MiniPrep kit? (suggestion: putting the modified protocol to https://www.protocols.io/ if deemed useful) Which parameters were used to perform the bwa alignment? What was the size of the reference database that was used for the mapping? How was the hierarchical clustering done that is described in the methods section? Which clustering method was used? Which distance measure was used?\n\nResults\n\"We obtained an average library size of 600K reads (min 350K, max 2400K)\" This is a rather large difference between the different libraries. Does the number of species found correlate with the sequencing depth? I.e. would you have found more species if you had sequenced more data for the smaller libraries? A rarefaction analysis would be useful to understand the impact of sequencing depth on species recovery. A minor, related suggestion: Having a table of sequencing statistics so that the reader can compare the samples. A major thing that is not mentioned in the results/discussion is the number of reads which could not be mapped against the reference database. How many reads of each library did not belong to any of the reference ITS sequences? And are the non-mapping reads similar to each other or can be clustered into OTUs? This would be needed to understand how many species/OTUs are in a given sample but could not be classified due to a lack of reference database. Without this the ITS diversity in a sample cannot be correctly estimated.\n\nMinor comments:\n\"we built the proof of concept for a targeted metagenome analysis pipeline for beer samples that can be used in high schools, citizen science laboratories, craft breweries or industrial plants\" It would be good to at least briefly discuss how this is currently limited by the need to have access to a high-throughput sequencer. It would be great if \"terroir\" could be defined in the introduction for those not too familiar with oenology \"a total of 88 fungal species were identified, including 52 unique occurrences\" are unique occurrences those species which are only found in a single beer? I'd suggest rephrasing it for a better understanding. \"Interestingly, most brews were found to contain low to medium presence of multiple other yeast species, including Saccharomyces bayanus (used in winemaking and cider fermentation), Saccharomyces kudriavzevii and Saccharomyces pastorianus (used in lager manufacturing), Saccharomyces eubayanus (a probable parent of Saccharomyces pastorianus) and Brettanomyces bruxellensis (typically used for the production of the Belgian beer styles)\" please include citations for these explanations of the different taxa. It's a matter of taste, but I recommend rethinking the use of \"microbial dark matter\", c.f. http://merenlab.org/2017/06/22/microbial-dark-matter/ for an explanation of why.\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? Yes",
"responses": [
{
"c_id": "3097",
"date": "20 Oct 2017",
"name": "Luc Henry",
"role": "Author Response",
"response": "We thank the referee for his constructive remarks, and in particular for his comments on our methods section. We have added the details he requested (see below). Regarding major comments: - The minor modifications to the ZR Fecal DNA MiniPrep kit instructions are already described in the methods section of the article, as well as on the methods description available in the GitHub repository. These consist of, instead of starting with 50-100 mg of fecal material as suggested by the manufacturer, using a sludge pellet obtained through centrifuging 50 mL of beer (as described in the methods). Similar to soil and fecal material, beer samples were found to contain PCR inhibitors (see Juvonen and Haikara, J. Inst. Brew. 115(3), 167–176, 2009). The ZR Fecal DNA MiniPrep kit can overcome this because it provides filter columns designed to remove PCR inhibitors. This choice was previously described by Bukolich and coworkers (eLife 2015;4:e04634 DOI: 10.7554/eLife.04634). - In order to do the alignment we have used BWA (version 0.7.13 ) with standards parameters with 300 bp paired-end reads. - The ITS reference database contains 5361 ITS sequences. The average size of ITS is 585 bp with a standard deviation of 90 bp. - Hierarchical clustering was done by applying the basic Ward clustering algorithm with the euclidian distance computed on the log10 read count. We modified the legend of the Figure 2 accordingly. - We have to acknowledge the relatively large size variability between the different libraries. The amount of ITS detected can be affected by the sequencing depth and by the richness of the beer ecosystem. Accordingly, if the sequencing is not deep enough, we will clearly miss some low abundance species. If the beer sample contains a low variety of fungal species, sequencing deeper will not however provide additional information. In our analysis, it does not seem to be any correlation between the library size and the variety of ITS detected. The sample producing the largest library (2,4 mio reads, “Les Trois Dames”) is not the one with the largest detected ITS variety (11 species). Also, the beer sample containing the largest ITS diversity (“Waldbier 2014 Schwarzkiefer”, with 38 fungal species) had a library size of only about 0.35 mio reads. A rarefaction analysis is beyond the scope of this dataset description. As suggested, we added a table (Table 1) with the different informations regarding the libraries such as the mapping percentage and the total number of reads. In this table we observe that more than 99% of reads map to the ITS database. Consequently, there is a limited interest to try to find missing species or Operational Taxonomic Units (OTUs), and we can reasonably conclude that the ITS database used is comprehensive enough. Regarding minor comments: - The current data-set is a proof of concept that sequencing beer metagenomic information can be done, at least partly, with the help of the public. For the current analysis, we indeed had to rely on high-throughput sequencing technology available to us through a partnership with the genomic facility at the University of Lausanne. In the future, we would like to overcome this limitation, e.g. by using a minION sequencer. A remark was added to the discussion. - The text was modified to clarify the notion of \"terroir\". - The text was modified to clarify the notion of \"unique occurrences\". - The text was modified (species were removed) to reflect changes due to an updated sensitivity of the analysis (based on another referee's comment), and a reference was added. - The so-called “microbial dark matter” concept is regularly used by microbiologist and we think that it points to an interesting hypothesis worth mentioning, although it has nothing to do with the dark matter in physics, as explained in the blog post mentioned."
}
]
},
{
"id": "25906",
"date": "18 Sep 2017",
"name": "Matthew L. Bochman",
"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, Sobel et al. present fungal microbiome data from 39 different beers as the culmination of a crowdfunded citizen science campaign. These data will be of interest to citizen scientists and financial backers of the project, as well as those in the fermentation (especially beer) industry. Overall, the data seem sound, but I have some concerns:\nMajor comments Were any controls for contamination used, i.e., are all of the fungi identified actually from the beer samples? The sequencing of a non-beer sample such as water that had been handled in the same way as the beer samples would help to determine if fungal DNA contamination occurred during sample processing.\nIn line with the comment above, the manuscript states that “…microorganisms, or their DNA, could be carried over from the ingredients to the final product.” Can the authors comment on whether they know if they are detecting the fungi themselves or DNA remnants from fungi that came from the raw ingredients of the beer? Again, one could add purified control DNA to a mash, brew and bottle a beer, and then try to detect that DNA by PCR in the end product (or even at various stages along the brewing and fermentation process). Attempting this with various concentrations of DNA would also yield information on how many cells of a particular species would be necessary on malted barley, for instance, to be detected in the final beer.\nMinor comments In the introduction, the authors state that “…sour beer…[is] produced without the controlled addition of known yeast cultivates.” Although this may be true for some types of sour beer like lambic and gueuze, many sour beers made in the U.S. are inoculated with known strains of yeast. In those cases, the souring bacteria are usually the unknowns.\nWhy was a fecal DNA prep kit used for DNA extraction?\nThe authors collected 120 beers from 20 countries but only sequenced the fungi from 39 (mostly from Switzerland). Is there an explanation for this attrition?\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? Yes",
"responses": [
{
"c_id": "3096",
"date": "20 Oct 2017",
"name": "Luc Henry",
"role": "Author Response",
"response": "Regarding major comments: - We did not include a water sample to account for any contaminant during the DNA extraction process. Processing such a control at this point would not reflect the original experimental conditions. We will include this control in the future when we will process new beers. - We did think of experiments to identify DNA remnants from raw ingredients but did not have the means to perform them. Indeed, the beer samples were sent from all over the world (Europe and Switzerland for the 39 reported in this data set) and we had no possibility to collect other related samples (raw ingredients, brewing environment, etc). We think that it is out of the scope of the current study. In this setup, it is not possible to know afterwards from which ingredient the DNA comes from and we comment on this in the text. Regarding minor comments: - We rephrased the sentence about sour beers accordingly. - Similar to soil and fecal material, beer samples were found to contain PCR inhibitors (see Juvonen and Haikara, J. Inst. Brew. 115(3), 167–176, 2009) that can interfere with the preparation of samples for sequencing analyses. The ZR Fecal DNA MiniPrep kit can overcome this because it provides filter columns designed to remove PCR inhibitors. This methodological choice was previously described by Bukolich and coworkers (eLife 2015;4:e04634 DOI: 10.7554/eLife.04634). - A majority of the samples we received were from industrial/filtered beers. Unfortunately the volumes we had at hand (typically a 330 mL bottle or below) did not yield enough material (DNA of good purity) to obtain sequencing results, as judged by QC of PCR products. In order to detect DNA in these beers, we would probably need to process a much larger volume of beer. We therefore did not include these samples in the final data set."
}
]
},
{
"id": "26415",
"date": "28 Sep 2017",
"name": "Kristoffer Krogerus",
"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 data note describes the fungal microbiome of 39 (commercial and homebrewed) beers as determined by next generation sequencing of ITS amplicons. The project was crowdfunded and many of the individual funders were also involved in providing beer samples and assistance during DNA extraction. While the results will be of interest, particularly for the brewing industry, I have some concerns with the analysis methods and the results presented in this first version of the manuscript.\nMajor comments:\nTo determine the microbiome of the beers, the authors align the raw sequencing reads to a concatenated dataset containing fungal ITS sequences. To my understanding, no quality control or filtering was performed prior to and after the alignment. This will cause a large number of false positives, as many of the intragenic ITS sequences are very similar. To demonstrate, I repeated the analysis on six samples I retrieved from the NCBI SRA:\nSRR5740352: Chimay Red Cap SRR5740353: Chimay Tripel SRR5740362: Waldbier 2014 Wienerwald SRR5740364: La Nebuleuse Chicha SRR5740374: Orval SRR5740375: Trois Dames\nAccording to the results presented in the manuscript, each of the samples contained traces of at least 11 different species (see Figure 1 and Figure 3).\nWhat I did to the sequencing reads was 1. Trim them using 'cutadapt' as follows (any similar tool would do the same job)\nRemove 20 first bases of each read Remove bases from end of read when quality score is less than 15 Remove any reads shorter than 200 base pairs Approximately 80% of the bases were retained from each set of reads after these steps\n2. After this the reads were aligned to the concatenated ITS sequence dataset using bwa mem with default settings as the authors had done. Reads mapping to the different sequences were then counted with the script used by the authors (obtainable from github). The results with no post alignment filtering: https://www.dropbox.com/s/llg94fgk23ag264/Beer_results_nofilter.txt\n3. After this I removed all reads that did not map to a unique location (i.e. could be mapped to the ITS sequences of several species) and reads where the two paired reads mapped to different sequences. This was done by removing all alignments with a MAPQ score below 4 and 'awk': https://www.dropbox.com/s/0iimh5fbb40qeh0/Beer_results_mapq4.txt\nAs can be seen, the diversity is reduced considerably, and if all hits where the read count is less than 10 are also removed (as the authors had done), most samples now contain only S. cerevisiae and/or B. bruxellensis.\n4. The amount of false positives can be further reduced by filtering by a higher MAPQ score (e.g. 30): https://www.dropbox.com/s/3x4gyylsiykyu7o/Beer_results_mapq30.txt\nI therefore suggest that the authors redo the analysis with proper filtering to remove poor quality alignments and false positives in the results. The results and conclusions will subsequently have to be rewritten accordingly.\nMinor comments:\nIn the Methods section, under Beer sample preparation: I assume the DNA was extracted from the frozen yeast pellet? Any reason why it was not attempted to extract DNA from the beer itself, e.g. using the method described in reference (23)? This would allow analysis of filtered beers as well.\nIs Figure 2 necessary, since Figure 3 shows the clustering as well? Also, why does the clustering in Figures 2 and 3 differ? Were these generated using different clustering methodologies?\nIt is mentioned that some of the beers contained speciality ingredients, such as pine cones. Do the authors know at what point in the production process these were added (i.e. pre- or post-boil)? This would have a large impact on how these ingredients affect the beer microbiome.\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? Yes",
"responses": [
{
"c_id": "3095",
"date": "20 Oct 2017",
"name": "Luc Henry",
"role": "Author Response",
"response": "Regarding the major comments: - We thank the referee for his thorough analysis of our results and for his comments. In our analysis, we initially choose to favor sensitivity over specificity and we did not filtered the reads based on their quality. We considered that, with 300 bp paired-end reads on ITS amplicons, we had enough specificity. But as referee 3 pointed out, we should take care of multiple mapping reads to for instance discriminate between the Saccharomyces sp. that are quite close to each other. Using a filter on MAPQ score > 30 is quite stringent, but the referee’s point on non-unique reads is critical. We have now re-performed the analysis using a series of MAPQ threshold. As expected, the higher the threshold, the more we lose some of the ITS detected, and some sensitivity. In our update of the article, we report results after using a filter with a MAPQ score of 3. This led to a reduction of the total fungal species identified, from 88 to 42, as well as of the unique occurrences, from 52 to 24. In addition we have performed quality control (QC) with samstat of our BAM files in order to see the quality score distribution of the reads of the alignment to the ITS database. The result of samstat has been added to our github repository. Samstat results show that about 3.2 % of reads are not aligned when reads with MAPQ score > 3 are taken into account. In addition we have checked the distribution of fragments size in the BAM files. We have an average fragment size of 383 bp with a sd of 10 bp. We have a very low fraction of fragments below 200 bp, or discordant pairs. Thanks to the comments of the Referee 3, we improved the specificity of the analysis and we may have excluded artifacts. Due to this re-analysis, we have updated Figure 1, Figure 2 and Figure 3, the code in our github repository as well as the main text. We have also added the QC informations. Regarding the minor comments: - With the goal to maximise our chances to obtain some preliminary results with the budget of the kickstarter campaign, we decided to use the pelleted material with the educated assumption that pelleted cells may protect DNA better than DNA left in solution. Reference (23) intrigued us: their DNA preparation method requires additional lyophilization, pulverization and digestion with amylase and we decided to start with an easier protocol to facilitate participation from the general public. - We included both Figure 2 and Figure 3 to provide two different representations of the same results. The differences in the tree are due to the different display of the dendrogram. The same three is underling based on the same distance (euclidian distance on log10 counts matrix, with ward clustering method). - The last comment is an interesting work hypothesis. We however do not know when these ingredients are added to the brewing process, and whether they were pre-treated before addition. If these ingredients contained living microorganisms at the moment of addition, they may indeed affect the brewing process and the taste of the final product. We believe collecting this metadata is outside the scope of this dataset description."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1676
|
https://f1000research.com/articles/6-1630/v1
|
04 Sep 17
|
{
"type": "Research Article",
"title": "Gender differences in the prevalence of nonalcoholic fatty liver disease in the Northeast of Thailand: A population-based cross-sectional study",
"authors": [
"Ueamporn Summart",
"Bandit Thinkhamrop",
"Nittaya Chamadol",
"Narong Khuntikeo",
"Metha Songthamwat",
"Christina Sunyoung Kim",
"Ueamporn Summart",
"Nittaya Chamadol",
"Narong Khuntikeo",
"Metha Songthamwat",
"Christina Sunyoung Kim"
],
"abstract": "Background. Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease. A large number of studies have strongly described larger proportions of men being afflicted with NAFLD than women; however, recent studies investigating the role of gender and NAFLD have exposed the contrary. Methods. This cross-sectional study utilized data from the baseline survey of an ongoing cohort study called the Cholangiocarcinoma Screening and Care Program (CASCAP), conducted in the northeastern region of Thailand between March 2013 and September 2015. Information regarding socio-demographic, including gender, was collected using a standardized self-administered questionnaire. NAFLD was diagnosed with ultrasonography by board-certified radiologists. A binomial regression was used for estimating the prevalence differences, odds ratios (OR) and the 95% confidence intervals (CI) of NAFLD between men and women. Results. A total of 34,709 participants (27,073 females and 7,636 males) were recruited. The prevalence of NAFLD in women was 22.9% (95% CI: 22.5 to 23.5), whereas it was only 18.3% (95% CI: 17.4 to 19.2) in men. After adjusting for age and presence of diabetes mellitus and other underlying diseases, the prevalence was significantly higher in women, with adjusted prevalence difference of 4.2% (95% CI: 3.2 to 5.2) and adjusted OR of 1.3 (95% CI: 1.2 to 1.4). Women had a higher prevalence of NAFLD than men in all age groups and the largest difference was found in those aged 56-60 years (prevalence = 27.4% versus 21.2%; adjusted prevalence difference = 9.4%; 95% CI: 7.9 to 10.9; adjusted OR = 1.8; 95% CI: 1.8 to 2.0). Conclusion. NAFLD is more likely to affect women more than men, in particular, among the population 56-60 years of age, which is the post-menopausal transitional period. Therefore, post-menopausal women should be the target for interventions or further investigation for NAFLD.",
"keywords": [
"Nonalcoholic fatty liver disease",
"Gender differences",
"Post-menopausal",
"Ultrasonography",
"Asian population"
],
"content": "Introduction\n\nNonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease and a major public health problem worldwide1. Its prevalence is increasing globally, and is currently estimated to be as high as 17–45% of the general population in Western countries2, while among Asian populations it is reported to be between 15 and 20%3. A progressive form of NAFLD called nonalcoholic steatohepatitis (NASH) can further progress to liver cirrhosis and hepatocellular carcinoma (HCC)4. Recent studies have found that HCC may complicate non-cirrhotic NAFLD with or without fibrosis5. In addition, NASH patients run an increased risk of cardiovascular mortality as a result of the metabolic risk factors that are common to both NAFLD and cardiovascular disease6.\n\nMajor risk factors of NAFLD include a sedentary lifestyle and diet with poor nutrition2,4,7. Other factors that influence the development of NAFLD include age, being a man between the ages of 40–65 years and Hispanic ethnicity1,8–11. In addition, insulin resistance (IR), metabolic syndrome (MS) and type 2 diabetes mellitus (T2DM) are considered to increase the risk of NAFLD2. NAFLD is closely associated with T2DM, and therefore T2DM is used as a determinant for the presence and severity of NAFLD12,13. Various studies have demonstrated that NAFLD is more prevalent in men, elderly populations, and post-menopausal women14.\n\nGender differences as a risk factor for NAFLD still need to be fully understood. There is controversy regarding gender and NAFLD; some studies claim that various gender-specific mechanisms, such as the effect of sex hormones and differences in lifestyles and physiology, have an influence on the prevalence of NAFLD15. In addition, a number of studies report NAFLD as being more frequently detected in men than women8,10,14,16,17. However, there are also some studies, both from Western and Asian populations, that suggest that the disease is generally more common in women9,11,18.\n\nUnderstanding the association between gender differences and NAFLD will allow us to target specific groups to improve health promotion and disease prevention activity, as well as provide proper treatment strategies in order to reduce the rates of morbidity and mortality associated with NAFLD and its associated pathologies. Therefore, this study investigated the gender differences in the prevalence of NAFLD from the general population of Northeast Thailand.\n\n\nMethods\n\nThis was a population-based cross-sectional study that retrieved the data from the baseline survey of an ongoing cohort research project called the Cholangiocarcinoma Screening and Care Program (CASCAP, www.cascap.in.th)19. All participants of CASCAP were enrolled between March 2013 and September 2015 were included into the study. In accordance with the CASCAP protocol, participants gave written informed consent and completed a baseline survey form. The standardized form included socio-demographic information including gender, behavioral factors, such as smoking status and alcohol consumption, and previous or current illnesses. After completion of the baseline survey, participants underwent hepatobiliary ultrasonography (US) performed by board-certified radiologists, who provided the participants with information on NAFLD. For the purpose of this study, subjects with alcohol consumption or those with incomplete information of US findings were excluded.\n\nThe primary outcome was the ultrasonographic diagnosis of NAFLD based on the presence of a diffuse increase of fine echoes in the liver parenchyma compared to the kidney or spleen parenchyma20. This was performed after excluding other causes of liver disease, such as viral hepatitis B or C and/or a history of current or past alcohol consumption15. In addition, the protocol also classified the severity of NAFLD as follows: absent, mild, moderate or severe steatosis20,21. Finally, the participatns were divided into those with and without NAFLD, according to the US results. Participants with absence of NAFLD were used as the comparison group for the study. The factor of interest in this study was gender.\n\nDemographic characteristics and other information of the participants serving as covariates that could have an effect on the association between gender and NAFLD were accounted for in the statistical analysis. These included age, the presence of diabetes mellitus (DM) and other underlying diseases. Age was initially treated as a continuous variable based on the assumption of a linear relationship. For practical purposes, age was then categorized into six groups comprising: <45 years, 46–50 years, 51–55 years, 56–60 years, 61–65 years and more than 65 years. Other confounder factors included: the presence of DM and presence of other underlying diseases. These were dichotomous variables, and were also analyzed for the relationship with NAFLD.\n\nThe characteristics of all enrolled participants were summarized by gender and the total number of study participants. All categorical variables were described by number and percentage of distributions. Continuous variables were expressed as means and standard deviation among male and female participants.\n\nTo answer the research questions, the prevalence of NAFLD was estimated separately for men and women. Univariate analysis using binomial regression was performed to explore the effect of gender and other clinical characteristics on NAFLD, ignoring the effect of other factors for better handling the covariates in a more sophisticated statistical modeling. In addition, we performed stratified analyses in pre-specific subgroups defined by age group and the presence of DM and other underlying diseases. The interaction of these stratified variables was investigated through a bivariate analysis performed by the Mentel-Haenszel extension of the chi-square test. Then, multivariable binomial regression was performed to quantify the effects of gender on NAFLD with the inclusion of age, DM, and other underlying diseases as covariates. The effect of gender on NAFLD was then obtained as adjusted prevalence differences and adjusted odds ratio (ORs) together with their 95% confidence intervals (CI). All analyses were done using Stata 13.1 (Stata Corp, College Station, TX, USA). The significance level was set at 0.05 and all statistical tests were two-sided.\n\nCASCAP was approved by Khon Kaen University Ethics Committee (HE551404), and was conducted according to the International Conference of Harmonization, Good Clinical Practice guidelines and the Declaration of Helsinki. The authors of the present study submitted a Data Analysis Plan Proposal to Khon Kaen University Ethics Committee in Human Research to request the data (approval number, HE591067).\n\n\nResults\n\nA total of 65,571 participants living in northeastern Thailand participated in CASCAP during the study period as shown in Figure 1. We excluded 30,661 participants who were known to consume alcohol, or to have viral hepatitis or alcoholism. Of the remaining participants, 201 were excluded because of incomplete data. Finally, a total of 34,709 participants were included for analysis.\n\nBaseline characteristics of the study populations are shown in Table 1. Of 34,709 participants enrolled in this study, 27,073 were women (78.0%), while 7,636 were men (22.0%). Both were predominantly middle-aged with a mean age of 55.5±38.3 years old. There were similar characteristics between men and women, except that women were older (57.8 versus 54.3 years old, and had a lower proportion of being a current smoker or previously having smoked than men (1.0% vs. 18.7%).\n\nOf 34,709 participants who underwent US, 7,584 had NAFLD; hence, the overall prevalence was 21.9% (95% CI: 21.4 to 22.3). The prevalence of NAFLD was 22.9% in women (95% CI: 22.5 to 23.5) and 18.3% in men (95% CI: 17.4 to 19.2) (Table 2). Based on an absolute effect represented by the prevalence difference, the prevalence of NAFLD was significantly higher in women than men by 4.6% (95% CI: 3.6 to 5.6). Similarly, based on a relative effect represented by the OR, women were 1.3 times (1.3; 95% CI: 1.2 to 1.4) as likely to have NAFLD compared with men. After adjusting for effect of other covariates, the prevalence difference was 4.2% (95% CI: 3.2 to 5.2), but adjusted OR remained unchanged (1.3; 95% CI: 1.2 to 1.4).\n\nDM, diabetes mellitus.\n\n* Both unadjusted and adjusted for age presence of DM and other underlying diseaes\n\nThe overall prevalence difference between gender was 4.6% (95%CI: 3.6 to 5.6), the prevalence difference in severity of ultrasonographic NAFLD with mild NAFLD was 3.8% (95%CI: 2.9 to 4.9) (Table 3). The majority of participants with NAFLD were also found to have periductal fibrosis (PDF), i.e., 1,143 with PDF out of 7,584 with NAFLD for women. After combining NAFLD with the PDF, the increased prevalence in women remained, but the gender difference was smaller (1.1%; 95% CI: 0.6 to 1.3).\n\nUS, ultrasound.\n\nBased on univariate analysis, prevalence differences stratified by age group demonstrated a noticeable pattern. That is, the difference in NAFLD prevalence tended to increase as the age increased. Moreover, while the overall prevalence difference was 4.6%, the stratified differences were 6.2% and 6.3% in the 56–60-year-old and 61–65-year-old age groups, respectively. The prevalence increased markedly in the age group up to 50 years, whereas both women and men at the age of 56–60 years have the highest prevalence of NAFLD (27.4% and 21.2%, respectively) (Figure 2). Results also showed that the prevalence of NAFLD increased from 16.8% to 21.5% in women younger than 45 versus women aged 45–50 years, and then peaked at 27.4% in women aged 55–60 years. However, the prevalence differences of NAFLD decreased slightly in the ≥65-year-old age group.\n\nResults of multivariable analysis, the prevalence difference of NAFLD between men and women stratified by age group exhibited similar patterns, but with larger differences than were found in the univariate analysis. That is, while the overall adjusted prevalence difference was 4.2%, the stratified adjusted differences were 9.4% and 8.8% in the 56–60-year-old and 61–65-year-old age groups, respectively. The largest prevalence difference was observed among participants with DM or underlying diseases (12.2%; 95% CI: 9.8 to 14.7) (Figure 3).\n\nEach was adjusted for all others.\n\n\nDiscussion\n\nWe investigated the inconsistencies in the literature regarding gender differences in NAFLD prevalence with a population-based study in Thailand. While many studies have indicated that NAFLD is more common in men than women10,14,16,17, others have suggested the opposite9,11,18. Our study supported the findings of a higher NAFLD prevalence in women, with a 4.2% (95% CI: 3.2 to 5.2) prevalence, compared with men (18.3%; 95%CI: 17.4 to 19.2), even after adjusting for the effects of other covariates. In addition, we found that these differences increased with age, where the largest difference was found in the age group 56–60 years old (prevalence difference = 9.4%; 95% CI: 7.9 to 10.9). Moreover, the largest gender difference in NAFLD prevalence was also found among DM participants (12.2%; 95% CI: 9.8 to 14.7).\n\nThis study utilized data from CASCAP, in which most female participants were over 50 years old (64.5%), with a mean age of 57.8 ±42.3 years. The largest group (35.9%) of the over-50-year group being between 51–60 years old, and most were likely post-menopausal. Although the prevalence of NAFLD tended to be higher in women for every age group, the largest difference was found among the 56–60-year-old age group. This suggests that sex hormones might play a role in NAFLD18,22, for the younger age group. However, the current study revealed a smaller difference in NAFLD prevalence. This might be because NAFLD in men tends to occur earlier, especially in middle age (40–49 years) than in women (>50 years), which could lead to a male predominance in younger and middle-aged populations14.\n\nThe gender differences on NAFLD prevalence became more pronounced as the age of the participants increased7,23,24. The highest NAFLD rate was found in 56–60-year-olds. This suggested a correlation between NAFLD and the various major risk factors commonly found in older people, such as MS, obesity, DM, and dyslipidemia. Moreover, older patients are more likely to have advanced fibrosis, cirrhosis, and HCC when compared with middle-aged patients9. Remarkably, the study found increased NAFLD prevalence in women in the transitional post-menopausal stage, especially among women aged 56–60 years. This statistic is consistent with a previous study that reported a 2 to 2.5 times higher prevalence in the 56–60-year-old age group compared with those aged less than 45 years25. Our study also found that an increased prevalence of NAFLD in female participants persisted in the post-menopausal group, while the prevalence trend declined in those aged more than 65 years compared with premenopausal women.\n\nThe results of this study suggest that women are at higher risk of NAFLD than men. This has been attributed to natural changes in female physiology, such as IR, central obesity, adipose distribution and sex hormones18. The gender differences in NAFLD observed in the study can be explained by the association of age and gender. Typically, younger-aged to middle-aged men tend to have a greater risk of acquiring NAFLD than women of the same age, as illustrated through an “inverted U-shaped curve”, in which the line begins to decline after the age of 50–60 years26. Accordingly, premenopausal women have a relatively low prevalence of NAFLD; however, the prevalence increases after the age of 50 years, peaks at 60–69 years, and declines after age 70 years5,22. After the age of 50 years, the protective effects of higher estrogen levels in women during pre-menopause are markedly eliminated in the transitional post-menopausal period25,26. These associations between age and gender can be explained by natural changes in female physiology that increase the risk of IR, hyperlipidemia, and visceral fat accumulation, which are known as risk factors for the development of NAFLD27. Estrogen is a powerful antioxidant that can inhibit hepatic stellate cell proliferation and fibrogenesis in experimental models22,25. These changes can reduce fatty acid oxidants, while increasing lipogenesis within the liver, which leads to a redistribution of subcutaneous fat and causes visceral fat accumulation22,26,27. Therefore, changes in body fat distribution resulting from declining levels of estrogen, relatively higher androgen levels and greater distribution of hormone receptors can lead to increased risk of NAFLD in post-menopausal women22,25,28. Subcutaneous and visceral compartmentalization of adipose tissue is influenced by age and gender18. Visceral adipose tissue accumulates more rapidly with age and weight gain in men and post-menopausal women than in younger women18. The NAFLD prevalence rate increases with age in all groups of younger to middle-aged men, and declines at the age of 50 to 60 years28. However, NAFLD prevalence becomes comparable between men and women at the age of 60 years8,14. Our results confirm results showing that the interrelation between aging in premenopausal women and the development of NAFLD is strongly associated with changes in the level of estrogen-related sex hormones5.\n\nOur data illustrate that T2DM patients had higher NAFLD prevalence compared with the general population12. After adjusting for the effects of other covariates, it was found that NAFLD prevalence in those with T2DM was significantly higher (12.2%). Because a healthy population is included at the community level, DM is believed to be distributed randomly in men and women. Confounding effects of T2DM would be minimal. Previous studies reported an association between T2DM and NAFLD that was particularly pronounced in post-menopausal women >50 years old22. This may be due to a decrease in estrogen in this group of women, which is a protective factor against DM29. Moreover, IR in the muscle, liver, and adipose tissue is a characteristic feature of T2DM and NAFLD. It is characterized not only by higher insulin circulation levels, but also by higher hepatic gluconeogenesis, reduction of insulin clearance, and impaired glucose uptake by muscles, all of which lead to elevated plasma glucose concentrations30. IR in adipose tissue can increase the release of free fatty acids and inflammatory cytokines31. Transaminase levels increase in patients with NAFLD; however, this does not commonly occur in subjects who also have T2DM. Despite this, over the years, many patients with NAFLD have also been classified as having T2DM4.\n\nThe study is a community-based study with a large sample size and a healthy population from the largest region of Thailand, the northeast region. In addition, the large size of the CASCAP database allows us to stratify the population by DM or non-DM, and to examine the interaction of different variables with adequate power. Second, the NAFLD diagnosis of all participants was performed by all board-certified radiologists. Finally, this study presented a strong link between gender and NAFLD presented with adjusted OR and absolute risk reduction (ARR). Using these statistical methods allowed us to properly measure the associations and determinants of certain health outcomes. It is important to note that, although the ARR varied according to event rates and the effects of other covariates, the adjusted OR remained unchanged. However, ARR is a valid index for healthcare providers because it demonstrates how certain risk factors impact the reduction of the overall prevalence of the disease.\n\nSeveral limitations associated with the present study warrant mention. First, there were insufficient data to distinguish alcoholic fatty liver disease from NAFLD, so this differentiation was based on self-reported alcohol intake. Therefore, we excluded all participants with any history of alcohol intake, which affected the total number of male participants compared with that of females. However, when all participants were included back into the analysis, the prevalence of NAFLD in women remained higher than men in all age groups. Second, it should be considered that the database utilized in this study did not provide certain variables that may support a better determination of NAFLD progression; for example, anthropometric variables, such as BMI. Further studies are required to minimize these possibly distorted associations and allow generalization of these findings to other sampling populations.\n\n\nConclusion\n\nNAFLD is more likely to affect women than men, in particular among the population 56–60 years of age, which is the post-menopausal transitional period. This suggests that post-menopausal women should be concerned about metabolic disorders that are exacerbated by changing hormonal status. Monitoring and prevention by dietary control, behavioral changes, and exercise may play an important role in preventing diseases, including NAFLD. We strongly recommend and encourage Thai health professionals promotion of the development of NAFLD targeted screening and prevention programs focusing on post-menopausal women and DM risk groups.\n\n\nData availability\n\nResearchers can request the CASCAP data by applying to the CASCAP Database Committee using a Data Analysis Plan Proposal. This can be found at http://www.cascap.in.th/damus/analysis_plan.php. More information can be requested from the corresponding author (bandit@kku.ac.th) and information about research proposals can be found at https://cloud.cascap.in.th/article/research/index",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by Khon Kaen University through CASCAP and the National Research Council of Thailand through the Medical Research Network of the Consortium of Thai Medical Schools.\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 is part of the first author’s thesis in partial fulfillment of the requirements for a Doctor of Public Health at Khon Kaen University, Thailand, and CASCAP. The authors thank for all participants and all participating institutions including the Ministry of Public Health, Ministry of Interior and Ministry of Education of Thailand.\n\n\nSupplementary material\n\nSupplementary File 1: Do file to generate variables from the main CASCAP dataset used in the present study.\n\nClick here to access the data.\n\nSupplementary File 2: Codes for variables from analysis.\n\nClick here to access the data.\n\n\nReferences\n\nYounossi ZM, Stepanova M, Negro F, et al.: Nonalcoholic fatty liver disease in lean individuals in the United States. Medicine (Baltimore). 2012; 91(6): 319–27. PubMed Abstract | Publisher Full Text\n\nEuropean Association for the Study of the Liver (EASL), European Association for the Study of Diabetes (EASD), European Association for the Study of Obesity (EASO): EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. Diabetologia. 2016; 59(6): 1121–40. PubMed Abstract | Publisher Full Text\n\nBuday B, Pach PF, Literati-Nagy B, et al.: Sex influenced association of directly measured insulin sensitivity and serum transaminase levels: Why alanine aminotransferase only predicts cardiovascular risk in men? Cardiovasc Diabetol. 2015; 14: 55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAgrawal S, Duseja AK: Non-alcoholic Fatty Liver Disease: East Versus West. J Clin Exp Hepatol. 2012; 2(2): 122–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHamaguchi M, Kojima T, Ohbora A, et al.: Aging is a risk factor of nonalcoholic fatty liver disease in premenopausal women. World J Gastroenterol. 2012; 18(3): 237–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVernon G, Baranova A, Younossi ZM: Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Aliment Pharmacol Ther. 2011; 34(3): 274–85. PubMed Abstract | Publisher Full Text\n\nAhmed A, Wong RJ, Harrison SA: Nonalcoholic Fatty Liver Disease Review: Diagnosis, Treatment, and Outcomes. Clin Gastroenterol Hepatol. 2015; 13(12): 2062–70. PubMed Abstract | Publisher Full Text\n\nXu C, Yu C, Ma H, et al.: Prevalence and risk factors for the development of nonalcoholic fatty liver disease in a nonobese Chinese population: the Zhejiang Zhenhai Study. Am J Gastroenterol. 2013; 108(8): 1299–304. PubMed Abstract | Publisher Full Text\n\nWang Z, Xu M, Peng J, et al.: Prevalence and associated metabolic factors of fatty liver disease in the elderly. Exp Gerontol. 2013; 48(8): 705–9. PubMed Abstract | Publisher Full Text\n\nLazo M, Hernaez R, Eberhardt MS, et al.: Prevalence of nonalcoholic fatty liver disease in the United States: the Third National Health and Nutrition Examination Survey, 1988–1994. Am J Epidemiol. 2013; 78(1): 38–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBedogni G, Miglioli L, Masutti F, et al.: Incidence and natural course of fatty liver in the general population: the Dionysos study. Hepatology. 2007; 46(5): 1387–91. PubMed Abstract | Publisher Full Text\n\nPappachan JM, Antonio FA, Edavalath M, et al.: Non-alcoholic fatty liver disease: a diabetologist's perspective. Endocrine. 2014; 45(3): 344–53. PubMed Abstract | Publisher Full Text\n\nKahn SE, Hull RL, Utzschneider KM: Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature. 2006; 444(7121): 840–6. PubMed Abstract | Publisher Full Text\n\nWang Z, Xu M, Hu Z, et al.: Sex-specific prevalence of fatty liver disease and associated metabolic factors in Wuhan, south central China. Eur J Gastroenterol Hepatol. 2014; 26(9): 1015–21. PubMed Abstract | Publisher Full Text\n\nPan JJ, Fallon MB: Gender and racial differences in nonalcoholic fatty liver disease. World J Hepatol. 2014; 6(5): 274–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBabusik P, Bilal M, Duris I: Nonalcoholic fatty liver disease of two ethnic groups in Kuwait: comparison of prevalence and risk factors. Med Princ Pract. 2012; 21(1): 56–62. PubMed Abstract | Publisher Full Text\n\nPark SH, Jeon WK, Kim SH, et al.: Prevalence and risk factors of non-alcoholic fatty liver disease among Korean adults. J Gastroenterol Hepatol. 2006; 21(1 Pt 1): 138–43. PubMed Abstract | Publisher Full Text\n\nAyonrinde OT, Olynyk JK, Beilin LJ, et al.: Gender-specific differences in adipose distribution and adipocytokines influence adolescent nonalcoholic fatty liver disease. Hepatology. 2011; 53(3): 800–9. PubMed Abstract | Publisher Full Text\n\nKhuntikeo N, Chamadol N, Yongvanit P, et al.: Cohort profile: cholangiocarcinoma screening and care program (CASCAP). BMC Cancer. 2015; 15: 459. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMahaling DU, Basavaraj MM, Bika AJ: Comparison of lipid profile in different grades of non-alcoholic fatty liver disease diagnosed on ultrasound. Asian Pac J Trop Biomed. 2013; 3(11): 907–12. Publisher Full Text | Free Full Text\n\nCuenza LR, Razon TL, Dayrit JC: Correlation between severity of ultrasonographic nonalcoholic fatty liver disease and cardiometabolic risk among Filipino wellness patients. J Cardiovasc Thorac Res. 2017; 9(2): 85–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlorentino GS, Cotrim HP, Vilar CP, et al.: Nonalcoholic fatty liver disease in menopausal women. Arq Gastroenterol. 2013; 50(3): 180–5. PubMed Abstract | Publisher Full Text\n\nKoehler EM, Schouten JN, Hansen BE, et al.: Prevalence and risk factors of non-alcoholic fatty liver disease in the elderly: Results from the Rotterdam study. J Hepatol. 2012; 57(6): 1305–11. PubMed Abstract | Publisher Full Text\n\nCaballeria L, Pera G, Auladell MA, et al.: Prevalence and factors associated with the presence of nonalcoholic fatty liver disease in an adult population in Spain. Eur J Gastroenterol Hepatol. 2010; 22(1): 24–32. PubMed Abstract | Publisher Full Text\n\nRyu S, Suh BS, Chang Y, et al.: Menopausal stages and non-alcoholic fatty liver disease in middle-aged women. Eur J Obstet Gynecol Reprod Biol. 2015; 190: 65–70. PubMed Abstract | Publisher Full Text\n\nYang JD, Abdelmalek MF, Pang H, et al.: Gender and menopause impact severity of fibrosis among patients with nonalcoholic steatohepatitis. Hepatology. 2014; 59(4): 1406–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuzuki A, Abdelmalek MF: Nonalcoholic fatty liver disease in women. Womens Health (Lond). 2009; 5(2): 191–203. PubMed Abstract | Publisher Full Text\n\nBertolotti M, Lonardo A, Mussi C, et al.: Nonalcoholic fatty liver disease and aging: epidemiology to management. World J Gastroenterol. 2014; 20(39): 14185–204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeko Y, Sumida Y, Tanaka S, et al.: Insulin resistance increases the risk of incident type 2 diabetes mellitus in patients with non-alcoholic fatty liver disease. Hepatol Res. 2017. PubMed Abstract | Publisher Full Text\n\nSima A, Timar R, Vlad A, et al.: Nonalcoholic fatty liver disease: a frequent condition in type 2 diabetic patients. Wien Klin Wochenschr. 2014; 126(11–12): 335–40. PubMed Abstract | Publisher Full Text\n\nSaponaro C, Gaggini M, Gastaldelli A: Nonalcoholic fatty liver disease and type 2 diabetes: common pathophysiologic mechanisms. Curr Diab Rep. 2015; 15(6): 607. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "25669",
"date": "04 Oct 2017",
"name": "Penprapa Siviroj",
"expertise": [
"Reviewer Expertise Community 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\nI would criticize and recommend the following:\nIntroduction\nThe review literature about Nonalcoholic fatty liver disease is not up to date.\nExample: Younossi et al., 20161\nMethods\nThe researcher should report the number of participants who were enrolled and excluded.\n\nThe word “participants” in the second paragraph of page 3 is wrong.\n\nIf there were comparison groups, the researcher should explain the purpose of the comparison group which was mentioned in the second paragraph of page 3.\n\nThe researcher should explain what “other underlying disease” is.\n\nFigure 1 (Flow of participants) should be written with inclusion criteria before exclusion criteria.\n\nResults and discussion\nWhat is the purpose of reporting educational level and occupation in table 1? Were these factors used for adjusting the result?\n\nThe age category should be similar in every part of the paper.\n\nIn the discussion, the researcher categorized into groups ranging 10 years each (the prevalence increases after the age of 50 years, peaks at 60–69 years) but in the methods the age was categorized into groups ranging 5 years each.\n\nIn the discussion, it was reported the prevalence of NAFLD declines after age 70, but in the results table reported the prevalence of age greater than 65.\nConclusion\nThis research studied only prevalence of NAFLD which is only one of many kinds of metabolic disorder, so it is not appropriate to conclude that “post- menopausal women should be concerned about metabolic disorders”.\n\nThis research surveyed a very specific population, so it is not appropriate to recommend NALFD screening and prevention programs focusing on post-menopausal women and DM risk groups because the prevalence of NAFLD in normal population might be different from this population.\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?\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? No",
"responses": []
},
{
"id": "26390",
"date": "16 Oct 2017",
"name": "Matthew J. Kelly",
"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\nThis paper was overall well presented and argued. The introduction is clear and makes a good rationale for the analyses in testing a contested area of the literature in gender differences in NAFLD. There are several points in the methods and discussion that could be made clearer however.\nMethods Firstly the description of the study population is inadequate. Who the cohort are in terms of age and gender is clear. But we need to know how and why the cohort was recruited. It does not diminish the importance of the findings if cohort participants are not representative of the general population but it is still necessary to explain how they are different. Were they recruited because they were at particular risk of certain health conditions? Or just from the general population? How were they contacted? This will have implications in the discussion of your findings.\n\nAlso there are several references to those who consumed alcohol being excluded from the analysis. But there is no explanation of how this alcohol status was determined, and whether there was a threshold for alcohol consumption over the lifecourse. That is, are even occasional social drinkers excluded?\nThe NAFLD itself was diagnosed using ultrasonography. But what about the other health conditions included in the analysis? Was diabetes also doctor diagnosed, or self-reported. And 'other underlying diseases are mentioned. What are they and how were they diagnosed?\nIn summary we need to know what questions were included in the baseline questionnaire that led to these categorizations.\nResults The results are well presented overall. There is one point that was a little unclear. In the 4th paragraph the authors mention interaction of the NAFLD with periductal fibrosis. No mention was made of this condition in the Methods. I am unclear what the importance of this interaction is or why it was mentioned.\n\nDiscussion This section is also well set out and argued. The main point missing here is again who the cohort are, and whether the prevalence figures reported are applicable to the general population. I will state again here that not being nationally representative does not reduce the value of the findings. But it needs to be mentioned.\n\nLastly a minor point in the discussion. In the 'Age-gender interaction' section the authors state that '..NAFLD becomes comparable between men and women at the age of 60 years.' This does not appear to be true from the results. This point could maybe be explained more 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? 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": [
{
"c_id": "3111",
"date": "23 Oct 2017",
"name": "Ueamporn Summart",
"role": "Author Response",
"response": "Thanks for all of your help and suggestion for my paper. I promise to approve my paper as possible as I can and also remember all of your suggestions and comments to improve my new manuscripts. Best regards Ueamporn"
}
]
},
{
"id": "26831",
"date": "17 Oct 2017",
"name": "Nirun Intarut",
"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 study aims to investigate the difference of NAFLD among gender. The author described the methodology results, and discussion clearly, but there have some point that need to clarify.\n\nThe author mentioned in the Introduction part “Various studies have demonstrated that NAFLD is more prevalent in men, elderly populations, and post-menopausal women 14”. Please add more evidence in this sentence!\n\nMethods\nadd more details of adjusted prevalence estimation, why the author use this methods for prevalence estimation. (according to the Figure 3) the author described this is a population-based cross-sectional study that was represented to (Northeast of Thailand population). Do they try to use weighted analysis? This wording might not be a reason for the analysis “ignoring the effect of other factors for better handling the covariates in a more sophisticated statistical modeling”\n\nResults\nTable 1, No definition of “Underlying disease” and “Other underlying disease”, so please clarify these terms. Table 2, Please specify “age presence of DM” in the methods For odds ratio or adjusted prevalence estimation, no reason was mentioned “why do the author select only age, DM or other underlying to be in the model” Figure 2 and 3, please check “Other disease”, this is might be “Other underlying disease”. Figure 3, The graph presented sounds, but it might be unclear for interpretation. For example, after accounted for the possible confounder, the odds of DM was increased risk to NAFLD 4 times of crude OR.\nMoreover, for the other disease, the adjusted OR was lower than crude OR about 3 times. This is means that if people having some other diseases, they tended to having lower NAFLD. But we don’t know what a disease they have!\n\nDiscussion\nPlease add more details for others age group. Based on the Figures 2 and 3, the author mentioned only age 56-60. However, among 61-65 was greater risk to NAFLD as well.\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/6-1630
|
https://f1000research.com/articles/6-1855/v1
|
18 Oct 17
|
{
"type": "Research Note",
"title": "Induced spawning of siban fish Cyclocheilichthys apogon using Ovaprim",
"authors": [
"Nuraini Nuraini",
"Afrizal Tanjung",
"Trisla Warningsih",
"Zainal A. Muchlisin",
"Afrizal Tanjung",
"Trisla Warningsih",
"Zainal A. Muchlisin"
],
"abstract": "Background: The objective of the present study was to examine the effect of Ovaprim dosage on the latency period, relative number of ovulated eggs, fertilization, hatching, and survival rates of the siban fish, Cyclocheilichthys apogon. Methods: Three dosages of Ovaprim were tested in this study, namely 0.3 ml kg-1 of broodfish body weight, 0.5 ml kg-1 body weight, and 0.7 ml kg-1 body weight, plus control (without Ovaprim). Results: The results showed that the best latency period, relative number of ovulated eggs, fertilization, hatching, and survival rates were obtained at a dosage of 0.7 ml kg-1 body weight. Conclusions: The best dosage of Ovaprim for siban fish from the dosages tested, was determined to be 0.7 ml kg-1 body weight.",
"keywords": [
"fish breeding",
"freshwater fish",
"pituitary",
"(sGnRH)",
"domperidone",
"hatchery"
],
"content": "Introduction\n\nSiban fish Cyclocheilichthys apogon is a commercial freshwater fish in Indonesia. This species is distributed in South East Asia regions such as Sumatra, Borneo, Malaysia, and Mekong, Thailand1. Siban fish is one of the main targets of local fishermen in Riau Province, Indonesia, resulting in decreasing wild populations over the last decade (Personal communication with local fishermen of the Kampar River). Therefore, the cultivation of siban fish needs to develop in relation to meeting the market demand, without disturbing the wild population.\n\nNurhusniah2 and Sari3 have studied the reproductive biology of the siban fish, but no other aspect has been studied yet. Nonetheless, the culturing of the siban fish has been initiated in Riau Province, Indonesia; the larvae were collected from the wild at low quality and quantity, with high seasonal dependence4, resulting in low production. Therefore, the breeding technology of siban fish is crucially needed to overcome these problems and to support the aquaculture business of the local people.\n\nInduced spawning is one of the common methods to stimulate ovulation of the fish in the hatchery. With this method, hormones are playing an important role5. Several natural and artificial hormones have been used to induce breeding of fish, while Ovaprim is one of the most popular and effective solutions to stimulate the maturation of male and female broodfish. Ovaprim contains combinations of salmon gonadotropin-releasing hormone (sGnRH) and domperidone. These hormones have been applied successfully to induce spawning of seurukan fish Osteochilus vittatus6, selais Ompok hypophthalmus7,8, common carp Cyprinus carpio9, mali-mali Labiobarbus festivus10, and lelan fish Osteochilus pleurotaenia11.\n\nOvaprim has several advantages, for example, it is cheap, has many practical uses, and it is easy to find in the local market in Indonesia. However, Ovaprim has never been used to induce spawning of siban fish. Hence, the objective of the present study was to determine an effective dosage of Ovaprim for siban female broodstock.\n\n\nMethods\n\nThe experiments were carried out within the ethical guidelines in animal research developed by NC3Rs. The study was conducted between April and June 2016, at the Fish Breeding Laboratory, Faculty of Fisheries and Marine Sciences, University of Riau, Pekanbaru, Indonesia. A total of 12 male and 12 female broodfish of the siban fish Cyclocheilichthys apogon species were collected from the Kampar River, Riau Province. The broodfish ranged between 10 and 15 cm in total length, and 18.42 and 31.15g in total weight. The fish were acclimatized for 24 hours prior to inducing with Ovaprim.\n\nThe experiment was carried out in compliance with the ethical guidelines provided by the Research Institution of Riau University (SOP/02/PL/LPPM/2016). Three dosages of Ovaprim (Syndel, Canada) were tested in this study: for females, 0.3 ml kg-1 body weight (BW), 0.5 ml kg-1BW, and 0.7 ml kg-1BW; for males, 0.15 ml kg-1BW. The broodfish in control groups were injected with physiological solution (0.9% NaCL) at the dosage of 0.25 ml kg-1BW.\n\nThe female broodfish were injected with their respective dosage two times; the first injection was at 8.00 PM with half of the tested dosage, and the second injection was conducted 6 hours after the first injection (at 2.00 AM), with the remainder of the tested dosage. The female broodfish was ovulated 6 hours after the second injection (at 8.00 AM). The eggs were collected by gentle finger pressure to the abdomen. The collected eggs were put in a plastic jar and kept in an ice box at 4°C6.\n\nThe males were injected with a single dose of 0.25 ml kg-1 BW at 8.00 PM. The male siban fish was sacrificed with MS-222 (Merck), prepared by dissolving 4g of MS-222 in 5L tap water12. The testes were removed and washed with physiological solution and then perforated and chopped with scissors. The semen was gently squeezed out and put in a tube, and kept in an icebox (4°C). The semen was mixed with physiological solution (0.9% NaCl) at dilution ratio 1: 100 (v/v).\n\nA total of 1 ml eggs and 1 ml of diluted sperm were mixed homogenously in a plastic basin (Calista, Volume 500 mL) and with approximately 1 ml of fertilization solution (contains 4 g urea and 3 g NaCl L-1) developed by The Laboratory of Fish Breeding, Faculty of Fisheries and Marine Sciences, Universitas Riau. The resulting mixture was left in contact for 5 minutes. The incubation basin was installed with 24-hour portable aerator and LED lamp.\n\nA total of 100 eggs were taken randomly and then incubated in a plastic basin (Calista, volume: 2L), with three replicates at a water temperature of 25–27°C in a water heater. Successful fertilization was observed 8 hours after incubation. Unfertilized eggs were identified by their opacity; the unfertilized eggs were removed from the jar, while hatching rate was monitored at two-hour intervals13. The larvae were fed on Tubifex sp. ad libitum on day 5 after being hatched and reared in the same jar for 40 days.\n\nThe latency period, the relative number of ovulated eggs, egg diameter, fertilization rate, hatching rate, and survival rate of larvae on day 40 after hatching were measured. The latency period was determined by calculating the time between the second injection and ovulation. The relative number of ovulated eggs was calculated by dividing the total number of released eggs after Ovaprim injection by the total body weight of the female broodfish. Fertilization, hatching, and survival rates were calculated based on Muchlisin et al6,14 and Adami et al15. All data were subjected to One-Way Analysis of Variance (ANOVA), followed by the Duncan multiple-range test.\n\n\nResults\n\nThe ANOVA test revealed that different Ovaprim dosages had a significant effect on latency period, relative number of ovulated eggs, fertilization, hatching, and survival rates (p<0.05). The result showed that the latency period was faster at a dosage of 7 ml kg-1BW; this value was significantly different from other dosages. A higher relative number of ovulated eggs and fertilization, hatching, and survival rates were also recorded at the Ovaprim dosage 7 ml kg-1 BW; these values are significantly different from other dosages (Table 1). In general, the relative number of ovulated eggs, fertilization, hatching, and survival rates were increased with increasing Ovaprim dosage. However, there were no significant differences between the values seen at 3 ml kg-1 BW and 5 ml kg-1 BW dosage.\n\n\nDiscussion\n\nThe results indicate a decreased latency period at increased Ovaprim dosage. On the contrary, the relative number of ovulated eggs, fertilization, hatching, and survival rates were increased at increased dosage. The best results for all parameters were recorded at the Ovaprim dosage of 7 ml kg-1 body weight. Therefore, the higher dosage of Ovaprim (7 ml kg-1 BW) was an effective dosage in inducing spawning of the siban fish compared to the lower dosage (3 ml kg-1 and 5 ml kg-1). This is probably because the combination of sGnRH and domperidon in Ovaprim solution at higher doses of 7 ml kg-1 BW stimulated gonadotropin (GTH II or LH) secretion in the pituitary gland of the broodfish to a greater extent, and then induced gonad maturation and ovulation. The higher levels of gonadotropin will induce ovulation faster16. According to Ithisom9, the hormone works optimally at a certain dose; and changing the dose will reduce effectiveness. Therefore, determining the optimum dose is crucial.\n\nThe influence of sGnRH and domperidon at the higher dosage most likely inhibited secretion of dopamine and stimulated the pituitary gland to secrete GTH. The GTH in this case, GTH II, would stimulate the theca cells to secrete hormone 17-alpha hydroxyprogesterone, which would then be converted into maturation-inducing steroid by the enzyme 20-beta-dihydroxy steroids, stimulating follicles to burst as the ooctes hydrate17,18.\n\nThe results showed that the high fertilization rate was obtained at doses of 0.7 ml kg-1 BW; this dosage probably gave the optimum effect of sGnRH and domperidone to increase the quantity and quality of eggs. This is indicated by the higher number of ovulated eggs as recorded at this dosage. According to Nuraini et al.19 the optimum dosage of stimulating hormone results in better ovulation and improves egg quality for the selais fish, Ompok hypophthalmus. Moreover, Woynarovich and Horvath20 stated that the fertilization rate is strongly influenced by the quality of eggs and spermatozoa. Prostaglandin plays an important role in inducing ovulation and significantly influences fertilization and hatching rates. This is because prostaglandin contains arachidonic acid derived from essential fatty acids that determine the egg quality21. In addition, the quality of eggs is influenced by several factors including the hormone levels22, nutrition23, genetics24 and the environment25. Bromage et al.26 stated that the quality of eggs is reflected in higher fertilization and hatching rates.\n\nBesides being influenced by hormonal factors, the hatching rate is also influenced by the temperature of the incubation media, dissolved oxygen, pH, and light intensity20. In general, the survival rate of siban larvae was categorized as being at a good level. According to Alikunhi et al.27 there are three levels of survival rate for the larvae: a good level when the survival is higher than 50%, moderate when between 30 and 50%, and low when the survival is lower than 30%. However, the survival of the larvae in this study was lower compared to seurukan fish Osteochilus vittatus and river catfish Mystus nigricep, which were injected with Ovaprim 0.5 ml kg-1 BW, resulting in a survival rate of between 80.66 and 90%6,28,29. It is assumed that the siban fish requires higher dosages of Ovaprim to induce gonad maturation as recorded in this study.\n\n\nConclusions\n\nIt is concluded that different doses of Ovaprim had a significant effect on the latency period, the relative number of ovulated eggs, fertilization, hatching, and survival rates of siban fish. The best Ovaprim dosage, from the ones tested, was 0.7 ml kg-1 BW.\n\n\nData availability\n\nDataset 1: Raw data collected for the latency period, relative numbers of ovulated eggs, fertilization, hatching and survival rates of the siban fish Cyclocheilichthys apogon. DOI, 10.5256/f1000research.12885.d18082530.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by Universitas Riau through thr PUPT scheme 2016, Grant number: 023/UN.5.1.3/PP/2016.\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 the rector of Universitas Riau for providing the reseach grant to support this study.\n\n\nReferences\n\nKottelat M, Whitten T, Kartikasari SN, et al.: Ikan air tawar Indonesia bagian barat dan Sulawesi. Periplus, Singapore. 1993. Reference Source\n\nNurhusniah: Biologi reproduksi ikan siban (Cyclocheilichthys apogon C.V) di Waduk PLTA Koto Panjang Kabupaten Kampar Provinsi Riau. Thesis, Faculty of Fisheries and Marine Sciences. Universitas Riau, Pekanbaru. 2007.\n\nSari IW: Biologi reproduksi ikan keperas (Cyclocheilicthys apogon) di Sungai Musi, Sumatera Selatan. Thesis, Faculty of Fisheries and Marine Sciences. Institut Pertanian Bogor, Bogor. 2007. Reference Source\n\nBrotowidjoyo, Mukayat D, Tribawono J, et al.: Pengantar lingkungan perairan dan budidaya air. Liberti Press, Yogyakarta. 1999. Reference Source\n\nElakkanai P, Francis T, Ahilan B, et al.: Role of GnRH, HCG and Kisspeptin on reproduction of fishes. Indian J Sci Technol. 2015; 8(17): 1–10. Publisher Full Text\n\nMuchlisin ZA, Arfandi G, Adlim M, et al.: Induced spawning of seurukan fish, Osteochilusvittatus (Pisces: Cyprinidae) using Ovaprim, oxytocin and chicken pituitary gland extracts. AACL Bioflux. 2014; 7(5): 412–418. Reference Source\n\nNuraini B, Hasan, Nasution S: Pengaruh penyuntikan Ovaprim dengan dosis yang berbeda terhadap ovulasi dan penetasan telur ikan selais danau (Kryptopterus limpok). Research Report of Universitas Riau, Pekanbaru. 2004.\n\nAryani N, Nuraini, Alawi H: Pengaruh sGnRH + Domperidon dengan dosis berbeda terhadap pembuahan dan penetasan telur ikan selais (Ompok rhadinurus). Research Center of Universitas Riau, Pekanbaru. 2012.\n\nItishom R: Pengaruh sGnRH + Domperidon dengan dosis pemberian yang berbeda terhadap ovulasi ikan mas (Cyprinuscarpio L) train Punten. Berkala Ilmiah Perikanan. 2008; 3(1): 9–16. Reference Source\n\nManik A: Pengaruh dosis Ovaprim berbeda terhadap pemijahan ikan mali-mali (Labeobarbus festivus). Thesis, Faculty of Fisheries and Marine Sciences, Universitas Riau, Pekanbaru. 2016.\n\nBakkara S: Pengaruh dosis ovaprim berbeda terhadap pemijajan ikan lelan (Osteochilus pleurotaenia).Thesis, Faculty of Fisheries and Marine Sciences, Universitas Riau , Pekanbaru. 2016. Reference Source\n\nMuchlisin ZA, Hashim R, Chong AS: Preliminary study on the cryopreservation of tropical bagrid catfish (Mystus nemurus) spermatozoa; the effect of extender and cryoprotectant on the motility after short-term storage. Theriogenology. 2004; 62(1–2): 25–34. PubMed Abstract | Publisher Full Text\n\nMuchlisin ZA, Nadiah WN, Nadiya N, et al.: Exploration of natural cryoprotectants for cryopreservation of African catfish, Clarias gariepinus, Burchell 1822 (Pisces: Clariidae) spermatozoa. Czech J Anim Sci. 2015; 60(1): 10–15. Publisher Full Text\n\nMuchlisin ZA, Nadiya N, Nadiah WN, et al.: Preliminary study on the natural extenders for artificial breeding of African catfish Clarias gariepinus (Burchell, 1822). AACL Bioflux. 2010; 3(2): 119–124. Reference Source\n\nAdami Y, Fadli N, Nurfadillah N, et al.: A preliminary observation on the effect of sperm extenders on the fertilization and hatching rates of seurukan fish (Osteochilus vittatus) eggs. AACL Bioflux. 2016; 9(2): 300–304. Reference Source\n\nLam TJ: Induce spawning in fish. In: LelanadCS, Lion IC (Eds). Reproduction and culture of milkfish. The Oceanic Institute, Hawai. 1985. Reference Source\n\nGoetz FW, Garczynski M: The ovarian regulation of ovulation in teleost fish. Fish Physiol Biochem. 1997; 17(1–6): 33–38. Publisher Full Text\n\nPatino R, Sullivan CV: Ovarian follicle growth, maturation, and ovulation in teleost fish. Fish Physiol Biochem. 2002; 26(1): 57–70. Publisher Full Text\n\nNuraini, Alawi H, Nurasiah, et al.: Pengaruh SGNRH + Domperidon dengan dosis yang berbeda terhadap pembuahan dan penetasan telur ikan selais (Ompokrhadinurus. Ng). Berkala Perikanan Terubuk. 2013; 41(2): 1–8. Reference Source\n\nWoynarovich E, Horvath L: The artificial propagation of warm-water finfishes. A manual for extension. FAO, Fisheris Technical Paper No. 201/FIR/T201, Rome. 1980. Reference Source\n\nGoodman S: Vitamin C, the master nutrient. Kients Publishing Inc., England. 1991. Reference Source\n\nBrooks S, Tyler CR, Sumpter JP: Egg quality in fish: what makes a good egg? Rev Fish Biol Fish. 1997; 7(4): 387–416. Publisher Full Text\n\nAbidin MZ, Hashim R, Chien AC: Influence of dietary protein levels on growth and egg quality in broodstock female bagrid catfish (Mystus nemurus Cuv. & Val.). Aquaculture Research. 2006; 37(4): 416–418. Publisher Full Text\n\nMuchlisin ZA: Factors affect gonadal development and eegs quality of female broodfish. Biologi. 2005; 4(6): 411–427. Reference Source\n\nLiley NR, Stacey NE: Hormones, Pheromones, and Reproductive Behavior in Fish. In: Hoar WS, Randall DJ, Donaldson EM (eds). Fish Physiology. Academic Press Inc., London. 1983; IX(Part B): 1–63. Publisher Full Text\n\nBromage NR: Propagation and stock improvement. In: Bromage NR, Stephard CJ(eds), Intensive fish farming. Block Well Scientific Publication Inc., USA. 1992. Reference Source\n\nAlikunhi KH, Chaudhary H, Ramchandran V: On the mortality of carp fry in nursery ponds and the role of plankton in their survival and growth*. Indian Journal of Fisheries. 1955; 2: 257–313. Reference Source\n\nSari EK: Pengaruh kombinasi pakan alami yang berbeda terhadap pertumbuhan dan kelulushidupan larva ikan pawas (Osteochilus hasselti CV). Thesis, Faculty of Fisheries and Marine Sciences, Universitas Riau, Pekanbaru. 2016. Reference Source\n\nYusuf M: Pemberian pakan alami yang berbeda terhadap pertumbuhan dan kelulushidupan larva ikan ingir-ingir (Mystusnigriceps). Thesis, Faculty of Fisheries and Marine Sciences, Universitas Riau, Pekanbaru. 2016.\n\nNuraini N, Tanjung A, Warningsih T, et al.: Dataset 1 in: Induced spawning of siban fish Cyclocheilichthys apogon using Ovaprim. F1000Research. 2017. Data Source"
}
|
[
{
"id": "27095",
"date": "30 Oct 2017",
"name": "Javad Ghasemzadeh",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is the outcome of a well designed and executed research plan. It has clearly reviewed the current literature and background of the research, and the objectives of their research, and clear explanation of materials and methods which can be used and replicated in similar research work on different fishes. The statistical analysis applied for this type of research is simple, logical and interpreted very well. The results are given based on the clear and measured outcomes of the methods applied in the experiment which is applicable and reproducible in similar research work on the induced and artificial propagation of fishes (especially on freshwater fishes). The conclusions are clearly based on the measured and practically approved results, with no particular limitations being addressed. This article is good enough to be indexed with its present condition, and I did not find any problem in the English language used,.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "27096",
"date": "14 Nov 2017",
"name": "Suparno Pranoto",
"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\nTitle: The title is good, brief and informative, you need to add the fish and the author name, also the year. Abstract: The conclusion still states the result of the research Introduction: In the introduction, give the information which is relevant with the Induced spawning of siban fish Cyclocheilichthys apogon using Ovaprim. Suggestion: The introduction should have information of previous research about siban fish reproduction with specifically,the latest reference and the novelty about the research. Methods: Please add the research design. Method of research must be added with the clear reference. Results: Please describe the table 1 and emphasize the findings. Findings on the latency period, relative number of ovulated eggs, fertilization, hatching, and survival rates of the siban fish. Discussion: Discussion must be based on the emphasis on the findings, and relation of the findings with the basic concepts. Is there any contradiction and suitability with other peoples research which is related with using ovaprim to siban fish? Conclusion: Please write the conclusion briefly and clearly to answer the purpose of the research. Conclusion is not the result of the research. References: Most of the references are published before 2012. References should be taken from the last 5 years.\n\nAn annotated Word version of the of the article is available here:\nhttps://f1000researchdata.s3.amazonaws.com/linked/184593.Surpano_Edt-Siban_Fish_%28Nuraini%29.docx\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": []
},
{
"id": "27098",
"date": "14 Nov 2017",
"name": "Agung Damar Syakti",
"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 studied the effect of Ovaprim dosage on the latency period, relative number of ovulated eggs, fertilization, hatching, and survival rates of the siban fish, Cyclocheilichthys apogon. Scientifically, overall quality of the manuscript is interesting and good. This work provides insight into the best latency period, relative number of ovulated eggs, fertilization, hatching, and survival rates of the optimum dosage of Ovaprim. Thus, from my point of view, this paper can provide some important scientific data and values. I can recommend the acceptance and indexing of this manuscript in F1000Research. However, there is some specific comment and queries to be considered and it is necessary to pay close attention to proper sentence structure and grammar in some sentences of the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1855
|
https://f1000research.com/articles/6-1850/v1
|
17 Oct 17
|
{
"type": "Review",
"title": "Zika virus reservoirs: Implications for transmission, future outbreaks, drug and vaccine development",
"authors": [
"Raj Kalkeri",
"Krishna K. Murthy",
"Krishna K. Murthy"
],
"abstract": "Zika virus (ZIKV) was recently declared as a ‘Global Health Emergency’ by the World Health Organization. Various tissue reservoirs of ZIKV in infected humans and animals models have been observed, the implications of which are not known. Compared to other Flaviviruses, sexual transmission and persistence in the genitourinary tract seem to be unique to ZIKV. ZIKV persistence and shedding in bodily secretions (e.g. saliva, semen) is a concern for potential disease spread and could pose challenges in diagnosis, regulatory guidelines and drug/vaccine development. Murine and non-human primate models could be useful to study the role of tissue reservoirs in the development of prophylactic or therapeutic strategies. There is a need for meta-analysis of the ZIKV infection and virus shedding data from infected patients and ZIKV animal models, and additional research is needed to fully comprehend the long term implications of tissue reservoirs on ZIKV disease pathogenesis and biology.",
"keywords": [
"Zika",
"Persistence",
"transmission",
"tissue reservoirs",
"drug and vaccine development"
],
"content": "Introduction\n\nAfter the recent outbreak of Zika virus (ZIKV) infection in Brazil and other countries, multiple studies have been published regarding various tissue reservoirs, including the eyes, nervous system, genitourinary tracts and placenta (Table 1). A recent report of ZIKV transmission in renal and liver transplant patients with abnormal graft functions1 poses a new challenge in screening organs intended for transplants and monitoring transplanted patients. Considering such new concerns for ZIKV, we have reviewed the existing literature about ZIKV tissue reservoirs in this article and provide our analysis of the potential impact of ZIKV reservoirs on transmission and challenges in development of medical countermeasures against ZIKV.\n\n\nZIKV tissue reservoirs\n\nThe role of immune-privileged sites (e.g. eyes, testes, fetal brain, and placenta) in harboring ZIKV has been previously reported2, contributing to the viral persistence in these tissues. Successful infection and amplification of ZIKV in vitro in primary cell cultures derived from the placenta3, kidney4, nervous tissue5, and amplification found in the brain of severely damaged fetus6 suggests the potential ability of these tissue reservoirs to support both infection and amplification of ZIKV. Furthermore, that the reservoir-derived virus is infectious has been confirmed by sexual- and or transplant-related transmission of infection from non-viremic partners or donors7. These studies raise the possibility that individuals with ZIKV reservoirs could serve as a potential source of endemic and recurrent outbreaks of infection. In addition, the prevalence of such reservoirs in immune compromised patients8 is a major concern as the disease could spread to other organs rapidly and run a more aggressive course, in the absence of immune defenses of the body.\n\n\nZIKV in body secretions\n\nInterestingly, besides tissue reservoirs, few reports have been successful in the detection of ZIKV in the bodily secretions of infected patients, such as saliva9,10 urine9,11, semen and vaginal secretions12–14, breast milk15 and conjunctival fluid16. Semen and vaginal secretions are already reported to be involved in sexual transmission of ZIKV7 in humans. Despite a recent report about the persistence of ZIKV in the semen for more than 6 months17, the duration of the persistence in other bodily secretions (urine, saliva etc) is unclear at this time. Careful evaluation of the duration of the ZIKV shedding and its role in non-sexual transmission needs to be conducted.\n\n\nIn vivo models for ZIKV tissue reservoirs\n\nAnimal models for ZIKV have further confirmed some of these tissue reservoirs18–25. Tang et al. demonstrated the persistence of ZIKV in the vaginal washes as late as 10 days post-infection in lethal and sub-lethal mouse models after intravaginal inoculation of ZIKV26. Ifnar1(-/-) mice showed sustained high viral loads in the brain, spinal cord and testes, confirming these tissue reservoirs21. AG129 mice showed severe pathologies in the muscle and brain27, confirming the presence of these reservoirs in mouse models.\n\nZIKV was shown to persist in the cerebrospinal fluid (CSF) and lymph nodes (LN) of infected rhesus monkeys weeks after virus clearance from the peripheral blood, urine and mucosal secretions28. Similar observation of longer persistence of ZIKV in the hemolymphatic tissue of non-pregnant rhesus macaques infected with 2015 Brazilian ZIKV isolate was observed by Coffey and colleagues29. ZIKV was also observed in the lacrimal fluid and parotid glands30 of rhesus macaques after subcutaneous infection. ZIKV RNA was detectable in saliva, semen, urine of both rhesus and cynomolgus macaques20. ZIKV infection in the pregnant rhesus macaques showed longer duration of persistence (57 days) compared to normal macaques (21 days)31, suggesting differential persistence depending upon the pregnancy status.\n\nA recent study showed the importance of autophagy on the vertical transmission of ZIKV in pregnant mice32. The authors demonstrated the activity of an autophagy inhibitor approved for use in pregnant women (hydroxychloroquine) in attenuating the placental and fetal ZIKV infection and ameliorated adverse placental and fetal outcomes. Thus animal models could prove useful to study the importance of tissue reservoirs in ZIKV biology and pathogenesis, as well as evaluation of experimental drugs and vaccines.\n\n\nRelationship to other Flaviviruses\n\nA review of the literature suggested similar tissue reservoirs observed for other Flaviviruses (Table 2). Tick-borne encephalitis, West Nile Virus and Japanese Encephalitis Virus have varying durations of persistence33. However, unlike other Flaviviruses, sexual transmission is unique to ZIKV infections7. This is not surprising due to ZIKV’s persistence in the genitourinary tract34. The presence of ZIKV in semen is without apparent disease symptoms35, and can be for a period of more than 6 months17. This indicates a need for inclusion of ZIKV in the screening panel for semen donors in addition to the current viruses in the panel (i.e. hepatitis B, C and HIV, as per the guidelines of American Society for Reproductive Medicine and Food and Drug Administration).\n\nAnother potential concern of ZIKV tissue reservoirs (with low levels of ZIKV) is triggering of sub-neutralizing antibodies leading to antibody-dependent enhancement (ADE) of subsequent Flavivirus infections and potential interplay with other mosquito born Flaviviruses. Recently, ADE of ZIKV by sub-neutralizing Dengue virus antibodies has been reported36,37. Considering the similar target organ affinity (e.g. nervous tissue) observed for other Flaviviruses (e.g. West Nile Virus, Dengue Virus)33, such a concern cannot be ruled out.\n\n\nChallenges for the development of countermeasures against ZIKV\n\nThe presence of tissue reservoirs and detection of the virus for varying periods of duration, after initial infection, in the absence of the apparent symptoms, poses new challenges for screening of populations. In 2013 during the French Polynesian Zika outbreak, differential virus positivity was observed between the serum, saliva and urine samples collected from 182 patients. Saliva but not serum, from 35 patients was Zika positive. In contrast, serum but not saliva from 16 other patients was Zika positive37. Considering such variability in different biological samples from patients, paired serum and urine samples are considered to be of primary diagnostic importance (CDC guidance for US laboratories testing for Zika virus infections).\n\nInterestingly, in animal studies, low levels of Zika were observed in the body secretions compared to the serum38. Such low levels of ZIKV in the body secretions near the detection limits and invasive processes needed to collect samples (eg. CSF, nervous tissue), suggest currently available detection assays may or may not be suitable to identify persistent ZIKV infections and quantify the tissue reservoirs. As per the CDC information, there are currently no FDA-authorized assays for Zika virus testing of tissue specimens, including fetal and placental tissue, suggesting the need for additional efforts towards such diagnostic assays.\n\nFDA has issued Emergency Use authorization of several diagnostic tools for Zika virus. These include both antibody based and nucleic acid testing (NAT) kits, which are currently distributed to qualified laboratories by the CDC (Table 3).\n\nNAT: Nucleic Acid based detection Test\n\nAntibody based ZIKV detection methods are suggestive of past or recent exposure to Zika and may not necessarily indicate the presence of the virus in the body. In addition, antibody based methods are suitable only for serum, whereas NAT might be suitable for all types of samples including serum and bodily fluids. Due to the direct detection of the virus, NAT-based detection kits might be well suited for the identification of persistent infections. A rapid and high-throughput method for detecting ZIKV RNA using transcription-mediated amplification technology, based Aptima Zika virus assay (Hologic, Marlborough, MA) is described to detect 11.5 to 17.5 genome copy equivalents in serum and urine39. A recent report suggests the utility of isothermal amplification based point-of-care diagnostic technology for rapid detection of ZIKV in the saliva40. The performance of such kits in the clinic remains to be seen. Additional research might shed some light on the acceptable standard for such an effort.\n\nZIKV tissue reservoirs bring in additional challenges for drug or vaccine development. According to the WHO vaccine pipeline tracker, there are many ZIKV vaccine candidates currently being tested. These include four DNA vaccines (one from GeneOne Life Sciences and three from NIAID), four inactivated whole virus vaccines (3 from NIAID and 1 from BIDMC), one peptide (NIH), one mRNA (Moderna), and one recombinant MV-Zika-101 (Themis Bioscience). Two of these candidates- VRC-ZKADNA090-00-VP (NIAID) and mRNA 1325 (Moderna) are currently recruiting patients for phase 2 trials.\n\nTo be effective, potential treatments for ZIKV might have to achieve appropriate tissue concentrations in these reservoirs. This might be limited by sufficient bioavailability of therapeutics in the tissue reservoirs and penetration of the blood brain barrier (BBB). Similar concerns for placental barrier and challenging anatomical locations (e.g. the eyes, kidney, and testes) create additional hurdles for elimination of viral reservoirs. Prophylactic vaccine strategies should be capable of not only preventing infection, but also prevent the establishment of viral reservoirs. In addition, potential vaccine candidates should be capable of inducing a potent neutralizing antibody response to mitigate the possibility of ADE mentioned above.\n\n\nConcluding remarks\n\nZIKV animal models (mice, monkeys) could serve as valuable tools in evaluating the efficacy of therapeutic and prophylactic strategies in eliminating ZIKV tissue reservoirs and prevent shedding of the virus. Additional questions such as relative difference between ZIKV strains to persist in the tissue reservoirs, relative size of different tissue reservoirs (brain, eyes, testes), role of host factors in persistence (tissue reservoir formation), kinetics of virus persistence, and phenotypic and genotypic characterization of the virus in tissue reservoirs, will require additional studies. Formation of ZIKV task force by the Global Virus Networks41 is a welcome move that might be helpful in addressing some of these issues. There is a need for a publically available database (suitable for meta-analysis) to compare long-term kinetics of ZIKV infections and persistence in both animal models and clinical settings. Together with additional research, this might reveal the impact of ZIKV tissue reservoirs on future outbreaks, and drug and vaccine development.",
"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\nNogueira ML, Estofolete CF, Terzian AC, et al.: Zika Virus Infection and Solid Organ Transplantation: A New Challenge. Am J Transplant. 2017; 17(3): 791–795. PubMed Abstract | Publisher Full Text\n\nMa W, Li S, Ma S, et al.: Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice. Cell. 2016; 167(6): 1511–1524.e10. PubMed Abstract | Publisher Full Text\n\nAagaard KM, Lahon A, Suter MA, et al.: Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication. Sci Rep. 2017; 7: 41389. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlcendor DJ: Zika Virus Infection of the Human Glomerular Cells: Implications for Viral Reservoirs and Renal Pathogenesis. J Infect Dis. 2017; 216(2): 162–171. PubMed Abstract | Publisher Full Text\n\nMladinich MC, Schwedes J, Mackow ER: Zika Virus Persistently Infects and Is Basolaterally Released from Primary Human Brain Microvascular Endothelial Cells. mBio. 2017; 8(4): pii: e00952-17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBhatnagar J, Rabeneck DB, Martines RB, et al.: Zika Virus RNA Replication and Persistence in Brain and Placental Tissue. Emerg Infect Dis. 2017; 23(3): 405–414. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFoy BD, Kobylinski KC, Chilson Foy JL, et al.: Probable non-vector-borne transmission of Zika virus, Colorado, USA. Emerg Infect Dis. 2011; 17(5): 880–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalvet GA, Filippis AM, Mendonça MC, et al.: First detection of autochthonous Zika virus transmission in a HIV-infected patient in Rio de Janeiro, Brazil. J Clin Virol. 2016; 74: 1–3. PubMed Abstract | Publisher Full Text\n\nBarzon L, Pacenti M, Franchin E, et al.: Infection dynamics in a traveller with persistent shedding of Zika virus RNA in semen for six months after returning from Haiti to Italy, January 2016. Euro Surveill. 2016; 21(32): 30316. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiuzzi G, Puro V, Lanini S, et al.: Zika virus and microcephaly: is the correlation causal or coincidental? New Microbiol. 2016; 39(2): 83–5. PubMed Abstract\n\nRossini G, Gaibani P, Vocale C, et al.: Comparison of Zika virus (ZIKV) RNA detection in plasma, whole blood and urine - Case series of travel-associated ZIKV infection imported to Italy, 2016. J Infect. 2017; 75(3): 242–245. PubMed Abstract | Publisher Full Text\n\nFroeschl G, Huber K, von Sonnenburg F, et al.: Long-term kinetics of Zika virus RNA and antibodies in body fluids of a vasectomized traveller returning from Martinique: a case report. BMC Infect Dis. 2017; 17(1): 55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNicastri E, Castilletti C, Liuzzi G, et al.: Persistent detection of Zika virus RNA in semen for six months after symptom onset in a traveller returning from Haiti to Italy, February 2016. Euro Surveill. 2016; 21(32): 30314. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNicastri E, Castilletti C, Balestra P, et al.: Zika Virus Infection in the Central Nervous System and Female Genital Tract. Emerg Infect Dis. 2016; 22(12): 2228–2230. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSotelo JR, Sotelo AB, Sotelo FJB, et al.: Persistence of Zika Virus in Breast Milk after Infection in Late Stage of Pregnancy. Emerg Infect Dis. 2017; 23(5): 856–857. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSun J, Wu, Zhong H, et al.: Presence of Zika Virus in Conjunctival Fluid. JAMA Ophthalmol. 2016; 134(11): 1330–1332. PubMed Abstract | Publisher Full Text\n\nAtkinson B, Thorburn F, Petridou C, et al.: Presence and Persistence of Zika Virus RNA in Semen, United Kingdom, 2016. Emerg Infect Dis. 2017; 23(4): 611–615. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGovero J, Esakky P, Scheaffer SM, et al.: Zika virus infection damages the testes in mice. Nature. 2016; 540(7633): 438–442. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdams Waldorf KM, Stencel-Baerenwald JE, Kapur RP, et al.: Fetal brain lesions after subcutaneous inoculation of Zika virus in a pregnant nonhuman primate. Nat Med. 2016; 22(11): 1256–1259. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOsuna CE, Lim SY, Deleage C, et al.: Zika viral dynamics and shedding in rhesus and cynomolgus macaques. Nat Med. 2016; 22(12): 1448–1455. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLazear HM, Govero J, Smith AM, et al.: A Mouse Model of Zika Virus Pathogenesis. Cell Host Microbe. 2016; 19(5): 720–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiner JJ, Cao B, Govero J, et al.: Zika Virus Infection during Pregnancy in Mice Causes Placental Damage and Fetal Demise. Cell. 2016; 165(5): 1081–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUraki R, Hwang J, Jurado KA, et al.: Zika virus causes testicular atrophy. Sci Adv. 2017; 3(2): e1602899. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYockey LJ, Varela L, Rakib T, et al.: Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection. Cell. 2016; 166(5): 1247–1256.e4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiner JJ, Sene A, Richner JM, et al.: Zika Virus Infection in Mice Causes Panuveitis with Shedding of Virus in Tears. Cell Rep. 2016; 16(12): 3208–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTang WW, Young MP, Mamidi A, et al.: A Mouse Model of Zika Virus Sexual Transmission and Vaginal Viral Replication. Cell Rep. 2016; 17(12): 3091–3098. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAliota MT, Caine EA, Walker EC, et al.: Characterization of Lethal Zika Virus Infection in AG129 Mice. PLoS Negl Trop Dis. 2016; 10(4): e0004682. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAid M, Abbink P, Larocca RA, et al.: Zika Virus Persistence in the Central Nervous System and Lymph Nodes of Rhesus Monkeys. Cell. 2017; 169(4): 610–620.e14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCoffey LL, Pesavento PA, Keesler RI, et al.: Zika Virus Tissue and Blood Compartmentalization in Acute Infection of Rhesus Macaques. PLoS One. 2017; 12(1): e0171148. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi XF, Dong HL, Huang XY, et al.: Characterization of a 2016 Clinical Isolate of Zika Virus in Non-human Primates. EBioMedicine. 2016; 12: 170–177. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDudley DM, Aliota MT, Mohr EL, et al.: A rhesus macaque model of Asian-lineage Zika virus infection. Nat Commun. 2016; 7: 12204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCao B, Parnell LA, Diamond MS, et al.: Inhibition of autophagy limits vertical transmission of Zika virus in pregnant mice. J Exp Med. 2017; 214(8): 2303–2313. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMlera L, Melik W, Bloom ME: The role of viral persistence in flavivirus biology. Pathog Dis. 2014; 71(2): 137–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaz-Bailey G, Rosenberg ES, Doyle K, et al.: Persistence of Zika Virus in Body Fluids - Preliminary Report. N Engl J Med. 2017. PubMed Abstract | Publisher Full Text\n\nCardona Maya WD, Du Plessis SS, Velilla PA: Semen as virus reservoir? J Assist Reprod Genet. 2016; 33(9): 1255–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCastanha PMS, Nascimento EJM, Braga C, et al.: Dengue Virus-Specific Antibodies Enhance Brazilian Zika Virus Infection. J Infect Dis. 2017; 215(5): 781–785. PubMed Abstract | Publisher Full Text\n\nMusso D, Roche C, Nhan TX, et al.: Detection of Zika virus in saliva. J Clin Virol. 2015; 68: 53–5. PubMed Abstract | Publisher Full Text\n\nKoide F, Goebel S, Snyder B, et al.: Development of a Zika Virus Infection Model in Cynomolgus Macaques. Front Microbiol. 2016; 7: 2028. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRen P, Ortiz DA, Terzian ACB, et al.: Evaluation of Aptima Zika Virus Assay. J Clin Microbiol. 2017; 55(7): 2198–2203. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMauk MG, Song J, Bau HH, et al.: Point-of-Care Molecular Test for Zika Infection. Clin Lab Int. 2017; 41: 25–27. PubMed Abstract | Free Full Text\n\nAliota MT, Bassit L, Bradrick SS, et al.: Zika in the Americas, year 2: What have we learned? What gaps remain? A report from the Global Virus Network. Antiviral Res. 2017; 144: 223–246. PubMed Abstract | Publisher Full Text\n\nKodati S, Palmore TN, Spellman FA, et al.: Bilateral posterior uveitis associated with Zika virus infection. Lancet. 2017; 389(10064): 125–126. PubMed Abstract | Publisher Full Text\n\nCalvet G, Aguiar RS, Melo ASO, et al.: Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study. Lancet Infect Dis. 2016; 16(6): 653–60. PubMed Abstract | Publisher Full Text\n\nGritsun TS, Frolova TV, Zhankov AI, et al.: Characterization of a siberian virus isolated from a patient with progressive chronic tick-borne encephalitis. J Virol. 2003; 77(1): 25–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRavi V, Desai AS, Shenoy PK, et al.: Persistence of Japanese encephalitis virus in the human nervous system. J Med Virol. 1993; 40(4): 326–9. PubMed Abstract | Publisher Full Text\n\nSharma S, Mathur A, Prakash V, et al.: Japanese encephalitis virus latency in peripheral blood lymphocytes and recurrence of infection in children. Clin Exp Immunol. 1991; 85(1): 85–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMontgomery SP, Brown JA, Kuehnert M, et al.: Transfusion-associated transmission of West Nile virus, United States 2003 through 2005. Transfusion. 2006; 46(12): 2038–46. PubMed Abstract | Publisher Full Text\n\nPrince HE, Lapé-Nixon M, Yeh C, et al.: Persistence of antibodies to West Nile virus nonstructural protein 5. J Clin Virol. 2008; 43(1): 102–6. PubMed Abstract | Publisher Full Text\n\nPealer LN, Marfin AA, Petersen LR, et al.: Transmission of West Nile virus through blood transfusion in the United States in 2002. N Engl J Med. 2003; 349(13): 1236–45. PubMed Abstract | Publisher Full Text\n\nBusch MP, Kleinman SH, Tobler LH, et al.: Virus and antibody dynamics in acute west nile virus infection. J Infect Dis. 2008; 198(7): 984–93. PubMed Abstract | Publisher Full Text\n\nMurray K, Walker C, Herrington E, et al.: Persistent infection with West Nile virus years after initial infection. J Infect Dis. 2010; 201(1): 2–4. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "27070",
"date": "30 Oct 2017",
"name": "Sunil K Joshi",
"expertise": [
"Reviewer Expertise Cellular immunology",
"Dendritic cell manipulations in Cancer and Infectious 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\nThis report is very important in terms of ZIKA as reservoir. Authors have done excellent work in writing this unique review and will enhance our understanding of ZIKA transmission via reservoir infection. Recent reports indicating that detection of ZIKV in the bodily secretions of infected patients, such as saliva, urine, semen and vaginal secretions, breast milk and conjunctival fluid is an important public health concern, particularly reservoir ZIKV still can cause spread of the disease.\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": "28041",
"date": "27 Nov 2017",
"name": "Michael P Busch",
"expertise": [
"Reviewer Expertise Diagnostics and pthogenesis of viral and parasitic infections"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nKalkeri and Murthy offer a valuable brief review of tissue reservoirs and its implications for transmission and treatment of Zika infection. Since studies of persistence of Zika virus infection in tissue reservoirs are limited in humans and hence not well understood, insights from rhesus macaque and mouse models of Zika infection are important to characterize persistence and its relevance to transmission and pathogenesis, as well as addressing challenges in treatment and diagnosis.\nNot mentioned in the review is the finding of persistence in whole blood, and in particular RBCs, after clearance from plasma (several publications) and the potential risk for transmission through bone marrow, cord blood and peripheral blood stem cell transplants. Transfusion transmission has been documented as another mode of transmission.\nCo-circulation of related Flaviviruses in Zika endemic areas is a concern for ADE of subsequent antigenically similar viruses as mentioned in the review. However evidence for ADE in humans is lacking. ADE from previous Dengue virus exposure has been demonstrated in vitro and in mouse models, but not in rhesus macaques and is not confirmed by human epidemiological studies based on several recent publications. Conclusions about ADE should therefore be made with caution.\nBriefly mentioned are issues with serological diagnostics, which are challenging due to cross reactivity and anamnestic response in serial infections by similar viruses as well as Arboviral vaccination. There are limitations with many existing assays, however newer assays have been more successful in differentiating Zika from Dengue. Access to samples from ZIKV/DENV infected macaques could allow for advances in this important area.\nReference 37 (ZIKV in saliva) is incorrectly placed in the context of ADE second paragraph of page 3.\nSuggest adding references at the bottom.\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": []
}
] | 1
|
https://f1000research.com/articles/6-1850
|
https://f1000research.com/articles/6-612/v1
|
02 May 17
|
{
"type": "Software Tool Article",
"title": "PubRunner: A light-weight framework for updating text mining results",
"authors": [
"Kishore R. Anekalla",
"J.P. Courneya",
"Nicolas Fiorini",
"Jake Lever",
"Michael Muchow",
"Ben Busby",
"Kishore R. Anekalla",
"J.P. Courneya",
"Nicolas Fiorini",
"Jake Lever",
"Michael Muchow"
],
"abstract": "Biomedical text mining promises to assist biologists in quickly navigating the combined knowledge in their domain. This would allow improved understanding of the complex interactions within biological systems and faster hypothesis generation. New biomedical research articles are published daily and text mining tools are only as good as the corpus from which they work. Many text mining tools are underused because their results are static and do not reflect the constantly expanding knowledge in the field. In order for biomedical text mining to become an indispensable tool used by researchers, this problem must be addressed. To this end, we present PubRunner, a framework for regularly running text mining tools on the latest publications. PubRunner is lightweight, simple to use, and can be integrated with an existing text mining tool. The workflow involves downloading the latest abstracts from PubMed, executing a user-defined tool, pushing the resulting data to a public FTP, and publicizing the location of these results on the public PubRunner website. This shows a proof of concept that we hope will encourage text mining developers to build tools that truly will aid biologists in exploring the latest publications.",
"keywords": [
"PubRunner",
"PubMed",
"biomedical text mining",
"text mining",
"natural language processing",
"BioNLP"
],
"content": "Introduction\n\nThe National Library of Medicine’s (NLM) PubMed database contains over 27 million citations and is growing exponentially (Lu, 2011). Increasingly, text mining tools are being developed to analyze the contents of PubMed and other publicly searchable literature databases. The goal of many of these tools is to enable a biologist to easily consume the latest relevant research and reduce the time searching for important results that would guide their research. These tools cover a wide variety of tasks, including improved searches across Pubmed (Tsai et al.), knowledge base construction (Xie et al.), and identification of concept association (Jelier et al.). Furthermore, there is significant interest in producing preprocessed sets of text with named entity annotation and part-of-speech tagging for use in further text mining analyses (for example, Hakala et al.).\n\nWith this huge rate of publication, it is commonly stated that text mining is becoming an essential research tool (Scherf et al.). However, molecular biologists rely on text mining experts to run these tools on the latest publications and openly share their results. Often the results of these text mining analyses and the code used are not shared, the analysis is not kept up-to-date with the latest publications, or the analysis is not publicized well to the biology community. These problems hinder the widespread use of text mining in scientific research.\n\nThe challenge of maintaining up-to-date results requires additional engineering, which often goes beyond a basic research project. Some research is beginning to look at methods to maintain updated analysis on PubMed (Hakala et al.), but a general framework is needed. In order to encourage biomedical text mining researchers to widely share their results and code, and keep analyses up-to-date, we present PubRunner. PubRunner is a small framework created during the National Center of Biotechnology Information Hackathon in January 2017. It wraps around a text mining tool and manages regular updates using the latest publications from PubMed. On a regular schedule, it downloads the latest Pubmed files, runs the selected tool(s), and outputs the results to an FTP directory. It also updates a public website with information about where the latest results can be located. We hope it will help the text mining community in producing robust and widely used text mining tools.\n\n\nMethods\n\nPubRunner manages monthly runs of text mining analyses using the latest publications from PubMed without requiring human intervention. The PubRunner framework has several key steps, outlined in Figure 1. First, it queries the PubMed FTP server to identify new XML files and downloads them. It currently downloads the Baseline dataset and then updates with the Daily Updates files (https://www.nlm.nih.gov/databases/download/pubmed_medline.html). It tracks which files are new and downloads the minimal required set to be up-to-date. Second, it executes the text mining tool(s) on the latest downloaded PubMed files. A JSON configuration file manages the set of tools to be run and determines whether they can be executed on only the new incremental files or require the full set of Pubmed XML files. These tools are then run as Python subprocesses and monitored for exit status. Furthermore, PubRunner uses a timeout parameter to kill processes that exceed a time limit. PubRunner runs on the same private server used for the text mining analysis, but moves results to a publicly visible FTP after the analysis is complete. It requires FTP login information to be able to copy files.\n\nPubMed abstract files in XML format are downloaded to the PubRunner framework, processed by the text-mining tools, the output pushed to a public FTP site and an update sent to the central PubRunner website.\n\nA central website was developed to track the status of different text mining analyses that are managed by PubRunner. These analyses may be executed on a variety of different researchers’ computers with results hosted on different FTPs. The website lists the tools with information about their latest run and where their code and results can be found. This allows text mining users to more easily find robust and up-to-date analyses on PubMed.\n\nA key design goal of PubRunner is to make installation as straightforward as possible. This is to encourage widespread use of the framework and release of both tool code and results data. Accordingly, a Docker image containing PubRunner has been produced, and installation from the Github code is also very straightforward. Also, each PubRunner component (server, website, and FTP) can be built by using the Docker file available for each in the GitHub repository. Deploying a specific component is thus made easy. Notably, there is not one central PubRunner FTP server. The output of PubRunner can be transferred to a pre-existing FTP server (e.g. an institution’s FTP server) or a new FTP server can be set up using the Docker image. After PubRunner is installed, configuration involves setting the paths to the tools to be run and the login information for the FTP.\n\nPubRunner currently has two dependencies: Python and R. The Docker file manages installation of these tools. The CPU and memory requirements required to run PubRunner depend on the associated text mining tools to be executed. PubRunner does require a reasonable amount of disk space, approximately 185GB, in order to download the full set of PubMed XMLs.\n\nIn order for a text mining tool developer to start using PubRunner, they first register their tool with the central website (http://www.pubrunner.org). Each tool should accept a set of Medline XML files as input and generate output files in a specific directory. The website gives them instructions on the necessary configuration settings so that their PubRunner instance can communicate with the central website. After each scheduled run of PubRunner on their remote server, an update message is sent to the website with a JSON packet of information. This information includes success status for the tools with URLs to the appropriate data. A potential extension to the website would hide tools that have failed for over three months and send notifications to the maintainers of each failed tool.\n\n\nUse case\n\nPubRunner was tested using three basic text mining tools that were developed specifically for testing the framework. These tools are also included in the Github repository. One of these tools, named CountWords, generated basic word counts for each abstract in a PubMed XML file. It takes as input a list of PubMed XML files, parses the XML for the AbstractText section, splits the text by whitespace and counts the resulting tokens to give a naïve word count. It then outputs the set of word counts along with the corresponding PubMed IDs to a tab-delimited file.\n\nIn order to test the robustness of the process management, two other tools that would fail were developed. The second tool, simply named Error, consistently failed. The third, named CountWordsError, uses the same code to calculate word counts as the first tool but would fail with a probability of 0.5. PubRunner successfully managed new runs of these test tools using updates from PubMed. At the time of publication, all three tools are deployed using PubRunner on a server hosted by the British Columbia Cancer Agency. PubRunner reruns the tools monthly and updates the results and status posted to the PubRunner website.\n\n\nConclusions and next steps\n\nThe PubRunner prototype reduces the additional engineering required for a text mining tool to be run on the latest publications. It will encourage the sharing of tool code and analysis data. At the moment, it can manage text mining runs using the latest Pubmed data. Future versions of the software will add additional corpora sources, such as PubMed Central, and allow easier integration of ontologies and other bioinformatics resources.\n\nWe hope to encourage more biomedical text mining developers to integrate their text mining tools into the PubRunner framework to develop an ecosystem of text mining tools running on the latest publications. This will certainly benefit biomedical researchers by allowing easier analysis of the latest publications so that new relevant knowledge is disseminated more easily.\n\n\nData and software availability\n\nPubRunner central website: http://www.pubrunner.org\n\nLatest source code for the pipeline is publically available on GitHub: https://github.com/NCBI-Hackathons/PubRunner.\n\nArchived source code as at time of publication: 10.5281/zenodo.556195 (Lever et al., 2017)\n\nLicense: MIT\n\nThe Docker image is available at https://hub.docker.com/r/ncbihackathons/pubrunner/.",
"appendix": "Author contributions\n\n\n\nAll of the authors participated in designing the study, carrying out the research, and preparing the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by the Intramural Research Program of the NIH, National Library of Medicine. JL is supported by a Vanier Canada Graduate Scholarship.\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 Lena Pons and Lisa Federer for their valuable input.\n\n\nReferences\n\nHakala K, Kaewphan S, Salakoski T, et al.: Syntactic analyses and named entity recognition for PubMed and PubMed Central—up-to-the-minute. ACL 2016, 2016; 102–107. Publisher Full Text\n\nJelier R, Schuemie MJ, Veldhoven A, et al.: Anni 2.0: a multipurpose text-mining tool for the life sciences. Genome Biol. 2008; 9(6): R96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLever J, Fiorini N, Anekalla KR, et al.: NCBI-Hackathons/PubRunner: Updated release for F1000 paper [Data set]. Zenodo. 2017. Data Source\n\nLu Z: PubMed and beyond: a survey of web tools for searching biomedical literature. Database (Oxford). 2011; 2011: baq036. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScherf M, Epple A, Werner T: The next generation of literature analysis: integration of genomic analysis into text mining. Brief Bioinform. 2005; 6(3): 287–297. PubMed Abstract | Publisher Full Text\n\nTsai RT, Dai HJ, Lai PT, et al.: PubMed-EX: a web browser extension to enhance PubMed search with text mining features. Bioinformatics. 2009; 25(22): 3031–3032. PubMed Abstract | Publisher Full Text\n\nXie B, Ding Q, Han H, et al.: miRCancer: a microRNA-cancer association database constructed by text mining on literature. Bioinformatics. 2013; 29(5): 638–44. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "22433",
"date": "26 May 2017",
"name": "Jin-Dong Kim",
"expertise": [
"Reviewer Expertise text mining",
"database",
"question-answering"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, I like the idea of PubRunner. However, as a potential user of the tool, I have three problems described below.\nWhile the motivation of the work is clear, what was not clear to me was the benefit of using a specialized tool like PubRunner instead of utilizing a more general scheduling tool like cron. I guess the authors know the benefits, but as it was not clear in the manuscript, I suggest the authors to explain it more clearly in the manuscript to better motivate potential users actually to choose to use PubRunner over alternatives.\n\nIn the manuscript, the protocol between an instance of PubRunner and the \"central website\" is not clearly described. It is even completely missing in Figure 1. I think designing such a protocol is not a trivial work, and it has be to clearly explained in the manuscript.\n\nFor the PubRunner to be more useful, I also think the method of publishing the results has to be more generalized. In fact, I have a big question about requiring the results to be uploaded to a FTP server. Can't it be a HTTP server? If PubRunner allows its user to choose the way of publishing the text mining results, it will become much more useful. This problem is actually related to the problem 1, e.g., if I choose to utilize cron, I can freely choose how to publish the result of my text mining pipeline.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "3025",
"date": "13 Oct 2017",
"name": "Ben Busby",
"role": "Author Response",
"response": "The reason to use the PubRunner framework over a simple CRON job is that PubRunner manages the download of files and upload of results files to a publicly available location. While many research groups have previously written code to manage the download of the PubMed baseline, there is incredible redundancy across these groups. It is worthwhile to create one implementation that can be used by existing groups or new groups as they start work in the biomedical text mining group.We have added further details of the communication protocol between the PubRunner tool and the website (http://www.pubrunner.org) which is used to update the current status and information about data location.You had also questioned the ability to only upload to FTP sites. Most HTTP websites will have an FTP access point which could be used by PubRunner. Furthermore, we have added functionality to upload to Zenodo which is a permanent data repository. It allows datasets up to 50GB, is hosted on the same infrastructure as the Large Hadron Collider and provides Digital Object Identifiers (DOIs) to allow PubRunner produced data to be easily citable."
}
]
},
{
"id": "24264",
"date": "07 Aug 2017",
"name": "Fabio Rinaldi",
"expertise": [
"Reviewer Expertise Biomedical text mining",
"ontologies",
"terminology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 idea presented in this short paper is clear: PubRunner is a software framework that might help to run text mining pipelines at regular intervals. Besides, Pubrunner is also capable of publishing their results on a reference web site.\nAlthough PubRunner does probably have some utility, the paper fails to address or even discuss other problems that hinder the reusability of text annotations produced by different text mining systems. On the most basic level, different annotation schemas might be used (e.g. different XML formats, or Json). On an higher levels, different annotation labels might be used for the same entity types, or there might even be partial overlappings between annotation categories used by different systems. Another problems is how to deal with errors and noise introduced by each system. And the list of potential obstacles to integration certainly does not end there.\nAnother severe limitation of the paper is that the framework has been tested using only three \"toy\" scenarios: a word counter, and two error-generating systems. It would have been more interesting to pick at least one real-world text mining system, which perhaps might have prompted the authors to consider other potential problems that the toy systems did not present.\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? No",
"responses": [
{
"c_id": "3024",
"date": "13 Oct 2017",
"name": "Ben Busby",
"role": "Author Response",
"response": "You raise one of the large challenges in biomedical text mining: the different annotation schemes used by different groups. We agree that is one of the limiting factors for the re-use of annotated data. However we argue that text mining tools are not reused because code is not often released, and any results from the tool are left static. We have added a section during the introduction discussing the challenge of reusing others research in biomedical text mining.We have also added an extra tool, word2vec, which is commonly used to generate word representation vectors for terms. Pyssalo et al (2013) created word vectors using the PubMed corpus, and these have been used by other researchers. However these vectors are now out-of-date. We added the word2vec tool to PubRunner. Furthermore we have added Zenodo as an option for hosting the resulting data. This allows the large files generated by word2vec to be hosted indefinitely."
}
]
},
{
"id": "24597",
"date": "07 Aug 2017",
"name": "Julien Gobeill",
"expertise": [
"Reviewer Expertise Text mining",
"natural language processing",
"bioinformatics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors' assumption is that many text mining tools are underused because their results are static (i.e. not updated with latest publications), and that biological experts could benefit from a centralized platform that would aggregate up-to-date outputs from various text mining tools. In this perspective, they propose and describe a framework: PubRunner.\n\nThe basic idea is simple and naive. Sometimes great things come from simple and naive ideas. I could imagine a centralized platform, where biological experts could explore up-to-date results of different tools, and occasionally download a dataset: it would be quite like Commoncrawl, but for text mining. This idea is quite interesting, as the authors say: \"an ecosystem of text mining tools running on the latest publications\". Moreover, this could help these developed tools to have a bigger visibility and a longer life.\n\nYet, I cannot imagine that this kind of platform could be integrated in a real curation workflow.\n\nFirst of all, the authors twice deal with the importance of sharing text mining results: \"molecular biologists rely on text mining experts to run these tools on the latest publications and openly share their results\". As a text miner, in contact with curators, this is not my life :) In my world, we text miners do not produce results, but tools and/or systems. Curators do not see us as data/results providers: they rather ask for quality-controlled and embedded assist in their curation workflow.\n\nIn a technical perspective, the presented idea has a lot of drawbacks.\n\nDealing with data format: what if the tool is not made for working from MEDLINE XML files as input? It seems that each tool would be free to deliver dataset in its own format (xml, json, csv...). How to describe the data content and format for the interested user? Moreover, computing data for all MEDLINE will result in huge dataset. Currently, there is a 260 Mo file just for word counts: would the dataset size still be manageable for more complex output? How to manage systems that are only relevant on a fraction of MEDLINE? (e.g. functional curation)\n\nDealing with data quality: how to know the quality of the dataset? This is extremely important for curators.\n\nDealing with computation: I don't think it is the job of Pubrunner to download MEDLINE updates (especially all the baseline, which will take days in a good server). \"The PubRunner prototype reduces the additional engineering required for a text mining tool to be run on the latest publications\": it can be true for text miners that are not used to work in biomedicine. For others, I assume they manage MEDLINE updates by their own. Moreover, I don't think that server managers will fully accept to have a third party platform running, especially on a production server.\n\nAs a conclusion, I think that this work could be the first step to a useful \"thing\", but it is far from being useful for the moment. The authors conclude that \"this will certainly benefit biomedical researchers by allowing easier analysis of the latest publications\", and this is not my opinion.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? No\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No",
"responses": [
{
"c_id": "3023",
"date": "13 Oct 2017",
"name": "Ben Busby",
"role": "Author Response",
"response": "We would like to thank you for taking the time to review this hackathon paper. We have read your comments carefully and have factored them into the changes.You provided your perspective on the application of biomedical text mining and its potential users. We would suggest that there are broadly three groups of users for text mining tools. Biocurators (those that curate biological databases) are the one that you discussed. They require customised tools that fit their workflows. While some groups do have a text mining expert on staff who is able to provide assistance through the lifetime of the project, other projects may only have an expert for a short-time to start up the initiative. The text mining tools used by these curators provide suggested papers (or more) to the curators. It would seem useful to those groups that their text mining expert could fit the tool in the PubRunner framework so that continued curation suggestions could be provided after the expert has left the project.However there are two other groups who benefit from text mining tools. There are biologists who use the output of text mining tools directly. These tools can be thought of as fully automated as they are not expertly curated. Understandably, these tools likely provide lower quality information but can be very useful in areas where expert curators cannot be found. Lastly there are text mining tools developed to be used by other text mining researchers (Hakala et al, Mikolov et al). We would suggest that these two groups would benefit greatly from automated updates using the latest publications.You raise a very valid point with the varied input formats used by different tools. PubRunner, currently expects a tool to accept the Medline XML files. However we have included code that extracts the title and abstract text from Medline XML files. Individual pipelines can make use of this code.The output data sizes of some text mining tools could be substantial. We have implemented an additional output hosting option with Zenodo. This allows datasets of up to 50GB to be hosted indefinitely. As an example of the ease of this, we added an additional tool (word2vec) that generates files of approximately 5GB.We agree that some text mining tools need only be run on part of Pubmed. This could be by filtering using MeSH terms, publication dates or other factors. We intend to add this functionality as a future feature and have added it to the paper.The quality of the output of the data is entirely dependent on the tool. PubRunner isn’t able to make evaluations on the quality of a tool. Users of any data output by a tool would need to refer to the tool’s documentation or associated publication in order to understand the expected quality of output data.You note that many biomedical text mining groups will already manage the download of MEDLINE baseline and update files. This exemplifies that each group has written their own code for this problem and shows the redundancy between groups. For new researchers, and particularly groups whose main focus is not biomedical text mining, they may not regularly download Pubmed. For a group that already manages the download of PubMed, we intend for a future version of PubRunner to accept the location of an existing download of Pubmed. It will then double check that all files are up-to-date and use that resource (thereby not duplicating downloading efforts).As a product of a hackathon, we understand that further development is needed in order to make PubRunner production-ready. We have noted this in the conclusion. Many server managers happily run third-party tools on a regular basis and with improved testing and documentation, we feel that a manager could happily run this framework on their own server."
}
]
}
] | 1
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https://f1000research.com/articles/6-612
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https://f1000research.com/articles/6-1836/v1
|
13 Oct 17
|
{
"type": "Research Article",
"title": "Socioeconomic disparities in income, education and geographic location for hypertension among Thai adults: Results from the National Socioeconomic Survey",
"authors": [
"Atthawit Singsalasang",
"Wongsa Laohasiriwong",
"Nattapong Puttanapong",
"Teerasak Phajan",
"Suwanna Boonyaleephan",
"Atthawit Singsalasang",
"Nattapong Puttanapong",
"Teerasak Phajan",
"Suwanna Boonyaleephan"
],
"abstract": "Background: Hypertension (HT) has been one of the leading global risk factors for health and the leading cause of death in Thailand for decades. The influence of socioeconomic factors on HT has been varied and inconclusive. The aim of this study was to determine the association between socioeconomic determinants and HT in Thailand. Methods: This study used data from the National Socioeconomic Survey, a cross-sectional study that was conducted by the National Statistical Office of Thailand in the years 2005, 2006 and 2007. In our analysis, data were collected on gender, age, marital status, smoking status, education, status of work, occupation, current liability (short-term debt), household monthly income, residential area, region and previously diagnosed HT by a physician. Results: The odds of having HT were significantly higher among those who had household monthly income, education, residential area and region. The participants who had monthly income of <10001 baht (2005: AOR = 3.19, 95%CI:1.47 - 6.92; 2006: AOR 2.53, 95%CI:1.37 - 4.69; 2007: AOR = 3.35, 95%CI: 1.97 - 7.00), were living in Bangkok compared with the Northeast region (2005: AOR = 1.72, 95%CI:1.37 - 2.17; 2006: AOR = 2.44, 95%CI: 1.89 - 3.13; 2007: AOR = 2.63, 95%CI 2.08 - 3.45), lived as an urban resident (2005: AOR= 1.32, 95%CI: 1.12 - 1.56; 2006: AOR= 1.21, 95%CI: 1.02 - 1.43; 2007: AOR= 1.47, 95%CI: 1.18 - 1.62), and finished primary education (2005: AOR =1.21, 95%CI: 1.03 - 1.43; 2006: AOR= 1.23, 95%CI: 1.04 - 1.46; 2007: AOR= 1.18, 95%CI: 1.01 - 1.38) when controlling for other covariates. Conclusion: This study indicated that socioeconomic disparity has an influence on HT. Those with low educational attainment, low income, lived in urban regions, and were metropolitan residents (Bangkok) were vulnerable to HT.",
"keywords": [
"Hypertension",
"socioeconomic disparity",
"income",
"education",
"geographic disparities",
"National Socioeconomic Survey",
"Thailand"
],
"content": "Introduction\n\nHypertension (HT) is one of the top modifiable risk factors for cardiovascular diseases (CVD), a cause of morbidity and mortality worldwide1,2. In Thailand, statistics for 2003, 2008 and 2013 indicated that the morbidity rate per 100,000 population for HT were 389.80, 860.53 and 1621.72, respectively, which shows an exponential increase3. In 2025, HT patients will likely to increase to 1.56 billion cases globally4. Moreover, half of HT patients die from ischemic heart disease and stroke caused by HT3,4. Many studies had found that there were several factors related to the occurrence of HT. There are some known individual factors consisting of the non-modifiable factors of age5–9, gender5,7–11, and having a family history of HT9,11,12, and some behavioural, modifiable factors, including being overweight/obese6,7,10,12–14, smoking11,15, physical inactivity5,11, high dietary salt intake5,15, alcohol consumption9,12,16,17, and stress6. There are also factors of socioeconomic status (SES) that are correlated with HT, namely education6–9,12,18–20, occupation6,9,15, economic status17,21,22, income17,20, and residential area6,11,15,16. Therefore, there are a variety of factors, both individual and SES, that have been previously associated with HT.\n\nPrevious studies on HT in Thailand5,11,17 were inconclusive regarding whether SES has any influence on HT. Studies on the association between SES and HT have been sparse, and the results are conflicting. Therefore, a large-scale study on HT and more focused research to determine whether disparity in socioeconomic effects on health status is needed. For these reasons, the objective of this study is to examine the association between the SES and HT among the Thai adult people.\n\n\nMethods\n\nThis study used data from the National Socioeconomic Survey (NSS), conducted in 2005, 2006 and 2007 by the National Statistical Office (NSO) of Thailand. The questionnaires collected information on gender, age, marriage status, smoking status, education, occupational, status of work, household monthly income, current liabilities (short-term debt), residential area, region and previously diagnosed HT by a physician. The outcome, HT, was classified into two categories: having HT and not having HT.\n\nThe cross-sectional survey was conducted by the NSO of Thailand. The survey used a stratified two-stage random sampling technique to select a nationally representative sample to respond to a structured questionnaire from all 76 provinces in Thailand. There were altogether 76 strata, each stratum was divided into two parts according to the type of local administration, namely, municipal areas and non-municipal areas. Selection of primary sampling, i.e. the sample selection of blocks/villages, was performed separately and independently in each part using probability proportional to the total number of households in that block or village. In the second step, the selection of secondary sampling units, i.e. private sampled households, were selected using the systematic method in each type of local administration (details of this sampling are available at http://web.nso.go.th/survey/house_seco/meth.pdf). Ultimately, there were a total of 16,306, 16,539 and 16,488 participants in 2005, 2006 and 2007, respectively, who met the inclusion criteria of Thai nationality and aged 15 years old and above were included in this analysis.\n\nThe characteristics of the participants were described using frequency and percentage for categorical variables and the mean and standard deviation for continuous variables. Crude odds ratios (OR), adjusted odds ratios (AOR) and 95% confidence intervals (CI) were calculated using bivariate and multiple logistic regression analysis to estimate the association between independent variables with HT. To obtain AOR for the effects of independent variables on HT, variables were placed in an initial model, and those with a p-value less than 0.25 were included in multivariate modelling. Backward elimination was used as the method for variable selection to obtain the final model. All analyses were performed using Stata version 13.0 (Stata Corp, College Station, TX). The magnitudes of effects were determined using AOR and 95% CI. A p-value less than 0.05 was considered statistically significant. All statistical tests were two-sided.\n\nThe NSS study obtained signed consent forms before enrolling participants. Confidentiality of the data was fully assured. The Ethical Committee of Khon Kaen University approved the exemption for ethical approval of this study (reference no. HE 582314). The NSO administrative board approved the research team to use the data (reference no.050601/1441).\n\n\nResults\n\nThe baseline characteristics of the 16,306 participants in 2005, the 16,539 participants in 2006 and the 16,488 participants in 2007 were as follows: The majority of the participants were women (53.53%, 53.61%, 53.58%, respectively); average ages were 42.23 ± 16.99 SD, 42.56 ± 17.17 SD and 43.04± 17.39 SD years old; most of the participants had monthly household income <10,001 baht (89.47%, 89.94%, 89.57%, respectively); about a half of participants completed primary education (55.08%, 54.10%, 53.27%, respectively); the majority lived in rural areas (60.70%, 61.72% and 62.77%, respectively); the highest proportion of participants was from the Northeast region (27.45%, 27.56% and 28.03%, respectively); prevalence of smoking was 28.92%, 28.27% and 26.98%, respectively (Table 1).\n\nThe bivariate analysis indicated that gender, age, marital status, smoking status, education, occupation, household monthly income, current liability, residential area and region were significantly (p-value <0.25) associated with HT in three consecutive years (Table 2).\n\nThis includes the odds ratio (OR) of having HT, with 95% confidence intervals (CI), for various characteristics of participants.\n\nThe final model of the multiple logistic regression analysis after adjusting for covariates, which included gender, age, and smoking status, indicated that in 2005, 2006 and 2007, the odds of having HT were significantly higher among those who had household monthly income <10001 baht1* (AOR = 3.19; 95%CI: 1.47 to 6.92, AOR = 2.53; 95%CI: 1.37 to 4.69, and AOR= 3.35; 95%CI: 1.97 to 7.00, respectively), lived in Bangkok when compared with the Northeast region (AOR = 1.72; 95%CI: 1.37 to 2.17, AOR= 2.44; 95%CI: 1.89 to 3.13 and AOR=2.63; 95%CI: 2.08 to 3.45, respectively), lived in urban areas (AOR= 1.32; 95%CI: 1.12 to 1.56, AOR= 1.21; 95%CI: 1.02 to 1.43 and AOR= 1.47; 95%CI: 1.18 to 1.62, respectively), and only finished primary education (AOR =1.21; 95%CI: 1.03 to 1.43, AOR = 1.23; 95%CI: 1.04 to 1.46, and AOR= 1.18; 95%CI: 1.01 to 1.38 respectively). Other covariates that were statistically significant associated with HT were smoking (AOR= 3.78; 95%CI: 3.29 to 4.34, AOR= 3.86; 95%CI: 3.35 to 4.45 and AOR= 4.00; 95%CI: 3.49 to 4.59, respectively), aged 35 to 44 years old when compared with younger age groups (AOR= 3.68; 95%CI: 2.65 to 5.09, AOR= 4.58; 95%CI: 2.99 to 7.01 and AOR= 3.89; 95%CI: 2.53 to 5.98, respectively), and women (AOR= 2.11; 95%CI:1.83 to 2.42, AOR= 2.11; 95%CI:1.81 to 2.44, and AOR= 2.05; 95%CI: 1.77 to 2.37, respectively) (Figure 1).\n\n\nDiscussion\n\nFrom the National Socioeconomic Survey data, we focused on examining the prevalence of HT and influences of SES on HT among Thai adults. HT is a common chronic disease and one of the most powerful contributors to CVD1,2. This study was conducted among the nationally representative samples with a large sample size; therefore, the results should be generalizable to represent the Thai population. In this study, SES factors, i.e. education, household monthly income, residential areas and region, were associated with HT even after adjusting for potential confounders.\n\nPrevious studies have revealed the association between the level of education and HT6–9,12,18–20,23, and they indicated that a low level of educational attainment was significantly associated with increased prevalence of HT. High education attainment is related to self-care and crucial to guarding against smoking. It can defeat related risk factors of HT by influencing a healthy lifestyle7,18,24. Moreover, sound knowledge on health can affect individual behaviour in several ways, such as involvement in health promotional activities and accessing health services24. Those with a higher education are provided with exponentially higher range and number of job opportunities and medical benefits packages, compared to those with lower levels of education. Another justification is that the higher education may promote the achievement of social gain, psychological support, and economic productivity by opening windows of opportunities. Thus, these performances can influence a person to socialize with peer groups that consequently promote god health behaviour, great self-esteem, and strong self-efficacy19,20,24.\n\nPrevious studies have reported that low income is associated with HT19,20. These findings are consistent with our study that participants with the low income had a higher risk of HT than those with a high income. As previously stated, a key factor is an income that can highly influence the behaviour. It can satisfy mental health, food behaviour, and make one aware of accessing health care to promote sound health19. Moreover, an income is essential to purchase better nutrition, high-quality education, healthy housing, and access to recreation. Previous studies also support these statements, saying that socioeconomic and psychosocial factors strongly affect individual health status20. Therefore, having a good income can be a useful measure to examine the variables that transform the health of the population19. A low income group have a higher tendency to develop HT and require treatment by changing lifestyles, such as weight loss, physical activity, and salt intake reduction20. It is highly important in terms of public health to identify these individuals so as to set up measures to delay or prevent HT progression or development.\n\nThe results of this study also showed that the residential area and region were significantly associated with HT. HT prevalence was highest among those who lived in the North than other regions and the lowest was in the Northeast. People who were living in Northeastern and Southern regions of Thailand were less likely to have HT than those living in Bangkok metropolitan area. Similarly, a previous study indicated that HT prevalence was correlated with geographical region25. For example, our study showed that there was the lowest prevalence of HT in the Northeast region, similar to the findings of a survey in Thai health working groups and health behaviour26 and the National Health Examination Survey25. In the Northeast, people were seldom aware that they had HT25, which is similar to the findings of the Health and Welfare Survey of the NSO, Thailand5. Residential areas where they lived could influence health behaviours in terms of lifestyle, social well-being and urbanization27. The results of this study corresponded to previous findings in Mae Hong Son province in Thailand11, Dehui City of Jilin province in China16, and Northwest in Ethiopia16, which reported a significantly higher tendency of HT in urban areas or cities rather than rural areas. Urbanization was associated with eating habit changes and obesity caused by reduced physical activity. Thai people have changed eating habits according to changing lifestyles between urban and rural residents28. Such lifestyle and eating habit changes are conducive to a high prevalence of abdominal obesity in the urban population, eventually resulting in increased prevalence of HT. Similar to the previous study28, residents in urban areas have a higher prevalence of being overweight or obese when compared with rural residents15,18. The differences in job opportunities and the quality of education in urban areas possibly impose an influence on the average socioeconomic accomplishment of its residents. The quality of the neighbourhood environment may be influenced by different levels of inequality regarding the distribution of social and economic resources across metropolitan areas. There have also been links of several aspects of the residential context to disparities in CVD risk and HT, including neighbourhood poverty and disadvantages, neighbourhood social cohesion, walking ability, availability of a healthy diet, and safety18. The differences in environmental exposures are possibly linked to HT29. In addition, neighbourhood-level SES could differently affect healthcare accessibility. Adverse neighbourhoods can increase levels of stress, and induce negative health behaviours, while failing to perform health promoting behaviours, possibly leading to HT. This study revealed that HT, which varied by each residential area, depended on the socio-environmental context at both metropolitan and neighbourhood levels29.\n\nIn addition, the results from the multivariate analysis performed in this study indicated that covariate factors, such as gender, age and smoking, were strongly associated with HT. Women had a higher prevalence of HT than men over all three years, similar to the findings of a previous study in Thailand5. In women, hormonal change after menopause has an effect on increasing blood pressure. The walls of a woman’s blood vessels can become less flexible when estrogen decreases, causing blood pressure (BP) to rise. The decline in estrogen levels can increase the risk for stroke and heart disease, especially due to high BP13. Moreover, an older age had significantly higher odds of having HT than younger individuals. These findings are consistent with previous studies5,6,9–12. In older individuals, arteries harden, kidney function decreases, the body has a greater sensitivity to salt and other factors, and there are hormonal changes, such as menopause. Furthermore, aging is also associated with a decrease in heart rate, intravascular volume, stroke volume, renal blood flow, plasma renin activity and cardiac output, and an increase in left ventricular mass index and renal vascular resistance, resulting in higher BP when a progressive decline in the ability of the kidneys to excrete salt loads efficiently30. In addition, elderly individuals are less likely to be physically active, which is also one of risk factor of HT. Thus, aging individuals are more likely to have an increasing risk of HT6,17. Additionally, this study indicated that HT was more prevalent among smokers. Indeed, smoking, in the form of cigarette or tobacco, can influence the deterioration of the overall health condition. Almost all physical systems, such as cardiovascular, cerebrovascular, respiratory, digestive, endocrine, urogenital and reproductive organs, can be affected by the harmful constituents of smoking. Smoking is an influential risk factor for developing cardiovascular-related diseases and morbidities, and discontinuation or cessation of smoking behavior can limit the process of initiating HT28. Thus, smoking causes a series of actions, such as loss of endothelial functionality, arterial stiffness causation, and recurrence of inflammation within the body28.\n\nThis study analysed nationally representative sample information. The findings indicated an increasing trend of HT and the association between the socioeconomic disparities and HT. It is noted that some variables, such as health behaviours, were not included in the study. However, these variables were found not strongly related to HT in previous studies when compared with demographics and smoking that were included in this study. Anyhow, we suggest that additional research focusing on biomolecular milieu, prenatal and early life exposures, historic SES conditions, health behaviours and their interplay in patients with HT may broaden the knowledge of associations among SES disparities and HT.\n\n\nConclusions\n\nThis study supports previous findings indicating that being a women, middle aged to elderly, and smoking are strongly associated with HT. The study also reported a new conclusion that socioeconomic factors had a significant influences on HT. Populations with low educational attainment, low income, urban, and metropolitan residents (Bangkok) were vulnerable to HT. Above all, the interaction between SES and biology combined to accelerate bio-molecular characteristics that could differently impose influences on HT. These findings deliver important implications for future research and healthcare provision in relation to the prevention of HT. Health personnel and other relevant sectors should be aware of the significant roles of these SES disparities on HT in order to develop appropriate policies aimed at preventing HT.\n\n\nData availability\n\nData used in this study were obtained from the NSO. Permission to use these data can be requested from the NSO. Researchers can request the NSS data by submitting an application form to the NSO Database Committee (the application form should be requested from the NSO Database Committee; services@nso.go.th). More details for submitting a request can be obtained from the NSO’s Statistical Information Service and Dissemination Group (services@nso.go.th).\n\n\nNote\n\n1* The amount was equal to 248.35 US dollar, 263.68 US dollar and 289.35 US dollar, in year 2005, 2006 and 2007, respectively. These conversions used the official exchange rates obtained from the Bank of Thailand.",
"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 (grant number, 6101/2015).\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 the contributors to this research, especially the National Statistical Office for the data, the Research and Training Center for Enhancing Quality of Life for Working Age People for the financial support and the Faculty of Public Health, Khon Kaen University, for financial and technical supports.\n\n\nReferences\n\nKayima J, Nankabirwa J, Sinabulya I, et al.: Determinants of hypertension in a young adult Ugandan population in epidemiological transition-the MEPI-CVD survey. BMC Public Health. 2015; 15: 830. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nPuavilai W, Laorugpongse D, Prompongsa S, et al.: Prevalence and some important risk factors of hypertension in Ban Paew District, Second report. J Med Assoc Thai. 2011; 94(9): 1069–1076. PubMed Abstract\n\nMinh HV, Byass P, Chuc NT, et al.: Gender differences in prevalence and socioeconomic determinants of hypertension: findings from the WHO STEPs survey in a rural community of Vietnam. J Hum Hypertens. 2006; 20(2): 109–115. PubMed Abstract | Publisher Full Text\n\nConen D, Glynn RJ, Ridker PM, et al.: Socioeconomic status, blood pressure progression, and incident hypertension in a prospective cohort of female health professionals. Eur Heart J. 2009; 30(11): 1378–1384. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaek TH, Lee HY, Lim NK, et al.: Gender differences in the association between socioeconomic status and hypertension incidence: the Korean Genome and Epidemiology Study (KoGES). BMC Public Health. 2015; 15: 852. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLam CS: The socioeconomics of hypertension: how $50 000 may buy a drop in blood pressure. Hypertension. 2011; 58(2): 140–141. PubMed Abstract | Publisher Full Text\n\nThonghong A, Thepsittha K, Jongpiriyaanan P, et al.: Chronic diseases surveillance report, 2012. Weekly Epidemiological Surveillance Report. 2013; 44: 800–808.\n\nJaddou HY, Batieha AM, Khader YS, et al.: Hypertension prevalence, awareness, treatment and control, and associated factors: results from a national survey, Jordan. Int J Hypertens. 2011; 2011: 828797. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLongtin Y, Sax H, Leape LL, et al.: Patient participation: current knowledge and applicability to patient safety. Mayo Clin Proc. 2010; 85(1): 53–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAekplakorn W, Abbott-Klafter J, Khonputsa P, et al.: Prevalence and management of prehypertension and hypertension by geographic regions of Thailand: the Third National Health Examination Survey, 2004. J Hypertens. 2008; 26(2): 191–198. PubMed Abstract | Publisher Full Text\n\nThe National Health Commission Office, Thailand: Thai health working group & health behavior. Institute for Population and Social Research. 2011; 18–19.\n\nMittal BV, Singh AK: Hypertension in the developing world: challenges and opportunities. Am J Kidney Dis. 2010; 55(3): 590–598. PubMed Abstract | Publisher Full Text\n\nVirdis A, Giannarelli C, Neves MF, et al.: Cigarette smoking and hypertension. Curr Pharm Des. 2010; 16(23): 2518–2525. PubMed Abstract | Publisher Full Text\n\nKershaw KN, Diez Roux AV, Burgard SA, et al.: Metropolitan-level racial residential segregation and black-white disparities in hypertension. Am J Epidemiol. 2011; 174(5): 537–545. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLogan AG: Hypertension in aging patients. Expert Rev Cardiovasc Ther. 2011; 9(1): 113–120. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "27016",
"date": "10 Nov 2017",
"name": "Limin Wang",
"expertise": [
"Reviewer Expertise I’m an epidemiologist and statistician on the field of chronic non-communicable diseases",
"including cardiovascular disease",
"diabetes",
"endocrine disorders and related risk factors(such smoking",
"alcohol drinking",
"obesity )."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments:\nLanguage in abstract is unreadable, especially in the results part.\n\nPlease provide the sample size for each stage of sampling, if equal sample size strategy was used.\n\nWhat is the ultimate sampling unit? If household, were all individuals in the selected household included in the survey?\n\nDid the analysis take into account the multistage sampling design? If not, the present estimations were not reliable, as sampling error might get probably underestimated.\n\nThe results for every study year are much similar, and the SES effects on HT should not change during short period. So, what is the point of annually independent analysis?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "30222",
"date": "06 Feb 2018",
"name": "Matthew J. Kelly",
"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 account of an important and growing health burden in Thailand, hypertension. Given the increasing prevalence of this condition and the associated increased risk for other chronic diseases this paper is timely and relevant. The identification of socio-economic drivers is particularly important in a country such as Thailand which has experienced rapid economic growth and change in the recent past.\nI have a few comments as follows:\nWhy are the authors using 2005 to 2007 data? These data are now more than 10 years old and given the rapid rise in HT prevalence described by the authors the most up to date data are important.\n\nThe results section of the abstract needs to be rewritten. It describes associations between for example income and education and HT but does not describe the direction of the association. That is, high income or low income that associates with hypertension.\n\nIn the Methods HT is measured by self-report of a physician diagnosis. Has any validation analysis been done to assess the accuracy of this type of self report? Also this leaves the strong possibility of undiagnosed HT being prevalent in the community. This cannot be measured using this study design but it should be mentioned in the limitation section of the discussion.\n\nAlso in the Methods the authors describe a sample of approximately 16,000 people in each study year. What proportion is this of the total study population after exclusions were made.\n\nWas there any non-response or refusal to participate rate that should be reported?\n\nIn the bivariate analysis variables were included where their association with hypertension was significant at p<0.25. Why was this value chosen?\n\nThe Discussion section of this paper needs some work.\n\na. The authors discuss education and income as indicators of socio-economic status and observe that low education and low income are associated with HT risk. This is the case in contemporary Thailand. It may be worth discussing here though that this is a modern phenomenon and the result of health and epidemiological transitions. Previously in most low and middle income countries chronic disease risk including obesity and HT was concentrated in the higher socio-economic groups.\n\nb. There is a little bit of a contradiction between the education/ income – HT association and the geographic association which needs some more consideration. Incomes are generally higher in urban areas which should according to the SES hypothesis mean that they have the lowest levels of HT. However in this study, the Northeast, the poorest area of the country has the lower prevalence. As the authors suggest this could be because in Isan the undiagnosed HT may be higher. But there also may be different directions of association between income and HT depending where you live. I am not sure there will be a clear answer to this but it deserves some discussion in the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "31475",
"date": "09 Apr 2018",
"name": "Abha Shrestha",
"expertise": [
"Reviewer Expertise Noncommunicable diseases"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have presented the growing burden of hypertension in Thailand and its association with the socioeconomic status. The paper is relevant to the current scenario of increasing burden of Noncommunicable diseases.\n\nI have a suggestion, please add the reference: Kearney et al. (2005)1 to the reference number 4.\n\nSince, this study is about hypertension, it would be nice to explain more on the diagnosis criteria used for hypertension.\n\nThe result section of the abstract is not very clear.\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/6-1836
|
https://f1000research.com/articles/6-1835/v1
|
13 Oct 17
|
{
"type": "Data Note",
"title": "Updated mtCOI reference dataset for the Bemisia tabaci species complex",
"authors": [
"Laura M. Boykin",
"Anders Savill",
"Paul De Barro",
"Anders Savill",
"Paul De Barro"
],
"abstract": "Members of the whitefly Bemisia tabaci species complex cause millions of dollars of damage globally and are considered one of the world’s most invasive species. They are capable of causing extensive damage to major vegetable, grain legume and fiber crops. All member of the species complex are morphologically identical therefore, data from the partial mitochondrial cytochrome oxidase subunit I (mtCOI) gene sequence has been used to identify the various species. The current reference dataset that is widely used is found on the CSIRO data portal. However, the reference set stored on the CSIRO data does not include newly added sequences (2013-2017), therefore an updated reference dataset is needed. All mtCOI data for the Bemisia tabaci species complex were downloaded on 22 May 2017 from GenBank and after quality checking, a dataset of 1,071 unique sequences and 696 base pairs was generated (https://doi.org/10.6084/m9.figshare.5437420.v1).",
"keywords": [
"species identification",
"whitefly",
"insect vector",
"mitochondrial cytochrome oxidase",
"DNA barcoding"
],
"content": "Introduction\n\nMembers of the Bemisia tabaci (whiteflies) species complex are among the world’s most devastating insect pests and cause billions of dollars (US) of damage each year, leaving farmers in the developing world food insecure (De Barro et al., 2011). As a species complex with at least 34 members, identification is based on the use of the 657 bp portion of the 3’ end of the mitochondrial COI (mtCOI) (Boykin et al., 2012, Boykin et al., 2013). In order to identify members of the complex correctly, a curated reference dataset is a useful resource. In 2012, a reference mtCOI dataset was made available on the CSIRO data portal (De Barro & Boykin, 2012). Errors in the dataset were subsequently identified and so the dataset was updated on 15 May 2017 (http://doi.org/10.4225/08/591a4018dfca8) (De Barro & Boykin, 2017), but did not include new additions from GenBank (post 2012). Therefore, the dataset described herein represents the most up-to-date reference resource for members of the complex.\n\n\nMethods\n\nThe CSIRO dataset (http://doi.org/10.4225/08/591a4018dfca8), updated 15 May 2017 was used as the starting point. The existing records were updated to include host plant data. New records post-2012 were then downloaded on 22 May 2017 directly from GenBank. All downloaded data was treated as follows:\n\n1) Data was classified with BLAST using the new CSIRO reference data set\n\n2) Sequences that caused gaps in the alignment were removed\n\n2) Sequences that had stop codons present were removed\n\n3) Clustal Omega (Sievers & Higgins, 2014) was used for preliminary alignment and fine tuning of the alignment was carried out with MAFFT (Katoh & Standley, 2013).\n\n4) Duplicate sequences were then removed using BBMAP Dedupe (Bushnell, 2017).\n\nIn addition, all MEAM2 sequences were removed as they have now been confirmed to be pseudogenes (Tay et al., 2017).\n\n\nData availability\n\nFigshare: Dataset 1. mtCOI reference data for species ID of Bemisia tabaci. DOI: 10.6084/m9.figshare.5437420 (Boykin et al., 2017)",
"appendix": "Competing interests\n\n\n\nThe authors have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBoykin LM, Armstrong KF, Kubatko L, et al.: Species delimitation and global biosecurity. Evol Bioinform Online. 2012; 8: 1–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoykin LM, Bell CD, Evans G, et al.: Is agriculture driving the diversification of the Bemisia tabaci species complex (Hemiptera: Sternorrhyncha: Aleyrodidae)?: Dating, diversification and biogeographic evidence revealed. BMC Evol Biol. 2013; 13: 228. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoykin L, Savill A, De Barro P: mtCOI reference data for species ID of Bemisia tabaci. figshare. 2017. Data Source\n\nBushnell B: BBmap. 2017. Reference Source\n\nDe Barro P, Boykin LM: Global Bemisia dataset release version 31 Dec 2012. CSIRO. 2012. Publisher Full Text\n\nDe Barro P, Boykin LM: Global Bemisia dataset release version 15 May 2017. CSIRO. 2017. Publisher Full Text\n\nDe Barro PJ, Liu SS, Boykin LM, et al.: Bemisia tabaci: a statement of species status. Annu Rev Entomol. 2011; 56: 1–19. 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\nSievers F, Higgins DG: Clustal Omega, accurate alignment of very large numbers of sequences. Methods Mol Biol. 2014; 1079: 105–116. PubMed Abstract | Publisher Full Text\n\nTay WT, Elfekih S, Court LN, et al.: The trouble with MEAM2: Implications of pseudogenes on species delimitation in the globally invasive Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species complex. Genome Biol Evol. 2017. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "26988",
"date": "23 Oct 2017",
"name": "Renate Krause Sakate",
"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 updated mtCOI reference dataset for the Bemisia tabaci species complex will add a valuable contribution to researches for a fast and accurate identification of members from the B. tabaci species complex based on the partial mitochondrial COI gene. The high quality data is easily accessible for download and gathers whiteflies collected globally. The availability of a reliable and updated reference dataset is an essential tool that will aid the scientific community to identify and classify correctly this pest, the first step in the crop management against whiteflies.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
},
{
"id": "26987",
"date": "08 Nov 2017",
"name": "Sharad Saurabh",
"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\nWhitefly (Bemisia tabaci) is becoming a global hazard for crop and ornamental plants. Identification of correct species is always better for the implication of best control strategy. In this regard, the effort made by Boykin et al for speedy and accurate identification of B. tabaci species complex is very significant. Additionally, the dataset developed with enriched quality is also very useful for whitefly biologist working in the area of evolution, mitochondrial genomics and crop management. All the bioinformatics tools used to generate this refined dataset are ideal to make such analysis.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1835
|
https://f1000research.com/articles/6-1833/v1
|
13 Oct 17
|
{
"type": "Systematic Review",
"title": "Comparison of school based and supplemental vaccination strategies in the delivery of vaccines to 5-19 year olds in Africa - a systematic review",
"authors": [
"Eposi C. Haddison",
"Leila H. Abdullahi",
"Rudzani Muloiwa",
"Gregory D. Hussey",
"Benjamin M. Kagina",
"Leila H. Abdullahi",
"Rudzani Muloiwa",
"Gregory D. Hussey",
"Benjamin M. Kagina"
],
"abstract": "Background: Some vaccine preventable diseases (VPDs) still remain a public health burden in many African countries. The occurrence of VPDs in all age groups has led to the realization of the need to extend routine immunisation services to school age children, adolescents and adults. Supplemental immunisation activities (SIAs) and school based vaccinations (SBVs) are common strategies used to complement the expanded programme on immunisation (EPI). This review aimed to assess the effectiveness of SIAs compared to SBVs in the administration of vaccines to 5-19 year olds in Africa. Methods: Systematic review methods were used to address our study aim. Several electronic databases were searched up to March 30, 2017 for primary studies investigating the delivery of vaccines via SIAs or SBVs to 5-19 year olds. This search was complemented by browsing reference lists of potential studies obtained from search outputs. Outcomes considered for inclusion were: vaccination coverage, costs of the strategy or its effect on routine immunisation services. Results: Out of the 4938 studies identified, 31 studies met the review inclusion criteria. Both SIAs and SBVs showed high vaccination coverage. However, the SIAs reported higher coverage than SBVs: 91% (95% CI: 84%, 98%) versus 75% (95% CI: 67%, 83%). In most settings, SBVs were reported to be more expensive than SIAs. The SIAs were found to negatively affect routine immunisation services. Conclusions: Both SIAs and SBVs are routinely used to complement the EPI in the delivery of vaccines in Africa. In settings where school enrolment is suboptimal, as is the case in many African countries, our results show SIAs may be more effective in reaching school age children and adolescents than SBVs. Our results re-iterate the importance of evaluating systematic evidence to best inform African authorities on the optimal vaccine delivery strategies targeting school age children and adolescents.",
"keywords": [
"Africa",
"systematic review",
"school based vaccination",
"supplemental immunisation activities",
"adolescents",
"school age children",
"routine immunisation"
],
"content": "Introduction\n\nThe Expanded Programme on Immunisation (EPI) was founded in 1974 to provide immunisation services to children, both nationally and globally1. The EPI has proven to be a cost effective public health strategy, with reports suggesting that because of the programme, millions of infants’ lives globally have been saved against vaccine preventable diseases (VPDs)1. Despite the widespread implementation of EPI, some VPDs still remain a public health burden in most of the African countries2,3. Suboptimal vaccination coverage rates in children targeted by the EPI and the inability to expand vaccination services to populations not targeted by the routine immunisation are some of the likely contributors to the high prevalence of VPDs in Africa4,5.\n\nRoutinely, school aged children and adolescents are not the primary target of EPI and as a result, an immunisation gap among these age groups has been observed in many settings6. To address the immunisation gap, the WHO has recommended the inclusion of several vaccines targeting school aged children and adolescents in national immunisation programmes (NIPs). The WHO recommended vaccines for older children include those against the following pathogens: human papillomavirus (HPV), diphtheria, tetanus, pertussis, measles, rubella, hepatitis B and meningococcus6,7. A majority of the High Income Countries (HICs) have implemented the WHO recommendations of vaccinating the older children, but this is not the case with the Low and Middle Income Countries (LMICs)8–10.\n\nSeveral reasons justify the inclusion of school aged children and adolescents into NIPs. First, infants who miss routine vaccinations remain susceptible to VPDs later in life6,11. Second, immunity acquired through infant immunisation for some VPDs like tetanus and pertussis wanes over time, thus requiring booster doses later in life12. Third, there are epidemiological changes of some VPDs like rubella where more infection rates have shifted from infancy to adolescence, thus requiring a review of immunisation policies13. Lastly, new vaccines under development such as against HIV and tuberculosis (TB) are likely to target older children and adolescents. In the absence of structured vaccine delivery programs for school age children and adolescents, many settings use school based vaccinations (SBVs) and supplementary immunisation activities (SIAs).\n\nSupplementary immunisation activities, also known as mass vaccination campaigns refer to an immunisation strategy where a large number of people are vaccinated within a defined geographical area and period, regardless of previous vaccination status14. The success of SIAs in disease outbreak control as well as in the eradication of smallpox is well documented14–17. However, there are reports suggesting negative effects of SIAs on the routine health services, including EPI18,19.\n\nSchool based vaccinations (SBVs) target school going children on school premises and within school hours. The SBVs delivery strategy is newer to the EPI compared to SIAs20, particularly in Africa. Currently, and in Africa, the main vaccine administered through SBVs is against HPV. Advantages of SBVs include high vaccination coverage and the possibility to extend other health services offered to school age children21–23. However, in Africa, there are millions of children not attending school24 and are missed by SBVs strategy.\n\nOur study aimed to compare the effectiveness of using SIAs or SBVs to deliver vaccines to 5–19 year olds in Africa.\n\n\nMethods\n\nThis review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Checklist (Supplementary File 1). The protocol was registered with PROSPERO (CRD42017057475)25.\n\nA search was carried out to identify all relevant studies. Both published and unpublished literatures were searched up to March 30, 2017. No restriction was placed on the publication language. The following electronic databases were searched using both medical subject headings (MeSH) and free text terms relating to vaccination, children, adolescents and Africa (Supplementary Table 1): PubMed, Africa Wide, Cochrane Central Register of Controlled trials (CENTRAL), Cumulative Index to Nursing and Allied Health Literature (CINAHL), World Health Organization Library Information System (WHOLIS), Web of Science, PDQ (Pretty Darn Quick)-Evidence and Scopus. The following grey literature databases were searched for reports, non-peer reviewed and non-indexed papers: WHO, The Global Vaccine Alliance (GAVI) and UNICEF. The reference lists of the included publications were evaluated to identify other potential studies.\n\nThe following criteria was used to select primary studies for inclusion:\n\n(1) the study was either a randomised controlled trial (RCTs), non-RCT, cluster-RCT, interrupted time series, controlled before-and-after, cohort, cross-sectional or case-control;\n\n(2) participants were school aged children or adolescents (5–19 years) living in Africa;\n\n(3) SIAs or SBVs were the vaccination strategies under investigation;\n\n(4) vaccination coverage, cost of vaccine delivery or effects of either strategy (SIAs or SBVs) on routine health services including EPI were reported as any of the outcomes. Retrieved articles were independently screened by two reviewers (HEC and LA). Where study eligibility was unclear, a review was carried out by a third independent study team member (BK).\n\nHEC and LA independently reviewed each included study and extracted data using a piloted data extraction form. Where discrepancies arose HEC, LA and BK reached a consensus by discussion. Corresponding authors were contacted for any missing data needed during the extraction process.\n\nExperimental studies were assessed using the Cochrane Collaboration’s tool for assessing risk of bias26, while the Hoy et al., modified tool was used for cross-sectional studies27. Using the Cochrane checklist, studies were scored as ‘High risk’ or ‘Low risk’. Using the Hoy et al., checklist, cross-sectional studies were scored as High risk’ or ‘Moderate risk’ or ‘Low risk’ of bias. Studies with high risk of bias were rated as poor quality while studies with moderate and low risk of bias were rated as moderate and high quality respectively. Where discrepancies in quality assessment occurred, HEC and LA discussed to arrive at a consensus. The quality of studies reporting the cost effectiveness was not assessed since our main interest was only the delivery costs.\n\nData was analysed using Stata v. 14.0. Results from the studies reporting vaccination coverage were expressed as percentages. Reported costs of the delivery strategies were standardised to United States Dollars (USD) if reported in a different currency. The costs of the strategies (SBVs or SIAs) and effects of the strategy on routine vaccination were presented in a narrative form.\n\nA meta-analysis for vaccination coverage using a random effects model with inverse variance proportion was carried out. Pooled statistics for vaccination coverage were expressed as proportions with 95% confidence interval (95% CI). Subgroup analyses were carried to evaluate vaccination coverage per strategy, stratified by study settings or type of vaccine. Missing values were imputed in order to enable a complete case analysis. A sensitivity analysis was then carried out where imputations were done to assess if the results significantly differed due to the data imputations.\n\nVisual inspection of the forest plots, the Chi-squared test for homogeneity with a significance level set at 10% and the I2 statistic were used to assess and quantify any heterogeneity between study results. Heterogeneity was rated as ‘low’ for ≤ 49% and ‘moderate’ for 50–74% and ‘high’ for ≥ 75% using the I2 statistic.\n\n\nResults\n\nThree thousand seven hundred and nineteen (3719) studies were identified through searching the electronic databases. A further 1461 were identified from grey literature. An additional five studies were identified from the reference lists of all the search outputs. After duplicates were removed, 4938 studies were left for screening. The titles and abstracts of the 4938 studies were screened and 4872 were not relevant and therefore excluded. The full text of the remaining 65 were retrieved and assessed for eligibility. From the 65 studies, 31 met our inclusion criteria (Supplementary File 2).\n\nA total of 31 studies were included in this review. There were 20 cross-sectional studies23,28–46, eight economic evaluation studies47–54, one cluster-randomised trial55, one epidemiological report56 and one interrupted time series57. The included studies were published between 1993 and 2016; only three studies were published before 200045,46,56. A total of 17 African countries (Figure 1) and five different vaccines were represented from the included studies. Except for four of the included studies that were written in French44,46,50,53, the rest were in English. One of the study team members (HEC) is French literate and translated the four articles. In terms of vaccine delivery strategy, 20 and 11 studies assessed SIAs and SBVs respectively. Out of these 32 studies, 20 reported on vaccination coverage (Table 1 and Table 2), nine on the cost of the vaccination strategy (Table 3) and three on the effect on routine immunisation (Table 4).\n\nUsing the Hoy et al., modified tool27, a 10 item scale was used to assess the internal and external validity of the 20 cross-sectional studies. Ninety-five percent (19) of the studies were of high quality (low risk of bias) meaning further research is unlikely to change our confidence in the estimate of the study outcomes. Five percent (1) of the studies were of moderate quality (moderate risk of bias) meaning further research is likely to have an impact on our confidence in the estimate of the outcomes (Figure 2b). For the internal validity, the included studies defined which participants were considered to have been vaccinated (by self-report or vaccination card) and used the same data collection tool for all the participants. However, five studies did not mention if the tool used was standardised29,34,42,45,46. Ten studies collected information from proxies (parents or guardians of vaccinated children)29–32,34,36,43–46. All the studies calculated vaccination coverage as the ‘number vaccinated divided by the number of the targeted population’. For the external validity, all the studies had representative samples in terms of age and sex. Random sampling was used in all except four studies33,35,37,39. Similarly, majority of the studies had a low non-response rate except three studies33,40,46.\n\nThe Cochrane checklist was used to assess the clustered-randomised trial55 and interrupted time series57 study (Figure 2a).\n\na. Risk of bias for experimental studies. b. Risk of bias for cross-sectional studies.\n\nSIAs: Twelve studies reported vaccination coverage for SIAs. Three studies reported coverage data on meningococcal serogroup A (PsA-TT) vaccine29,30,44, five on measles vaccine31,32,34,56,57 and two on yellow fever vaccine36,43. Each of the remaining two studies reported on one vaccine; cholera38 and meningococcal bivalent polysaccharide A/C vaccine46. Vaccination coverage for SIAs ranged from 48.5% to 98% (Table 1).\n\nA meta-analysis of a pooled estimate showed high vaccination coverage for SIAs 86% (95% CI: 80%, 93%) (Figure 3a). Three studies were excluded from the pooled meta-analysis due to missing sample sizes31,32,38. However, after imputation of the sample sizes based on vaccination coverage, no major difference was seen in the pooled coverage (Figure 3b).\n\na. Forest plot showing vaccination coverage for SIAs. b. Forest plot showing vaccination coverage for SIAs with imputed data.\n\nSBVs: Eight studies reported vaccination coverage for SBVs (Table 2). All the SBVs reported coverage of the HPV vaccine among girls aged 9–19 years. Four HPV vaccine studies reported a combination of a grade and age based approach for identifying the target girls for the vaccination28,33,35,55 while three studies reported a grade based only approach23,40,41. The remaining study identified girls for HPV vaccination by age42, (Table 2). Vaccination coverage of completed doses among the targeted population ranged from 42.4 – 97.4%. A meta-analysis of SBVs showed a pooled vaccination coverage of 75% (95% CI: 67, 83) (Figure 4).\n\nComparison of vaccination coverage: To compare the two vaccine delivery strategies, pooled estimates for SIAs and SBVs were evaluated. For this comparison and subsequent analyses, Huhn et al., study36 was not included since its setting (displaced) is not similar to the other studies reporting SIAs. The SIAs showed a higher vaccination coverage than and SBVs (Figure 5).\n\nSubgroup analyses were carried out to evaluate if vaccination coverage varied by the study setting or by vaccine type (for SIAs).\n\nStudy setting: The settings evaluated were: urban only, rural and urban mixed, or rural only. For SIAs, vaccination coverage did not vary irrespective of the three study settings (Figure 6). However, vaccination coverage was highest in urban areas (97%, 95% CI: 96, 98) and lowest in a mixed setting (88%, 95% CI: 79, 98).\n\nSimilarly for SBVs, there was no variation in vaccination coverage across the three settings. Vaccination coverage in urban, rural and mixed settings were as follows: 79% (95% CI: 77, 81), 74% (95% CI: 63, 85) and 75% (95% CI: 53, 97) respectively.\n\nVaccine type: There were variations in vaccine coverage dependent on the type of vaccine delivered via SIAs (Figure 7). Of all the vaccinations, measles SIAs reported the lowest coverage 83% (95% CI: 72, 93). Highest vaccination coverage were reported for SIAs against meningitis and yellow fever at 96%, (95% CI: 94, 98) and 98% (95% CI: 95, 99) respectively.\n\nSIAs: Seven studies reported the costs of conducting SIAs (Table 3). The costs represent monies spent to deliver the vaccines to the total target population during the SIAs. The available data on costs of the SIAs was not age specific and therefore, the costs for 5–19 year olds only could not be calculated. The SIAs targeted children aged 6 months and older age groups with the number of vaccinated people across all studies ranging from 23921 to 12,649,448.\n\nMajority of the total cost in SIAs was used to procure vaccines and consumables. The highest proportion (86%) for vaccines and consumables costs’ was reported by da Silva et al., for the combined yellow fever and meningitis A/C campaign in Senegal53. Costs for salaries of health staff and international supervisors were another high expense during SIAs. Other reported minor costs were: training of personnel, social mobilisation, transport, maintenance of equipments, cold chain and management of adverse events following immunisation (AEFI).\n\nSBVs: Two studies reported the cost of HPV vaccination in Tanzania and Uganda47,48 (Table 3).In Tanzania, 4211 girls were vaccinated with three doses of HPV vaccine either based on age or class. The total economic costs for the SBVs were 349,400 USD. The total cost per fully immunised girl in urban areas were 66 USD and 100 USD for class-based and age-based approach respectively; and in rural areas, 78 USD and 107 USD for class-based and age-based approach respectively.. Administration and supervision (salaries) of the project and procurement of vaccines accounted for the major expenses. The reported minor expenses in the study included training, cold chain, waste management and social mobilisation. The observed difference between costs per fully immunised girl for class-based and aged-based selection may be due to the fact that more time and logistics were needed to select girls based on their age. In Uganda, 3038 girls in a rural setting were vaccinated with three doses of HPV vaccine using a class based approach. The total economic cost (cost of all resources used regardless of who paid) was 30,646 USD excluding the cost of the vaccine. The total economic cost per fully immunised girl was 9.5 USD. Salaries of staff accounted for the greatest part of the cost followed by micro-planning, staff training, community mobilisation (start-up costs). Other expenses included supplies, cold chain, vehicles and transportation.\n\nSIAs: Two studies reported the effect of SIAs on routine immunisation and health services. Mounier-Jack et al., reported on a 10 day meningitis A campaign in Mali, in 201037 while Verguet et al., reported on a 3 weeks measles campaign in South Africa in the same year57. Both SIAs reported a negative effect on routine immunisation (Table 4).\n\nLow attendance: Both studies reported a decrease in the number of children attending the child clinics during the vaccination period. Mounier-Jack et al., reported a 71–74% decrease in the number of children vaccinated during the campaign37 while Verguet et al., reported an 8% decrease in the number of children who were weighed in the postnatal clinic57.\n\nRedeployment of health staff: During the SIAs, staff in charge of routine immunisations were either deployed as supervisors for the campaign. This led to the closure of routine immunisation services in some districts during the campaign period.\n\nCold chain management: In Mali, routine vaccines were relocated and stored at the regional and district level so fridges could be made available to store the campaign vaccine37.\n\nSBVs: One study in Rwanda reported the effect of HPV vaccination on routine immunisation39 (Table 4). According to Torres-Rueda et al., the vaccination activity which lasted 2 days had no effect on routine immunisation services39. There was no change in the demand for routine immunisation and health services.\n\n\nDiscussion\n\nBoth SIAs and SBVs are supplementary EPI programs in many settings, including Africa. Our results show both strategies attain high coverage with SIAs showing greater vaccination coverage than SBVs. However, this coverage should be interpreted with caution since single dose vaccines were administered during the SIAs as opposed to two or three HPV doses during SBVs. Nonetheless, this review showed SIAs negatively affect the provision of routine health services, particularly the EPI. In settings like Africa where many resource challenges prevail, the simultaneous use of both SIAs and SBVs to reach school age and adolescents is questionable.\n\nThe high vaccination coverage achieved by SIAs reported in this review corroborates with the past successes of smallpox eradication, achieved by complementing EPI with SIAs58. Interestingly, coverage of the SIAs was high irrespective of the vaccine and setting, and this attests to the robustness of the strategy. Despite being a new strategy in Africa and being used for the introduction of HPV vaccines, SBVs was able to achieve a high but variable coverage. Our findings are similar to those obtained in HICs where the SBVs strategy is more established and used for routine immunisation to this age group59.\n\nIn terms of vaccine coverage, our review supports what is already known: both SIAs and SBVs are good options to complement the EPI. However, other factors such as the costs of the strategy, logistical requirements, the number of vaccine doses to be administered, school attendance and existing immunisation policies have to be taken into consideration when deciding which of the two strategies to use in any given setting. Local evidence should be used to evaluate which vaccine delivery strategy is more optimal to reach school age children and adolescents in Africa.\n\nSBVs are likely to be more cost-effective than SIAs in countries with high school enrolment. Similarly, SBVs are likely to be optimal in countries with strong inter-ministerial collaboration. Collaboration between health and education sectors is crucial to ensure smooth implementation of SBV strategy60. Conversely, SBVs are unlikely to be optimal in countries without sufficient financial commitment. School based vaccinations have been reported to be an expensive strategy which may be feasible on a small scale but not sustainable at a national level61. Our findings support the reports that SBVs are an expensive strategy although this was based on limited data of newer and more expensive HPV vaccines.\n\nSupplemental immunisation activities, could be the preferred strategy in countries where campaigns are regularly used to complement infant immunisation. The experience in conducting SIAs and community awareness of the strategy can be used to extend the vaccination services to school age children and adolescents. Additionally, the SIAs are able to reach non-school going children. A majority of African countries have millions of non-school going children24 who will miss vaccines delivered by SBV programs. The negative impacts of SIAs on routine immunisation and health services due to the overlapping of resources (financial and human) is a key concern that should be minimized wherever SIAs are used.\n\nWe comprehensively and systematically searched for the relevant literature: both peer-reviewed and non-reviewed records were obtained. This study adhered to the PRISMA guidelines of conducting systematic reviews. Nonetheless, our review had several limitations. First, age specific coverage was not reported in some of the retrieved studies. Second, we observed a high heterogeneity during the meta-analysis. The heterogeneity was likely due to differences across studies of factors such as the age groups included, study settings and period. Third, the only vaccine administered via SBVs was the HPV vaccine, a new and more expensive vaccine. Lastly, few studies reported the effect of SIAs or SBVs on routine immunisation and health services and therefore, our findings may not accurately reflect the effects.\n\nLocal evidence is crucial in the review and development of immunisation policies. Both SIAs and SBVs are routinely used in Africa to vaccinate older children. In settings where school enrolment is low as is the case in many African countries, our results show SIAs may be more effective in reaching school age children and adolescents than SBVs. However, caution needs to be exercised to mitigate the negative effect of SIAs on the routine health services. In Africa, the SBV has mainly been tested in the delivery of two or three dose HPV vaccine to adolescent girls while SIAs have been used for diverse vaccines and on a larger scale. Further research is therefore needed to assess the sustainability of SBVs for nationwide delivery of vaccines to school age children and adolescent in resource constraint settings as is the case in Africa. In the event that SBVs are chosen as the main delivery strategy, complementary community activities can be set up to target out of school children. Our results re-iterate the importance of systematic evidence to best inform African authorities on the optimal delivery strategies of vaccines targeted at school age children and adolescents for immunisation.\n\n\nData availability\n\nDataset 1: Characteristics of included studies reporting vaccination coverage. DOI, 10.5256/f1000research.12804.d18061662.",
"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: PRISMA Checklist.\n\nClick here to access the data.\n\nSupplementary File 2: PRISMA flowchart, showing the number of records identified, included and excluded.\n\nClick here to access the data.\n\nSupplementary Table 1: Search strategy.\n\nClick here to access the data.\n\n\nReferences\n\nShen AK, Fields R, McQuestion M: The future of routine immunization in the developing world: challenges and opportunities. Glob Health Sci Pract. 2014; 2(4): 381–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMwenda J, Mihigo R, Tevi-Benissan C, et al.: Rotavirus disease burden in Africa and the need to accelerate introduction of vaccines. African Health Monitor. 2015; 19. 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PubMed Abstract | Publisher Full Text\n\nMeyer SA, Kambou JL, Cohn A, et al.: Serogroup A meningococcal conjugate (PsA-TT) vaccine coverage and measles vaccine coverage in Burkina Faso--implications for introduction of PsA-TT into the Expanded Programme on Immunization. Vaccine. 2015; 33(12): 1492–8. PubMed Abstract | Publisher Full Text\n\nTall H, Yaro S, Kpoda HB, et al.: Meningococcal Seroepidemiology 1 Year After the PsA-TT Mass Immunization Campaign in Burkina Faso. Clin Infect Dis. 2015; 61(Suppl 5): S540–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuquero FJ, Pham-Orsetti H, Cummings DA, et al.: A long-lasting measles epidemic in Maroua, Cameroon 2008–2009: mass vaccination as response to the epidemic. J Infect Dis. 2011; 204 Suppl 1: S243–51. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatagwa VN, Opio RO, Niwasasira DN, et al.: Acceptability of human papilloma virus vaccination among primary school girls in Minakulu sub-county, northern Uganda. Euro J Cancer Prev. 2014; 23(4): 294–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBagonza J, Rutebemberwa E, Mugaga M, et al.: Yellow fever vaccination coverage following massive emergency immunization campaigns in rural Uganda, May 2011: a community cluster survey. BMC Pub Health. 2013; 13: 202. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOuatara S, Faye A, Leye MM, et al.: [Study of the immunization coverage determinants of vaccination campaign against meningococcal A meningitis in Burkina Faso]. Rev Epidemiol Sante Publique. 2015; 63(6): 347–53. PubMed Abstract | Publisher Full Text\n\nLegros D, Paquet C, Perea W, et al.: Mass vaccination with a two-dose oral cholera vaccine in a refugee camp. Bull World Health Org. 1999; 77(10): 837–42. PubMed Abstract | Free Full Text\n\nSpiegel A, Greindl Y, Lippeveld T, et al.: [Effect of 2 vaccination strategies on developments during the epidemic of meningococcal A meningitis in N'Djamena (Chad) in 1988]. Bull World Health Organ. 1993; 71(3–4): 311–5. PubMed Abstract | Free Full Text\n\nQuentin W, Terris-Prestholt F, Changalucha J, et al.: Costs of delivering human papillomavirus vaccination to schoolgirls in Mwanza Region, Tanzania. BMC Med. 2012; 10: 137. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLevin CE, Van Minh H, Odaga J, et al.: Delivery cost of human papillomavirus vaccination of young adolescent girls in Peru, Uganda and Viet Nam. Bull World Health Organ. 2013; 91(8): 585–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVerguet S, Jassat W, Bertram M, et al.: Supplementary immunization activities (SIAs) in South Africa: comprehensive economic evaluation of an integrated child health delivery platform. Glob Health Action. 2013; 6(1): 20056. PubMed Abstract | Publisher Full Text\n\nZengbe-Acray P, Douba A, Traore Y, et al.: [Estimated operational costs of vaccination campaign to combat yellow fever in Abidjan]. Sante Publique. 2009; 21(4): 383–91. PubMed Abstract | Publisher Full Text\n\nWallace AS, Masresha BG, Grant G, et al.: Evaluation of economic costs of a measles outbreak and outbreak response activities in Keffa Zone, Ethiopia. Vaccine. 2014; 32(35): 4505–14. PubMed Abstract | Publisher Full Text\n\nSchaetti C, Weiss MG, Ali SM, et al.: Costs of illness due to cholera, costs of immunization and cost-effectiveness of an oral cholera mass vaccination campaign in Zanzibar. PLoS Negl Trop Dis. 2012; 6(10): e1844. 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}
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[
{
"id": "28681",
"date": "11 Dec 2017",
"name": "Rosemary J. Burnett",
"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:\n\nThis systematic review comparing SIAs to SBVs in Africa has been well designed and conducted, and the conclusions reached are well-founded. The manuscript can however be improved, with some minor editing. This includes deleting the repetition of phrases / words within a sentence / paragraph, as these make some sentences / paragraphs clumsy and increase the word count unnecessarily. Also, abbreviations must (a) only be introduced if they will be used again; (b) be introduced the first time the term is used and used only thereafter; and (c) never reintroduced. An edited version of the PDF has been sent to the supervisor to assist with improving and shortening the manuscript.\n\nSpecific comments:\n\nIntroduction:\n\nPage 3, paragraph 3: The third point does not adequately explain the issue. The shift in age at infection has occurred because of the introduction of the MMR into the infant vaccination schedule followed by low coverage. Is this a problem in Africa? Surely not, since the MMR is not widely used. The third point here should thus be about vaccines needed before girls become sexually active, and should include both HPV and rubella.\n\nPage 3, paragraph 3, last sentence: School-based vaccinations are offered through structured vaccination delivery programs, but this sentence makes it sound as if they are not.\n\nPage 3, paragraph 5, 2nd sentence: This suggests that in other regions, SBVs preceded SIAs, which is not the case. Page 3, paragraph 5, 3rd sentence: Reference?\n\nPage 3, paragraph 6: The aim should be unpacked into objectives to clarify exactly what aspects of effectiveness are being measured.\n\nMethods:\n\nPage 3, last part of study selection: The last 2 sentences are not unique to (4), so should start on a new line.\n\nPage 4, end of paragraph 1: Because the objectives have not been clearly stated, the last sentence is not clear.\n\nResults: In general, what is clearly presented in the tables and figures should not be repeated in the text, as this makes the results section repetitive and too long. Also, be consistent in the way the 95% CI is reported. Conventionally it is xx-xx%, but whichever format is used, use it consistently throughout. Page 5, 1st sentence under “risk of bias”: This sentence is part of the methods, not the results. Page 5, end of 3rd sentence in paragraph under risk of bias: Figure 2b is mentioned in the text before Figure 2a.\n\nPage 5, Table 1 and Table 2: The country where the study was conducted should be included in these tables. Page 6, Table 3: The country where the study was conducted should be included in this table. Page 6, Table 3: Vergeut et al, column on major expenses: Does the word “Personnel” include more than salaries? If not, then this should be “salaries” to be consistent.\n\nPage 6, Table 3: Legros et al, column on major expenses: Transport is mentioned as a minor cost in the text, but vaccine transport is listed as a major cost here. Or is the transport being referred to in the text, transport of personnel? Clarity is needed.\n\nPage 6, Table 3: Uzicanin et al, column on major expenses: In the text, vaccine administration is summarised as salaries. Page 6, Table 3: Schaetti et al, column on major expenses: The salary detail for international staff is given here, which immediately raises a question about whether or not staff are local or international in the other studies. Page 7, Figures 2a and 2b: More details in the captions would add clarity, eg: number of studies, followed by reference numbers. Page 11 paragraph 2: Can be summarised as personnel salaries being the second major cost driver. Also, transport of vaccines is listed in table 3 in the column on major costs. Or is this transport of personnel? Page 11 paragraph 3: In the text, vaccine administration is summarised as salaries. See comment about this in table 3. Terms need to be used consistently for clarity.\n\nPage 11, 1st paragraph under SIAs: Very clear in the table, thus unnecessary\n\nPage 11, 1st sentence under “low attendance”: Again, this is clear both from the table and the text below this sentence.\n\nPage 11, 2nd sentence under “redeployment of health staff”: Not stated in the table, and not clear if this was for both studies.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\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": "28889",
"date": "10 Jan 2018",
"name": "Eddy Bresnitz",
"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\nWell written report assessing the issue. Methods clearly described. Agree with general comments of the first reviewer. In addition to Dr. Burnett's editorial comments, I have several additional suggestions.\nOn page 4 in the Characteristics section, please clarify whether it's 31 or 32 studies reviewed.\n\nIn Tables 2 and 3, I suggest the column entitled \"Targeted/Vaccinated\" be inverted so that the numerator and denominator are in the appropriate relationship in the column.\n\nI find that the provision of costs in Table 3 is not really helpful to policy makers. As written, the text just describes facts but does not provide an interpretation of the implications for other programs. Also, I'm not sure comparing SIV programs that use different vaccines, in different countries, is very helpful either. The cost per immunized person is very contextual.\n\nClearly, the SBV programs are limited to HPV whereas the SIV studies are a mélange of vaccines and approached. Perhaps there can be more discussion of how this (and the related social issues) could skew 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": []
},
{
"id": "28617",
"date": "16 Jan 2018",
"name": "Ifedayo Adetifa",
"expertise": [
"Reviewer Expertise Paediatrics",
"infectious diseases",
"vaccines",
"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\nGeneral Comments This is a useful article that reviews two essential approaches to the delivery of vaccines in older children/age groups in Africa\n\nAbstract\nMethods\nplease list the electronic databases and add a line describing how quality assessment of retrieved articles.\n\nResults\nPlease include number of countries represented by the 32 studies included in the review\n\nConclusions\nThe authors say, ‘Both SIAs and SBVs are routinely used to complement…’. This is incorrect based on the results reported in this review. SIAs are definitely more commonly/routinely used not SBVs. While the assertion in the 2nd sentence is well known truth, authors have not provided any results in this review to support this.\n\nIntroduction\n2nd paragraph, 1st sentence- it is not entirely true that the immunisation gap results from the EPI not primarily targeting school aged children and adolescents. Gaps may result from poor coverage of childhood vaccinations, changing epidemiology, waning immunity requiring booster doses, etc. I suggest this paragraph should be revised to highlight the need to find suitable platforms reach older children/adolescents with vaccines primarily targeted at them, to address immunisation gaps and to deliver boosters. This is mentioned in the 3rd paragraph but can come earlier and merged with the 2nd paragraph 3rd paragraph, 4th sentence- The shift in susceptibility to older ages for rubella will be the consequence of introducing rubella vaccine with suboptimal coverage like is currently seen for measles in Africa and as already reported elsewhere. I suggest a revision here to make this clear given the setting for the review. 5th paragraph, 3rd sentence- HPV is not the main vaccine administered through SBVs in Africa. The few pilot HPV SBV do not compare to the use of SBV for MR, YF and MeningA campaigns. 5th paragraph, 4th sentence- is high coverage with SBVs a given considering its dependence on school enrollment? In the few cases where high coverage is reported, is the denominator population all school-age children or just enrolled children?\n\nMethods\nStudy selection, item 2- are school age children/adolescents the same as those enrolled? Should authors not consider differentiating between both or clarify that they mean the same thing for purposes of this review? Study selection, item 4- it is unclear why outcomes around cost of vaccine delivery were not extended to cost effectiveness if the data was reported in retrieved articles. In my opinion, not doing this is a missed opportunity to increase the relevance of results here to policy makers.\n\nThis will increase the relevance of results here to policy makers. The last sentence under study selection should be started as a new paragraph Quality assessment of included studies- as earlier I mentioned earlier, not including papers reporting cost effectiveness is a significant omission. Data synthesis and analyses-\nIt is my understanding that a complete case analyses what you do when you restrict analyses to complete datasets followed by imputation for missing values and analyses of datasets with imputed data for comparison? Please clarify It would be helpful to have some more description of how imputation was done and a summary of the extent of missingness so readers can assess the extent to which the results depend on imputed data. Why did authors use a 10% significance threshold? Are the categories of inconsistency arbitrary selections by authors or supported by a reference?\n\nResults\nLiterature search- Most of this text can be left out or shortened since all of this information is displayed in SF2. Characteristics of included study\n1st sentence is a repetition What is an epidemiological report? Last sentence page 4, are there 31 or 32 studies?\n\nRisk of bias and quality assessment- a table showing the quality rating for each included study would be helpful. Perhaps this table can replace figures 2a and 2b (move to supplementary file) or quality is reported in an additional column in tables 1-4 Comparison of vaccination coverage\nI think it is more useful to compare the SIA vs SBV for the same vaccines. Coverage for vaccines requiring multiple doses in SIA or SBV should not be compared to those requiring a single dose. There are also other sociocultural and religious issues associated with the uptake of HPV vaccination in particular. Figure 5 may be more useful if pooled coverage estimates by vaccine are included\n\nSubgroup analyses for vaccination coverage\nPlease revise the 2nd sentence “For SIAs, …”\n\nCost of vaccine delivery strategy\nSBVs-Some information about the unit cost of the vaccine would be useful. Also, whether the vaccine procurement was subsidized by GAVI or not. Summaries of the total economic cost per fully immunised child cannot be correct if economic costs do not include vaccine costs.\n\nFor the pooled coverage estimates, authors do not comment on heterogeneity/I-square results at all yet there is significant heterogeneity for many of the results. Is there an explanation for this?\n\nDiscussion\n1st paragraph, 2nd sentence- Pooled coverage estimates by vaccine is given in the results so beyond the caveat given for single vs. multiple dose vaccines, it would be useful to read authors’ opinion about the impact of heterogeneity studies in these estimates. 2nd paragraph-\nI am not sure I agree measles coverage by SIA should be described as high. As I alluded to earlier, HPV coverage in SBVs has to be interpreted with caution\n\n4th paragraph- I am not convinced, at least by the results presented here that SBVs are likely to be more cost-effective even with high school enrolment. Authors need to provide more data and vaccine delivery costs are not the same as cost effectiveness of the strategy Strength and limitations\nWhile it is good to see comments about heterogeneity, it is important to mention this in the results according to cut-offs given in the methods. While school enrolment in general is lower than it should be in Africa, one other limitation is data on school enrolment in the settings covered by included studies was not available\n\nImplication for policy and research\nI think the conclusion here regarding enrolment and effectiveness of SIAs compared to SBV is speculative. There is simply not enough data in this review especially for SBV to support this.\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? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1833
|
https://f1000research.com/articles/6-1831/v1
|
13 Oct 17
|
{
"type": "Research Article",
"title": "Nutritional and micronutrient status of adolescent schoolgirls in eastern Sudan: A cross-sectional study",
"authors": [
"Hyder M. Mahgoub",
"Omar E. Fadlelseed",
"Ammar H. Khamis",
"Jalal A. Bilal",
"Ishag Adam",
"Hyder M. Mahgoub",
"Omar E. Fadlelseed",
"Ammar H. Khamis",
"Jalal A. Bilal"
],
"abstract": "Background: Adolescents, especially girls, are susceptible to malnutrition and their diet must be adequate to support their very rapid growth and development. Currently, there is little published data on the nutritional state amongst adolescent girls in Sudan. Methods: A cross sectional study was conducted to assess the nutritional and micronutrient status of adolescent schoolgirls in eastern Sudan during the period of January-February 2015. Weight and height were measured using standard methods. Haemoglobin and ferritin levels were measured using blood samples, and blood films for malaria and stool samples for Schistosoma mansoni were investigated. Nutritional status was assessed according to the WHO 2006 reference values. Copper and zinc concentrations were measured by atomic absorption. Results: Twenty-five (13.7%) out of 183 girls were stunted. Seventy (38.3%) were thin; 17.5, 9.3 and 11.5% had mild, moderate and severe thinness, respectively. Only 10 (5.5%) and six (3.3%) girls were overweight and obese, respectively. The prevalence of anaemia was 77.0%. While there was no significant difference in the haemoglobin, ferritin, copper levels and thinness; thin children had significantly lower zinc (P=0.007). Conclusions: There is a high rate of stunting, thinness and anaemia among adolescent schoolgirls in eastern Sudan. More care has to be taken in order to provide a better nutrition status in the area.",
"keywords": [
"adolescent",
"anthropometric",
"micronutrient",
"schoolgirls",
"Sudan"
],
"content": "Introduction\n\nAdolescents (individuals aged 10 to 19 years) make up approximately one fifth of the world’s population1. During adolescence there is an increase in nutrient demand that is needed for intense growth, where individuals can gain 15% of their ultimate adult height and half of their adult weight2–4. Adolescents are vulnerable groups and their diet must be adequate to support their very rapid growth and development5. Well-nourished adolescents can have optimum cognitive and learning skills, and energies, as well as be healthy for future parenthood1 Unfortunately, the required optimum nutrition remains unmet for a vast numbers of adolescents, who are thus unable to achieve their full genetic developmental potential2. In particular, adolescents girls in developing countries are at risk, since they may become pregnant at an early age and have a greater risk of pregnancy-associated morbidity and mortality6,7. Furthermore, malnourished adolescent girls are at a higher risk of being stunted mothers, who are likely to suffer obstetric complications, such as delivering low birth weight babies8.\n\nResearch on the nutritional status of adolescent girls is of paramount importance to health planners, as well as to practicing physicians. There is little published data on the nutritional status of adolescent girls in countries with few resources, including Sudan, where malnutrition is a major health problem9–14. Consequently, the current study was conducted to assess the nutritional and micronutrient status of adolescent schoolgirls in eastern Sudan.\n\n\nMethods\n\nA cross-sectional study was conducted in the New Halfa area in eastern Sudan during the period of January–February 2015. Adolescent schoolgirls aged from 11 to 18 years were selected through a two-stage random (using computer generated number) cluster sample of adolescent girls attending three primary schools (AlHara Aloula, Tania and AlHara Althalta) in the area. Stage one, simple random sampling of the schools to randomly identify classes within the specific age range; stage two, random sampling of the class in identify individuals.\n\nThose with chronic illnesses were excluded.\n\nA sample size of 183 subjects was calculated using the assumption of the prevalence of stunting (9.45%) and thinness (23.7%) that was recently reported among school children in Northern Sudan9. This sample would provide 80% power to detect a 5% difference at α = 0.05, with an assumption that complete data might not be available for 10% of participants.\n\nNutritional status. Age of the adolescent schoolgirls was taken and double checked with that in the school records, which had been completed using the birth certificate.\n\nWeight was measured using a digital scale to the nearest 0.1 kg. Height was measured using a stadiometer with a moveable headboard to the nearest 0.1 cm, while the participant was barefoot. Body mass index (BMI) was computed as weight (kg) divided by the square of height (m2).\n\nHeight-for-age (HAZ) and BMI-for-age z-scores (BAZ) were calculated according to the WHO reference15. Stunting was defined by HAZ <−2 z-scores. Overweight was defined by BAZ between 1 z-scores and 2 z-scores, and obesity by BAZ >2 z-scores. The WHO defines mild, moderate and severe thinness by z-scores: −2, −1; −3, −2; and <−3, respectively15\n\nMicronutrient status. Venous blood (5 mls) was collected from each participant and allowed to clot in plain tubes, and serum was stored at −20°C until analysed in the laboratory in Khartoum for measurement of serum ferritin, copper and zinc. Concentrations of ferritin were determined by immunofluorescent assay using IMMULITE1000 (SIEMENS, CA, USA), according to the manufacturer’s instructions.\n\nCopper and zinc concentrations were measured by atomic absorption spectrophotometry (SOLAAR 2.0, atomic absorption spectrophotometer (iCE 3000), Thermo Fisher Scientific, Cambridge, UK, 3000 Serie), according to the manufacturer’s instructions.\n\nHaemoglobin level was measured by HemoCue haemoglobinometer (HemoCue AB, Ängelhom, Sweden), according to the manufacturer’s instructions. Anaemia and severe anaemia were defined as haemoglobin <12 and <8 /dl g/dl, respectively. Iron deficiency was defined as serum ferritin <12 μg/l; iron deficiency anaemia as haemoglobin <12 g/dl and s-ferritin<12 μg/l.\n\nParasite status. Blood films for malaria (Plasmodium falciparum) were prepared and Giemsa-stained.\n\nSchistosoma mansoni infection was investigated in a single stool sample that was collected from each participant, and a Kato-Katz slide prepared and used to determine the infection intensity, if any16.\n\nData were entered using EpiData 3.4 and then exported to SPSS 20 for analysis. Anthropometric indices (HAZ and BAZ) were calculated using WHO child growth references for Z score15. Haemoglobin, ferritin, zinc and copper were tested for normality using Kolmogorov –Smirnov test. Mann-Whitney and Kruskal –Wallis tests were used to compare the continuous non-parametric data between two and more than two groups, respectively. Age was normally distributed and compared between two and more than two groups with t-test and ANOVA, respectively. Spearman’s (non-parametric) correlation was used to investigate the correlations between the different variables. A P value of < 0.05 was considered significant.\n\nThe study received ethical clearance from the Research Board at the Faculty of Medicine University of Khartoum (approval# 2012,18). After explaining the purpose of the study, written permission to perform the study was obtained from the local health and education office (New Halfa Head Office for Education). Written informed consent was obtained from the parents/guardians of the school girls before data collection.\n\n\nResults\n\nTwo hundred adolescent schoolgirls were initially screened; 183 had complete data and were analysed. The nutritional status of the girls is shown in Table 1. Twenty-five (13.7%) out of the 183 girls were stunted. Seventy (38.3%) were thin, 17.5%, 9.3% and 11.5% had mild, moderate and severe thinness, respectively. Only 10 (5.5%) and six (3.3%) children were overweight and obese, respectively. The prevalence of anaemia, iron deficiency and iron deficiency anaemia was 77.0%, 18.0% and 17.5% respectively. Four children (2.2%) had severe anaemia.\n\nThere was no significant difference in the mean (SD) age between girls with a normal anthropometric status (14.2 [1.3] years), and girls who had mild (13.7 [0.9] years), moderate (13.4 [1.3] years) and severe thinness (13.8 [1.3] years), overweight (13.7 [1.4] years) and obese (13.6 [1.2] years) girls (P=0.161). Likewise the mean (SD) age was not significantly different between stunted and non-stunted girls (14.2 [1.0] vs. 13.9 [1.3] years; P=0.318).\n\nOnly two girls had P. falciparum infections. Eleven girls had S. mansoni infection, but the rate was not significantly different between the stunted and non-stunted group (3/25 (12.0%) vs. 8/158 (5.1%); P=0.167).\n\nWhile there was no significant difference in the haemoglobin, ferritin, copper levels and thinness, thin children had a significantly lower zinc level compared to normal, overweight and obese children (P=0.007; Table 2). No significant difference was found between age, haemoglobin and micronutrient levels, and stunting (Table 3).\n\nValues show the median (interquartile range).\n\nValues show the median (interquartile range).\n\nWhile there was no correlation between BMI, height, ferritin, zinc and copper, a significant positive correlation was found between haemoglobin, zinc and copper (Table 4).\n\n\nDiscussion\n\nThe current study found a high prevalence of stunting (13.7%), thinness (38.3%) and anaemia (77.0%) among adolescent schoolgirls in this setting. This supports the findings of a previous study where a high prevalence of underweight (41.0%) and stunting (21.4%) was reported among primary school children in Khartoum, Capital of Sudan17. These two studies should be compared with caution because of the different study techniques used. Unlike the WHO standard used in the current study, weight-for-age, HAZ and skin-fold thickness of triceps muscle was used by the later study. Recently, Sarar and Diab reported9 a lower prevalence of stunting (9.4%), thinness (23.1%), and anaemia (29.7%) among 835 school-aged children in Northern Sudan.\n\nThe overall prevalence of thinness (38.3%) in the current study was much higher than reports of studies conducted in other neighbouring African countries, e.g. Ethiopia (1.4-8.9%)18, Kenya (4.5%)19 and Uganda (10.1%)20.\n\nWasting, stunting and thinness are indicators of acute nutritional deficiency. Malnutrition in all age groups is caused by inadequate food intake or poor food utilization caused by infections15,21. Six (3.3%) adolescent girls in the current study were obese. Being overweight and obesity have been reported among 10.7% of urban Sudanese adolescents22.\n\nThough there was no significant difference in haemoglobin, ferritin, and copper levels, a significantly lower zinc level among thin adolescent girls was found compared with girls with normal BMIs in the current study. Contrary to our findings, Bemnet and colleagues showed no association between zinc and HAZ. However, they observed a significant positive correlation between HAZ and copper levels18. Both zinc and copper have an important role in growth, and participate in numerous enzyme systems23. Zinc deficiency is common in developing countries and can delay linear growth24,25. Unfortunately, recent research failed to show any benefit of zinc supplement in decreasing stunting in Malawian children26.\n\nIn this study, we aimed to assess the nutritional and micronutrient status of adolescent schoolgirls in eastern Sudan. A limitation of this study was that adolescent boys were not investigated, which might have under-estimated the rate of malnutrition among adolescents (both males and females), as adolescent girls have been shown to be less likely to be stunted than boys20,27. In addition, only schoolgirls were investigated, which might have missed the more vulnerable group of adolescent girls who do not attend school. Another limitation of the current study was the lack of puberty history, since the onset of the menarche might have effects on the nutritional status of the adolescent girls. Moreover, there was a lack of dietary intake history, since physical growth of adolescent girls is generally related to their dietary intake. Another point that should be remembered is the lack of investigation of acute inflammatory markers, e.g. C-reactive protein, which has been to be associated with anaemia in eastern Sudan28\n\n\nConclusions\n\nThere is a high rate of stunting, thinness and anaemia among adolescent schoolgirls in eastern Sudan. More care has to be taken in order to provide a better nutrition status in the area.\n\n\nData availability\n\nDataset 1: Raw data collected as the basis for this study. doi, 10.5256/f1000research.12721.d18082429",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWorld Health Organization: Adolescent nutrition: a review of the situation in selected South-East Asian countries. 2006, [cited 2016 Nov 12]. Reference Source\n\nCordeiro L, Lamstein S, Mahmud Z, et al.: Adolescent malnutrition in developing countries a close look at the problem and at two national experiences. United Nations SCN. 2005, [cited 2016 Nov 12]. Reference Source\n\nDiMeglio G: Nutrition in adolescence. Pediatr Rev. 2000; 21(1): 32–3. PubMed Abstract\n\nDoustmohammadian A Jr, Dorostymotlagh AR, Keshavarz A, et al.: Socio-demographic Factors Associated with Body Mass Index of Female Adolescent Students in Semnan City, Iran. Malays J Nutr. 2009; 15(1): 27–35. PubMed Abstract\n\nSpear BA: Adolescent growth and development. J Am Diet Assoc. 2002; 102(3 Suppl): S23–9. PubMed Abstract\n\nWorld Health Organization: Programming for adolescent health and development: report of a WHO/UNFPA/UNICEF Study Group on Programming for Adolescent Health. 1999, [cited 2016 Nov 12]. Reference Source\n\nAdam GK, Elhassan EM, Ahmed AM, et al.: Maternal and perinatal outcome in teenage pregnancies in Sudan. Int J Gynecol Obstet. 2009; 105(2): 170–1. PubMed Abstract | Publisher Full Text\n\nThame M, Wilks RJ, McFarlane-Anderson N, et al.: Relationship between maternal nutritional status and infant’s weight and body proportions at birth. Eur J Clin Nutr. 1997; 51(3): 134–8. PubMed Abstract\n\nMohamed S, Hussein MD: Prevalence of Thinness, Stunting and Anemia Among Rural School-aged Sudanese Children: A Cross-sectional Study. J Trop Pediatr. 2015; 61(4): 260–5. PubMed Abstract | Publisher Full Text\n\nMahgoub HM, Adam I: Morbidity and mortality of severe malnutrition among Sudanese children in New Halfa Hospital, Eastern Sudan. Trans R Soc Trop Med Hyg. 2012; 106(1): 66–8. PubMed Abstract | Publisher Full Text\n\nAbdelrahim II, Mahgoub HM, Mohamed AA, et al.: Anaemia, folate, zinc and copper deficiencies among adolescent schoolgirls in eastern Sudan. Biol Trace Elem Res. 2009; 132(1–3): 60–6. PubMed Abstract | Publisher Full Text\n\nSeal A: Adopting the WHO Growth Standards for Diagnosis and Classification of Acute Malnutrition in Emergencies: An Assessment of Resource Implications. Growth (Lakeland). 2006.\n\nFiorentino M, Bastard G, Sembène M, et al.: Anthropometric and micronutrient status of school-children in an urban West Africa setting: A cross-sectional study in Dakar (Senegal). PLoS One. 2013; 8(12): e84328. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTeji K, Dessie Y, Assebe T, et al.: Anaemia and nutritional status of adolescent girls in Babile District, Eastern Ethiopia. Pan Afr Med J. 2016; 24: 62. PubMed Abstract | Free Full Text\n\nde Onis M, Onyango AW, Borghi E, et al.: Development of a WHO growth reference for school-aged children and adolescents. Bull World Health Organ. 2007; 85(9): 660–7. PubMed Abstract | Free Full Text\n\nWHO: Basic Laboratory Methods in Medical Parasitology. Geneva, Switzerland: World Health Organization; Google Scholar, 1991. Reference Source\n\nNabag F: Comparative Study of Nutritional Status of Urban and Rural School Girl’s Children Khartoum State, Sudan. J Sci Technol. 2011, [cited 2016 Nov 13]. Reference Source\n\nAmare B, Moges B, Fantahun B, et al.: Micronutrient levels and nutritional status of school children living in Northwest Ethiopia. Nutr J. 2012; 11(1): 108. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChesire EJ, Orago AS, Oteba LP, et al.: Determinants of under nutrition among school age children in a Nairobi peri-urban slum. East Afr Med J. 2008; 85(10): 471–9. PubMed Abstract | Publisher Full Text\n\nAcham H, Kikafunda J, Tylleskar T: Nutritional and health status primary schoolchildren in rural Uganda. African J Food. 2012, [cited 2016 Nov 12]. Reference Source\n\nFriis H, Michaelsen KF, Wells JC: Choice of design and outcomes in trials among children with moderate acute malnutrition. Food Nutr Bull. 2015; 36(1 Suppl): S35–40. PubMed Abstract | Publisher Full Text\n\nMusaiger AO, Nabag FO, Al-Mannai M: Obesity, Dietary Habits, and Sedentary Behaviors Among Adolescents in Sudan: Alarming Risk Factors for Chronic Diseases in a Poor Country. Food Nutr Bull. 2016; 37(1): 65–72. PubMed Abstract | Publisher Full Text\n\nSolomons NW: Update on zinc biology. Ann Nutr Metab. 2013; 62 Suppl 1: 8–17. PubMed Abstract | Publisher Full Text\n\nKrebs NF, Miller LV, Hambidge KM: Zinc deficiency in infants and children: a review of its complex and synergistic interactions. Paediatr Int Child Health. 2014; 34(4): 279–88. PubMed Abstract | Publisher Full Text\n\nKrebs NF: Update on zinc deficiency and excess in clinical pediatric practice. Ann Nutr Metab. 2013; 62 Suppl 1: 19–29. PubMed Abstract | Publisher Full Text\n\nWang AZ, Shulman RJ, Crocker AH, et al.: A Combined Intervention of Zinc, Multiple Micronutrients, and Albendazole Does Not Ameliorate Environmental Enteric Dysfunction or Stunting in Rural Malawian Children in a Double-Blind Randomized Controlled Trial. J Nutr. 2017; 147(1): 97–103. PubMed Abstract | Publisher Full Text\n\nHioui M, Azzaoui F, Ahami A: Nutritional Status and School Achievements in a Rural Area of Anti-Atlas, Morocco. Food Nutr. 2011; 2(8): 7890. Publisher Full Text\n\nMohamed AA, Ali AA, Ali NI, et al.: Zinc, parity, infection, and severe anemia among pregnant women in Kassla, eastern Sudan. Biol Trace Elem Res. 2011; 140(3): 284–90. PubMed Abstract | Publisher Full Text\n\nMahgoub HM, Fadlelseed OE, Khamis AH, et al.: Dataset 1 in: Nutritional and micronutrient status of adolescent schoolgirls in eastern Sudan: A cross-sectional study. F1000Research. 2017. Data Source"
}
|
[
{
"id": "27612",
"date": "13 Nov 2017",
"name": "Youssef Aboussaleh",
"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\nMany parts of the report need good English editing and avoidance of long sentences.\n\nThe discussion does not report comparisons between anaemic and non anaemic in terms of anthropometry. Nor is there any emphasis on iron deficiency without anaemia.\n\nAre there any interactions between iron, zinc and cooper?, needs speculating!\n\nMay be above the scope of this study but no data is reported on food intake or at least food behaviour in terms of frequency (skipping breakfast for example?).\n\nThe parasite status is not brought significantly in the results with their interaction with micronutrient status. They are also omitted totally in the discussion.\n\nThe conclusion is very weak and general. It needs to reports the main contributions of this 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? Partly",
"responses": []
},
{
"id": "29436",
"date": "08 Jan 2018",
"name": "David Z. Munisi",
"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\nLine 8, you write “energies”. There is no plural form of energy, so this should be written energy.\n\nMETHODS\nTo make the section clearer and easy to follow, study design, study population inclusion and exclusion criteria, sample size and sampling procedures should be written under separate subheadings. You wrote “.........through a two-stage random cluster sample of adolescent girls attending three primary schools in the area.” Use the word sampling to indicate that it is a procedure. So you can instead write “Two stage cluster random sampling”. You have only mentioned exclusion criteria with no inclusion criteria mentioned. Kindly provide inclusion criteria as well. You have not mentioned which formula or statistical package you used to calculate the sample size using the values you have supplied. Kindly supply this information to make your methodology section easy to follow.\nData collection Nutritional status:\nYou mention that “Age of the adolescent schoolgirls was taken....” without stating it was taken from where; kindly state. .......“The WHO defines mild, moderate and severe thinness by z-scores: −2, −1; −3, −2; and <−3, respectively”....Kindly state which Z-scores are you referring to here. To state thinness is not enough to make everyone understand which z-scores you are referring to.\nMicronutrient status:\n\"serum was stored at −20°C until analysed in the laboratory in Khartoum for measurement of serum ferritin, copper and zinc.”. Kindly supply the specific name of the laboratory in which this analysis was done. Please provide the reference for the cut-off points for anemia that you used in your study.\n\nParasite status: ...Change this to “Parasite infection status”\n...\"Blood films for malaria (Plasmodium falciparum)”.... Why have you specifically mentioned Plasmodium falciparum here? State which blood smear did you prepare? Why did you only asses Schistosoma mansoni infection and ignored other intestinal nematodes (Soil transmitted helminths in particular i.e. Hookworms, Trichuris trichura and Ascaris lumbricoides) which are well known to significantly affect nutritional status and haemoglobin levels?\n\nRESULTS Table 1:\nStunting rate doesn’t appear in this table. To make this table more useful, you can redraw by including nutritional status against school/or grade.\nParagraph two\nIn the paragraph two of the results section, you report findings after comparing means, but it is not clear which test you used to compare the means. Please indicate this in a tabular form and write its summary in the text referring to the table. Tables make it easier for the reader to understand your analysis and findings.\nTable 2 and 3\nUnder each of the table in which you indicate a p-value, indicate as a footnote the test on which the reported p-values are based.\nTable 4\nAs a footnote indicate what ‘r’ stands for.\n\nDISCUSSION\nYou may need to start your discussion with a brief statement as to why it was important to conduct this study where it was conducted. In paragraph three you claim that wasting, stunting and thinness are indicators of acute nutritional insult. Kindly revisit the literature with a particular attention on stunting, then rephrase accordingly. “.........C-reactive protein, which has been to be associated with anaemia in eastern Sudan”..It appears there is a missing word between the word been....and to.\n\nCONCLUSION\nThe conclusion is very insufficient. Beef up your conclusion by reflecting on what you have found in your 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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1831
|
https://f1000research.com/articles/4-621/v1
|
25 Aug 15
|
{
"type": "Opinion Article",
"title": "Null hypothesis significance testing: a short tutorial",
"authors": [
"Cyril Pernet"
],
"abstract": "Although thoroughly criticized, null hypothesis significance testing (NHST) is the statistical method of choice in biological, biomedical and social sciences to investigate if an effect is likely. In this short tutorial, I first summarize the concepts behind the method while pointing to common interpretation errors. I then present the related concepts of confidence intervals, and discuss what should be reported in which context. The goal is to clarify concepts, present statistical issues that researchers face using the NHST framework and propose reporting practices.",
"keywords": [
"null hypothesis significance testing",
"tutorial",
"p-value",
"reporting"
],
"content": "The Null Hypothesis Significance Testing framework\n\nNull hypothesis significance testing (NHST) is a method of statistical inference by which an observation is tested against a hypothesis of no effect or no relationship. The method as practiced nowadays is a combination of the concepts of critical rejection regions developed by Neyman & Pearson (1933) and the p-value developed by Fisher (1959).\n\nThe method developed by Fisher (1959) allows computation of the probability of observing a result at least as extreme as a test statistic (e.g. t value), assuming the null hypothesis is true. This p-value thus reflects the conditional probability of achieving the observed outcome or larger, p(Obs|H0), and is equal to the area under the null probability distribution curve, for example [-∞ -t] and [+t +∞] for a two-tailed t-test (Turkheimer et al., 2004). Following Fisher, the smaller the p-value, the greater the likelihood that the null hypothesis is false. This was however only intended to be used as an indication that there is something in the data that deserves further investigation. The reason for this is that only H0 is tested whilst the effect under study is not itself being investigated.\n\nThe p-value is not the probability of the null hypothesis of being true, p(H0) (Krzywinski & Altman, 2013). This common misconception arises from confusion between the probability of an observation given the null p(Obs|H0) and the probability of the null given an observation p(H0|Obs) (see Nickerson (2000) for a detailed demonstration). The p-value is not an indication of the strength or magnitude of an effect. Any interpretation of the p-value in relation to the effect under study (strength, reliability, probability) is indeed wrong, since the p-value is conditioned on H0. Similarly, 1-p is not the probability to replicate an effect. Often, a small value of p is considered to mean a strong likelihood of getting the same results on another try, but again this cannot be ascertained from the p-value because it is not informative on the effect itself (Miller, 2009). If there is no effect, we should replicate the absence of effect with a probability equal to 1-p. The total probability of false positives can also be obtained by aggregating results (Ioannidis, 2005). If there is an effect however, the probability to replicate is a function of the (unknown) population effect size with no good way to know this from a single experiment (Killeen, 2005). Finally, a (small) p-value is not an indication favouring a hypothesis. A low p-value indicates a misfit of the null hypothesis to the data and cannot be taken as evidence in favour of a specific alternative hypothesis more than any other possible alternatives such as measurement error and selection bias (Gelman, 2013). The more (a priori) implausible the alternative hypothesis, the greater the chance that a finding is a false alarm (Krzywinski & Altman, 2013; Nuzzo, 2014). As Nickerson (2000) puts it ‘theory corroboration requires the testing of multiple predictions because the chance of getting statistically significant results for the wrong reasons in any given case is high’.\n\nNeyman & Pearson (1933) introduced the notion of critical intervals over which the probability of observing a test statistic is less than a stipulated significance level, α. If the statistic value falls within those intervals, it is deemed significantly different from that expected under the null hypothesis. For instance, we can estimate that the probability of a given F value to be in the critical interval [+2 +∞] is less than 5%. Because the space of results is dichotomized, we can distinguish correct results (rejecting H0 when there is an effect and not rejecting H0 when there is no effect) from errors (rejecting H0 when there is no effect and not rejecting H0 when there is an effect). When there is no effect (H0 is true), the erroneous rejection of H0 is known as type I error and is equal to the p-value.\n\nThe significance level α is defined to be the maximum probability that a test statistic falls into the rejection region when the null hypothesis is true (Johnson, 2013). When the test statistics falls outside the critical region(s), all we can say is that no significant effect is observed, but one cannot conclude that the null hypothesis is true, i.e. we cannot accept H0. There is a profound difference between accepting the null hypothesis and simply failing to reject it (Killeen, 2005). By failing to reject, we simply continue to assume that H0 is true, which implies that one cannot, from a non-significant result, argue against a theory. We cannot accept the null hypothesis, because all we have done is not disprove it. To accept or reject equally the null hypothesis, Bayesian approaches (Dienes, 2014; Kruschke, 2011) or confidence intervals must be used.\n\nConfidence intervals (CI) have been advocated as alternatives to p-values because (i) they allow judging the statistical significance and (ii) they provide estimates of effect size. CI fail to cover the true value at a rate of alpha, the type I error rate (Morey & Rouder, 2011) and therefore indicate if values can be rejected by a two tailed test with a given alpha. CI also indicates the precision of the estimate of effect size, but unless using a percentile bootstrap approach, they require assumptions about distributions which can lead to serious biases in particular regarding the symmetry and width of the intervals (Wilcox, 2012). Assuming the CI (a)symmetry and width are correct, this gives some indication about the likelihood that a similar value can be observed in future studies, with 95% CI giving about 83% chance of replication success (Lakens & Evers, 2014). Finally, contrary to p-values, CI can be used to accept H0. Typically, if a CI includes 0, we cannot reject H0. If a critical null region is specified rather than a single point estimate, for instance [-2 +2] and the CI is included within the critical null region, then H0 can be accepted. Importantly, the critical region must be specified a priori and cannot be determined from the data themselves.\n\nAlthough CI provide more information, they are not less subject to interpretation errors (see Savalei & Dunn, 2015 for a review). People often interpret X% CI as the probability that a parameter (e.g. the mean) will fall into that interval X% of the time. The (posterior) probability of an effect can however not be obtained using a frequentist framework. The CI represents the bounds for which one has X% confidence. The correct interpretation is that, for repeated measurements with the same sample sizes, taken from the same population, X% of times the CI obtained will contain the same parameter value, e.g. X% of the times the CI contains the same mean (Tan & Tan, 2010). The alpha value has the same interpretation as when using H0, i.e. we accept that 1-alpha CI are wrong in alpha percent of the times. This implies that CI do not allow to make strong statements about the parameter of interest (e.g. the mean difference) or about H1 (Hoekstra et al., 2014). To make a statement about the probability of a parameter of interest, likelihood intervals (maximum likelihood) and credibility intervals (Bayes) are better suited.\n\n\nThe (correct) use of NHST\n\nNHST has always been criticized, and yet is still used every day in scientific reports (Nickerson, 2000). Many of the disagreements are not on the method itself but on its use. The question one should ask is what is the goal of a scientific experiment at hand? If the goal is to establish the likelihood of an effect and/or establish a pattern of order, because both requires ruling out equivalence, then NHST is a good tool (Frick, 1996). If the goal is to establish some quantitative values, then NHST is not the method of choice. Because results are conditioned on H0, NHST cannot be used to establish beliefs. To estimate the probability of a hypothesis, a Bayesian analysis is a better alternative. To estimate parameters (point estimates and variances), alternative approaches are also better suited. Note however that even when a specific quantitative prediction from a hypothesis is shown to be true (typically testing H1 using Bayes), it does not prove the hypothesis itself, it only adds to its plausibility.\n\n\nWhat to report and how?\n\nConsidering that quantitative reports will always have more information content than binary (significant or not) reports, we can always argue that effect size, power, etc. must be reported. Reporting everything can however hinder the communication of the main result(s), and we should aim at giving only the information needed, at least in the core of a manuscript. Here I propose to adopt minimal reporting in the result section to keep the message clear, but have detailed supplementary material. When the hypothesis is about the presence/absence or order of an effect, it is sufficient to report in the text the actual p-value since it conveys the information needed to rule out equivalence. When the hypothesis and/or the discussion involve some quantitative value, and because p-values do not inform on the effect, it is essential to report on effect sizes (Lakens, 2013), preferably accompanied with confidence, likelihood or credible intervals depending on the question at hand. The reasoning is simply that one cannot predict and/or discuss quantities without accounting for variability. For the reader to understand and fully appreciate the results, nothing else is needed.\n\nBecause science progress is obtained by cumulating evidence (Rosenthal, 1991), scientists should also consider the secondary use of the data. With today’s electronic articles, there are no reasons for not including all of derived data: mean, standard deviations, effect size, CI, Bayes factor should always be included as supplementary tables (or even better also share raw data). It is also essential to report the context in which tests were performed – that is to report all of the tests performed (all t, F, and p values) because of the increase in type I error rate due to selective reporting (multiple comparisons problem - Ioannidis, 2005). Providing all of this information allows (i) other researchers to directly and effectively compare their results in quantitative terms (replication of effects beyond significance, Open Science Collaboration, 2015), (ii) to compute power to future studies (Lakens & Evers, 2014), and (iii) to aggregate results for meta-analyses.",
"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\nDienes Z: Using Bayes to get the most out of non-significant results. Front Psychol. 2014; 5: 781. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFisher RA: Statistical methods and scientific inference. (2nd ed.). New-York: Hafner Publishing, 1959. Reference Source\n\nFrick RW: The appropriate use of null hypothesis testing. Psychol Methods. 1996; 1(4): 379–390. Publisher Full Text\n\nGelman A: P values and statistical practice. Epidemiology. 2013; 24(1): 69–72. PubMed Abstract | Publisher Full Text\n\nHoekstra R, Morey RD, Rouder JN, et al.: Robust misinterpretation of confidence intervals. Psychon Bull Rev. 2014; 21(5): 1157–1164. PubMed Abstract | Publisher Full Text\n\nIoannidis JP: Why most published research findings are false. PLoS Med. 2005; 2(8): e124. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson VE: Revised standards for statistical evidence. Proc Natl Acad Sci U S A. 2013; 110(48): 19313–19317. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKilleen PR: An alternative to null-hypothesis significance tests. Psychol Sci. 2005; 16(5): 345–353. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKruschke JK: Bayesian Assessment of Null Values Via Parameter Estimation and Model Comparison. Perspect Psychol Sci. 2011; 6(3): 299–312. PubMed Abstract | Publisher Full Text\n\nKrzywinski M, Altman N: Points of significance: Significance, P values and t-tests. Nat Methods. 2013; 10(11): 1041–1042. PubMed Abstract | Publisher Full Text\n\nLakens D: Calculating and reporting effect sizes to facilitate cumulative science: a practical primer for t-tests and ANOVAs. Front Psychol. 2013; 4. 863. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLakens D, Evers ER: Sailing From the Seas of Chaos Into the Corridor of Stability: Practical Recommendations to Increase the Informational Value of Studies. Perspect Psychol Sci. 2014; 9(3): 278–292. PubMed Abstract | Publisher Full Text\n\nMiller J: What is the probability of replicating a statistically significant effect? Psychon Bull Rev. 2009; 16(4): 617–640. PubMed Abstract | Publisher Full Text\n\nMorey RD, Rouder JN: Bayes factor approaches for testing interval null hypotheses. Psychol Methods. 2011; 16(4): 406–419. PubMed Abstract | Publisher Full Text\n\nNeyman J, Pearson ES: On the problem of the most efficient tests of statistical hypotheses. Philos Trans R Soc Lond Ser A. 1933; 231(694–706): 289–337. Publisher Full Text\n\nNickerson RS: Null hypothesis significance testing: a review of an old and continuing controversy. Psychol Methods. 2000; 5(2): 241–301. PubMed Abstract | Publisher Full Text\n\nNuzzo R: Scientific method: statistical errors. Nature. 2014; 506(7487): 150–152. PubMed Abstract | Publisher Full Text\n\nOpen Science Collaboration. Estimating the reproducibility of psychological science. Science. in press. 2015. Reference Source\n\nRosenthal R: Cumulating psychology: an appreciation of Donald T. Campbell. Psychol Sci. 1991; 2(4): 213–221. Publisher Full Text\n\nSavalei V, Dunn E: Is the call to abandon p-values the red herring of the replicability crisis? Front Psychol. 2015; 6: 245. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan SH, Tan SB: The Correct Interpretation of Confidence Intervals. Proceedings of Singapore Healthcare. 2010; 19(3): 276–278. Publisher Full Text\n\nTurkheimer FE, Aston JA, Cunningham VJ: On the logic of hypothesis testing in functional imaging. Eur J Nucl Med Mol Imaging. 2004; 31(5): 725–732. PubMed Abstract | Publisher Full Text\n\nWilcox R: Introduction to Robust Estimation and Hypothesis Testing. (3rd ed.). Oxford, UK: Academic Press, Elsevier, 2012. Reference Source"
}
|
[
{
"id": "10159",
"date": "30 Oct 2015",
"name": "Daniel Lakens",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI appreciate the author's attempt to write a short tutorial on NHST. Many people don't know how to use it, so attempts to educate people are always worthwhile. However, I don't think the current article reaches it's aim. For one, I think it might be practically impossible to explain a lot in such an ultra short paper - every section would require more than 2 pages to explain, and there are many sections. Furthermore, there are some excellent overviews, which, although more extensive, are also much clearer (e.g., Nickerson, 2000). Finally, I found many statements to be unclear, and perhaps even incorrect (noted below). Because there is nothing worse than creating more confusion on such a topic, I have extremely high standards before I think such a short primer should be indexed. I note some examples of unclear or incorrect statements below. I'm sorry I can't make a more positive recommendation.“investigate if an effect is likely” – ambiguous statement. I think you mean, whether the observed DATA is probable, assuming there is no effect?The Fisher (1959) reference is not correct – Fischer developed his method much earlier.“This p-value thus reflects the conditional probability of achieving the observed outcome or larger, p(Obs|H0)” – please add 'assuming the null-hypothesis is true'.“p(Obs|H0)” – explain this notation for novices.“Following Fisher, the smaller the p-value, the greater the likelihood that the null hypothesis is false.” This is wrong, and any statement about this needs to be much more precise. I would suggest direct quotes.“there is something in the data that deserves further investigation” –unclear sentence.“The reason for this” – unclear what ‘this’ refers to.“not the probability of the null hypothesis of being true, p(H0)” – second of can be removed?“Any interpretation of the p-value in relation to the effect under study (strength, reliability, probability) is indeedwrong, since the p-value is conditioned on H0” - incorrect. A big problem is that it depends on the sample size, and that the probability of a theory depends on the prior.“If there is no effect, we should replicate the absence of effect with a probability equal to 1-p.” I don’t understand this, but I think it is incorrect.“The total probability of false positives can also be obtained by aggregating results (Ioannidis, 2005).” Unclear, and probably incorrect.“By failing to reject, we simply continue to assume that H0 is true, which implies that one cannot, from a nonsignificant result, argue against a theory” – according to which theory? From a NP perspective, you can ACT as if the theory is false.“(Lakens & Evers, 2014”) – we are not the original source, which should be cited instead.“ Typically, if a CI includes 0, we cannot reject H0.” - when would this not be the case? This assumes a CI of 1-alpha.“If a critical null region is specified rather than a single point estimate, for instance [-2 +2] and the CI is included within the critical null region, then H0 can be accepted.” – you mean practically, or formally? I’m pretty sure only the former.The section on ‘The (correct) use of NHST’ seems to conclude only Bayesian statistics should be used. I don’t really agree.“we can always argue that effect size, power, etc. must be reported.” – which power? Post-hoc power? Surely not? Other types are unknown. So what do you mean?The recommendation on what to report remains vague, and it is unclear why what should be reported.",
"responses": [
{
"c_id": "2060",
"date": "13 Jul 2016",
"name": "Cyril Pernet",
"role": "Author Response",
"response": "“investigate if an effect is likely” – ambiguous statement. I think you mean, whether the observed DATA is probable, assuming there is no effect? This sentence was changed, following as well the other reviewer, to ‘null hypothesis significance testing is the statistical method of choice in biological, biomedical and social sciences to investigate if an effect is likely, even though it actually tests whether the observed data are probable, assuming there is no effect’ The Fisher (1959) reference is not correct – Fischer developed his method much earlier. Changed, refers to Fisher 1925 “This p-value thus reflects the conditional probability of achieving the observed outcome or larger, p(Obs|H0)” – please add 'assuming the null-hypothesis is true'. “p(Obs|H0)” – explain this notation for novices. I changed a little the sentence structure, which should make explicit that this is the condition probability. “Following Fisher, the smaller the p-value, the greater the likelihood that the null hypothesis is false.” This is wrong, and any statement about this needs to be much more precise. I would suggest direct quotes. This sentence has been removed “there is something in the data that deserves further investigation” –unclear sentence. “The reason for this” – unclear what ‘this’ refers to. This has been changed to ‘[…] to decide whether the evidence is worth additional investigation and/or replication (Fisher, 1971 p13)’ “not the probability of the null hypothesis of being true, p(H0)” – second of can be removed? my mistake – the sentence structure is now ‘not the probability of the null hypothesis p(H0), of being true,’ ; hope this makes more sense (and this way refers back to p(Obs>t|H0) “Any interpretation of the p-value in relation to the effect under study (strength, reliability, probability) is indeed wrong, since the p-value is conditioned on H0” - incorrect. A big problem is that it depends on the sample size, and that the probability of a theory depends on the prior. Fair enough – my point was to stress the fact that p value and effect size or H1 have very little in common, but yes that the part in common has to do with sample size. I left the conditioning on H0 but also point out the dependency on sample size. “If there is no effect, we should replicate the absence of effect with a probability equal to 1-p.” I don’t understand this, but I think it is incorrect. Removed “The total probability of false positives can also be obtained by aggregating results (Ioannidis, 2005).” Unclear, and probably incorrect. Removed “By failing to reject, we simply continue to assume that H0 is true, which implies that one cannot, from a nonsignificant result, argue against a theory” – according to which theory? From a NP perspective, you can ACT as if the theory is false. The whole paragraph was changed to reflect a more philosophical take on scientific induction/reasoning. I hope this is clearer. “(Lakens & Evers, 2014”) – we are not the original source, which should be cited instead. done “ Typically, if a CI includes 0, we cannot reject H0.” - when would this not be the case? This assumes a CI of 1-alpha. “If a critical null region is specified rather than a single point estimate, for instance [-2 +2] and the CI is included within the critical null region, then H0 can be accepted.” – you mean practically, or formally? I’m pretty sure only the former. Changed to refer to equivalence testing The section on ‘The (correct) use of NHST’ seems to conclude only Bayesian statistics should be used. I don’t really agree. I rewrote this, as to show frequentist analysis can be used - I’m trying to sell Bayes more than any other approach. “we can always argue that effect size, power, etc. must be reported.” – which power? Post-hoc power? Surely not? Other types are unknown. So what do you mean? The recommendation on what to report remains vague, and it is unclear why what should be reported. I’m arguing we should report it all, that’s why there is no exhausting list – I can if needed."
}
]
},
{
"id": "11036",
"date": "10 Nov 2015",
"name": "Marcel A.L.M. van Assen",
"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\nNull hypothesis significance testing (NHST) is a difficult topic, with misunderstandings arising easily. Many texts, including basic statistics books, deal with the topic, and attempt to explain it to students and anyone else interested. I would refer to a good basic text book, for a detailed explanation of NHST, or to a specialized article when wishing an explaining the background of NHST. So, what is the added value of a new text on NHST? In any case, the added value should be described at the start of this text. Moreover, the topic is so delicate and difficult that errors, misinterpretations, and disagreements are easy. I attempted to show this by giving comments to many sentences in the text.Abstract: “null hypothesis significance testing is the statistical method of choice in biological, biomedical and social sciences to investigate if an effect is likely”. No, NHST is the method to test the hypothesis of no effect.Intro: “Null hypothesis significance testing (NHST) is a method of statistical inference by which an observation is tested against a hypothesis of no effect or no relationship.” What is an ‘observation’? NHST is difficult to describe in one sentence, particularly here. I would skip this sentence entirely, here.Section on Fisher; also explain the one-tailed test.Section on Fisher; p(Obs|H0) does not reflect the verbal definition (the ‘or more extreme’ part).Section on Fisher; use a reference and citation to Fisher’s interpretation of the p-valueSection on Fisher; “This was however only intended to be used as an indication that there is something in the data that deserves further investigation. The reason for this is that only H0 is tested whilst the effect under study is not itself being investigated.” First sentence, can you give a reference? Many people say a lot about Fisher’s intentions, but the good man is dead and cannot reply… Second sentence is a bit awkward, because the effect is investigated in a way, by testing the H0.Section on p-value; Layout and structure can be improved greatly, by first again stating what the p-value is, and then statement by statement, what it is not, using separate lines for each statement. Consider adding that the p-value is randomly distributed under H0 (if all the assumptions of the test are met), and that under H1 the p-value is a function of population effect size and N; the larger each is, the smaller the p-value generally is.Skip the sentence “If there is no effect, we should replicate the absence of effect with a probability equal to 1-p”. Not insightful, and you did not discuss the concept ‘replicate’ (and do not need to).Skip the sentence “The total probability of false positives can also be obtained by aggregating results (Ioannidis, 2005).” Not strongly related to p-values, and introduces unnecessary concepts ‘false positives’ (perhaps later useful) and ‘aggregation’.Consider deleting; “If there is an effect however, the probability to replicate is a function of the (unknown) population effect size with no good way to know this from a single experiment (Killeen, 2005).”The following sentence; “ Finally, a (small) p-value is not an indication favouring a hypothesis. A low p-value indicates a misfit of the null hypothesis to the data and cannot be taken as evidence in favour of a specific alternative hypothesis more than any other possible alternatives such as measurement error and selection bias (Gelman, 2013).” is surely not mainstream thinking about NHST; I would surely delete that sentence. In NHST, a p-value is used for testing the H0. Why did you not yet discuss significance level? Yes, before discussing what is not a p-value, I would explain NHST (i.e., what it is and how it is used). Also the next sentence “The more (a priori) implausible the alternative hypothesis, the greater the chance that a finding is a false alarm (Krzywinski & Altman, 2013; Nuzzo, 2014).“ is not fully clear to me. This is a Bayesian statement. In NHST, no likelihoods are attributed to hypotheses; the reasoning is “IF H0 is true, then…”.Last sentence: “As Nickerson (2000) puts it ‘theory corroboration requires the testing of multiple predictions because the chance of getting statistically significant results for the wrong reasons in any given case is high’.” What is relation of this sentence to the contents of this section, precisely?Next section: “For instance, we can estimate that the probability of a given F value to be in the critical interval [+2 +∞] is less than 5%” This depends on the degrees of freedom.“When there is no effect (H0 is true), the erroneous rejection of H0 is known as type I error and is equal to the p-value.” Strange sentence. The Type I error is the probability of erroneously rejecting the H0 (so, when it is true). The p-value is … well, you explained it before; it surely does not equal the Type I error.Consider adding a figure explaining the distinction between Fisher’s logic and that of Neyman and Pearson.“When the test statistics falls outside the critical region(s)” What is outside?“There is a profound difference between accepting the null hypothesis and simply failing to reject it (Killeen, 2005)” I agree with you, but perhaps you may add that some statisticians simply define “accept H0’” as obtaining a p-value larger than the significance level. Did you already discuss the significance level, and it’s mostly used values?“To accept or reject equally the null hypothesis, Bayesian approaches (Dienes, 2014; Kruschke, 2011) or confidence intervals must be used.” Is ‘reject equally’ appropriate English? Also using Cis, one cannot accept the H0.Do you start discussing alpha only in the context of Cis?“CI also indicates the precision of the estimate of effect size, but unless using a percentile bootstrap approach, they require assumptions about distributions which can lead to serious biases in particular regarding the symmetry and width of the intervals (Wilcox, 2012).” Too difficult, using new concepts. Consider deleting.“Assuming the CI (a)symmetry and width are correct, this gives some indication about the likelihood that a similar value can be observed in future studies, with 95% CI giving about 83% chance of replication success (Lakens & Evers, 2014).” This statement is, in general, completely false. It very much depends on the sample sizes of both studies. If the replication study has a much, much, much larger N, then the probability that the original CI will contain the effect size of the replication approaches (1-alpha)*100%. If the original study has a much, much, much larger N, then the probability that the original Ci will contain the effect size of the replication study approaches 0%.“Finally, contrary to p-values, CI can be used to accept H0. Typically, if a CI includes 0, we cannot reject H0. If a critical null region is specified rather than a single point estimate, for instance [-2 +2] and the CI is included within the critical null region, then H0 can be accepted. Importantly, the critical region must be specified a priori and cannot be determined from the data themselves.” No. H0 cannot be accepted with Cis.“The (posterior) probability of an effect can however not be obtained using a frequentist framework.” Frequentist framework? You did not discuss that, yet.“X% of times the CI obtained will contain the same parameter value”. The same? True, you mean?“e.g. X% of the times the CI contains the same mean” I do not understand; which mean?“The alpha value has the same interpretation as when using H0, i.e. we accept that 1-alpha CI are wrong in alpha percent of the times. “ What do you mean, CI are wrong? Consider rephrasing.“To make a statement about the probability of a parameter of interest, likelihood intervals (maximum likelihood) and credibility intervals (Bayes) are better suited.” ML gives the likelihood of the data given the parameter, not the other way around.“Many of the disagreements are not on the method itself but on its use.” Bayesians may disagree.“If the goal is to establish the likelihood of an effect and/or establish a pattern of order, because both requires ruling out equivalence, then NHST is a good tool (Frick, 1996)” NHST does not provide evidence on the likelihood of an effect.“If the goal is to establish some quantitative values, then NHST is not the method of choice.” P-values are also quantitative… this is not a precise sentence. And NHST may be used in combination with effect size estimation (this is even recommended by, e.g., the American Psychological Association (APA)).“Because results are conditioned on H0, NHST cannot be used to establish beliefs.” It can reinforce some beliefs, e.g., if H0 or any other hypothesis, is true.“To estimate the probability of a hypothesis, a Bayesian analysis is a better alternative.” It is the only alternative?“Note however that even when a specific quantitative prediction from a hypothesis is shown to be true (typically testing H1 using Bayes), it does not prove the hypothesis itself, it only adds to its plausibility.” How can we show something is true?I do not agree on the contents of the last section on ‘minimal reporting’. I prefer ‘optimal reporting’ instead, i.e., the reporting the information that is essential to the interpretation of the result, to any ready, which may have other goals than the writer of the article. This reporting includes, for sure, an estimate of effect size, and preferably a confidence interval, which is in line with recommendations of the APA.",
"responses": [
{
"c_id": "2059",
"date": "13 Jul 2016",
"name": "Cyril Pernet",
"role": "Author Response",
"response": "Null hypothesis significance testing (NHST) is a difficult topic, with misunderstandings arising easily. Many texts, including basic statistics books, deal with the topic, and attempt to explain it to students and anyone else interested. I would refer to a good basic text book, for a detailed explanation of NHST, or to a specialized article when wishing an explaining the background of NHST. So, what is the added value of a new text on NHST? In any case, the added value should be described at the start of this text. Moreover, the topic is so delicate and difficult that errors, misinterpretations, and disagreements are easy. I attempted to show this by giving comments to many sentences in the text. The idea of this short review was to point to common interpretation errors (stressing again and again that we are under H0) being in using p-values or CI, and also proposing reporting practices to avoid bias. This is now stated at the end of abstract. Regarding text books, it is clear that many fail to clearly distinguish Fisher/Pearson/NHST, see Glinet et al (2012) J. Exp Education 71, 83-92. If you have 1 or 2 in mind that you know to be good, I’m happy to include them. Abstract: “null hypothesis significance testing is the statistical method of choice in biological, biomedical and social sciences to investigate if an effect is likely”. No, NHST is the method to test the hypothesis of no effect. I agree – yet people use it to investigate (not test) if an effect is likely. The issue here is wording. What about adding this distinction at the end of the sentence?: ‘null hypothesis significance testing is the statistical method of choice in biological, biomedical and social sciences used to investigate if an effect is likely, even though it actually tests for the hypothesis of no effect’. Intro: “Null hypothesis significance testing (NHST) is a method of statistical inference by which an observation is tested against a hypothesis of no effect or no relationship.” What is an ‘observation’? NHST is difficult to describe in one sentence, particularly here. I would skip this sentence entirely, here. I think a definition is needed, as it offers a starting point. What about the following: ‘NHST is a method of statistical inference by which an experimental factor is tested against a hypothesis of no effect or no relationship based on a given observation’ Section on Fisher; also explain the one-tailed test. Section on Fisher; p(Obs|H0) does not reflect the verbal definition (the ‘or more extreme’ part). Section on Fisher; use a reference and citation to Fisher’s interpretation of the p-value Section on Fisher; “This was however only intended to be used as an indication that there is something in the data that deserves further investigation. The reason for this is that only H0 is tested whilst the effect under study is not itself being investigated.” First sentence, can you give a reference? Many people say a lot about Fisher’s intentions, but the good man is dead and cannot reply… Second sentence is a bit awkward, because the effect is investigated in a way, by testing the H0. The section on Fisher has been modified (more or less) as suggested: (1) avoiding talking about one or two tailed tests (2) updating for p(Obs≥t|H0) and (3) referring to Fisher more explicitly (ie pages from articles and book) ; I cannot tell his intentions but these quotes leave little space to alternative interpretations. Section on p-value; Layout and structure can be improved greatly, by first again stating what the p-value is, and then statement by statement, what it is not, using separate lines for each statement. Consider adding that the p-value is randomly distributed under H0 (if all the assumptions of the test are met), and that under H1 the p-value is a function of population effect size and N; the larger each is, the smaller the p-value generally is. Done Skip the sentence “If there is no effect, we should replicate the absence of effect with a probability equal to 1-p”. Not insightful, and you did not discuss the concept ‘replicate’ (and do not need to). Done Skip the sentence “The total probability of false positives can also be obtained by aggregating results (Ioannidis, 2005).” Not strongly related to p-values, and introduces unnecessary concepts ‘false positives’ (perhaps later useful) and ‘aggregation’. Done Consider deleting; “If there is an effect however, the probability to replicate is a function of the (unknown) population effect size with no good way to know this from a single experiment (Killeen, 2005).” Done The following sentence; “ Finally, a (small) p-value is not an indication favouring a hypothesis. A low p-value indicates a misfit of the null hypothesis to the data and cannot be taken as evidence in favour of a specific alternative hypothesis more than any other possible alternatives such as measurement error and selection bias (Gelman, 2013).” is surely not mainstream thinking about NHST; I would surely delete that sentence. In NHST, a p-value is used for testing the H0. Why did you not yet discuss significance level? Yes, before discussing what is not a p-value, I would explain NHST (i.e., what it is and how it is used). Also the next sentence “The more (a priori) implausible the alternative hypothesis, the greater the chance that a finding is a false alarm (Krzywinski & Altman, 2013; Nuzzo, 2014).“ is not fully clear to me. This is a Bayesian statement. In NHST, no likelihoods are attributed to hypotheses; the reasoning is “IF H0 is true, then…”. The reasoning here is as you state yourself, part 1: ‘a p-value is used for testing the H0; and part 2: ‘no likelihoods are attributed to hypotheses’ it follows we cannot favour a hypothesis. It might seems contentious but this is the case that all we can is to reject the null – how could we favour a specific alternative hypothesis from there? This is explored further down the manuscript (and I now point to that) – note that we do not need to be Bayesian to favour a specific H1, all I’m saying is this cannot be attained with a p-value. Last sentence: “As Nickerson (2000) puts it ‘theory corroboration requires the testing of multiple predictions because the chance of getting statistically significant results for the wrong reasons in any given case is high’.” What is relation of this sentence to the contents of this section, precisely? The point was to emphasise that a p value is not there to tell us a given H1 is true and can only be achieved through multiple predictions and experiments. I deleted it for clarity. Next section: “For instance, we can estimate that the probability of a given F value to be in the critical interval [+2 +∞] is less than 5%” This depends on the degrees of freedom. This sentence has been removed “When there is no effect (H0 is true), the erroneous rejection of H0 is known as type I error and is equal to the p-value.” Strange sentence. The Type I error is the probability of erroneously rejecting the H0 (so, when it is true). The p-value is … well, you explained it before; it surely does not equal the Type I error. Indeed, you are right and I have modified the text accordingly. When there is no effect (H0 is true), the erroneous rejection of H0 is known as type 1 error. Importantly, the type 1 error rate, or alpha value is determined a priori. It is a common mistake but the level of significance (for a given sample) is not the same as the frequency of acceptance alpha found on repeated sampling (Fisher, 1955). Consider adding a figure explaining the distinction between Fisher’s logic and that of Neyman and Pearson. A figure is now presented – with levels of acceptance, critical region, level of significance and p-value. “When the test statistics falls outside the critical region(s)” What is outside? “There is a profound difference between accepting the null hypothesis and simply failing to reject it (Killeen, 2005)” I agree with you, but perhaps you may add that some statisticians simply define “accept H0’” as obtaining a p-value larger than the significance level. Did you already discuss the significance level, and it’s mostly used values? “To accept or reject equally the null hypothesis, Bayesian approaches (Dienes, 2014; Kruschke, 2011) or confidence intervals must be used.” Is ‘reject equally’ appropriate English? Also using Cis, one cannot accept the H0. I should have clarified further here – as I was having in mind tests of equivalence. To clarify, I simply states now: ‘To accept the null hypothesis, tests of equivalence or Bayesian approaches must be used.’ Do you start discussing alpha only in the context of Cis? It is now presented in the paragraph before. “CI also indicates the precision of the estimate of effect size, but unless using a percentile bootstrap approach, they require assumptions about distributions which can lead to serious biases in particular regarding the symmetry and width of the intervals (Wilcox, 2012).” Too difficult, using new concepts. Consider deleting. Done “Assuming the CI (a)symmetry and width are correct, this gives some indication about the likelihood that a similar value can be observed in future studies, with 95% CI giving about 83% chance of replication success (Lakens & Evers, 2014).” This statement is, in general, completely false. It very much depends on the sample sizes of both studies. If the replication study has a much, much, much larger N, then the probability that the original CI will contain the effect size of the replication approaches (1-alpha)*100%. If the original study has a much, much, much larger N, then the probability that the original Ci will contain the effect size of the replication study approaches 0%. Yes, you are right, I completely overlooked this problem. The corrected sentence (with more accurate ref) is now “Assuming the CI (a)symmetry and width are correct, this gives some indication about the likelihood that a similar value can be observed in future studies. For future studies of the same sample size, 95% CI giving about 83% chance of replication success (Cumming and Mallardet, 2006). If sample sizes differ between studies, CI do not however warranty any a priori coverage”. “Finally, contrary to p-values, CI can be used to accept H0. Typically, if a CI includes 0, we cannot reject H0. If a critical null region is specified rather than a single point estimate, for instance [-2 +2] and the CI is included within the critical null region, then H0 can be accepted. Importantly, the critical region must be specified a priori and cannot be determined from the data themselves.” No. H0 cannot be accepted with Cis. Again, I had in mind equivalence testing, but in both cases you are right we can only reject and I therefore removed that sentence. “The (posterior) probability of an effect can however not be obtained using a frequentist framework.” Frequentist framework? You did not discuss that, yet. Removed “X% of times the CI obtained will contain the same parameter value”. The same? True, you mean? “e.g. X% of the times the CI contains the same mean” I do not understand; which mean? “The alpha value has the same interpretation as when using H0, i.e. we accept that 1-alpha CI are wrong in alpha percent of the times. “ What do you mean, CI are wrong? Consider rephrasing. “To make a statement about the probability of a parameter of interest, likelihood intervals (maximum likelihood) and credibility intervals (Bayes) are better suited.” ML gives the likelihood of the data given the parameter, not the other way around. corrected “Many of the disagreements are not on the method itself but on its use.” Bayesians may disagree. removed “If the goal is to establish the likelihood of an effect and/or establish a pattern of order, because both requires ruling out equivalence, then NHST is a good tool (Frick, 1996)” NHST does not provide evidence on the likelihood of an effect. “If the goal is to establish some quantitative values, then NHST is not the method of choice.” P-values are also quantitative… this is not a precise sentence. And NHST may be used in combination with effect size estimation (this is even recommended by, e.g., the American Psychological Association (APA)). Yes, p-values must be interpreted in context with effect size, but this is not what people do. The point here is to be pragmatic, does and don’t. The sentence was changed. “Because results are conditioned on H0, NHST cannot be used to establish beliefs.” It can reinforce some beliefs, e.g., if H0 or any other hypothesis, is true. “To estimate the probability of a hypothesis, a Bayesian analysis is a better alternative.” It is the only alternative? Not for testing, but for probability, I am not aware of anything else. “Note however that even when a specific quantitative prediction from a hypothesis is shown to be true (typically testing H1 using Bayes), it does not prove the hypothesis itself, it only adds to its plausibility.” How can we show something is true? Cumulative evidence is, in my opinion, the only way to show it. Even in hard science like physics multiple experiments. In the recent CERN study on finding Higgs bosons, 2 different and complementary experiments ran in parallel – and the cumulative evidence was taken as a proof of the true existence of Higgs bosons."
}
]
}
] | 1
|
https://f1000research.com/articles/4-621
|
https://f1000research.com/articles/6-1222/v1
|
25 Jul 17
|
{
"type": "Research Article",
"title": "Logarithmic distributions prove that intrinsic learning is Hebbian",
"authors": [
"Gabriele Scheler"
],
"abstract": "In this paper, we document lognormal distributions for spike rates, synaptic weights and intrinsic excitability (gain) for neurons in various brain areas, such as auditory or visual cortex, hippocampus, cerebellum, striatum, midbrain nuclei. We find a remarkable consistency of heavy-tailed, specifically lognormal, distributions for rates, weights and gains in all brain areas. The difference between strongly recurrent and feed-forward connectivity (cortex vs. striatum and cerebellum), neurotransmitter (GABA (striatum) or glutamate (cortex)) or the level of activation (low in cortex, high in Purkinje cells and midbrain nuclei) turns out to be irrelevant for this feature. Logarithmic scale distribution of weights and gains appears as a functional property that is present everywhere. Secondly, we created a generic neural model to show that Hebbian learning will create and maintain lognormal distributions. We could prove with the model that not only weights, but also intrinsic gains, need to have strong Hebbian learning in order to produce and maintain the experimentally attested distributions. This settles a long-standing question about the type of plasticity exhibited by intrinsic excitability.",
"keywords": [
"neural coding",
"synaptic weights",
"Hebbian learning",
"intrinsic excitability",
"rate coding",
"spike frequency",
"neural circuits",
"neural networks",
"lognormal distributions."
],
"content": "1 Introduction\n\nIndividual neurons have very different, but mostly stable, mean spike rates under a variety of conditions1,2. To report on behavioral results, spike counts are often normalized with respect to the mean for each neuron. But this obscures an important question: Why do neurons within a tissue operate at radically different levels of output frequency? In order to answer this question our approach is twofold: (a) we try to document this phenomenon for different neural tissues and behavioral conditions. We also examine neural properties for their distribution, namely intrinsic gains and synaptic weights; (b) we build a very generic neural model to explore the conditions for generating and maintaining these distributions. First, we give examples for the distribution of mean spike rates for principal neurons under spontaneous conditions, as well as in response to stimuli. We furthermore document distributions for intrinsic excitability3–5 for cortical and striatal neurons, as well as synaptic weight distributions6–11.\n\nWith the current data, we show that the distribution of spike rates within any neural tissue follows a power-law distribution, i.e. a distribution with a ‘heavy tail’. There is also a small number of very low-frequency neurons, so that we have a lognormal distribution2. This lognormal distribution is present in spontaneous spike rates as well as under behavioral stimulation. For each neuron, the deviation from the mean rate attributable to a stimulus is small (CV = 0.3–1, standard deviation = 1–4 spikes/s), when compared to the variability in mean spike rate over the whole population (5-7-fold), cf. Table 1 and Table 3.\n\nThis work refers back to data initially reported in 1. At the time, we only had data on spike rates of cortical neurons available, plus independent evidence on intrinsic properties of striatal neurons. The observation on cortical data was taken up by2,12, and led to a number of papers13,14 focusing on the power-law distribution of spike rates as a cortical phenomenon, seeking explanations in the recurrent excitatory connectivity of cortical tissue2,13,15. However,we find the same spike rate distributions for midbrain nuclei, medium striatal neurons and cerebellar Purkinje cells, which do not have this kind of connectivity. We extended the data search for intrinsic excitability and found that lognormal distributions are ubiquitous there as well, at least in cortical as well as in striatal tissues. Finally, lognormal distributions have also been found for synaptic weights6–11. The explanation for this universal phenomenon must lie elsewhere.\n\nWe have constructed a generic model for neuronal populations with adaptable weights and gains. We initialized both weights and gains with uniform, Gaussian or lognormal distributions. We then employed either Hebbian or homeostatic adaptation rules on both. Under a variety of conditions we could show that lognormal distributions develop from any initial distribution only with Hebbian (positive) adaptivity. Additional homeostatic adaptation stabilized learning but erased the lognormal distributions if it was stronger than Hebbian adaptation. We could even show that the widths of the distributions from the model match with the experimental data for rates, weights, and gains (Table 1 and Table 3) that we have available. Lognormal distributions develop robustly from Hebbian learning rules, and lognormal distributions can only be maintained by Hebbian-style, not homeostatic adaptation.\n\nThis finally answers the question that experimental researchers have investigated for some time: Is intrinsic plasticity mostly homeostatic, i.e. adjusts values inversely to use, or is there Hebbian, positive learning involved: when a neuron fires, does its gain increase? The answer is that the attested distribution of intrinsic gain can only derive from a Hebbian style adjustment rule, even though additional homeostatic adaptation is possible. Intrinsic plasticity is Hebbian.\n\n\n2 Methods\n\nIn this section we report on data collection for spike rates, intrinsic properties and synaptic weights. Secondly, we explain the simulation model we constructed to explore the generation and persistence of the attested distributions.\n\nWe analyze five data sets for spike rates from principal neurons under behavioral activation:\n\n1. inferior temporal (IT) cortex from monkeys16\n\n2. primary auditory cortex (A1) from monkeys17\n\n3. primary auditory cortex (A1) of rats2\n\n4. Purkinje cells in cerebellum18–21\n\n5. midbrain principal cells from inferior colliculus (IC) from the guinea pig22\n\nIn monkey IT, single unit activity was recorded over 200ms for passive viewing of 77 different natural stimuli for 100 neurons, each stimulus shown 10 times16. This yielded 770 spike rate response data points per neuron. For these data, we show mean spike rate, standard deviation, max-min values, coefficient of variation (CV) and Fano factor (FF) (Figure 1), (cf. 1). What is remarkable is that the dispersion for each neuron (variance related to mean, FF) is fairly constant, and not related to the rank of a neuron as high or low-frequency. In other words, neurons have roughly similar behavioral responses relative to their average spike rate. For this reason,many behavioral experiments have reported percentage of increase/decrease of spiking as the relevant parameter.\n\n100 neurons, passive viewing of 77 stimuli, 10 trials (770 data points per neuron), data collected over 200ms. The data are shown for each neuron, where neurons are sorted by mean spike count. A: Mean spike rates (blue), standard deviation (red), and minimum/maximum absolute values (green). B: Mean spike rates histogram shows a lognormal distribution (σ*=2.32). C: Distributions of standard deviation (green) and CV (blue) have linear slopes, with small variation. The Fano factor (red), measuring the dispersion for each neuron, is nearly constant at about 2.\n\nAdditionally, we show data from primary auditory cortex (A1) from awake monkeys, which were recorded for spike responses to a 50ms, 100ms, or 200ms pure tone (17, Figure 2A and B) and data for spike rates from the primary auditory cortex of rats for four different conditions, which were recorded as cell-attached in vivo recordings (2, Figure 2C).\n\nA: Distribution of spike rates to a 50ms tone (n = 119, red) 100ms tone (n = 115, blue), or 200ms tone (n = 23, green) in primary auditory cortex in monkeys17. B: Histogram for spike rate distribution for the 100ms tone response (n = 115) fitted by an exponential (red) or lognormal (blue) distribution17. C: Spontaneous spike rate distribution from primary auditory cortex in unanesthetized rats2 fitted by an exponential (red) or lognormal (blue) distribution. Note that the spontaneous firing rates are much lower and narrower distributed than evoked spikes in response to stimuli at short time scales (B), but that they still follow a lognormal distribution.\n\nFor midbrain nuclei neurons (IC), we re-analyzed spike rates in response to tones (for 200ms after stimulus onset) under variations of binaural correlation22. The frequency ranking of neurons by mean spike rate, standard deviation, min-max values, CV and FF are shown in (Figure 3A–C). CV and FF are similar to the cortical data. Data from GABAergic cerebellar Purkinje cells offer some difficulty for this analysis since they have regular single spikes at high frequencies, and in addition, calcium-based complex spikes20. Complex spike rates however are low (< 1Hz). This can therefore be regarded as a form of multiplexing, with two separate codes, where single spike rates can be separately assessed in their distribution. Here we report data for single spikes from in vivo recordings in anesthetized rats20 (Figure 4A)and data from spontaneous spiking (in the absence of synaptic stimulation) under in vitro conditions (18, 19, Figure 4B and C).\n\nThe data are shown for each neuron, with neurons sorted by mean spike count. A: Mean spike rates (blue), standard deviation (red), and minimum/maximum absolute values (green). B: Mean spike rates histogram shows a lognormal distribution. C: Distributions of standard deviation (green), CV (blue) and FF (red). Again, the dispersion is fairly constant.\n\nA: Data for single spikes for Purkinje cells recorded from anesthetized rats (spontaneous in vivo)20 (n = 346). B: Data for spike frequencies of isolated cell bodies of mouse Purkinje cells in vitro18 (n = 34). C: Spontaneous spike rates for Purkinje cells in slices (n = 106)19. D: Spike counts per neuron from 18 (n = 34), together with variability data from21 (n = 2).\n\nIn order to show values for standard deviation and variance, data for two Purkinje cells from a behavioral experiment,21, i.e. single spike rates during arm movements from monkeys, have been added to the ranking of spontaneously firing neurons by mean spike rate from 18 (Figure 4D).\n\nThe logarithmic (heavy-tailed) distribution of spike rates is evident under all conditions.\n\nThe distribution of spike rates for neurons spiking in the absence of synaptic input shows that there are differences in the intrinsic excitability of neurons. To further explore this we looked at three additional datasets, which report the action potential firing of a cell due to injected current, such as by a constant pulse. This can be defined as the neuronal gain parameter (spike rate divided by current, [Hz/nA]).\n\n1. medium striatal neurons in slices from rat dorsal striatum and nucleus accumbens shell (NAcb shell)3, cf. 1, Figure 5.\n\n2. cortical neurons in cat area 17 in vivo23, Figure 6A and B\n\n3. striatal neurons from globus pallidus (GP) from awake rats5, Figure 6C\n\nA: Spike rate in response to a 300ms constant current pulse at 180pA (blue), 200pA (green), and 220pA (red) for neurons in dorsal striatum (n = 28, solid lines) and nucleus accumbens shell (n = 24, dashed lines). B: Gain (Hz/nA) for neurons in dorsal striatum (n = 28). C: Gain (Hz/nA) for neurons in NAcb shell (n = 24).\n\nA: Gain for all types of cortical neurons in vivo (n = 220)23. B: Gain for fast spiking cortical neurons only (n = 33)23. C: Gain for neurons in globus pallidus (GP) in response to a +100pA current pulse (n = 145)5.\n\nIn 1, we already presented the data from striatum, which show that the spike response to a constant current follows a heavy-tailed distribution3. Figure 5A shows the spike rate in response to current pulses of different magnitude in two different areas, nucleus accumbens (NAcb) shell and dorsal striatum. Figure 5B and C show the distribution of rheobase (current-to-threshold) for dorsal striatal and NAcb shell neurons. Distributions appear mostly lognormal, with the exception of the 200pA current pulse response and the data in Figure 5C, which appear normally distributed.\n\nWe extend this dataset by recordings from different types of cortical neurons in cat area 17 in vivo (23, Figure 6A and B) and from GP in awake rats (5, Figure 6C).\n\nA lognormal distribution of intrinsic gain is clearly apparent, except for fast-spiking interneurons, which, however, may be a sampling error (n=33).\n\nSynaptic weight distributions have been investigated starting with10 in hippocampus by measuring EPSC magnitude6,24–27 (Figure 7). There is also a review paper available28 to summarize the findings. Recently, the expression of AMPA receptor subunit GluA1, which is correlated with spine size, has also been measured29, Figure 8. We used five datasets from cortex, hippocampus and cerebellum:\n\nA: Cortex: Deep-layer (L5) pyramidal-pyramidal cell connections6,24. B: Cortex: Deep-layer (L5) pyramidal-pyramidal cell connections26. C: Hippocampus: CA1 to CA3 connections10. D: Cerebellum: Granule cells to Purkinje cells27.\n\nA: Expression of labeled GluA1 AMPA receptor subunit in layer 2/3 mouse barrel cortex in vivo follows a lognormal distribution (σ* = 2.59, µ* = 0.32, n = 560). B: GluA1 density for control (black) and after 1 hour whisker stimulation (red). Stimulation leads to an increase of GluA1 in ≈ 30% of neurons29.\n\n1. EPSPs for deep-layer pyramidal-pyramidal cell connections in rat visual cortex24,6\n\n2. EPSP amplitudes for deep-layer excitatory neuron connections in somatosensory cortical slices of juvenile rats26\n\n3. EPSP amplitudes for CA1 to CA3 connections in guinea pig hippocampal slices10\n\n4. EPSCs for granule cells to Purkinje cells in adult rat cerebellar slices27\n\n5. labeled GluA1 AMPA receptor subunit in mouse somatosensory barrel cortex29\n\nIn 6, EPSP magnitude was measured for L5 pyramidal neurons in slices from rat visual cortex, averaged over 45–60 responses, and peak amplitude recorded, (Figure 7A). Similar data were used in 26 for slices from a single barrel column in rats (Figure 7B). A 30-fold variation of coupling strength was noted. In 10, EPSPs between CA3 and CA1 in hippocampal slices were recorded, by detecting somatic membrane potential changes in response to presynaptic neuron stimulation (Figure 7C). There are also synaptic weight data on granule cell to Purkinje cell connections11,27,28, which show a similar distribution, but have an order of magnitude weaker connections than cortical connections (Figure 7D). Finally, a different type of evidence was obtained in 29, namely labeling for a subunit of AMPA receptors in layer 2/3 mouse barrel cortex in vivo both before and after whisker stimulation. The AMPA intensity is distributed lognormally over the spines, corresponding to the observations on the strengths of EPSPs. It is noticeable that stimulation leads to increase of on average 200% (two-fold) in about 30% of spines29. Yet as we know, over time the overall distribution of synaptic strengths remains stable.\n\nFor synaptic weights, just as for intrinsic gains and spike rates, lognormal distributions have been found for both EPSPs and AMPA receptor distribution in a highly consistent manner.\n\nIn many cases, the data were only available in the form of histograms. The parameters of the lognormal distribution were then obtained by fitting the data using a Nelder-Mead optimization method. A number of parameters were derived from these fits and reproduced in Table 1–Table 3, cf. 3.2.\n\nGiven are two neuron populations I, J with each n = 1000 neurons and variable random connectivity C between I and J. C determines the density between I and J. The input population I always has excitatory output onto J. Inhibitory input to J is modeled by a population H with n = 200. The output neuron population J may also have recurrent excitatory connectivity. Figure 9 shows the architecture for the generic neural network used. The model (GNN) was programmed in Matlab, and is available in GitHub https://github.com/gscheler/GNN. The input population I is modeled according to2 for pyramidal cortical neurons with a spike rate distribution of µ* = 4.95 and σ* = 1.98 (Table 1). The goal is to generate a spike rate distribution RJ for J, given a gain distribution G for the target neurons, and the weight distribution WIJ such that RJ is similar to RI.\n\nLognormal distributions occur for gain G, rates RI, RJ,RH, and weight distributions WIJ. J may have recurrent connectivity.\n\nFor each neuron j in J the spike rate rj is calculated by applying its gain gj to the weighted sum of its connected excitatory inputs and its inhibition. Cj is the set of neurons from I that have excitatory connections to neuron j.\n\n\n\nwhere the rate ri is taken from the distribution RI, rjH from RH, wij from WIJ, and gj from G. gj is modeled as a factor for a linear gain function. It is possible to use a sigmoidal gain function instead, but this makes no difference for the conclusions from the model (Section 3.3). The output RJ may used as input to I with a matrix WI,J for tests of the adaptation rules.\n\nFor the adaptation of weights WIJ and gains G, we use Hebbian or homeostatic rules, as described in Section 3.3. The system described in this way is sufficient for all the calculations on the shape of distributions used in this paper.\n\n\n3 Results\n\nWe have documented the distribution of spike rates, gains, and weights for different types of neurons (Figure 1–Figure 8). The distribution in all cases follows a lognormal shape. In some cases, we had data on the variability of spike rates and analyzed them for dispersion (CV, FF) under behavioral stimulation. While the fold-change from low spiking neurons to high spiking neurons is high, 5- to 7-fold, the variability for each neuron is comparatively low. It also seems to be adequately described by a percentage change over the whole population. This means that a low spiking neuron never reaches the same rate as a high spiking neuron, even when fully activated.\n\nThe similarities across neural systems are striking. For instance, in a midbrain nucleus (inferior colliculus) which is essentially an ’output’ site for auditory and somatosensory cortex, spike rates are high overall22, nonetheless the distribution of mean spike rate, and the variability are comparable to cortical data (Figure 3). The distribution of mean spike rate is essentially the same under spontaneous and under behavioral conditions.\n\nLognormal rate distributions appear to be an essential property of neural tissues that occur in areas with very different neuron types and connectivity, and different absolute spike frequencies. They are present during spontaneous activity, and under activation of a network, in vivo as well as in vitro. They have a counterpart in a lognormal distribution of intrinsic excitability, and lognormal synaptic connectivity. This type of distribution seems to be an essential component of the functional structure of a mature network, which is not affected by learning, plasticity, or processing of information.\n\nA lognormal distribution is characterized by parameters µ*and σ*. µ* = eµ is the median, a scale parameter, which determines the height of the distribution. σ* = eσ is the multiplicative standard deviation, a shape parameter which determines the width of the distribution. For distributions with small σ*(approximately σ* < 1.2 or σ < 0.182) a lognormal distribution is essentially identical to a normal distribution. (The coefficient of variation CV ∼ σ* − 1, so that for CV < 0.18, a lognormal equals normal distribution.) We collected data on spike rate, gain and synaptic weight distribution for a number of tissues in different experimental conditions (Table 1–Table 3). For the height of the spike rate distribution, there are known differences, e.g. with lower values for cortex (µ* ≈ 4.5) and higher values for Purkinje cell (µ* ≈ 30) and midbrain nuclei (cf. Table 1). In other words, spike rates differ between brain areas such as cortex and cerebellum by a factor of 10.\n\nIn contrast to that, the width of spike rate distributions is more similar, with an average at σ* ≈ 2.2, with one outlier. The gain has a smaller σ*, i.e. a more normal, less heavy-tailed distribution than the spike rate. Minus the outlier (3.46), the mean for σ* is only 1.86, considerably lower than the width of the spike rate distribution (Table 2). For weight distributions (Table 3), the width σ* is consistently larger, with an average of almost 3 (2.91). The synaptic strength (µ*) varies over at least one order of magnitude between cortex and cerebellum.\n\nIt turns out that σ* values are significantly different for rates, gains and weights, lowest for gains (σ* ≈ 1.8), higher for rates (σ* ≈ 2.2) and highest,(σ* ≈ 3), for weights. The data that we have are not precise enough to draw quantitative conclusions, but no large distinctions are apparent between the tissues (Table 1–Table 3). We use a generic neural network to recreate lognormal distributions by adaptation rules and we will also show that distribution widths are structural properties which follow from general network properties.\n\nSince not only mean spike rates but also both components, intrinsic excitability and synaptic weights, have lognormal distribution, this raises the question of how the functional system that we observe is generated. It is obvious, if the data are accurate, that these are basic parameters of any simulation and need to be reproduced in any model to make it biologically realistic.\n\nWe set up a generic neural network model (cf. Section 2.2) to explore the mechanisms of generating and maintaining rate, weight and gain distributions. The model consists of a source neuron group I, a target group J, a population of inhibitory neurons H, which are connected with J, and potentially recurrent excitation in the target group J. The spike rate distribution RI acts through a weight distribution W onto a gain distribution G, where inhibition H is subtracted, and a spike rate output distribution RJ is produced (Figure 9).\n\nIn the simplest case, we look at two sets of neurons, the source and the target. The source sends excitatory connections to the target, and exhibits variable weights at outgoing synapses. The input that a target neuron receives is fed through a linear filter G to produce an output rate RJ according to Equation (1). The distribution for RJ depends on G and W as well as on RI. The system is sufficient for calculations on the shape of distributions, as well as the effects of Hebbian and homeostatic plasticity.\n\nWe have explored the dependencies between gain, weight and rate distributions in simulations. First, we found that the width of the output spike rate distribution RJ depends heavily on the gain distribution, but only slightly on the input weight distribution (Figure 10). It does depend on the overall connectivity C, where σRJ* is wider for lower connectivity, but not very much (Figure 10). Secondly, the width of the output distribution RJ does not depend on RI or RH either (Figure 11). The most important factor for a spike rate distribution remains the gain σG*.\n\nThere is a slight effect of connectivity (upper sheet C=5%,lower sheet C=10%). (μW*=0.7,μG*=30,N=1000,σRI*=2.74,μRI*=4.5.)\n\n(μW*=0.7,μG*=30,C=10%,N=1000,μRI*=4.5.)\n\nWe may now ask, where do lognormal spike rate distributions come from? How is the system set up, i.e. what rules of adaptation generate lognormal distributions in weights and gains?\n\nIn the case of cortical networks, there are excitatory recurrent interactions that constitute a significant part of total input. In the case of cerebellar or striatal neurons, there are no recurrent excitatory interactions, only inhibitory interneurons and excitatory input. The generation of lognormal distributions must therefore be independent of recurrent excitation. It requires a system where continuous input shapes the weights and gains of a target network J. We start with the system that we described before, with random assignment of weights and gains. We employ adaptivity for weights, and also for gains, by positive Hebbian learning, or by negative homeostatic learning. The output of I is fed into J, and W and G are adaptive. Additionally, J may have excitatory recurrent connectivity, and learning takes place within the network J.\n\nFrom any given initial spike rate distribution (Gaussian, uniform, lognormal) for I, we calculate W assuming a positive learning (Hebbian) adjustment rule, which is dependent on input and output frequencies. Each individual weight wij is updated by\n\n\n\nWe use parameters λ and µ, such that the generated spike rate output RJ is compatible in strength with the input rate RI.\n\nUsing Hebbian learning, we generate a weight distribution W that is lognormally distributed, independent of the initial configuration or the distribution of the gains in the system (Figure 12). The lognormal distribution also develops independently of the rate distribution of the inputs, it only develops faster with lognormal rather than normally distributed spike rate input (not shown). It makes no difference whether we use a recurrent system J, or a non-recurrent population J with input from a population I with a spike rate distribution, as long as we use a Hebbian weight adaptation rule. For the shape of the distribution, it also does not matter whether we route the output of J back to I, or whether we use local or no recurrence. To show the effect of the adaptation rule, we also used homeostatic synaptic plasticity to adjust the weights. This means that the weight is adjusted inversely to the spike rate of input and output neurons.\n\nGiven is a lognormal input rate σRI*=2,μRI*=4.95. A: Initial weight configurations: Gaussian (grey) or uniform (blue). B: After Hebbian learning using Gaussian gain distribution (grey, blue as before). C: After Hebbian learning using uniform gain distribution (grey, blue as before). D: After Hebbian learning using lognormal gain distribution. (σG*=1.37,μG*=32.7) (grey, blue as before).\n\n\n\nIn this case, it is very clear that with any input or initial configuration and any gain distribution, only a normal distribution of weights results (Figure 13). Again, a lognormal input spike rate slows the process of adaptation, but the end result is the same, a normal distribution.\n\nGiven is a lognormal input rate σRI*=2,μRI*=4.95. A: Initial Configuration: Gaussian (grey) or uniform (blue). B: Homeostatic weight learning using Gaussian gain distribution (grey, blue as before). C: Homeostatic weight learning using uniform gain distribution (grey, blue as before). D: Homeostatic weight learning using lognormal gain distribution (σG*=1.37,μG*=32.7) (grey, blue as before).\n\nSince gain distributions are also lognormal, we may ask in the same way how they develop and are maintained by plasticity rules. We adapt the linear gain G by either Hebbian or homeostatic learning. Each gain can be adjusted by a Hebbian rule\n\n\n\nor a homeostatic rule\n\n\n\nwith parameters λ and µ.\n\nWe start with uniform or normally distributed G in an environment where W is lognormal, normal or uniform, and RI is normal or lognormal. If we adapt only G for any initial configuration, using any distribution for RI, including the lognormal distribution, and a lognormal or normal weight distribution, we arrive at a normal distribution for G with homeostatic learning and a lognormal distribution with Hebbian learning (Figure 14).\n\nGiven is a lognormal input rate σRI*=2,μRI*=4.95. A: Initial Configuration: Gaussian (grey) or uniform (blue). B and C: Hebbian learning using lognormal or Gaussian weights, resulting gain distribution is lognormal. D and E: Homeostatic learning using lognormal or Gaussian weights, resulting gain distribution is Gaussian.\n\nLognormal distributions develop from Hebbian plasticity, and homeostatic plasticity generates only normal distributions. The explanation lies in the nature of random statistical events, which generate normal distributions when the underlying mechanisms are sums of many small events, but lognormal distributions when the underlying mechanisms are multiplicative30. We also wanted to understand the observed widths of the distributions. We hypothesized that the differences for σ* between W, R and G result from the network structure. Accordingly we started a simulation with initial uniform values for G and W and Hebbian update rules using the same learning rate λ for both (Figure 15). We find that gain, rate and weight distributions match the experimental values, and that this is true for any tested constellation. We also found that Hebbian learning alone quickly escalates values, which develop exponentially, and that additional rounds of homeostatic adaptation are required to stabilize the system. Homeostatic learning pushes the system back towards a normal distribution.\n\nGrey, experimental measurements (s. Tables); red, generated with 100% Hebbian learning; or blue, 80% Hebbian and 20% homeostatic learning combined. The basic distinction in distribution width for gains, rates, weights is reproduced with Hebbian learning, additional homeostatic learning matches experimental values best.\n\nOur data, in the most general way, allowing for various conditions and architectures, show that Hebbian learning is required both for intrinsic gain and for weights in order to generate the attested lognormal distributions. This is an interesting result, because it shows that we need prominent Hebbian intrinsic learning to explain the gain distributions that we find experimentally. Intrinsic learning is not just homeostatic adaptation, it follows the same rules as synaptic weight learning.\n\n\n4 Discussion\n\nSpike rates of neurons seem to be universally distributed according to a lognormal distribution, with many neurons at low spike rates, and a small number at successively higher spike rates (heavy-tail)1. The same distributions are found for synaptic weights6, and intrinsic properties associated with excitability (gain). The neurons that we reported on are of very different types, and they are embedded in different kinds of connectivity. Medium spiny neurons and Purkinje cells are GABAergic (inhibitory), while cortical and IC neurons are glutamatergic (excitatory), but this is not reflected in a distinct spike rate distribution. They also fire with very different average spike rates. IC neurons operate at very high frequencies, and Purkinje neurons at much higher frequencies than cortical or striatal projection neurons. But they all have the same spike rate distribution. It has been suggested2 that lognormal spike distributions are only a feature of cortical tissue and arise from the strong excitatory recurrent connectivity, but this is neither experimentally nor theoretically correct. While cortical pyramidal neurons exist in a heavily excitatory recurrent environment, medium spiny neurons, cerebellar Purkinje cells and IC neurons act mostly in a feed-forward environment, i.e. have no significant recurrent connectivity.\n\nBeyond spike rate distribution, we also gathered data on weight and gain distributions. Again the observation of lognormal distributions is ubiquitous. We find synaptic weight distributions for cortex6 and cerebellum that are lognormal, with characteristic width of distributions. For intrinsic properties, striatal projection neurons and cortical neurons23 show responses to constant current and current-to-threshold (gain) distributions, which again appear lognormally distributed, with smaller widths than spike rate distributions.\n\nOur models show that lognormal distributions arise even in a purely input-output environment, and that they are a result of Hebbian learning of weights and gains, quite independent of the overall magnitude of the spike rates.\n\nMean spike rates, as well as intrinsic excitability and synaptic weights, have lognormal distributions.\n\nIt has often been assumed that variability in intrinsic excitability is a source of noise in neural computation31, even though others have argued that intrinsic variability contributes to neural coding32,33 and that intrinsic plasticity follows certain rules34. An excellent overview of the experimentally attested forms of intrinsic plasticity is contained in 35, cf. 36–38. Many other detailed observations are contained in 39–42.\n\nRecently, Mahon and Charpier4 have shown that intrinsic excitability is stable in individual neurons under control conditions, while stimulation protocols (e.g. in barrel cortex of anesthetized rats) change intrinsic excitability by at least 50–100%. However, the conclusions drawn from the experimental research are often contradictory. Intrinsic plasticity is sometimes assumed to act in a negative, homeostatic way, i.e. opposite to synaptic plasticity4, but sometimes in a ’Hebbian’, positive way, i.e. cooperative with synaptic plasticity41,43. There is evidence for (short-term) negative or homeostatic plasticity, which has been previously investigated4.\n\nOur work has now shown that any kind of neural system with linear gains requires positive, Hebbian intrinsic plasticity to produce and maintain a lognormal distribution of gains. We also could show that the observed widths of the distributions, i.e. the differences for σ* between W, R and G, naturally result from the network structure and are built into the system simply by Hebbian adaptation.\n\nLognormal distributions may arise as stable properties of the system during early development (the set-up of the system), i.e. before actual pattern storage or event memory develops, and they are maintained during processing by a Hebbian type of positive adaptation events. Homeostatic plasticity consists in downregulating gains or weights with increases in firing rates. Purely homeostatic learning results in normal distributions, and erases existing lognormal distributions. By combining homeostatic and Hebbian adaptation we can achieve and maintain stable lognormal distributions.\n\nA lognormal distribution means that values are normally distributed on a logarithmic scale. From an engineering perspective, basic Hebbian plasticity for synapses and intrinsic properties is sufficient to generate stable logarithmic distributions. If there is random variation of multiplicative events, as in Hebbian plasticity, a lognormal distribution will be the result30.\n\nThis is related to principles of sensory coding, where logarithmic scale signal processing enhances perception of weak signals, while also being able to respond to large signals - effectively increasing the perceptual range compared to linear coding14. In an interconnected network logarithmic coding may turn into a property for the access of representations. Feature clusters, or event traces could be accessed by targeted connections to the top-level neurons, which then activate lower level neurons in their immediate vicinity. By accessing high frequency neurons preferentially, a whole feature area can be reached, and local diffusion will provide any additional computation. Similarly, the results of a local computation can be efficiently distributed by high frequency neurons to other areas. Fast point-to-point communication using only high frequency neurons may be sufficient for fast responses in many cases. Scale-free networks in general support synchronization, which is also a useful feature for rapid information transfer and access44.\n\nRecently, publications45,46 have shown that there is indeed a difference between high-frequency and low-frequency neurons in their connectivity: high-frequency neurons have short delays, strong connections, and directed targets, while low-frequency neurons have long delays, weak connections and diffuse targets.\n\nThe lognormal distribution of spike rates has significant implications for neural coding. Logarithmic spike rates are coupled with linear variance for responses to behavioral stimulation. In other words, the greatest part of the coding results already from the frequency rank of the neuron itself, such that high frequency neurons have the largest impact. A fixed mean rate for each neuron allows stable expectation values for network computations.\n\nLogarithmic, hierarchical coding does not need to be sparse. The low frequency neurons may matter the most in terms of input response. With lognormal synaptic weight distributions, if strong synapses are kept stable, they may transmit an input neuron’s mean firing rate to targets and in this way provide stability to the system. All other synapses could be arbitrary. This would allow for continued pattern learning to be implemented by the bulk of low weight synapses, while the framework of neuronal interactions, e.g., the ensemble structure, could be unchanged. Such a division of labor between strong synapses and weaker ones could have many advantages in a complex, modular network.\n\nExperimental data have often shown that sampling of neuronal responses from a large population (105 or more neurons), which become activated at 30% or more, yields accuracy for a stimulus already for small samples (100–200 neurons or 1–2%) (e.g., 47). We may suggest that this happens when we sample from a highly modular structure, and we have been able to replicate the effect with lognormal networks48.\n\nIn our earlier work1, we found that intrinsic excitability manifested by spike response to current injection and rheobase in vitro for dorsal striatal and nucleus accumbens neurons seems to have the same distribution as the firing rate in cortex under in vivo conditions. Approximately at the same time6, had observed a heavy-tailed distribution of synaptic weights in cortical tissue.\n\nIn this article, we have done three things: (a) collected data to show that rate, weight and gain distributions in different brain areas all follow a heavy-tailed, specifically a lognormal distribution; (b) created a generic neural network model to show that these distributions arise from Hebbian learning, and specifically that intrinsic plasticity must be Hebbian as well; and (c) shown that the width of the distributions, as experimentally attested, arise naturally from the network structure and the role of its components, in a very robust way. We have also discussed what the lognormal distribution means for neural coding: a division of labor between fast transmission by high-frequency neurons and low-level computation by low-frequency neurons in a modular structure, and possibly a division of labor between stable components (strong synapses, high-frequency neurons) and more variable components (weak synapses, low-frequency neurons).\n\nThe GNN simulation software was programmed in Matlab, and is available in GitHub: https://github.com/ gscheler/GNN/tree/v0.1\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.82994949\n\nOSS approved license: Apache 2.0.\n\nAll the data required for re-analysis of the study have been referenced throughout the manuscript.",
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PubMed Abstract | Publisher Full Text\n\nScheler G: Extreme pattern compression in log-normal networks [version 1; not peer reviewed]. F1000Res. (poster), 2016; 5: 2177. Publisher Full Text\n\nScheler G: gscheler/GNN: Initial version. Zenodo. 2017. Data Source"
}
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[
{
"id": "25048",
"date": "29 Aug 2017",
"name": "Rune W Berg",
"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 the author investigate various distributions of neuronal networks, and suggest that lognormality is ubiquitous. The author constructs a Hebbian model to better understand the mechanisms behind these distributions. The analyses are detailed, well-done and interesting. The manuscript is not so well--organized and the writing can be improved. There are many statements, which are stronger than the data can support. I suggest a thorough rewrite - incorporating the issues below.\n\nMajor concerns: Methods section is full of results, discussions and conclusions. I suggest a thorough rewrite of the manuscript to better separate out the methods, results and discussion.\nFrom the discussion: “Again the observation of lognormal distributions is ubiquitous. We find synaptic weight distributions for cortex6 and cerebellum that are lognormal, with characteristic width of distributions. For intrinsic properties, striatal projection neurons and cortical neurons23 show responses to constant current and current- to-threshold (gain) distributions, which again appear lognormally distributed, with smaller widths than spike rate distributions. “\nThe author keeps stating that she documents lognormal distributions. But I did not find any statistical test. It should be clear how this has been assessed. Do we know it is not e.g. a gamma distribution?\nConclusion: “(b) created a generic neural network model to show that these distributions arise from Hebbian learning, and specifically that intrinsic plasticity must be Hebbian as well; “\nThis \"Hebbian finding\" has been repeated many times in the manuscript, and generally there is a problem with the logic. Just because Hebbian learning results in lognormal distributions, it does not imply that neuronal networks must be Hebbian. Other types of learning may also result in lognormal distribution. Can we really make such an exclusive statement based on this model? I suggest a milder version of the manuscript, where it is suggested that Hebbian learning could explain the lognormality seen in real networks.\n\nMinor issues:\nAbstract:\n“In this paper, we document lognormal distributions for spike rates,… “ -> In this paper, we investigate distributions for spike rates,…\n\nRegarding the statement: “Secondly, we created a generic neural model to show that Hebbian learning will create and maintain lognormal distributions.” If there is a “secondly…” there should also be a “Firstly,…” Maybe put that before “The difference between….”? I also suggest the replacement: “Secondly, we created a generic neural model to test whether Hebbian learning will create and maintain lognormal distributions.”\nI also suggest replacing: “We could prove with the model that not only weights, but also intrinsic gains, need to have strong Hebbian learning in order to produce and maintain the experimentally attested distributions. This settles a long-standing question about the type of plasticity exhibited by intrinsic excitability. “ with “In our model we found that not only weights, but also intrinsic gains, need to have strong Hebbian learning in order to produce and maintain the experimentally attested distributions. This suggest a solution to a long-standing question about the type of plasticity exhibited by intrinsic excitability. “\nIntroduction: In the first paragraph I suggest citing Wohrer, A., Humphries, M. D., and Machens, C. K. (2013). Population-wide distributions of neural activity during perceptual decision-making. Prog Neurobiol, 103, 156–193.\nbecause they discuss exactly this issue of distributions and mean of spiking.\n\nRegarding: “With the current data, we show that the distribution of spike rates within any neural tissue follows a power-law distribution, i.e. a dis- tribution with a ‘heavy tail’. There is also a small number of very low-frequency neurons, so that we have a lognormal distribution2. This lognormal distribution is present in spontaneous spike rates as well as under behavioral stimulation. For each neuron, the devia- tion from the mean rate attributable to a stimulus is small (CV = 0.3–1, standard deviation = 1–4 spikes/s), when compared to the variability in mean spike rate over the whole population (5-7-fold), cf. Table 1 and Table 3. “ First I suggest replacing “any neural tissue” with “many neural tissues”, since no one has looked at all tissue in the nervous system.\n\nFurther I suggest including the reference: Petersen, P. C. and Berg, R. W. (2016). Lognormal firing rate distribution reveals prominent fluctuation-driven regime in spinal motor networks. eLife, 5, e18805. since this will make the statement stronger.\nAnother relevant paper which should be discussed and included is: Ikegaya Y, Sasaki T, Ishikawa D, Honma N, Tao K, Takahashi N, Minamisawa G, Ujita S, Matsuki N. 2013. Interpyramid spike transmission stabilizes the sparseness of recurrent network activity. Cerebral Cortex 23:293– 304 And also the detailed analysis of firing rate versus irregularity for different brain regions and animals should be included: http://www.jneurosci.org/content/36/21/5736\n\nRegarding this statement: “We have constructed a generic model for neuronal populations with adaptable weights and gains. “ I suggest writing a sentence before that giving a motivation for constructing a model, which seem to come out of the blue as it is. Perhaps something like this: “In order to understand these lognormal distributions we constructed a model….”\nDiscussion: Suggest modifying: “It has been suggested that lognormal spike distributions are only a feature of cortical tissue and arise from the strong excitatory recurrent connectivity, but this is neither experimentally nor theoretically correct. “ -> “It has been suggested that lognormal spike distributions are only a feature of cortical tissue and arise from the strong excitatory recurrent connectivity, but this has not been substantiated neither experimentally nor theoretically. “\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "25241",
"date": "25 Sep 2017",
"name": "Tomáš Hromádka",
"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 manuscript the author documents various instances of lognormal distributions of several neuronal parameters across brain areas. The description then serves as motivation for an intriguing investigation of possible underlying mechanisms responsible for creating such distributions. In particular, combination of Hebbian learning with homeostatic adaptation in a simple model is sufficient to recreate the observed distributions of firing rates, synaptic weights, and gains.\nThe presented combination of data and modeling is interesting. The model itself is presented in sufficient detail. However, some statements throughout the text seem to be too strong given the evidence. I suggest rephrasing the strong statements and conclusions to better reflect the presented data and model. After minor changes the manuscript certainly will be of interest to experimenters as well as modelers.\n\nComments:\n\n1. Strong statements\n\nMany a strong statement is sprinkled throughout the manuscript. I suggest modifying such statements to reflect evidence presented in the manuscript itself and the cited literature.\n\nFor example: Abstract: \"Logarithmic scale distribution of weights and gains appears as a functional property that is present everywhere.\" The author probably meant to say \"everywhere we looked.\"\n\nIntroduction: \"...lognormal distributions can only be maintained by Hebbian-style, not homeostatic adaptation.\" There might be a plethora of other mechanisms (not studied in the manuscript) that could lead to lognormal distributions. The fact that Hebbian learning is sufficient does not mean it is also necessary.\n\nSimilarly, here: \"The answer is that the attested distribution of intrinsic gain can only derive from a Hebbian style adjustment rule, even though additional homeostatic adaptation is possible. Intrinsic plasticity is Hebbian\".\n\n2. Methods section\n\nThe Methods section presents a mix of Methods, Introduction, and Results. I suggest refining the section and moving some results into Results. For example, descriptions of various datasets could be easily incorporated into Results, especially when they actually contain new data - \"We extend this dataset by recordings from different types of cortical neurons...\" (p. 3).\n\n3. Lognormal fits\n\nPresented distributions of various neuronal parameters do appear lognormal, with a few exceptions, as correctly pointed out in the text. It is also unlikely that exponential distribution would provide a better fit to any of presented examples. There are, however, other distributions which might, or might not, fit the data better? Was the goodness-of-fit evaluated in any way, or were other types of distributions considered? A brief statement along these lines would further improve this interesting manuscript.\n\n4. Minor comments\n\np. 3 - \"The distribution of spike rates for neurons spiking in the absence of synaptic input...\". I believe it is highly unlikely that spontaneous rates in-vivo arise in the absence of synaptic inputs. Unless controlled---such as in slices, in neuronal cultures, etc.---neurons will receive barrages of input activity even in the absence of experimental stimulation.\n\np. 7 - \"...populations I, J with each n = 1000 neurons...\" should be \"...populations I, J each with n = 1000 neurons...\"\n\np. 8 - \"The output R^J may BE used as input...\"\n\n5. Minor changes to figures\n\nFig. 9 caption needs description of color arrows. A simple statement, such as \"J (excitatory, blue arrows) and H (inhibitory, red arrows)\", would suffice.\n\nWhat is the meaning of colors in Figs. 10 and 11? If there is any, please state so in the caption. If there isn't, please state so as well. The two 'sheets' in Fig. 10 are almost indistinguishable. If colors have no results-related meaning, it might be helpful to plot the bottom sheet (for example) in slightly different colors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1222
|
https://f1000research.com/articles/6-1826/v1
|
11 Oct 17
|
{
"type": "Research Article",
"title": "Development of pulmonary arterial hypertension and diffuse alveolar damage in 2-month old Holstein dairy calves following an acute episode of bloody scours",
"authors": [
"Kerry Gonyeau",
"Seenivasan Subbiah",
"David Klein",
"Dee Church",
"Joseph M. Neary",
"Kerry Gonyeau",
"Seenivasan Subbiah",
"David Klein",
"Dee Church"
],
"abstract": "Background: The goal of this study was to evaluate the effect of hypoxia on intestinal permeability and cardiopulmonary physiology in 2-month old calves. Methods: Calves were exposed to normoxic (975 m altitude; controls) or hypoxic (4,570 m altitude) conditions for 2 weeks. Pulmonary arterial pressures and intestinal permeability to mannitol and lactulose were assessed on Days 0 and 14. Calves were euthanized on Day 15. Two control calves shed occult fecal blood on Day 3; consequently, all calves were treated for coccidiosis. Results: Control calves tended to have greater mean pulmonary arterial pressure than hypoxic calves at Day 0 (P = 0.17), but there was no difference between groups at Day 14 (P = 0.47). On average, mean pulmonary arterial pressure increased by 16 ± 2 mm Hg from Day 0 to 14 (P < 0.001). Serum lactulose was 0.8 ± 0.4 mg/L greater in the control group than the hypoxic group on Days 0 and 14 (P = 0.08). Serum mannitol was 2.0 ± 0.8 mg/L greater in control calves than hypoxic calves on Day 0 (P = 0.009) but there was no difference between groups at Day 14 (P = 0.61). Conclusions: Hypoxia did not affect intestinal permeability, but the results were confounded by intestinal disease. Interestingly, the two calves that had bloody scours had the greatest pulmonary arterial pressures and diffuse alveolar damage. The findings of this study provide preliminary evidence that intestinal disease may contribute to the development of pulmonary diseases in cattle.",
"keywords": [
"endotoxin",
"acute lung injury",
"interstitial pneumonia",
"heart failure"
],
"content": "Introduction\n\nBovine pulmonary hypertension is associated with arterial hypoxemia, systemic arterial hypotension, and increased central venous pressure1,2. These physiological changes are conducive to tissue ischemia. This may be particularly deleterious to tissues, such as the intestinal mucosa, that must function in low oxygen environments under normal physiological conditions3. Impaired intestinal mucosal barrier function may, therefore, be a sequela of rising mean pulmonary arterial pressures in cattle progressing through the confined feeding period of production. If so, mucosal ischemia may promote the translocation of bacteria and toxins across the intestinal wall and into the mesenteric lymph or portal venous circulations. Reduced mucosal barrier function may be a component cause for numerous diseases of feedlot cattle approaching slaughter weight such as liver abscess formation. The goal of this study was, therefore, to evaluate the effect of hypoxia-induced pulmonary hypertension on intestinal mucosal barrier function in a Holstein calf model. We hypothesized that hypoxia-induced pulmonary hypertension would be associated with increased intestinal permeability.\n\n\nMaterials and methods\n\nSix, 2-month old male, intact, clinically healthy Holstein dairy calves were collected from a farm in West Texas. Calves were housed for 2-weeks under normoxic (975 m altitude) or hypoxic (4,570 m altitude) conditions. Pulmonary arterial pressures were measured on Days 0 and 14 of the study. Calves were euthanized on Day 15, and lung tissue was collected for histology. Pulmonary arteriolar remodeling was semi-quantitatively scored. The study was approved by the Texas Tech University Institutional Animal Care and Use Committee (Protocol 16109-12). All efforts were made to ameliorate any suffering experienced by the animals in this study through daily observations and recording of animal health.\n\nMale, intact Holstein calves were obtained from one commercial dairy farm in West Texas (n = 6). Calves were born and raised on the dairy farm until collection at 2 months of age. The calves were clinically healthy, but the farm calf manager reported a recent outbreak of bloody scours among other calves on the farm. Calves were fed 4 to 5 liters of colostrum within 24 hours of birth and provided with 2.8 L of milk twice per day until weaning at 60 days of age. From 2 weeks of age they were provided with ad libitum access to a pelleted complete ration calf starter (≥ 20% crude protein, dry matter basis).\n\nCalves were weighed on arrival at the Texas Tech University farm (altitude: 975 m) and randomly allocated to one of two pens stratified by body mass (Agri-Plastics, Grassie, ON, Canada). The hypoxic group (n = 3) was housed on a raised slatted floor inside a temperature-controlled chamber (temperature 17 ± 3 °C) (dimensions: 1.8 m × 2.3 m). The normoxic control group (n = 3) was housed in a shaded outdoor pen (1.8 m × 3.5 m) with straw bedding on a sloped concrete floor. The pen was moved to a new location every 3 days, and the inside of the pen cleaned was cleaned with a virucidal disinfectant (Virkon S, DuPont, Wilmington, DE). Soiled straw was removed daily and all straw was replaced every 3 days. Maximum and minimum daily temperatures experienced by the control calves during the study ranged from 11 to 30°C and from 5 to 15°C, respectively. After a 5-day acclimation period, the air within the chamber housing the hypoxic group was reduced to 14% oxygen, simulating an altitude of 4,570 m (Day 1 of the study). Calves were provided ad libitum access to water and a pelleted complete ration calf starter (≥ 20% crude protein, dry matter basis).\n\nTwo control calves (calves 2 and 3) started shedding occult fecal blood on Day 3; consequently, all calves were treated for coccidiosis over a 5-day period starting on Day 5. Calves were given amprolium in drinking water at a rate of 47 mL per 10 gallons of water, providing approximately 10 mg amprolium per kg body mass at the usual rate of water consumption. Fecal shedding of blood ceased on Day 8 and feces returned to normal color and consistency on Day 9. Calves did not show signs of tenesmus and maintained a normal appetite.\n\nPulmonary arterial pressure testing was performed on Days 0 and 14. The neck was cleaned with chlorhexidine solution before a 12 gauge, 8.9 cm hypodermic needle was inserted into the jugular vein. Flexible, saline-filled polyethylene catheter tubing (external and internal diameter of 17 and 12 mm, respectively) was then fed through the needle and into the jugular vein. A pressure transducer (TranStar DPT, Smiths Medical ASD, Inc., Dublin, OH) connected the catheter and oscilloscope (BM5Vet, Bionet America, Inc. Tustin, CA, U.S.A.). The change in the pressure waveform that occurred as the catheter tip was advanced through the right atrium, right ventricle, and finally into the pulmonary artery was monitored on the oscilloscope. The jugular vein, right atrium, right ventricle and pulmonary artery have distinct pressure waveforms.\n\nArterial blood-gas analyses were performed to verify the hypoxic status of the calves exposed to hypoxic and normoxic conditions. Samples were collected from all calves on Days 0 and 14. Approximately 1 to 3 mL of blood was collected from the auricular artery using a 20 gauge, 2.5 cm hypodermic needle attached to a pre-heparinized 3 mL syringe. Air bubbles were immediately expelled and the first several drops of blood discarded before analysis on a portable analyser (VetScan i-STAT 1, Abaxis, Union City, CA, USA). Blood-gas tensions were adjusted according to rectal temperature.\n\nIntestinal permeability to the synthetic substances D-Mannitol (100%) (Fisher Scientific, Bridgewater, NJ) and lactulose (99%, Alfa Aesar, Ward Hill, MA) were evaluated twice: on Days 0 and 14. Poly-vinyl tube (outer diameter 0.64cm) was used as a nasogastric tube to administer the substances dissolved in 60 mL of warm water (15 g lactulose and 5 g mannitol). The calf’s head was restrained to one side before the nasogastric tube was inserted into the nostril and advanced caudo-ventrally so that it passed along the ventral meatus, through the nasopharyngeal opening and into the esophagus. The lactulose and mannitol was syringed into the tube. Approximately, 5 mL of air was used to clear all remaining fluid from the nasogastric tube before it was removed.\n\nA 16 gauge, 5 cm catheter was placed in the jugular vein to facilitate the collected of blood at 0, 2, 4, 6, and 8 hours following the administration of the lactulose and mannitol. Blood was collected in red stopper blood tubes (10 mL) and the serum stored (-20 °C) within approximately 1 hour of collection.\n\nTo extract serum proteins in preparation for Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis, samples were thawed at room temperature and 100 µL of homogenized serum sample transferred to a microcentrifuge tube (2 mL, Eppendorf, Hauppage, NY, USA). Next, 300 µL of solvent mixture containing acetonitrile (LC/MS Optima Grade, Fisher Scientific, Bridgewater, NJ, USA) and water (LC/MS Optima Grade, Fisher Scientific, Bridgewater, NJ, USA) (80:20; v/v) was transferred to the tube. After 5 minutes at room temperature, the tube, now containing serum and solvent solution, was vortexed for 30 seconds prior to centrifugation at 21,130 × g for 10 min (5424 R, Eppendorf, Hauppage, NY, USA). The supernatant was transferred into a syringeless filter vial (PTFE, 0.45 µm, GE Healthcare UK Ltd., UK) for LC-MS/MS analysis. Standards were prepared using 10 mg of the standard in 10 mL (mannitol) or 15 mL (lactulose) of solvent (50:50; v/v; methanol:water). From these, serial dilutions of 1, 50, 10, 25, 50, 100 µg/mL in acetonitrile/water mixture (80/20; v/v) were prepared.\n\nUltra-High Pressure Liquid Chromatography-Mass Spectrometry (UPLC-MS) was performed on a liquid chromatograph with triple staged quadrupole mass spectrometer (TSQ-MS) (Ultimate 3000, TSQ Endura, Thermo Fisher Scientific, Waltham, MA). Serum extract (5 µL) was injected into an RP-Amide column (Accentis RP-Amide; 5 µm; 50 × 2.1 mm, Sigma-Aldrich, St. Louis, MO, USA). The column and autosampler tray temperatures were 45°C and 10°C, respectively. Mobile phases A and B consisted of 0.1% formic acid (LC/MS Optima Grade, Fisher Scientific, Bridgewater, NJ, USA) in water and 0.1% formic acid in acetonitrile, respectively. The flow rate was 0.4 mL/min. Mobile phase gradient information, mass spectrometry parameters, and transitions monitored for lactulose and mannitol analysis are provided (Table 1 to Table 3).\n\nCalves were euthanized with intravenous pentobarbital sodium (85 mg/kg) on Day 15 of the study. 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 right diaphragmatic lung 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. Tissue from the caudate liver lobe was also preserved in formalin (10%). Tissue sections (4 µm) were stained with hematoxylin and eosin. Pulmonary arterioles (< 500 μm) were semi-quantitatively scored for medial hypertrophy and adventitial fibrosis (0 = no lesion; + 1 = mild; + 2 = moderate; + 3 = severe). The liver was evaluated for congestion, hydropic degeneration, lipidosis, and other miscellaneous lesions.\n\nStatistical analyses were performed using a commercially available software (Stata version 12.1, College Station, TX). Summary statistics are presented as mean ± SE unless otherwise specified. 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 large4. Generalized estimating equations with an exchangeable correlation structure were used to evaluate the effect of hypoxia (hypoxic versus normoxic) on serum levels of lactulose, mannitol, and the lactulose to mannitol ratio. Two-way interactions were evaluated between group and test and between group and time from lactulose and mannitol administration.\n\n\nResults\n\nOn Day 0, calf body masses ranged from 75.0 to 86.4 kg with a median of 76.4 kg. Mean body masses for hypoxic and control calves were 78.9 ± 2.8 kg and 79.2 ± 3.6 kg, respectively (P = 0.95). On Day 14, calf body masses ranged from 82.7 to 94.0 kg with a median of 90.4 kg. By the end of the study, the body mass of all calves had increased (P = 0.01), but the controls (93.1 ± 0.5 kg) were significantly heavier than the hypoxic calves (85.9 ± 1.7 kg) (P = 0.02).\n\nControls had greater mean right atrial pressures than hypoxic calves at Day 14 (P = 0.001) but not at Day 0 (P = 0.53) (Table 4). There was no difference in mean right ventricular pressures at Day 0 (P = 0.81) or Day 14 (P = 0.30). Control calves tended to have greater mean pulmonary arterial pressures than hypoxic calves on Day 0 (P = 0.17) but there was no difference between groups on Day 14 (P = 0.47). On average, mean pulmonary arterial pressure increased by 16 mm Hg (± 2 mm Hg) from Day 0 to 14 (P < 0.001).\n\nArterial pCO2 did not differ between control and hypoxic calves on Day 0 (P = 0.99) or Day 14 (P = 0.71) of the study (Table 5). Arterial pO2 did not differ between control and hypoxic calves on Day 0 (P = 0.99), but pO2 was significantly lower in hypoxic calves than controls on Day 14 (P = 0.005).\n\nThe ratio of right ventricular mass to total ventricular mass was greater in hypoxic calves than controls indicating greater work hypertrophy (P = 0.04). Both hypoxic and control calves showed histologic lesions of mild (1+) to moderate (2+) medial hypertrophy of the pulmonary arterioles and zero to moderate adventitial fibrosis (Table 6). Two control calves (calves 2 and 3) had gross and histologic lesions consistent with interstitial pneumonia: heavy, wet lungs that failed to collapse (Figure 1) and diffuse alveolar damage (Figure 2). One hypoxic calf (calf 4) had extensive (3+) bronchial associated lymphoid tissue (BALT) and two control calves (calves 1 and 3) had moderate (2+) BALT proliferation. In all cases the BALT was only found around large bronchioles.\n\nKey:\n\n*: Right ventricular mass: total ventricular mass\n\nGross lesions observed in a control calf (calf 2) that developed bloody scours 13 days prior to postmortem examination showing (A) reddened, inflamed intestines next to healthy intestine, (B) wet lungs that failed to collapse, and (C) heart showing a dilated right ventricle.\n\nH&E staining. Bar = 0.25 mm.\n\nAll calves showed histologic evidence of hepatic congestion, which primarily affected hepatic lobule zones 1 and 2. Two hypoxic calves and one control calf showed hydropic degeneration. Two control calves had hepatic lipidosis. Only one calf, a control (calf 3), had liver microabscesses.\n\nControl calves had greater intestinal permeability to mannitol than the hypoxic calves on Day 0 but had similar intestinal permeability at Day 14 (Figure 3). Control calves tended to have greater permeability to lactulose than hypoxic calves throughout the study. Serum lactulose levels were 0.8 ± 0.4 mg/L greater in the control group than the hypoxic group (P = 0.08). Serum lactulose levels decreased 1.0 ± 0.4 mg/L from Day 0 to Day 14 (P = 0.02). Serum lactulose levels did not significantly vary among sampling time points (P = 0.29).\n\nSerum concentrations of lactulose and mannitol in 2-month old Holstein calves at 0 and 14 days of exposure to hypoxic (4,570 m altitude) or normoxic (975 m altitude) conditions.\n\nSerum mannitol levels were 2.0 ± 0.8 mg/L greater in control calves relative to hypoxic calves on Day 0 (P = 0.009). There was a significant interaction between group and Day (P = 0.04). Serum mannitol decreased by 1.4 ± 0.6 mg/L in control calves from Day 0 to Day 14 (P = 0.01), but there was no change in serum mannitol in the hypoxic calves (P = 0.76). There was no difference in serum mannitol between groups at Day 14 (P = 0.61). Mannitol levels tended to decrease by 0.2 ± 0.1 mg/L per hour from administration (P = 0.09).\n\nThe serum lactulose to mannitol ratio decreased by 2.7 ± 1.1 from Day 0 to 14 in the hypoxic group (P = 0.01) but there was no change in the ratio between Days 0 and 14 in the control group (P = 0.85). Calves in the hypoxic group had a ratio that was 2.8 ± 0.9 mg/L greater than the control calves on Day 0 (P = 0.003). There was a tendency for an interaction between group and Day (P = 0.10). The ratio did not significantly vary among over time from administration (P = 0.86).\n\n\nDiscussion\n\nThe findings of this study provide preliminary evidence that intestinal inflammation may be associated with pulmonary disease in cattle. Unfortunately, we were unable to test our proposed hypothesis because control calves developed bloody scours. Because of this unforeseen event, however, the findings are all the more notable. The control calves showed a similar increase in mean pulmonary arterial pressure as the calves housed under hypoxic conditions, but they had significantly greater arterial oxygen tensions indicating that the increase in mean pulmonary arterial pressure was not attributable to hypoxia-induced pulmonary hypertension. Furthermore, the two calves that developed bloody scours had gross and microscopic pathology consistent with diffuse alveolar damage, the histologic counterpart of acute lung injury. In concert, these findings provide preliminary evidence that intestinal inflammation may contribute to the development of pulmonary disease in cattle.\n\nGiven the small study size, our findings are not robustly supported by statistical analyses. There is, however, considerable supporting evidence for an inter-relationship between the pulmonary and gastrointestinal systems. This is to be expected given that the pulmonary and gastrointestinal systems share a common embryonic origin: the lungs evolved as an outgrowth from the primitive gut. In one study of feedlot cattle, the incidence of acute interstitial pneumonia (AIP) was reported to be 70% greater in pens in which at least one animal had died from a digestive disorder than pens in which digestive disorder death loss did not occur5. There is also accumulating evidence that dietary intervention with probiotics may have a favorable effect on the incidence and recovery of cattle from respiratory diseases6,7. Evidence for a link between inflammatory diseases in the respiratory and gastrointestinal systems in humans is also mounting8. Crohn’s Disease sufferers, for example, are approximately 3-times more likely to die from chronic obstructive pulmonary disease (COPD) than none sufferers9,10. It is plausible that inflammatory mediators released into the circulation by inflamed bowel mucosa triggers a secondary inflammatory event within the lung11.\n\nInflammatory mediators likely contributed to the rise in mean pulmonary arterial pressure in the control calves through either a direct effect of the pulmonary vasculature or indirectly by inducing diffuse alveolar damage. Alveolar hypoxia was unlikely the primary because arterial oxygen tensions were significantly greater in the controls than the calves housed under hypoxic conditions. Gut-derived gram-negative sepsis likely contributed to the development of pulmonary hypertension in the control calves in our study. Intravenous injection of calves with endotoxin was reported to increase pulmonary arterial resistance and pressure mediated, in part, by prostaglandin F12–15. Most notably, calves with large pressor responses had increased pulmonary arterial wedge pressures, which may have reflected pulmonary venous hypertension and interstitial edema formation14. Furthermore, pulmonary edema was observed on gross and histological examination of calves given heat killed Pseudomonas aeruginosa organisms14 or endotoxin15. Similarly, studies of broiler chickens intravenously16 or intratracheally17 injected with lipopolysaccharide (LPS) reported a significant but short-lived increase in mean pulmonary arterial pressure. In humans, pulmonary hypertension is commonly reported in association with acute lung injury; however, it is unknown if the hypertension is merely a consequence of increased arterial resistance secondary to diffuse alveolar damage, if the increased arterial pressure contributes to the development of alveolar damage, or if they share a common etiology18.\n\nThe etiologic agent of the bloody scours was not investigated in our study, but we believe that it was most likely attributable to coccidiosis due to the age of the calves, the positive response to treatment, and the potential for parasite accumulation in the straw bedding. Given that the calves were not housed under identical conditions there are other potential confounding factors, such as environmental temperature and straw bedding that need to be considered; however, other than the development of bloody scours, there is no evidence, to our knowledge, that any of these factors could have contributed to the findings of this study.\n\nIn conclusion, we report the development of pulmonary arterial hypertension and diffuse alveolar damage in 2-month old Holstein calves following an acute episode of bloody scours at an altitude of 975 m. The findings of this study provide preliminary evidence that inflammatory gastrointestinal-pulmonary cross-talk may contribute to pulmonary arterial remodeling and hypertension in cattle.\n\n\nData availability\n\nThe full UPLC-MS results for mannitol and lactulose for all calves and times of collection is available at: http://dx.doi.org/10.7910/DVN/OU5YC119.\n\nHistology images of the liver and lung for all calves are available at: http://dx.doi.org/10.7910/DVN/FB6GXV20.\n\nPhotographs were only taken of the heart, lungs, and intestine of calf 2. They are available at: http://dx.doi.org/10.7910/DVN/TMS3AE21.",
"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 would like to thank Dr. Todd Anderson, Director of the The Institute of Environmental and Human Health, Texas Tech University, for the provision of resources and use of the UPLC-MS facilities.\n\n\nReferences\n\nHecht HH, Kuida H, Lange RL, et al.: Brisket disease. III. Clinical features and hemodynamic observations in altitude-dependent right heart failure of cattle. Am J Med. 1962; 32: 171–183. 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: 172–177. PubMed Abstract | Publisher Full Text\n\nZheng L, Kelly CJ, Colgan SP: Physiologic hypoxia and oxygen homeostasis in the healthy intestine. A Review in the Theme: Cellular Responses to Hypoxia. Am J Physiol Cell Physiol. 2015; 309(6): C350–360. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Winter J: Using the Student’s t-test with extremely small sample sizes. Practical Assessment, Research & Evaluation. 2013; 18(10): 1–12. Reference Source\n\nLoneragan GH: Epidemiological characteristics of AIP in feedlot cattle. In: Academy of Veterinary Consultants. Colorado Springs. 2002.\n\nKeyser SA, McMeniman JP, Smith DR, et al.: Effects of Saccharomyces cerevisiae subspecies boulardii CNCM I-1079 on feed intake by healthy beef cattle treated with florfenicol and on health and performance of newly received beef heifers. J Anim Sci. 2007; 85(5): 1264–1273. PubMed Abstract | Publisher Full Text\n\nTimmerman HM, Mulder L, Everts H, et al.: Health and growth of veal calves fed milk replacers with or without probiotics. J Dairy Sci. 2005; 88(6): 2154–2165. PubMed Abstract | Publisher Full Text\n\nKeely S, Talley NJ, Hansbro PM: Pulmonary-intestinal cross-talk in mucosal inflammatory disease. Mucosal Immunol. 2012; 5(1): 7–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuricova D, Pedersen N, Elkjaer M, et al.: Overall and cause-specific mortality in Crohn's disease: a meta-analysis of population-based studies. Inflamm Bowel Dis. 2010; 16(2): 347–353. PubMed Abstract | Publisher Full Text\n\nJess T, Loftus EV Jr, Harmsen WS, et al.: Survival and cause specific mortality in patients with inflammatory bowel disease: a long term outcome study in Olmsted County, Minnesota, 1940–2004. Gut. 2006; 55(9): 1248–1254. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang H, Liu JS, Peng SH, et al.: Gut-lung crosstalk in pulmonary involvement with inflammatory bowel diseases. World J Gastroenterol. 2013; 19(40): 6794–6804. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnderson FL, Tsagaris TJ, Jubiz W, et al.: Prostaglandin F and E levels during endotoxin-induced pulmonary hypertension in calves. Am J Physiol. 1975; 228(5): 1479–1482. PubMed Abstract\n\nReeves JT, Daoud FS, Estridge M: Endotoxin: a cause of spontaneous pulmonary hypertension in cattle? Am J Vet Res. 1973; 34(12): 1573–1576. PubMed Abstract\n\nReeves JT, Daoud FS, Estridge M: Pulmonary hypertension caused by minute amounts of endotoxin in calves. J Appl Physiol. 1972; 33(6): 739–743. PubMed Abstract\n\nTikoff G, Kuida H, Chiga M: Hemodynamic effects of endotoxin in calves. Am J Physiol. 1966; 210(4): 847–853. PubMed Abstract\n\nWideman RF, Bowen OT, Erf GF: Broiler pulmonary hypertensive responses during lipopolysaccharide-induced tolerance and cyclooxygenase inhibition. Poult Sci. 2009; 88(1): 72–85. PubMed Abstract | Publisher Full Text\n\nLorenzoni AG, Wideman RF Jr: Intratracheal administration of bacterial lipopolysaccharide elicits pulmonary hypertension in broilers with primed airways. Poult Sci. 2008; 87(4): 645–654. PubMed Abstract | Publisher Full Text\n\nRyan D, Frohlich S, McLoughlin P: Pulmonary vascular dysfunction in ARDS. Ann Intensive Care. 2014; 4: 28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeary J: Analytical Report on Lactulose and Mannitol by LC-MS/MS. Harvard Dataverse, V1. 2017. Data Source\n\nNeary J: Hepatic and pulmonary histology of 2-month old, male, intact, Holstein calves. Harvard Dataverse, V1.2017. Data Source\n\nNeary J: Heart, liver, and intestines of a 2-month old Holstein calf. Harvard Dataverse, V1. 2017. Data Source"
}
|
[
{
"id": "27300",
"date": "09 Nov 2017",
"name": "Dale Grotelueschen",
"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 are to be congratulated for conducting this work and submitting it for publication. It contributes to the very limited body of knowledge about pulmonary arterial hypertension in the bovine species. This clinically relevant work, although with very small numbers of animals, relates on a preliminary basis, potential influence of intestinal disease on pulmonary disorders. The authors aptly discuss limitations due to small sample size as well as the confounding that occurred during the experiment. Results testing the original hypothesis were confounded by intestinal disease in two of the three control animals. However, findings from the two diseased animals provided preliminary evidence that might prove to be a basis for further exploration. Results are clinically plausible and warrant further investigation. There is not evidence in the manuscript that any attempts to identify the etiologic agent or agents involved with the bloody scours cases. Coccidiosis should be considered a presumptive diagnosis rather than confirmed.\nThe authors clearly defined their hypotheses and carried out the study as planned with the addition of the adaptations required by confounding disease cases in control animals, which led to unexpected findings. Results appear to be fully reported and are very informative to the reader.\nAs the authors state, this report should be interpreted as preliminary evidence.\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": "26946",
"date": "05 Feb 2018",
"name": "Daniel Gould",
"expertise": [
"Reviewer Expertise Animal 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\nTwo groups of 3 calves were maintained under either normoxic or hypoxic conditions. The goal was to determine if hypoxia-induced pulmonary hypertension would be associated with increased intestinal permeability. After 14 days the hypoxia calves had reduced arterial pO2 and elevated pulmonary arterial pressure, but not increased intestinal permeability. During the study 2 of the normoxic calves developed “bloody scours” which compromised their use as controls for intestinal permeability.\n\nEvaluation of the data from the normoxic control calves provided preliminary evidence that intestinal inflammation may contribute to the development pulmonary diseases. The pulmonary disease is assumed to be manifested as elevated pulmonary arterial pressure, diffuse alveolar damage, and pulmonary vascular remodeling (medial hypertrophy and adventitial hyperplasia). The data supporting the pulmonary arterial pressure change seems clear. However, the evidence for diffuse alveolar damage and pulmonary vascular remodeling is less clear. The gross image of the lung of calf 2 does have the features consistent with interstitial pneumonia. But evidence of diffuse alveolar damage is equivocal in the microscopic images provided. Thickening of alveolar walls and hyaline membranes are not present. Changes indicative of pulmonary vascular remodeling are also equivocal. The basis of this discrepancy is unclear but may be due to variations in sampling locations. With regards to the fixation of lung tissue, the route of the fixative perfusion should be indicated.\n\nEvidence for intestinal inflammation seems to be based solely on the observation of bloody scours, a term used in the title of the paper. This implies grossly evident blood in the feces. As stated in the methods, the first indication of the intestinal problem was the presence of occult blood. Were the investigators testing for occult blood on a regular basis? Later, after the initiation of treatment, it is stated that fecal shedding of blood stopped and the feces returned to normal color and consistency. How frequently was there overt blood in the feces? How severe was the diarrhea. The intestinal disease was assumed to be coccidiosis. Other diagnostic tests and histopathology on the intestine would have been helpful in assessing the nature of the inflammation.\n\nIt seems to me that it would be beneficial to emphasize the original goal of this study, ie to test the hypothesis that hypoxia–induced pulmonary hypertension would be associated with increased intestinal permeability. There is good evidence that this prediction failed. The intestinal permeability was unaltered even though the calves had decreased pO2 and increased pulmonary arterial pressure. The rejection of the hypothesis appears to be valid even though the controls were compromised. They were not needed.\n\nThe present title of the paper directs attention to an ad hoc aspect of the study. Since the bloody scours in the control group was unanticipated, collection of a robust data set was difficult for fully investigating the relationship between the pneumonia and enteritis. To the extent that more data could be available, this part of the study could be improved. Refocusing the paper on the hypothesis would emphasize that the original question was answered, but still allow presentation of findings observed and implications raised in the control calves.\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/6-1826
|
https://f1000research.com/articles/6-243/v1
|
09 Mar 17
|
{
"type": "Research Article",
"title": "In silico analysis of single nucleotide polymorphisms (SNPs) in human FOXC2 gene",
"authors": [
"Mohammed Nimir",
"Mohanad Abdelrahim",
"Mohamed Abdelrahim",
"Mahil Abdalla",
"Wala eldin Ahmed",
"Muhanned Abdullah",
"Muzamil Mahdi Abdel Hamid",
"Mohanad Abdelrahim",
"Mohamed Abdelrahim",
"Mahil Abdalla",
"Wala eldin Ahmed",
"Muhanned Abdullah",
"Muzamil Mahdi Abdel Hamid"
],
"abstract": "Introduction: Lymphedema is abnormal accumulation of interstitial fluid, due to inefficient uptake and reduced flow, leading to swelling and disability, mostly in the extremities. Hereditary lymphedema usually occurs as an autosomal dominant trait with allelic heterogeneity. Methods: We identified single nucleotide polymorphisms (SNPs) in the FOXC2 gene using dbSNP, analyzed their effect on the resulting protein using VEP and Biomart, modelled the resulting protein using Project HOPE, identified gene – gene interactions using GeneMANIA and predicted miRNAs affected and the resulting effects of SNPs in the 5’ and 3’ regions using PolymiRTS. Results: We identified 448 SNPs - 429 were nsSNPs and 44 SNPs were in the 5’ and 3’ UTRs. In total, 2 SNPs have deleterious effects on the resulting protein, and a 3D model confirmed those effects. The gene – gene interaction network showed the involvement of FOXC2 protein in the development of the lymphatic system. hsa-miR-6886-5p, hsa-miRS-6886-5p , hsa-miR-6720-3p, which were affected by the SNPs rs201118690, rs6413505, rs201914560, respectively, were the most important miRNAs affected, due to their high conservation score. Conclusions: rs121909106 and rs121909107 were predicted to have the most harmful effects, while hsa-miR-6886-5p, hsa-miR-6886-5p and hsa-miR-6720-3p were predicted to be the most important miRNAs affected. Computational biology tools have advantages and disadvantages, and the results they provide are predictions that require confirmation.",
"keywords": [
"Primary lymphedema",
"FOXC2",
"SNP",
"In silico",
"Bioinformatics",
"miRNA"
],
"content": "Introduction\n\nLymphedema is abnormal accumulation of interstitial fluid, due to inefficient uptake and reduced flow, leading to swelling and disability, which mostly affects the extremities. It can be divided into primary and secondary lymphedema according to the underlying cause. Primary or hereditary lymphedema results from genetic damage, while secondary or acquired lymphedema is caused by lymphatic system malfunction, resulting from trauma, including surgery, radiotherapy, tumors, or infections (for example, parasitic infections)1.\n\nHereditary lymphedema usually occurs as an autosomal dominant trait with allelic heterogeneity. The most common type of primary hereditary lymphedema, Milroy disease, can develop due to mutations in the vascular endothelial growth–factor receptor-3 gene (VEGFR-3; FLT4)2.\n\nForkhead box (Fox) proteins are a family of transcription factors (TFs) that play a key role in cell development, cell cycle regulation, and other important biological processes3. FOXC2, was first identified as a TF that plays a key role in the morphogenesis of the cardiovascular system4. Further studies revealed that FOXC2 was involved in lymphatic vascular development. In both humans and mice, FOXC2 is expressed in large amounts in the developing lymphatic vessels and in adult lymphatic valves5. FOXC2-deficient mice were demonstrated to have abnormal lymphatic patterning and failure to form lymphatic valves, which reveals the critical role of FOXC2 in lymphatic vascular development6. Truncating and missense mutations of FOXC2 have been discovered in patients with late-onset lymphedema (hereditary lymphedema II; OMIM 153200), often associated with distichiasis (double row of eyelashes), and sometimes ptosis (Lymphedema Distichiasis Syndrome [LDS]; OMIM 153400), and/or yellow nails (OMIM 153300)7–9. LDS patients develop defects characterized by lymphatic and venous blood reflux, which means failure and/or absence of lymphatic and venous valves10,11. On a molecular level, FOXC2 DNA binding sites are enriched in NFATC1 consensus sequences, and the two TFs work in tandem during lymphatic vascular morphogenesis12. FOXC2 controls the expression of proteins that are essential for lymphatic valve development, such as connexins. Thus, adequate control of their activity is extremely important for proper lymphatic vessel development and function13.\n\nThe aetiology governing the phenotypic variability remains unclear14. Lymphatic malformations could also result from slightly mutated germline alleles, which are difficult to access (and so to identify), mutations in regulatory regions of the DNA, epigenetic changes, or a combination of the three. Epigenome sequencing and whole exome sequencing may be needed for in-depth study of these mutations. The part genetics play in the development of lymphatic anomalies is highly complex, shown by the discovery of 23 mutated human genes13.\n\nFew studies addressing FOXC2 mutations from a bioinformatics point of view have been published. Out of those, none have specifically focused on single nucleotide polymorphisms (SNPs). SNPs are located in vital regions of the gene, which may code for proteins located at the forkhead active domain of the protein or other sites, may severely affect the function of the TF. Furthermore, SNPs are feasible and cost effective to study using in silico analysis via available bioinformatics tools.\n\nThis study aimed to analyze all SNPs in the human FOXC2 gene and predict their effect on the structure, function, stability and regulation of its respective protein. The results of our study can be used in population studies to screen patients with hereditary lymphedema, and more importantly in phenotypic variations in lymphatic malformations of affected individuals.\n\n\nMethods\n\nWe selected the National Center for Biotechnology Information (NCBI) database, dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP) for the retrieval of SNPs and their related protein sequence of FOXC2 gene. We used “FOXC2” as our search term and used filters to narrow down our search results into two categories: 3’ + 5’ UTR SNPs only and all SNPs in FOXC2, except for those in the 3’ + 5’ UTR. This gene was chosen because it’s the one that is known to be associated with LDS, used for our computational analysis.\n\nWe chose three complementary algorithms for functional impact prediction of nsSNPs: Sorting Intolerant From Tolerant (SIFT; http://sift.bii.a-star.edu.sg/), Polymorphism Phenotyping (PolyPhen; http://genetics.bwh.harvard.edu/pph/) and CONsensus DELeteriousness (Condel; http://bg.upf.edu/fannsdb/)15–17.\n\nSIFT version 2.0 was used to distinguish between tolerant and intolerant coding mutations, and is used to predict whether an amino acid substitution in a protein will have a phenotypic effect. SIFT is based on the premise that protein evolution is correlated with protein function. Variants that occur at conserved alignment positions are expected to be tolerated less than those that occur at diverse positions. PolyPhen is a computational tool for identification of potentially functional nsSNPs. Predictions are based on a combination of phylogenetic, structural and sequence annotation information characterizing a substitution and its position in the protein.\n\nWe uploaded the SNP accession numbers of all the SNPs into VEP (http://www.ensembl.org/Tools/VEP)18 and enabled “SIFT”, “PolyPhen” and “Condel” to retrieve the corresponding predictions of the functional significance of each SNP from all of the three algorithms. We repeated this step using Biomart (http://www.ensembl.org/biomart/martview/3beeb39b00d4f063ebfe8016f3a3b4ba)19, which is similar to VEP and part of Ensembl. It provides a SIFT and PolyPhen prediction and score, like VEP, but it does not provide a Condel prediction. We took the SNPs predicted by both databases to be deleterious and pathogenic (2 SNPs; rs121909106 and rs121909107) to perform the next steps in the analysis.\n\nProject HOPE (http://www.cmbi.ru.nl/hope/) is an online web-server used to search protein 3D structures by collecting structural information from a series of sources. We input the two SNPs (rs121909106 and rs121909107) that we obtained from the last step together with the primary structure of the FOXC2 protein (obtained from the Protein Data Bank http://www.rcsb.org/pdb/explore/explore.do?structureId=1D5V) into HOPE20. HOPE lets you choose the amino acid that was affected but the mutation and allows you to change that specific amino acid and then analyse the resulting change in the 3D (tertiary/quaternary) structure and outputs the change predicted, plus the explanation of such a change (in both structure and function of the protein).\n\nGeneMANIA ver. 3.1.2.8 (http://genemania.org/) finds other genes that are related to a set of input genes, using a very large set of functional association data21. Association data include protein and genetic interactions, pathways, co-expression, co-localization and protein domain similarity. We input FOXC2 as our query gene and the website generated a network of genes along with their gene-gene interactions, according to gene ontology terms.\n\nPolymiRTS v3.0 (http://compbio.uthsc.edu/miRSNP/) is an integrated platform for analyzing the functional impact of genetic polymorphisms in miRNA seed regions and miRNA target sites22. We input a second set of SNPs (containing only 5’ and 3’ SNPs) and acquired a list of the miRNAs affected by these mutations. The affected miRNAs might lead to a decrease/increase of the expression of FOXC2.\n\nThe analysis steps performed are summarized in Figure 1.\n\n\nResults\n\nSNP information for FOXC2 was retrieved from dbSNP. For our investigations, we selected SNPs in coding and UTR (5’and 3’) regions. Among the 448 SNPs, 429 were nsSNPs and 44 SNPs were in the 5’ and 3’ UTRs of FOXC2.\n\nWe found two SNPs, rs121909106 and rs121909107, which correspond to S125L and R121H, to have significant deleterious effect on the structure and function of FOXC2 protein. We summarized the information of the two SNPs predicted by the two databases in Table 1.\n\nFigure 2 shows a 3D model of rs121909107 and rs121909106 and the spatial effects of each on the respective domains they are a part of, in addition to their effects on neighbouring domains.\n\nOverview shows the protein in grey and the affected amino acid in purple, the close-up shows the side chains of both the wild-type (Arginine) and the mutant residue (Histidine) at position 121 and colored green and red respectively. For rs121909106; overview shows the protein in grey and the affected amino acid in purple, the close-up shows the side chains of both the wild-type (Serine) and the mutant residue (Leucine) at position 125 are shown and colored green and red, respectively.\n\nFigure 2 shows the gene-gene interactions of FOXC2. The most important interactions are with FOXB1, FOXD1, FOXD2 and FOXD3 (Figure 3) (additional data in Supplementary material).\n\nTable 3–Table 5 show the effect that SNPs in the 3’ and 5’ region of FOXC2 might have, along with the conservation score (CS) of the site in question. The higher the CS, the more profound the effect of the SNP is predicted to be.\n\n\nDiscussion\n\nThis study aimed to analyze the SNPs identified in the FOXC2 gene. We found a total of 472 SNPs, 2 of which were predicted to adversely affect the function of the resulting protein. The mutation leads to transcription of a histidine instead of an arginine at position 121, and the mutant residue is located near a highly conserved region20. The mutation was reported previously in a patient suffering fromlymphedema-distichiasis syndrome by Berry et al. (2005), and the effects that the R121H mutation would have on the DNA-binding part of the forkhead domain (FHD) of FOXC2 was predicted. It was predicted that R121H would impair FOXC2 protein’s ability to bind DNA and act as a transcription activator, and this was confirmed by biochemical studies. Also, the mutation resulted in the mislocalization of FOXC2. Berry et al. (2005) determined that the mutation results in a non-functional protein and that this leads to hereditary LDS23. The residue affected by rs121909107 is part of an inter pro-domain named Fork Head Domain Conserved Site 2 (Interpro, IPR030456), and it is annotated with the following Gene-Ontology (GO) terms to indicate its function: sequence-specific DNA binding (GO:0043565) and sequence-specific DNA binding transcription factor activity (GO:0003700).\n\nThe mutation of a serine into a leucine at position 125 means that each amino acid has its own specific size, charge, and hydrophobicity-value. The original wild-type residue and newly introduced mutant residue often differ in these properties. The mutation results in incorporating as amino acid with a different level of hydrophobicity, this will affect hydrogen bond formation, and the mutant residue is located near a highly-conserved region20. This mutation matches a previously described variant in affected members of families with lymphedema-distichiasis syndrome, previously reported by Mangion et al.24 and Bell et al.7. The mutation was not identified in 100 normal chromosomes. This mutation results in a wide range of phenotypes from minimal distichiasis to severe lymphoedema and congenital heart disease7. The residue affected by rs121909106 is part of an inter prodmain named Winged Helix-Turn-Helix Dna-Binding Domain (IPR011991) and it is annotated with the following GO terms to indicate its function: nucleic acid binding (GO:0003676) and nucleic acid binding transcription factor activity (GO:0001071).\n\nOur results from GeneMANIA showed that FOXC2 interacts with a lot of genes (Figure 2), mainly functioning to control connective tissue, tube and epithelial tissue development. These results were proved important by Mangion et al.24 and Bell et al.7, which showed that mutations in FOXC2 lead to developing LDS.\n\nPolymiRTS results showed SNPs and INDELs in miRNA target sites: target sites disrupted by SNPs and INDELs in miRNA seeds and target sites created by SNPs and INDELs in miRNA seeds (Table 3–Table 5). Three miRNAs that are worth noting are hsa-miR-6886-5p (with a CS of 7), hsa-miR-6886-5p (with a CS of 7) and hsa-miR-6720-3p (with a CS of 6), which are affected by the SNPs rs201118690, rs6413505, rs201914560, respectively. This points to possibility that the areas affected by those SNPs have an evolutionary important function.\n\n\nConclusions\n\nIn conclusion, there are many SNPs that affect FOXC2 gene; some are predicted to be harmful, such as rs121909106 and rs121909107, but most are not. miRNAs were affected by SNPs in the 3’ and 5’ untranslated regions of FOXC2 gene and three are noteworthy, hsa-miR-6886-5p, hsa-miR-6886-5p and hsa-miR-6720-3p, due their high conservation score.\n\nComputational biology tools are very powerful, especially when provided with good data and used by experts. However, bioinformatics tools have their limitations; most importantly the fact that their results are but predictions, meaning that the information they provide us with requires confirmation by probing.\n\n\nData availability\n\nDataset 1: SNPs used for PolymiRTS analysis. doi, 10.5256/f1000research.10937.d15306425\n\nDataset 2: SNPs used for other analyses. doi, 10.5256/f1000research.10937.d15306526",
"appendix": "Author contributions\n\n\n\nMN conceived the study, contributed to data collection and analysis, and preparation of the manuscript. MA, MA, MA, WA and MA contributed to data collection. MH contributed to preparation of the manuscript and supervised the whole process. All authors were involved in the revision of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nMany thanks to Prof. Muntasir Ibrahim, Sarmad Haydar and Mutaz Amin for their encouragement and help.\n\n\nSupplementary material\n\nSupplementary File 1: GeneMANIA network as an Excel file.\n\nClick here to access the data.\n\nSupplementary File 2: Print-out of the full report obtained from the GeneMANIA website for FOXC2 gene.\n\nClick here to access the data.\n\n\nReferences\n\nKurland LT, Molgaard CA: The patient record in epidemiology. Sci Am. 1981; 245(4): 54–63. PubMed Abstract | Publisher Full Text\n\nKarkkainen MJ, Ferrell RE, Lawrence EC, et al.: Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nat Genet. 2000; 25(2): 153–9. PubMed Abstract | Publisher Full Text\n\nWijchers PJ, Burbach JP, Smidt MP: In control of biology: of mice, men and Foxes. Biochem J. 2006; 397(2): 233–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIida K, Koseki H, Kakinuma H, et al.: Essential roles of the winged helix transcription factor MFH-1 in aortic arch patterning and skeletogenesis. Development. 1997; 124(22): 4627–38. PubMed Abstract\n\nPetrova TV, Karpanen T, Norrmen C, et al.: Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat Med. 2004; 10(9): 974–81. PubMed Abstract | Publisher Full Text\n\nKriederman BM, Myloyde TL, Witte MH, et al.: FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome. Hum Mol Genet. 2003; 12(10): 1179–85. PubMed Abstract | Publisher Full Text\n\nBell R, Brice G, Child AH, et al.: Analysis of lymphoedema-distichiasis families for FOXC2 mutations reveals small insertions and deletions throughout the gene. Hum Genet. 2001; 108(6): 546–51. PubMed Abstract | Publisher Full Text\n\nFang J, Dagenais SL, Erickson RP, et al.: Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome. Am J Hum Genet. 2000; 67(6): 1382–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFinegold DN, Kimak MA, Lawrence EC, et al.: Truncating mutations in FOXC2 cause multiple lymphedema syndromes. Hum Mol Genet. 2001; 10(11): 1185–9. PubMed Abstract | Publisher Full Text\n\nBrice G, Mansour S, Bell R, et al.: Analysis of the phenotypic abnormalities in lymphoedema-distichiasis syndrome in 74 patients with FOXC2 mutations or linkage to 16q24. J Med Genet. 2002; 39(7): 478–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMellor RH, Brice G, Stanton AW, et al.: Mutations in FOXC2 are strongly associated with primary valve failure in veins of the lower limb. Circulation. 2007; 115(14): 1912–20. PubMed Abstract | Publisher Full Text\n\nIvanov KI, Agalarov Y, Valmu L, et al.: Phosphorylation regulates FOXC2-mediated transcription in lymphatic endothelial cells. Mol Cell Biol. 2013; 33(19): 3749–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrouillard P, Boon L, Vikkula M: Genetics of lymphatic anomalies. J Clin Invest. 2014; 124(3): 898–904. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYu Z, Wang J, Peng S, et al.: Identification of a novel VEGFR-3 missense mutation in a Chinese family with hereditary lymphedema type I. J Genet Genomics. 2007; 34(10): 861–7. PubMed Abstract | Publisher Full Text\n\nGonzález-Pérez A, López-Bigas N: Improving the assessment of the outcome of nonsynonymous SNVs with a consensus deleteriousness score, Condel. Am J Hum Genet. 2011; 88(4): 440–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRamensky V, Bork P, Sunyaev S: Human non-synonymous SNPs: server and survey. Nucleic Acids Res. 2002; 30(17): 3894–900. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNg PC, Henikoff S: Predicting deleterious amino acid substitutions. Genome Res. 2001; 11(5): 863–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcLaren W, Pritchard B, Rios D, et al.: Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor. Bioinformatics. 2010; 26(16): 2069–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKinsella RJ, Kähäri A, Haider S, et al.: Ensembl BioMarts: a hub for data retrieval across taxonomic space. Database (Oxford). 2011; 2011: bar030. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVenselaar H, Te Beek TA, Kuipers RK, et al.: Protein structure analysis of mutations causing inheritable diseases. An e-Science approach with life scientist friendly interfaces. BMC Bioinformatics. 2010; 11: 548. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWarde-Farley D, Donaldson SL, Comes O, et al.: The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res. 2010; 38(Web Server issue): W214–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBhattacharya A, Ziebarth JD, Cui Y: PolymiRTS Database 3.0: linking polymorphisms in microRNAs and their target sites with human diseases and biological pathways. Nucleic Acids Res. 2014; 42(Database issue): D86–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBerry FB, Tamimi Y, Carle MV, et al.: The establishment of a predictive mutational model of the forkhead domain through the analyses of FOXC2 missense mutations identified in patients with hereditary lymphedema with distichiasis. Hum Mol Genet. 2005; 14(18): 2619–27. PubMed Abstract | Publisher Full Text\n\nMangion J, Rahman N, Mansour S, et al.: A gene for lymphedema-distichiasis maps to 16q24.3. Am J Hum Genet. 1999; 65(2): 427–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNimir M, Abdelrahim M, Abdelrahim M, et al.: Dataset 1 in: In silico analysis of single nucleotide polymorphisms (SNPs) in human FOXC2 gene. F1000Research. 2017. Data Source\n\nNimir M, Abdelrahim M, Abdelrahim M, et al.: Dataset 2 in: In silico analysis of single nucleotide polymorphisms (SNPs) in human FOXC2 gene. F1000Research. 2017. Data Source"
}
|
[
{
"id": "21906",
"date": "18 Apr 2017",
"name": "Pascal Brouillard",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes an elegant bioinformatic analysis of variations in a known gene (FOXC2) for primary lymphedema. It uses numerous freely available tools to identify not only variants that can possibly alter the function of the protein, but also affect miRNA (binding) sites. However, one weakness is that it only focuses on changes present in dbSNP, which lacks known amino acid-changing mutations such as e.g. those identified by Van Steenstel et al. or Sholto-Douglas-Vernon et al.1,2. The results of the PolymiRTS should also be explained a bit more, especially on how to interpret the scores and what to consider as important. Indeed, contrary to the two known amino acids that have been previously characterized, these have not been proven. Focusing on the conservation score is probably not enough because some scores of 2 have a stronger \"context+score change\" effect than the scores of 7.\n\nThere are several mistakes that need to be corrected:\nThe number of SNPs is unclear. The abstract and results mention a total of 448 and the discussion 472. Yet, neither of these corresponds to 429 nsSNPs + 44 in UTRs (= altogether 474) Page 3, right column, second line, SNPs may \"code for amino acids\", not proteins. Page 3, last paragraph, the Biomart link can stop after /martview/. The alphanumerical term after is a changing each time you open a session. Page 4, line 10: ... affected BY the mutation ..., not \"but\" Page 4, GeneMANIA is Figure 3, not 2, and PolymiRTS reffers to table 2 to 4, not 3-5. The bolded title of Figure 2 cannot be solely rs121909107. Page 6, Discussion, 4th line \"the mutation leads to TRADUCTION of a histidine ..., not transcription. Page 6, right column, the first sentence is strange. It is not the mutation into a leucine at position 125 that means that \"each amino acid has its own specific size, charge and hydrophobicity-value\". This is a general fact! Page 6, right col, line 5: AN amino acid, not as. Line 14: interpro domain. Line 15, DNA in capital letters. Second paragraph, GeneMania links to Figure 3, not 2. Second-last line, refer to Tables 2-4.",
"responses": []
},
{
"id": "22567",
"date": "19 May 2017",
"name": "Fengkai Zhang",
"expertise": [
"Reviewer Expertise Systems biology",
"bioinformatics",
"genomics",
"immunology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript presents nice work in analyzing the SNPs in the human FOXC2 gene with a set of publicly available bioinformatics tools and identified 2 nsSNPs and several other UTR region SNPs with potential significance in Lymphedema. The pipeline of tools has been well described and it could benefit research and analysis of other important genes associated with diseases. As the authors pointed out, the findings need further confirmation. I would suggest the authors to address the following aspects: (1) Is there any relationship or coordintation among the identified SNPs? Do they belong to the same haplotype(s)? (2) What is the consideration or motivation to select the tools used for this study? For example, why did the authors choose GeneMANIA? All other tools in this study are used to analyze the SNPs in FOXC2 gene. GeneMANIA gives the result of the network involving FOXC2 with other genes. However, FOXC2 has been well introduced with multiple references at the beginning of this manuscript about its importance in Lymphedema. GeneMANIA does not show obvious help in SNP analysis focused in this study. (3) What is the criteria to identify the positive and negative SNPs for FOXC2, and why? How can false negative be avoided?\n\nBesides the above major reservations, I would like to ask the authors to clarify/correct the following:\nAs pointed by the first referee, what is the total number of SNPs? The abstract and result sections indicated 448 SNPs and the discussion indicated 472 SNPs. How can the two numbers be calculated from 429 nsSNPs and 44 UTR region SNPs (429+44=473)?\n\nIn both abstract and discussion sections, the authors indicate the results require further confirmation. Can the authors describe some possible methods, like experimental, clinical or statistical?\n\nPage 3, spell out NFATC1 when it appears at the first time . Page 3, rephrase “SNPs are located in vital regions of the gene, which may code for proteins located at the forkhead active domain of the protein or other sites, may severely affect the function of the TF.”\n\nPage 6, figure 3, the color codes are hard to distinguish in the network visualization. Use alternative method, or give a more detailed explanation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-243
|
https://f1000research.com/articles/6-1505/v1
|
17 Aug 17
|
{
"type": "Research Article",
"title": "Towards a systematic assessment of assay interference: Identification of extensively tested compounds with high assay promiscuity",
"authors": [
"Erik Gilberg",
"Dagmar Stumpfe",
"Jürgen Bajorath",
"Erik Gilberg",
"Dagmar Stumpfe"
],
"abstract": "A large-scale statistical analysis of hit rates of extensively assayed compounds is presented to provide a basis for a further assessment of assay interference potential and multi-target activities. A special feature of this investigation has been the inclusion of compound series information in activity analysis and the characterization of analog series using different parameters derived from assay statistics. No prior knowledge of compounds or targets was taken into consideration in the data-driven study of analog series. It was anticipated that taking large volumes of activity data, assay frequency, and assay overlap information into account would lead to statistically sound and chemically meaningful results. More than 6000 unique series of analogs with high hit rates were identified, more than 5000 of which did not contain known interference candidates, hence providing ample opportunities for follow-up analyses from a medicinal chemistry perspective.",
"keywords": [
"Biological screening data",
"statistical analysis",
"active compounds",
"assay frequency",
"hit rate distribution",
"assay interference"
],
"content": "Introduction\n\nCompounds with false-positive signals in biological assays cause substantial problems for biological screening and medicinal chemistry1. Assay artifacts often remain undetected or are unveiled only at later stages of compound development efforts, leading to substantial loss of time and resources. Moreover, once published, artificial activities spread through the scientific literature and potentially cause even more harm by inspiring follow-up investigations that are doomed to fail. Known assay interference compounds include colloidal aggregators2–7 and many other compound classes that can react in different ways or are fluorescent under assay conditions6–15. Systematic efforts to identify interference compounds include the compilation of aggregators2–4 and pan-assay interference compounds (PAINS)8,9. The latter comprise a set of 480 classes of compounds originally identified in AlphaScreen assays8. PAINS are typically contained as substructures in larger compounds. However, the assessment and prediction of assay interference is far from being a trivial exercise. For example, analysis of screening data from PubChem16 has revealed that many compounds containing PAINS, including most reactive chemical entities, have very different hit rates or might be consistently inactive17,18. Moreover, analogs or different series of analogs containing the same PAINS substructure often have distinct activity profiles and are active against different targets19. Thus, interference characteristics of related compounds frequently differ and a substructure with interference potential does not necessarily give rise to false-positive assay signals. To further complicate matters, promiscuous compounds may also have true multi-target activities20 that are relevant for polypharmacology20–22. Moreover, even highly promiscuous screening hits include molecules with no apparent liabilities, in addition to obvious interference compounds12.\n\nWithout doubt, judging assay interference and candidate compounds requires profound chemical knowledge and experience. It is equally relevant, however, to strive for a data-driven assessment of promiscuity by exploring compound activity data on a large scale20, aiming to identify compounds with interference potential for further analysis. Therefore, we have carried out a statistical analysis of hit rates of compounds that were extensively tested in screening assays. A special feature of this study has been its focus on pairs or larger series of analogs, rather than single compounds, which provides additional confidence criteria for activity assessment and further increases the information content of activity data analysis. Many series of analogs with much higher than typically observed hit rates and largely consistent activity profiles across many different assays were identified. This collection of series provides a basis for further investigating compounds with interference potential or true multi-target activities.\n\n\nMethods\n\nAll calculations were carried out using in-house scripts and implementations.\n\nFrom the PubChem BioAssay database16, 437,257 compounds were pre-selected that were tested in both primary and confirmatory assays, representing extensively assayed screening compounds23. Approximately 95% of these compounds were evaluated in more than 50 primary and/or confirmatory assays23. Primary PubChem assays report compound activity (e.g., percentage activity) for a single dose, while confirmatory assays are dose-response assays yielding titration curves and IC50 values. Our current analysis focused on primary assays, for which much larger data volumes were available than for confirmatory assay. Primary assays also included assays for which no target was specified (such as cell-based assays). For pre-selected compounds, hit rate statistics were determined.\n\nA matched molecular pair (MMP) is a pair of compounds that are only distinguished by a chemical change at a single site24, termed a chemical transformation25. As an extension of the MMP concept, a matched molecular series (MMS) was defined as the union of all MMP compounds that are only distinguished by chemical modifications at a given site26. Accordingly, an MMS represents a series of analogs sharing a single substitution site. To generate MMPs, exocyclic single bonds in screening compounds were systematically fragmented25 following retrosynthetic fragmentation rules27, yielding so-called RECAP-MMPs28. These MMPs were subject to transformation size restrictions in order to limit chemical changes to modifications typically observed in series of analogs29. An MMS was designated as redundant if it was a subset of a larger MMS or if there was another MMS representing the same series of analogs but having a larger MMP core. For screening compounds with high hit rates, non-redundant MMS were systematically determined.\n\nFor each MMS, three parameters were calculated. First, the MMS hit rate (HR) was obtained from the union of all assays (i.e., the number of unique assays in which one or more analogs were tested in) and assays with activity signals (active assays, i.e., the number of unique assays in which one or more analogs were found to be active). Second, assay overlap was determined as the proportion of assays in which all MMS compounds were tested in (shared assays, i.e., the intersection of assays) relative to the union of assays. Third, from assay overlap, assays with inconsistent activity were calculated as the proportion of shared assays in which different MMS compounds were active or inactive.\n\n\nResults and discussion\n\nA statistical analysis of hit rates of extensively tested screening compounds is presented taking assay frequency into account. On the basis of the hit rate distribution, ranges of unusually high hit rates were determined. From compounds with high hit rates, analog series with single substitution sites (MMSs), i.e., “minimal” chemical modifications within series, were systematically extracted, which provided structural context information and hit rate controls for closely related compounds. For MMSs, different parameters were calculated, making it possible to compare and prioritize these series. The collection of MMSs with high hit rates provides a basis for investigating assay interference candidates, as well as chemical entities with potential multi-target activities.\n\nFigure 1 (boxplot on the left) shows the global distribution of primary assays for 437,257 extensively tested PubChem compounds, with a median value of 347 assays per compound. From these, a subset of 327,532 compounds was selected that were tested in more than 257 primary assays, corresponding to the lower quartile boundary of the global distribution. For this subset, the assay distribution was separately monitored (Figure 1, boxplot on the right), yielding a median of 426 (and a maximum of 626) assays per compound. Hence, half of these compounds were tested in more than 426 primary assays.\n\nThe frequency distribution of primary assays is shown in a boxplot format for 437,257 pre-selected PubChem compounds and a subset of 327,532 compounds. The plot gives the smallest number of primary assays (lower whisker), first quartile (lower boundary of the box), median value (thick line), third quartile (upper boundary of the box), and largest number of assays (upper whisker). Outliers are not displayed. The dashed blue line indicates the selection criterion for the compound subset (i.e., tested in more than 257 primary assays).\n\nFor 327,532 compounds tested in more than 257 assays, hit rates were determined. The distribution is reported in Figure 2 (boxplot on the left), resulting in a median hit rate of 0.4%. The lower quartile boundary and lower whisker of the boxplot were identical and represented consistently inactive compounds, which were not of interest for our current analysis. On the basis of the distribution, the interval of “bulk hit rates” (b_hr) for these extensively assayed PubChem compounds was defined as 0% < b_hr ≤ 1.0%, covering the lower quartile, median, and upper quartile (and hence the “bulk” of the distribution). There were 80,495 compounds with hit rates ≥ 1.0%. The hit rate distribution of this compound subset is shown in Figure 2 (middle), yielding a median of 1.8%. This value was set as the hit rate threshold for most active screening compounds. The threshold was exceeded by 41,609 compounds, representing 12.7% of the initial compound pool. The hit rate distribution of these compounds is reported in Figure 2 (right), resulting in a median of 2.9%. We determined that 93.1% of the compounds with hit rates greater than 1.8% in primary assays were also active in confirmatory assays (yielding IC50 values). Hence, their activity was not confined to primary assays.\n\nFor three different subsets of PubChem compounds, hit rate distributions are shown in boxplots according to Figure 1. The subsets are characterized by increasing hit rates (marked by dashed blue lines).\n\nFrom the 41,609 compounds with highest hit rates, MMSs were systematically extracted on the basis of RECAP-MMPs. After removal of redundant MMSs (see Methods), 6941 unique MMSs were obtained comprising 14,646 compounds, which represented our final hit rate- and series-based selection set. Table 1 reports the size distribution of the MMSs, ranging from two to 17 analogs per series. With 6111 instances, compound pairs and triplets dominated the distribution, but more than 800 larger MMSs were also obtained. As further discussed below, compound pairs and triplets already provide informative controls for activity analysis and enable a more confident assessment compared to the analysis of individual compounds. This was a major motivation for focusing the analysis on MMSs.\n\nThe distribution of 6941 frequently active MMSs (#MMSs) over increasing numbers of compounds (#CPDs) is reported.\n\nFigure 3a illustrates the derivation of three parameters for the characterization and comparison of MMSs (rationalized in the Methods section). The cumulative MMS hit rate is a direct measure for the activity of a series. In addition, assay overlap represents a confidence criterion for MMS assessment, i.e., large assay overlap of compounds comprising a series assigns high confidence to hit rate comparisons. By contrast, the proportion of assays with inconsistent activity should best be minimal to draw firm conclusions. Figure 3b reports the distribution of these three parameters for the 6941 MMSs. Assay overlap (upper left plot) and MMS hit rates (lower left) were generally high, with median values of 79.3% and 5.8%, respectively. By contrast, the proportion of inconsistent assays (upper right) was overall low, with a median of only 3.7%. Thus, the distributions of MMS parameters indicated that the set of MMSs was suitable for the analysis of series-based hit rates and hit rate comparison of compounds comprising individual MMSs. We note that MMSs can be ranked in the order of decreasing assay overlap and MMS hit rates and increasing inconsistent assays and prioritized, for example, on the basis of rank fusion calculations.\n\n(a) An exemplary MMS comprising three analogs is shown. The MMS core and varying substituents are colored in black and orange, respectively. For each compound, the number of assays it was tested and active in is reported, respectively. Furthermore, the assay union, intersection, and MMS hit rate (purple) are given. From these data, the assay overlap (green) of MMS analogs was determined as well as the proportion of assays with consistent activity, inactivity, and inconsistent activity (blue). (b) Boxplots are shown reporting the distribution of assay overlap, assays with inconsistent activity as well as assay- or target-based MMS hit rates for PubChem compounds with greater than 1.8% hit rate.\n\nOur analysis was intentionally focused on hit rates over assays (i.e., assay promiscuity) to take as many activity readouts as possible into account. Therefore, as a control, assay- and target-based hit rates were also compared. Compounds forming the 6941 MMS were evaluated in a total of 1213 assays. For 255 of these assays, no individual target was specified. The remaining 958 assays covered 426 different targets. Figure 3b reports the distributions of MMS hit rates over assays (lower left plot) and targets (lower right). The distributions were overall similar, with median values of assay- and target-based hit rates of 5.8% and 4.9%, respectively. Hence, despite the presence of multiple assays for a subset of targets, assay-based hit rates were only slightly higher than target-based rates, indicating that corresponding conclusions would be drawn from the analysis of these distributions.\n\nThe computational aggregation advisor4 and compound strings taken from PAINS filters30,31 (http://www.rdkit.org) were used to search the MMSs for known assay interference candidates. The 14,646 MMS compounds contained 783 aggregators (on the basis of 100% similarity) and 2381 compounds with PAINS substructures. There were 611 MMSs with one or more aggregators, 1139 MMSs with one or more PAINS, and 126 MMSs including aggregators and PAINS. However, 5065 MMSs with high hit rates did not contain known compounds with aggregation potential or PAINS substructures. Thus, the MMSs provide a large source of analogs for the exploration of other interference candidates, as well as compounds with true multi-assay/target activities.\n\nFigure 4 shows exemplary compound pairs and triplets with high assay promiscuity. The two analogs in Figure 4a were tested in more than 380 assays with 93.5% assay overlap and only 1.6% inconsistent assays, yielding comparable hit rates of 2.8% and 3.2%, respectively, resulting in an MMS hit rate of 4.5%. These analogs contained a classical PAINS substructure (ene_rhodanine)8,9. Furthermore, compounds in Figure 4b were analogs of a molecule with aggregation potential4. They were tested in more than 300 and 400 assays, respectively, yielding a relatively low assay overlap of 59%, and had hit rates of 2.2% and 2.6%, respectively, resulting in a low MMS hit rate of 2.9%. Thus, these analogs were far from being consistently active, as one might assume for strong aggregators. In Figure 4c, a pair of thieno[2,3-d]pyrimidine-2-acetic acid ethyl ester analogs is shown that were tested in 442 assays with large overlap. These compounds had high hit rates of 5.9% and 7.9%, respectively, resulting in a high MMS hit rate of 9.8%. Moreover, Figure 4d shows a triplet of sulfonylpyrimidines that were tested in 357–361 assays with 89.7% overlap, having very high hit rates of 8.7% (one analog) and more than 13% (two analogs). Compounds forming each of the MMSs in Figure 4 displayed consistent hit rate characteristics, hence assigning confidence to their observed activity phenotype. The analogs in Figure 4c and Figure 4d have previously not been classified as interference candidates. However, they might well be reactive under assay conditions. Taken together, these examples of analog pairs and triplets (i.e., minimally sized MMSs) are indicative of the potential of well characterized MMSs for follow-up investigations focusing on assay interference and multi-target activities.\n\n(a–d) Four exemplary MMSs (core, black; substituents, orange) are shown and the MMS hit rate, assay overlap, and proportion of assays with inconsistent activity are reported. In addition, for each individual analog, its assay frequency and hit rate are provided.\n\n\nConclusions\n\nHerein, a detailed analysis of hit rates of nearly 440,000 extensively assayed screening compounds has been presented. On the basis of hit rate distributions, 12.7% of the compounds with highest hit rates were selected. From these compounds, analog series with single substitution sites were systematically extracted to complement hit rate statistics with the assessment of structural relationships between active compounds. A total of 6941 unique MMSs were obtained comprising 14,646 compounds. These MMSs were characterized using different parameters prioritizing high-confidence series for activity analysis. A major goal of our study has been the data-driven generation of a pool of analog series for the evaluation of assay interference potential and multi-target activities. More than 5000 MMSs did not contain known interference candidates, providing an opportunity to evaluate compounds with interference potential on a large scale. In the next step, analog series will be evaluated from a medicinal chemistry perspective to complement and further extend statistical considerations. Annotated series and associated assay/target information will then be made freely available. The statistics and selection steps reported herein also make it possible to regenerate compound subsets at different hit rate levels and subject them to further analysis. In addition, large numbers of compounds with high hit rates that were not part of MMSs are also available. For reasons discussed, our preferred approach is taking compound series information into account when judging assay promiscuity.\n\n\nData availability\n\nThe data sets used in this study are freely available in PubChem and can be generated following the selection protocol reported in the Methods.",
"appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nDS is supported by Sonderforschungsbereich 704 of the Deutsche Forschungsgemeinschaft.\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\nAldrich C, Bertozzi C, Georg GI, et al.: The Ecstasy and Agony of Assay Interference Compounds. ACS Cent Sci. 2017; 3(3): 143–147. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcGovern SL, Caselli E, Grigorieff N, et al.: A Common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J Med Chem. 1996; 45(8): 1712–1722. PubMed Abstract | Publisher Full Text\n\nShoichet BK: Screening in a spirit haunted world. Drug Discov Today. 2006; 11(13–14): 607–615. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIrwin JJ, Duan D, Torosyan H, et al.: An Aggregation Advisor for Ligand Discovery. 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J Med Chem. 2016; 59(22): 10285–10290. PubMed Abstract | Publisher Full Text\n\nBaell JB: Feeling Nature’s PAINS: Natural Products, Natural Product Drugs, and Pan Assay Interference Compounds (PAINS). J Nat Prod. 2016; 79(3): 616–628. PubMed Abstract | Publisher Full Text\n\nBisson J, McAlpine JB, Friesen JB, et al.: Can Invalid Bioactives Undermine Natural Product-Based Drug Discovery? J Med Chem. 2016; 59(5): 1671–1690. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNelson KM, Dahlin JL, Bisson J, et al.: The Essential Medicinal Chemistry of Curcumin. J Med Chem. 2017; 60(5): 1620–1637. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Y, Xiao J, Suzek TO, et al.: PubChem’s BioAssay Database. Nucleic Acids Res. 2012; 40(Database issue): D400–D412. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCapuzzi SJ, Muratov EN, Tropsha A: Phantom PAINS: Problems with the Utility of Alerts for Pan-Assay INterference CompoundS. J Chem Inf Model. 2017; 57(3): 417–427. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJasial S, Hu Y, Bajorath J: How Frequently Are Pan-Assay Interference Compounds Active? Large-Scale Analysis of Screening Data Reveals Diverse Activity Profiles, Low Global Hit Frequency, and Many Consistently Inactive Compounds. J Med Chem. 2017; 60(9): 3879–3886. PubMed Abstract | Publisher Full Text\n\nGilberg E, Stumpfe D, Bajorath J: Activity profiles of analog series containing pan assay interference compounds. RSC Adv. 2017; 7(57): 35638–35649. Publisher Full Text\n\nHu Y, Bajorath J: Compound promiscuity: what can we learn from current data? Drug Discov Today. 2013; 18(13–14): 644–650. PubMed Abstract | Publisher Full Text\n\nPaolini GV, Shapland RH, van Hoorn WP, et al.: Global mapping of pharmacological space. Nat Biotechnol. 2006; 24(7): 805–815. PubMed Abstract | Publisher Full Text\n\nBoran AD, Iyengar R: Systems approaches to polypharmacology and drug discovery. Curr Opin Drug Discov Devel. 2010; 13(3): 297–309. PubMed Abstract | Free Full Text\n\nJasial S, Hu Y, Bajorath J: Determining the Degree of Promiscuity of Extensively Assayed Compounds. PLoS One. 2016; 11(4): e0153873. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGriffen E, Leach AG, Robb GR, et al.: Matched molecular pairs as a medicinal chemistry tool. J Med Chem. 2011; 54(22): 7739–7750. PubMed Abstract | Publisher Full Text\n\nHussain J, Rea C: Computationally efficient algorithm to identify matched molecular pairs (MMPs) in large data sets. J Chem Inf Model. 2010; 50(3): 339–348. PubMed Abstract | Publisher Full Text\n\nWawer M, Bajorath J: Local structural changes, global data views: graphical substructure-activity relationship trailing. J Med Chem. 2011; 54(8): 2944–2951. PubMed Abstract | Publisher Full Text\n\nLewell XQ, Judd DB, Watson SP, et al.: RECAP--retrosynthetic combinatorial analysis procedure: a powerful new technique for identifying privileged molecular fragments with useful applications in combinatorial chemistry. J Chem Inf Comput Sci. 1998; 38(3): 511–522. PubMed Abstract | Publisher Full Text\n\nde la Vega de León A, Bajorath J: Matched molecular pairs derived by retrosynthetic fragmentation. Med Chem Commun. 2014; 5(1): 64–67. Publisher Full Text\n\nHu X, Hu Y, Vogt M, et al.: MMP-Cliffs: systematic identification of activity cliffs on the basis of matched molecular pairs. J Chem Inf Model. 2012; 52(5): 1138–1145. PubMed Abstract | Publisher Full Text\n\nSterling T, Irwin JJ: ZINC 15--Ligand Discovery for Everyone. J Chem Inf Model. 2015; 55(11): 2324–2337. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaulton A, Bellis LJ, Bento AP, et al.: ChEMBL: a large-scale bioactivity database for drug discovery. 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}
|
[
{
"id": "25140",
"date": "18 Aug 2017",
"name": "John A. Lowe III",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article makes an important contribution to addressing a serious complication in screening for new biological activity, especially in the context of new drug discovery. Several researchers continue to point out the waste of time and money in following up on false positive hits from biological screening programs. While paradigms to filter out false positives, such as aggregators or other “frequent hitters” such as PAINs, are becoming mainstream techniques, these may not be sufficient, either because they miss some false positives or because they mistakenly classify legitimate hits as false positives. By carrying out a statistical analysis of a large set of screening data for hit rates using matched molecular pairs and series, this article offers a valuable perspective on this issue.\n\nThere are several aspects of this article that are particularly valuable. For example, Figure 3b is an important control showing that using assay hit rate does not overstate promiscuity, in that target hit rate is similar, linking the results to a biological mechanism of action. In Figures 4c and 4d, the exemplified compounds appear to be Michael acceptors, and could react with Cys residues covalently, or sequester thiol reagents used in the assay, which could explain their promiscuity. It is worth pointing these out, as they would not be picked up in PAINs filters and only by a knowledgeable chemist. Overall, this work is as important contribution to the ongoing effort to reduce the occurrence of false positive hits serving as starting points for discovery programs.\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": "25674",
"date": "12 Sep 2017",
"name": "Jose L. Medina-Franco",
"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 addresses a timely topic and is part of an effort to understand the multitarget activities of small molecules and their potential utility or drawbacks for drug discovery.\nUsing the MMs formalism, the authors developed a protocol to identify analog series with high hit rates. The MMs approach responds to the need to generate SAR information of interference compounds. This approach allows for more reliable evaluation compared to the analysis of individual compounds.\nThe analogue series identified with data from PubChem should be useful for medicinal chemistry programs. Similarly, the protocol developed in this work using MMPs should be useful to mine other large screening data sources (either public or proprietary data sets).\nMinor suggestions to further improve the quality of the manuscript:\nComment on the manuscript the effect of the compound concentration that is used to define a “hit” compound in a given assay. In other words, since it is unlikely that the same compound concentration is used across all assays, how this variable influences the “hit rates” and conclusions of the study?\n\nThe current analysis is made based primarily in primary assays in PubChem. In the Methods authors justify that there are larger data volumes for primary assays. We agree but would be nice to see in the manuscript a comment regarding the balance between volume vs. quality of the data.\n\nWe found it very pertinent that the study focused on compounds with high rates in primary assays, since more than 90% of these compounds are also active in confirmatory trials. It is interesting to comment how the hit rate is related to the quality of the data.\n\nAn earlier study 1 mentions that the assay frequency is not correlated with increased promiscuity. It is desirable to include a commentary in the manuscript when discussing the frequency of the test distribution and the generation of the first subset of 327,532 compounds. Three parameters for the characterization and comparison of MMSs were determined (hit rate (HR), assay overlap, assays with inconsistent activity). Does the MMS size affect these determinations?\nOther suggestions (per section):\nIntroduction, last paragraph: briefly summarize the major findings of previous works related to this study (e.g., refs. 18, 19, 23) and emphasize the novelty of this work.\n\nShorten the title (and include the concept of MMPs).\n\nIn the Introduction (last paragraph), include figure numbers for expressions such as “extensively”, “larger’, “many”, “much higher”. For instance, when the authors mention “extensively tested” in the title and through the manuscript, they mean “>10,000”, “>100,000”, “>400,000”, etc.\n\nMethods: elaborate a bit more on the in-house scripts, e.g., the program language.\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": "25426",
"date": "02 Oct 2017",
"name": "Michael Walters",
"expertise": [
"Reviewer Expertise assay interference",
"medicinal 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\nThis is an excellent manuscript that will certainly help researchers further understand the liabilities of \"good-actors\"; compounds that provide structure-interference relationships (SIR) that may not be pertinent to the biology being studied.\nNotes:\nInconsistent is misspelled in Panel B of Figure 3.\n\nThough this will need to be experimentally-verified, the sulfonylpyrimidines are almost certainly interfering by SnAr reactions. A literature search to gauge this potential reactivity of the core structure retrieved >100 articles and >1000 reactions. This group is certainly not in the PAINS definitions, but its assay interference potential can readily be ascertained by a quick reaction substructure search. Most importantly, this observation reinforces that lack of PAINS \"flagging\" is not sufficient to ensure that compounds don't have assay interference potential. The authors may wish to highlight this in their 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
|
https://f1000research.com/articles/6-1505
|
https://f1000research.com/articles/6-1817/v1
|
09 Oct 17
|
{
"type": "Research Article",
"title": "Impact of effective contact rate and post treatment immune status on population tuberculosis infection and disease using a mathematical model",
"authors": [
"Chacha M. Issarow",
"Nicola Mulder",
"Robin Wood",
"Nicola Mulder",
"Robin Wood"
],
"abstract": "Background: Tuberculosis (TB) disease burden is determined by both infection and progression rate to disease. Progression rate varies by immune status, with prior infection in high burdened settings significantly reducing the progression to disease from subsequent reinfections and completion of successful treatment associated with increased risk of subsequent TB disease. Novel studies of TB vaccines are now underway targeting high risk individuals who have completed successful combination TB chemotherapy for active TB. Methods: In our study, we explored the impact of effective contact rate (β) and post-treatment immune status on population TB burden using a mathematical model incorporating five immunological states; susceptible, newly infected, reinfected, active TB and treated TB. Results: We found that the number of newly infected individuals increased with increasing values of β< 10yr-1, but declined when β> 10yr-1. Corresponding numbers of reinfected individuals increased with increasing values of β irrespective of post-treatment immune status. Furthermore, we noted that the number of active TB cases decreased by 7 - 17% when treated individuals moved to either newly infected or reinfected immune states, respectively, rather than to the fully susceptible state at values of β< 10yr-1. The corresponding declines in TB burden were only 2 - 7% at values of β> 10yr-1. Results show that TB prevalence in high burden settings is primarily driven by effective contact rates, which are significantly modified by pre- and post-treatment immune factors. Conclusions: The observation that impact of post-treatment immune status modification on population burden may be diminished in very high burdened settings will be important for vaccine design.",
"keywords": [
"Contact rate",
"Immunological memory",
"Mathematical model",
"Eigenvalue Stability point"
],
"content": "Introduction\n\nTuberculosis (TB) transmission results from air exchange between infective sources and susceptible individuals within enabling environments. Enabling environments are frequently poorly ventilated congregate settings, such as households, public transport, schools, and work places, resulting in high effective contact rate between infectious and susceptible individuals1,2.\n\nEffective contact number is determined by the effective contact rate and the mean period of infectiousness of source cases. Progression from TB infection to disease depends on the interaction between the immunological state of the host and the virulence of the pathogen strain3. TB disease may arise either early or late following a new infection and also from reinfection of previously infected cases. Substantial epidemiological evidence suggests that reinfection is among the factors hampering successful TB control4,5 and is a major contributor to active disease in both high and low TB burden settings6.\n\nIndividuals treated with combination chemotherapy for active disease have high rates of re-treatment, due to both reactivation and reinfection, and contribute 30% of total TB notifications in high burdened settings7. Treated TB cases therefore constitute an attractive target for vaccine studies because of a high new TB event frequency and ease of identification. Two recent studies of TB vaccines are currently exploring a novel strategy of vaccination in individuals who have had successfully treated active TB8,9. However, the biological mechanisms for high rates of re-treatment and the immune status following successful treatment remain unclear.\n\nThis paper describes a mathematical model that incorporates susceptible, newly infected, reinfected, active TB and treated individuals to study the population impact of effective contact rate and post-treatment immune status on TB epidemiology. An infectious individual with varying effective contact rate (ranging from 5 to 30 y r–1) was introduced among 100, 000 fully susceptible individuals and we observed the number of individuals in all five states at stability point of a TB epidemic. This range of effective contact rate was selected because β = 5−10 y r–1 is related to TB transmission in the United Kingdom in 1900 − 19504, β = 15 y r–1 is related to TB transmission in schools in South Africa2, and β = 30 y r–1 is related to TB transmission in prisons in South Africa10. Here, we explore the population impact of effective contact rate by comparing the number of newly infected and that of reinfected individuals at stability point of a TB epidemic, with increasing effective contact rate. The study was divided into three parts and simulated the model numerically when all treated individuals move to: (i) susceptible (ii) newly infected or (iii) reinfected state, and we observed the number of individuals in all five states. We explore the population impact of post-treatment immune status by comparing the number of active TB cases when all treated individuals move to either a susceptible, newly infected or reinfected state with increasing effective contact rate. This is an important study population as the incidence of TB can be higher in people recently cured of the disease. We compute the basic reproduction number and eigenvalues from the model when treated individuals move to all three states of susceptible, newly infected and reinfected to examine the stability of the disease free equilibrium.\n\n\nMethods\n\nThe model consists of five states of susceptible individuals (S) who are not yet infected, but at a high risk of acquiring TB infection or disease if exposed to an infectious individual(s); newly infected individuals (L1) who have been infected once in their lifetime with Mycobacterium tuberculosis (MTB) and if exposed, they move to either reinfected or active TB state through exogenous reinfection, depending on the host immune response and virulence of the pathogen strain; reinfected individuals (L2) who have been infected multiple times with MTB of either the same or different strains and remain latently infected or move to active TB state through endogenous reactivation; active TB individuals (I) who are infectious and can transmit TB; treated or recovered individuals (T) who were previously infectious and were cured by chemotherapy, though some are still on treatment. Note that the difference between newly infected and reinfected individuals is that newly infected individuals are latently infected who have been infected once in lifetime with MTB and they can develop active disease through either exogenous reinfection or endogenous reactivation, while reinfected individuals are latently infected individuals who are multiply infected with MTB of either the same or different virulent strains and they can only develop active disease through endogenous reactivation or remain latently reinfected, depending on the virulence of the pathogen strain and immune response of the host.\n\nThe design and development of the model was based on an actual population in Cape Town, South Africa11, where many already infected individuals are repeatedly exposed to different infectious sources and become reinfected, and start treatment after acquiring TB disease11.\n\nFurthermore, since natural MTB infection and chemotherapy induce an immune response that reduces TB susceptibility12, we take into consideration the fact that treated individuals move to either a newly infected or reinfected state if they maintain immunological memory after successful treatment, and if they become susceptible due to waning of the immune response, they move to a susceptible state. This is because the average duration for the waning of induced immune response by MTB and chemotherapy is not well understood. However, if treatment fails or is not completed, they can move back to active disease and become infectious. As it has been reported that TB disease incidence in TB treated individuals is several-fold higher than in latently infected13, we explored post-treatment cases returning to either the susceptible, newly infected or reinfected immunological state. This approach makes our model unique and different from the prior studies, which apply infection force for treated individuals moving to latent TB states without considering immunological memory of the host. In order to explore the population impact of effective contact rate and post treatment immune status on TB epidemiology in high TB burden settings, such as Cape Town, the model includes human immunodeficiency virus (HIV) infection, but it does not incorporate: (i) age stratification or (ii) multi-drug resistant TB (MDR-TB).\n\nIn the development of the model, we assume that the population consists of fully susceptible individuals, hence, the number of births or recruitment individuals become susceptibles at a rate π y r–1. When susceptible individuals become infected with MTB, a proportion p in this group develop active TB very fast and it’s complement proportion 1−p, which constitute the majority become latently infected and move to a newly infected state depending on the virulent strains of the pathogen and host immunological factors. Newly infected individuals are at a high risk of developing active disease through either endogenous reactivation or exogenous reinfection. Here, we take into consideration that one group of newly infected individuals develop active TB slowly through endogenous reactivation at a rate c y r–1, and a fraction q of the second group of newly infected individuals develop active TB very fast after infection through exogenous reinfection, while a remaining fraction 1-q of this group move to a reinfected state. Reinfected individuals develop active TB through endogenous reactivation either fast or slowly at a rate y y r–1, depending on the immunological state of the host and the virulent strains of the infecting pathogen. Individuals with active TB receive medical treatment and move to a treated state at a rate k, while some die of active TB at a rate µt y r–1. Treated and successfully cured individuals may become susceptible and move to a susceptible state at a rate r or retain immunological memory and move to either a newly infected at a rate g or a reinfected state at a rate z y r–1, without exposure to the force of infection14. This is because latently infected and treated individuals acquire an immune response that reduces TB susceptibility though it may wane and individuals become susceptible. As mentioned earlier, we only implement treatment for active TB cases because in the real world, many TB patients only start treatment after active disease development. However, if TB cases are not well treated, treatment fails or chemotherapy is incomplete, they incubate the infection and relapse back to active TB at a rate α y r–1. As stated earlier. it has been noted that latently infected individuals acquire immunity that reduces TB susceptibility5,15, though they can be (re)infected and develop active disease if they become frequently exposed to infectious individuals in confined spaces. Thus, ω(0 < ω < 1) is the preventive factor due to prior natural MTB infection, which partially prevents latently infected individuals from TB susceptibility5. In each of the five states, there is a natural mortality rate µ y r–1 as demonstrated in Figure 1.\n\nParameters are described in Table 1.\n\nFrom the model (Figure 1), we developed a mathematical model as follows:\n\n\n\nwhere θ=βIN, which is the force of infection, β is the effective contact rate, IN is the prevalence, I is the number of infectious individuals and N is the total number of individuals in the population. Other parameters are described in Table 1.\n\nDisease free equilibrium is defined as the point at which there is no existence of the disease in the population. By computing S, L1, L2, I and T at disease free equilibrium (i.e., L1 = L2 = I = T = 0) and assuming that birth rate is equal to death rate, we obtain the disease free equilibrium point as (So,L1o,L2o,Io,To) = (N, 0, 0, 0, 0). This implies that at disease free equilibrium, the total number of individuals in the population is equal to that of susceptible individuals, such that N = S. However, during an epidemic of active disease at any time, t, we have N(t) = S(t)+L1(t)+L2(t)+I(t)+T(t), implying that the population is comprised of all five states and disease free equilibrium might be unstable. The model is epidemiologically balanced. From the mathematical model (Equation 1), we compute the basic reproduction number to determine the stability of the disease free equilibrium as discussed below.\n\nThe values of parameters used in this study were estimated from published literature. We also computed and estimated other values by matching infection and incidence data in South Africa.\n\nComputed values: In the design of the model (Figure 1), we take into consideration that treated or recovered individuals may become either susceptible or immune, depending on the host immunological status. Thus, successful treatment to either g, r or z = 1 - treatment failure (α).\n\nBasic reproduction number (Ro) is the average number of secondary cases generated by a single case connected with the fully susceptible population16,20,21. We use the next generation method to determine the basic reproduction number, which is an important figure in the epidemiology of infectious diseases. It is among the most frequently estimated quantities to predict the outbreak of infectious diseases, and its value provides information for designing control interventions for the infectious disease burden16,21. Ro thus plays an important role in the analysis of infectious disease models, such as TB17. If Ro < 1, the disease free equilibrium is asymptotically stable, and it is unstable if Ro > 116,20,21.\n\nThe next generation method is used extensively to compute the basic reproduction number, though numerous other methods are discussed in the literature21. It generates a next generation matrix, and the basic reproduction number is the spectral radius of this matrix. The next generation matrix is defined as a matrix that relates the numbers of newly infected individuals in the various states in consecutive generations21. The dominant eigenvalue in a next generation matrix is referred to as the basic reproduction number. The next generation matrix (M) is defined mathematically as\n\n\n\nand the basic reproduction number is the dominant eigenvalue of M. Where F(So,L1o,L2o,Io,To) and V(So,L1o,L2o,Io,To) are the transmission and transition matrices (n × n matrix) at disease free equilibrium, respectively. These are defined mathematically as\n\n\n\nwhere xo is the disease free equilibrium, fi is the rate of appearance of the new infection in classes, vi is the transfer of the infection from one class to another and n is the number of infective classes.\n\nHowever, prior to the formation of the next generation matrix, we generate the transmission (f) and transition (v) subsystems from Equation (1). We then linearise subsystems f and v by applying the Jacobian (Jc) and obtain matrices F(S, L1, L2, I, T) and V(S, L1, L2, I, T), which are n × n transmission and transition matrices, respectively. The transmission subsystem is an infected subsystem that describes the production of newly infected individuals and changes in the states of existing infected individuals. The transition subsystem is the transfer of the infection from one class to another. On the other hand, the transmission matrix describes the production of new infections, and the transition matrix describes changes of infected states, such as the immunity acquisition or removal by death21. Considering Equation (1), the Jacobian method that is used to linearise subsystems f and v is described mathematically as\n\n\n\nHence, from Equation (1), we form the transmission, f and transition, v subsystems as\n\n\n\nIn Figure 1, there are three infected states, which are L1, L2 and I, giving a 3×3 matrix. Hence, we form the transmission matrix, F(S, L1, L2, I, T) by using Jacobian (see Equation (2)) in f for L1, L2 and I as\n\n\n\nSubstituting S, L1, L2, I and T at disease free equilibrium point (So,L1o,L2o,Io,To) = (N, 0, 0, 0, 0) into Equation (3), we obtain the transmission matrix at disease free equilibrium, F(So,L1o,L2o,Io,To), as\n\n\n\nBy applying the Jacobian with respect to L1, L2 and I in v above, we obtain the transition matrix, V(S, L1, L2, I, T), as\n\n\n\nSubstituting disease free equilibrium point into Equation (5), we obtain the transition matrix at disease free equilibrium, V(So,L1o,L2o,Io,To), as\n\n\n\nSince the transition matrix consists of many variables, to be consistent we used a computational tool (Sage version 8.0; http://www.sagemath.org/) to compute the inverse. Hence the inverse of Equation (6), V−1(So,L1o,L2o,Io,To), is demonstrated as\n\n\n\nWe obtain the next generation matrix, which is the product of F(So,L1o,L2o,Io,To) and V−1(So,L1o,L2o,Io,To) as\n\n\n\nThus, the basic reproduction number, which is defined as the dominant eigenvalue in the next generation matrix, was obtained from Equation (8) as\n\n\n\nThe computed basic reproduction number has a positive real part, implying that it might be greater than 1. We further examined the stability of the disease free equilibrium by computing eigenvalues as discussed below.\n\nSince the basic reproduction number computed in this study has a positive real part, it is likely to be greater than 1, implying that TB transmission is increasing, regardless of the implementation of medical treatment16,20,21. Here, we explore the stability of the disease free equilibrium by computing eigenvalues in Equation (1). Disease free equilibrium is stable if all eigenvalues have negative real parts, and unstable if any of the eigenvalues is positive20. Hence, applying the Jacobian in Equation (1), gives\n\n\n\nwhere H=pβSN+ωβL1N−(k+μ+μt)\n\nThe characteristic equation of the model at disease free equilibrium is expressed as\n\n[J0–λI] = 0\n\nwhere J0 is the Jacobian matrix (from the model) at disease free equilibrium, λ is the eigenvalue and I is the identity matrix.\n\nSubstituting disease free equilibrium point (So,L1o,L2o,Io,To) = (N, 0, 0, 0, 0) into Equation (9), minus the product of eigenvalue and identity matrix diagonally, we obtain the model matrix at disease free equilibrium as follows:\n\n\n\nFrom Equation (10), we form the characteristic equation, [J0−λI] = 0, as\n\n(–μ – λ)[(–c – μ – λ)(–y – μ – λ)(pβ – k – μ – μt – λ)(–r – α – z – g – μ – λ)]\n\n–c[(–y – μ – λ)(pβ – k – μ – μt – λ)(–r – α – z – g – μ – λ)] = 0\n\nSimplifying the characteristic equation further, gives\n\n(–y – μ – λ)(pβ – k – μ – μt – λ)(–r – α – z – g – μ – λ)[μ(c + μ) + λ(2μ + c + λ) – c] = 0\n\nFrom the simplified characteristic equation, we form the following four equations:\n\n–(y + μ) – λ = 0 (11)\n\npβ – (k + μ + μt) – λ = 0 (12)\n\n–(r + α + z + g + μ) – λ = 0 (13)\n\nλ2 + (2μ + c)λ + μ2 + cμ – c = 0 (14)\n\nUsing Equation (11) to Equation (14), we can determine the eigenvalues that predict the stability of the disease free equilibrium. As mentioned earlier, disease free equilibrium is stable if all eigenvalues have negative real parts, and unstable if any of the eigenvalues is positive. Additionally, if one of the eigenvalues is zero then we cannot tell from stability analysis whether the disease free equilibrium is stable or unstable. Thus, considering Equation (11) to Equation (14), the eigenvalues are:\n\nλ1 = –(y + μ) < 0\n\nλ2 = pβ – (k + μ + μt)\n\nλ3 = –(r + α + z + g + μ) < 0\n\n\n\nHere, we noted that λ1 and λ3 have negative real parts, and λ2 has a positive real part if pβ > (k + µ + µt). For stability of the disease free equilibrium, we assume that λ4,5 have negative real parts (i.e., λ4 < 0 and λ5 < 0). Disease free equilibrium is stable if pβ < (k + µ + µt) and unstable if pβ > (k + µ + µt), where pβ is the effective contact rate for susceptible individuals moving directly to active disease. On the other hand, disease free equilibrium is stable if the effective contact rate for susceptible individuals moving directly to active disease is less than the summation of case detection, natural death and disease death rates. Otherwise disease free equilibrium is unstable.\n\n\nResults from numerical analysis\n\nUsing data estimated from published literature, varying effective contact rate (ranging from β = 5 to β = 30 y r–1) and computed or estimated values by matching infection and incidence data in South Africa (Table 1), we simulated Equation (1) numerically to explore the population impact of effective contact rate and post treatment immune status on TB epidemiology. As mentioned earlier, the study was divided into three parts where we simulated the model when all treated individuals move to: (i) susceptible (i.e., z = g = 0, r = 0.99 y r–1) (ii) reinfected (i.e., r = g = 0, z = 0.99 y r–1) or (iii) newly infected state (i.e., r = z = 0, g = 0.99 y r–1). In the model simulation, we used the initial assumption that S = 100, 000, L1 = L2 = T = 0 and I = 1, implying that an infectious individual was introduced among 100, 000 fully susceptible individuals. The epidemic was observed for 700 years when β = 5 y r–1 and 200 years when β ≥ 10 y r–1 so that the TB epidemic stability point could be attained.\n\nHere, we observed the number of individuals in all five states at stability point of a TB epidemic with increasing effective contact rate, and compared the number of newly infected and that of reinfected individuals. We also compared the number of active TB cases schematically when all treated individuals move to a susceptible, newly infected or reinfected state at stability point of a TB epidemic. We found that the number of newly infected cases increases when β = 5 to β = 10 y r–1, then decreases with increasing effective contact rate, while that of reinfected individuals increases in all values of effective contact rate (Figure 2, Figure 4 and Figure 5), implying that a large number of active TB cases might be contributed by repeatedly exposed individuals. The number of active TB individuals was increasing with increasing effective contact rate, but lower than that of newly infected and reinfected individuals. Of all five states, the number of treated individuals was noted to be the lowest, probably because treated individuals become either susceptible and move to a susceptible state or retain immunological memory and move to either a newly infected or reinfected state. We noted that as the effective contact rate increases, the number of susceptible individuals decreases dramatically with a steep slope until a stability point is attained, implying that effective contact rate is an important factor, which determines the likelihood of TB infection and disease in the community. Table 2, Table 3 and Table 4 summarise simulation results in Figure 2, Figure 4 and Figure 5, which are magnified and visualised in Figure 7, Figure 8 and Figure 9 with increasing effective contact rate, while Figure 6 compares active TB cases.\n\nThe number of individuals in all five states from β = 5 to β = 30 y r–1 at stability point of a TB epidemic are shown in Table 2. Note: S = susceptible, L1 = newly infected, L2 = reinfected, I = active TB, T = treated.\n\nObserving the number of active TB cases when treated individuals move to a susceptible, newly infected or reinfected state, we noted the highest number of active TB cases when all treated individuals move to a susceptible state (Table 2, Table 3 and Table 4). We went on to compare the number of active TB cases schematically when treated individuals move to a susceptible, newly infected or reinfected state and found that when all treated individuals become susceptible, the number of active TB cases becomes higher than moving to either a newly infected or reinfected state (Figure 6), perhaps because individuals moving to a newly infected or reinfected state have more protection due to immunological memory retention than susceptible individuals.\n\nWe separated and compared the number of active TB cases due to new infection (new active cases) and reactivation (reactivation cases) when treated individuals move to a susceptible, newly infected and reinfected state with increasing effective contact rate. We noted that new active cases are very dependent on the effective contact rate and highest when treated individuals move to a susceptible state, while reactivation cases plateau when β > 10 y r–1 (Figure 3).\n\nIt shows that new active cases are very dependent on effective contact rate and highest when treated individuals move to a susceptible state, but reactivation cases plateau at the effective contact rate greater than 10 y r–1.\n\nThe number of individuals in all five states from β = 5 to β = 30 y r−1 at stability point of a TB epidemic are shown in Table 3. Note: S = susceptible, L1 = newly infected, L2 = reinfected, I = active TB, T = treated.\n\nThe number of individuals in all five states from β = 5 to β = 30 y r–1 at stability point of a TB epidemic are shown in Table 4. Note: S = susceptible, L1 = newly infected, L2 = reinfected, I = active TB, T = treated individuals.\n\nThe number of active TB becomes higher when all treated individuals become susceptible than moving to either a newly infected or reinfected state.\n\nSusceptible (S), newly infected (L1) and reinfected (L2) are located on the left side of y-axis, while active TB (I ) and treated (T) are located on the right side of y-axis.\n\nSusceptible (S), newly infected (L1) and reinfected (L2) are located on the left side of y-axis, while active TB (I) and treated (T) are located on the right side of y-axis.\n\nSusceptible (S), newly infected (L1) and reinfected (L2) are located on the left side of y-axis, while active TB (I) and treated (T) are located on the right side of y-axis.\n\nThe time taken for a TB epidemic to reach the peak and stability points was noted to decrease with increasing effective contact rate in all cases, implying that effective contact rate is directly correlated with the force of infection. Here, we show that the number of active TB cases in the community is contributed by both newly infected and reinfected individuals after developing active disease. However, since the number of reinfected individuals becomes higher than that of newly infected individuals at stability point of a TB epidemic (if β ≥ 20 y r–1), TB prevalence might be driven by reinfection force.\n\n\nDiscussion\n\nUsing a mathematical model developed in this study, we investigated the impact of effective contact rate and post-treatment immune status on TB epidemiology. A caveat is that this is a simple model assuming a homogeneous population without age stratification and HIV. However, the model has important findings: Susceptible (S), newly infected (L1), reinfected (L2), active TB (I), and treated (T) individuals are all affected in a non-linear relationship with effective contact rate. While it is intuitive that the number of susceptible individuals declines with increasing effective contact rate, the other states are more complex. When β < 20 then L1 > L2 if T move to either S or L1, and when β ≥ 20 then L2 > L1. Treatment and active TB rates are also non-linear, if effective contact rate decreases from 30 to 25 y r–1 then the decline in TB cases is 5/100, 000, but a reduction of 10 to 5 y r–1 produces a decline of 177(range, 173−180)/100, 000.\n\nThis saturation effect resulting from high effective contact rate may explain the difficulty in TB control in very high burdened settings, such as mines and prisons.\n\nThis may also explain the empiric finding that TB notification rates are relatively insensitive to moderate improvements in case finding in high burden settings. E.g. the Zamstar study and the World Health Organization observation of very little data on the impact of enhanced case finding in high burdened settings22. This is also compatible with the modelling of TB transmission in South African prisons10. At high effective contact rate (i.e., β ≥ 20), there is a high proportion of cases from L2 rather than L1. This is compatible with the observation that 20 − 25% of TB cases are re-treatment cases in high TB burden settings, such as Cape Town. Post-treatment immunological boosting from susceptible status (S) to latently infected status (L2) could decrease population TB by 17% at lower effective contact rate but only 7% at high effective contact rate. Post-treatment vaccination impact would therefore be reduced in very high transmission settings. This finding is of considerable importance for post TB vaccine study design, requiring a balance between high event frequency and reduced vaccination effect.\n\nThe early peak values in TB rates demonstrate that there may be temporary peaks followed by apparent decline in notifications when TB disease is introduced into a new susceptible population, as seen in studies of the Alaskan Inuit population23. The time course of TB epidemiology is long, and the status of the post-treatment state is something that prior models have ignored. Loss of protective immunity in newly infected individuals following treatment (or prophylaxis which is a form of treatment) could have major negative epidemiological consequences.\n\nThe findings in this study suggest that TB prevalence is driven by reinfection force, particularly in high TB burden settings, such as Cape Town. Although prior natural MTB infection induces a partial immune protection, which reduces TB susceptibility12, the number of reinfected individuals continues to increase dramatically. The source of reinfection strains is not clearly understood, but the increasing number of reinfected individuals might be accelerated by the force of infection due to high effective contact rate and duration of exposure in congregate areas in particular. In conclusion, enhancing post-treatment immune status by vaccination may significantly augment medical treatment. However, an increased focus on diminishing the effective contact rate will require greater focus on environmental interventions.\n\n\nData availability\n\nAll the data sources used in the analysis are referenced in Table 1.",
"appendix": "Competing interests\n\n\n\nThe authors declare no competing interests.\n\n\nGrant information\n\nThis work is funded by grants from the South African Medical Research Council with funds from National Treasury under the Economic Competitiveness and Support Package (MRC-RFA-UFSP-01-2013/CCAMP) and the Bill & Melinda Gates Foundation (OPP1116641).\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 article is based on the PhD thesis of CI at the University of Cape Town, South Africa.\n\n\nReferences\n\nAndrews JR, Morrow C, Walensky RP, et al.: Integrating social contact and environmental data in evaluating tuberculosis transmission in a South African township. J Infect Dis. 2014; 210(4): 597–603. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWood R, Morrow C, Ginsberg S, et al.: Quantification of Shared Air: A Social and Environmental Determinant of Airborne Disease Transmission. PLoS One. 2014; 9(9): e106622. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNyabadza F, Winkler D: A simulation age-specific tuberculosis model for the Cape Town metropole. S Afr J Sci. 2013; 109(910): 1–7. Publisher Full Text\n\nVynnycky E, Fine PE: The natural history of tuberculosis: the implications of age-dependent risks of disease and the role of reinfection. Epidemiol Infect. 1997; 119(2): 183–201. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGabriela M Gomes M, Rodrigues P, Hilker FM, et al.: Implications of partial immunity on the prospects for tuberculosis control by post-exposure interventions. J Theor Biol. 2007; 248(4): 608–617. PubMed Abstract | Publisher Full Text\n\nCohen T, Colijn C, Finklea B, et al.: Exogenous re-infection and the dynamics of tuberculosis epidemics: local effects in a network model of transmission. J R Soc Interface. 2007; 4(14): 523–531. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiddelkoop K, Bekker LG, Shashkina E, et al.: Retreatment tuberculosis in a South African community: the role of re-infection, HIV and antiretroviral treatment. Int J Tuberc Lung Dis. 2012; 16(11): 1510–1516. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndrews JR, Noubary F, Walensky RP, et al.: Risk of progression to active tuberculosis following reinfection with Mycobacterium tuberculosis. Clin Infect Dis. 2012; 54(6): 784–791. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFletcher HA, Schrager L: TB vaccine development and the End TB Strategy: importance and current status. Trans R Soc Trop Med Hyg. 2016; 110(4): 212–218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnstone-Robertson S, Lawn SD, Welte A, et al.: Tuberculosis in a South African prison - a transmission modelling analysis. S Afr Med J. 2011; 101(11): 809–813. PubMed Abstract | Free Full Text\n\nHermans S, Horsburgh CR Jr, Wood R: A Century of Tuberculosis Epidemiology in the Northern and Southern Hemisphere: The Differential Impact of Control Interventions. PLoS One. 2015; 10(8): e0135179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGomes MG, Franco AO, Gomes MC, et al.: The reinfection threshold promotes variability in tuberculosis epidemiology and vaccine efficacy. Proc Biol Sci. 2004; 271(1539): 617–623. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVerver S, Warren RM, Beyers N, et al.: Rate of reinfection tuberculosis after successful treatment is higher than rate of new tuberculosis. Am J Respir Crit Care Med. 2005; 171(12): 1430–1435. PubMed Abstract | Publisher Full Text\n\nDye C, Gamett GP, Sleeman K, et al.: Prospects for global tuberculosis control under the WHO dots strategy. Distr: General WHO/TB 98.251. 1998. Reference Source\n\nFennelly KP, Jones-López EC: Quantity and quality of inhaled dose predicts immunopathology in tuberculosis. Front Immunol. 2015; 6: 313. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlower SM, Mclean AR, Porco TC, et al.: The intrinsic transmission dynamics of tuberculosis epidemics. Nat Med. 1995; 1(8): 815–821. PubMed Abstract | Publisher Full Text\n\nSanchez MA, Blower SM: Uncertainty and sensitivity analysis of the basic reproductive rate. Tuberculosis as an example. Am J Epidemiol. 1997; 145(12): 1127–1137. PubMed Abstract | Publisher Full Text\n\nSouth Africa Demographics Profile. 2014.\n\nWorld Health Organization: Global tuberculosis report. 2015. Reference Source\n\nvan den Driessche P, Watmough J: Reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission. Math Biosci. 2002; 180(1–2): 29–48. PubMed Abstract | Publisher Full Text\n\nDiekmann O, Heesterbeek JA, Roberts MG: The construction of next-generation matrices for compartmental epidemic models. J R Soc Interface. 2010; 7(47): 873–885. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKranzer K, Houben RM, Glynn JR, et al.: Yield of HIV-associated tuberculosis during intensified case finding in resource-limited settings: a systematic review and meta-analysis. Lancet Infect Dis. 2010; 10(2): 93–102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nComstock GW, Baum C, Snider DE Jr: Isoniazid prophylaxis among Alaskan Eskimos: a final report of the bethel isoniazid studies. Am Rev Respir Dis. 1979; 119(5): 827–830. PubMed Abstract"
}
|
[
{
"id": "28025",
"date": "05 Feb 2018",
"name": "M. Gabriela M. Gomes",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a mathematical model for the transmission of tuberculosis in human populations, and describes how endemic equilibria change with effective contact rate and post treatment immune status.\n\nThe novelty of the model is the way in which the latent infection compartment is slip into L1 and L2. The primary infection compartment (L1) consists of individuals who have been infected from a susceptible TB-free state (S) and were able to contain the infection in a latent state. These individuals are subject to reactivation and progression to active disease at a rate 0.0026 per year, and to reinfection at a rate 0.21 times the force of infection, where 0.21 accounts for reduced susceptibility due to prior infection. The reinfection compartment (L2) consists of those individuals who have been reinfected from L1 and were again able to contain the infection in a latent state. These individuals are now subject to reactivation and progression to active disease at an increased rate 0.0053 per year, but are no longer susceptible to subsequent reinfections. The model also provides the option for individuals who had active TB disease to return to S, L1 or L2 upon successful treatment. This seems sensible to me and so do the results, although I have not seen TB transmission models formulated in exactly this way before.\n\nAfter defining the model, the authors proceed to calculating the basic reproduction number R0, the threshold parameter which determines whether the disease-free equilibrium is stable (R0<1) or unstable to perturbations that will take the system to an endemic state. This is followed by a section dedicated to the stability analysis of the disease-free equilibrium (R0>1). My first reaction to this was to say that this section is redundant given that R0 has already been calculated. Closer inspection, however, shows contradiction between the two sections. While the R0 section implies that the disease-free state loses stability when beta*(mu*p+c)/(mu+c)>(k+mu+muI), the stability analysis section concludes that this happens when p*beta>(k+mu+muI). I have not verified the calculations in full, but this cross check shows that there must be an error somewhere needing to be corrected. I think the authors will want to verify their calculation.\n\nMinor comments:\n\nIn the abstract and other parts in the text, the authors refer to the 5 host states as “immunological states”. I don’t think that immunity is always the factor that differentiates the states. For example, the difference between “active TB” and “treated TB” is not whether individuals are immune or not, but rather whether they are under treatment or not. This can be fixed by simply using the denomination “host states”.\n\nIn page 3, 3rd paragraph, I got stuck where it reads “because of a high ‘new’ TB event frequency”. Consider saying just “because of a high TB event frequency”.\n\nIn the last sentence of the same page, the authors say that “the model includes HIV infection”. I don’t think it does!\n\nAt the end of page 5, there is a period “M. Where” where should be a comma “M, where”.\n\nAt the end of page 6, “it is likely to be greater than 1” is awkward. How about “it can be greater than 1”?\n\nIn page 7, “we assume that lambda4,5 have negative real parts”. Stability analyses do not accommodate such assumptions. Can the authors calculate the real parts of lambda4,5 and determine under what conditions (in terms of model parameters) these are negative? Can they show that these are always negative?\n\nFigures and tables are typically displayed in the order they are cited in the text. This is not the case in this paper.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "30286",
"date": "07 Feb 2018",
"name": "James M. Trauer",
"expertise": [
"Reviewer Expertise Tuberculosis",
"modelling",
"respiratory infections"
],
"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 strengths of the manuscript by Issarow et al. include that the article is clearly written, the large majority of the mathematical results appear to be correct to me and the work does build upon previous research. Unfortunately, the main weakness is that the compartmental structure of the model fundamentally does not answer the question that has been posed.\nThe main reason for this is that transition from T to one of either S, L1 or L2 does not only capture a change to the relative immunity status of a person who has completed treatment, but also affects their infection status. That is, S, L1 and L2 also have different rates of endogenous reactivation to I (zero for S), as well as different rates of (re-)infection (zero for L2) and different proportions that progress to active disease immediately on infection. Therefore, the authors are really exploring the idea that treated individuals become fully susceptible and clear their infection (transition to S), behave as for once infected persons (transition to L1) or behave as for multiply infected persons (transition to L2 and remain immune from further infection) - in all respects. Perhaps it may be possible to argue that this is an exploration that is of interest and has a sound basis in epidemiology, but it is not the argument that the authors make and not the question they are seeking to answer. Also, it is unclear which of the multiple epidemiological differences differences between S, L1 and L2 is driving the observations.\nFurther, the reason for persons spending one year in the T compartment is unclear. If T is intended to represent persons under treatment (which is not what the authors argue) then this might be reasonable. However, in this case, the sojourn time in T should probably be six months, the alpha parameter should better represent relapse rates (and so be greater than 1% for Cape Town). Therefore, it is unclear what this state represents, other than that patients are fully immune from reinfection during their stay in T and are waiting to transition to a new susceptibility state.\nThe result for the basic reproductive number is doubtless correct and the method used to obtain it is well accepted. However, the formula can be calculated much more easily by simply taking the product of the number of infections per unit time in I (beta), the duration of time in I (1 / (k + mut + mu)) and the proportion of infections reaching I (which requires a small amount of algebra only). Similarly, stability analysis for the disease free equilibrium seems correct (although the calculations are a little beyond my mathematical ability), but there is no reason to doubt that the disease-free equilibrium would be stable. (Finding a second equilibrium at R0=1 and hence a backward bifurcation might perhaps be of more interest - however, as omega remains < 1 throughout the calculations, I do not believe such a phenomenon would be present.)\nI have suggested reject as a decision for this manuscript, but am on the borderline between reject and major revision. I have also read the other reviewer's comments, which would also strengthen the paper and note her suggestion for a major revision. Therefore, I would be happy to review another version of the manuscript if the editors feel this is appropriate.\nMINOR COMMENTS Value of k for case detection is set at 0.68/year, but rationale is not described. Presumably this is the 2014 CDR estimate for South Africa. However, CDR is a proportion, so it cannot be simply incorporated into the model as a rate. In the Methods it is stated that susceptible individuals have never been previously infected and L1 compartment individuals have only been infected once - however, this is only true if g=r=0, which is only the case in one configuration that is explored. Therefore these statements are misleading. Prefer high-burden to high-burdened Space between y and r-1 is unclear, should be just yr-1 Definitions of the transmission and transition matrices are unclear and seem incorrect. Prefer lower limit of vertical axis of Figure 3 to be zero.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1817
|
https://f1000research.com/articles/6-317/v1
|
24 Mar 17
|
{
"type": "Research Article",
"title": "Improving agricultural knowledge management: The AgTrials experience",
"authors": [
"Glenn Hyman",
"Herlin Espinosa",
"Paola Camargo",
"David Abreu",
"Medha Devare",
"Elizabeth Arnaud",
"Cheryl Porter",
"Leroy Mwanzia",
"Kai Sonder",
"Sibiry Traore",
"Herlin Espinosa",
"Paola Camargo",
"David Abreu",
"Medha Devare",
"Elizabeth Arnaud",
"Cheryl Porter",
"Leroy Mwanzia",
"Kai Sonder",
"Sibiry Traore"
],
"abstract": "Background: Opportunities to use data and information to address challenges in international agricultural research and development are expanding rapidly. The use of agricultural trial and evaluation data has enormous potential to improve crops and management practices. However, for a number of reasons, this potential has yet to be realized. This paper reports on the experience of the AgTrials initiative, an effort to build an online database of agricultural trials applying principles of interoperability and open access. Methods: Our analysis evaluates what worked and what did not work in the development of the AgTrials information resource. We analyzed data on our users and their interaction with the platform. We also surveyed our users to gauge their perceptions of the utility of the online database. Results: The study revealed barriers to participation and impediments to interaction, opportunities for improving agricultural knowledge management and a large potential for the use of trial and evaluation data. Conclusions: Technical and logistical mechanisms for developing interoperable online databases are well advanced. More effort will be needed to advance organizational and institutional work for these types of databases to realize their potential.",
"keywords": [
"Crop trial",
"variety",
"trial site",
"metadata",
"CGIAR"
],
"content": "Introduction\n\nAgricultural research produces thousands of technology evaluations, most of which are crop variety trials. Such trials are carried out in small plots, where researchers can evaluate different plant materials across an entire growing season. Many have been conducted by researchers at the 15 Centers of the CGIAR system, a leader in international agriculture for research and development. National agricultural research institutes, universities, nongovernmental organizations (NGOs), the private sector, and farmers also conduct agronomic and breeding trials in efforts to improve farming system productivity and profitability. There is substantial variability in the quantity of experiments and the quality of data produced among these different actors.\n\nThe potential uses of agricultural trial data are enormous. Genotypes can be targeted to the environments where they are most likely to succeed based on their performance in crop trials (Hyman et al., 2013). Appropriately-targeted cultivars can improve yields substantially (Annicchiarico et al., 2005; Annicchiarico et al., 2006), particularly when combined with site similarity methods, yielding information on analogous sites (Jarvis et al., 2014; Jones et al., 2005; Ramírez et al., 2011). Agronomic data can also be used as a benchmark in yield gap studies for what farmers might be able to achieve under improved conditions and management (Gustafson et al., 2014). Trial data collected across a large geographic extent and over decades can be useful to monitor climate change or the spread of pests and diseases (Gourdji et al., 2012; Lampe et al., 2014; Lobell et al., 2011), to understand the drivers of technology adoption, to set research and development priorities and to conduct both ex-ante and ex-post impact analysis (Badu-Apraku et al., 2011; Hyman et al., 2016; Renkow & Byerlee, 2010; Setimela et al., 2005). One of the most obvious uses of agricultural trial data is to calibrate crop models, for a single location or for spatially explicit models covering countries, regions or the entire globe.\n\nFuture use of agricultural trial databases will likely be driven by the increased linking of genotype and phenotype to improve selection and use of germplasm, a growing trend driven by advances in molecular biology and site-specific agriculture. These possibilities suggest great potential for the growing “big data” movement in agriculture to use trial data as part of its larger goal to transform the sector. Combination of agronomic data from field trials with genomic data shows promise for developing next generation breeding and selection tools using models (Hwang et al., 2016). However, the dispersion, lack of organization and inaccessibility of agricultural trial data hinder their use and applicability for resolving problems in agriculture.\n\nIn an effort to make datasets of agricultural trials publicly and widely available, the AgTrials initiative received startup funds from the Gates Foundation to design and populate an online database, mostly the evaluation of crop varieties. Project researchers developed a website and a network of data providers and users. After an initial startup period, the AgTrials agricultural data repository (http://agtrials.org) was further supported and developed by the Climate Change, Agriculture, and Food Security (CCAFS) Collaborative Research Program of CGIAR, in collaboration with a number of national and international partners. AgTrials provides access to standardized agronomic trial information for the benefit of future climate change analyses, multi-environment trials and research and development in international agriculture, supporting increased collaboration between countries and institutions across the developing world.\n\nOne of the AgTrials approaches to standardization is to leverage the Crop Ontology Curation Tool (http://www.cropontology.org/). AgTrials includes a dynamic link to the Crop Ontology so that traits or variables measured in trials appear with hyperlinks to their definitions in the Crop Ontology. This capacity allows users to search for a variable measured in any trial in the AgTrials database, and combine it with other trials produced by different data providers. For example, two CGIAR Centers, CIAT and IITA, both conduct large numbers of trials on cassava, the former concentrating work in Latin America and Asia, while the latter focuses on sub-Saharan Africa. In a hypothetical example, a researcher working on resistance to green mites may be searching for germplasm tested in both regions by different organizations. The implementation of Crop Ontology in AgTrials permits this researcher to evaluate the same cassava green mite severity variables from different data providers, as long as data providers have ensured that their trait names are standardized to the Crop Ontology. AgTrials data still needs a great deal of work to ensure standardization, but without standardized terms, the utility of a global trial database is dramatically reduced.\n\nAn initial effort to link trial data to crop modeling was developed with researchers of the Agricultural Model Inter-comparison Project (AgMIP; Rosenzweig et al., 2013). Project researchers have used application programming interfaces (APIs) that permit AgMIP systems to view AgTrials data and vice versa. AgMIP has also developed a protocol for downloading AgTrials data to a suite of crop models (Porter et al., 2014). The link between agricultural trial data providers and the crop modeling community shows the potential for combining initiatives for analysis of crop improvement potential.\n\nThe developments of the AgTrials initiative described above have provided us with an initial experience in the construction of an interoperable agricultural trial database. To date, our experience with the AgTrials initiative suggests that research and development advances from using agricultural trial databases will require increased collaboration among and within public and private sectors. The private sector may not share data because their business case depends on not making it widely accessible. Public sector research and development faces a number of obstacles for increased collaboration. Researchers may refuse to make their data available because they have not yet published their findings, their data is not well organized, or they have no incentive to share. Organizational and access issues include the frequent lack of consistent metadata and standards to enable interoperability, resulting in problems of data integration, which is a key requirement to addressing global agricultural challenges.\n\nThus, our experience to date in the use of agricultural trial data across organizations, countries and continents presents both problems and opportunities. This evaluation assesses the experience of the AgTrials initiative in greater depth, reporting on the results of a survey administered via its network of users. Our aim is to consider prospects for developing a larger initiative to promote the development and use of agricultural trial data in the future.\n\n\nMethods\n\nThe objective of our analysis was to understand what worked and what did not in an effort to develop a global database of trials and evaluations of agricultural technology. Our approach was to review user experience in the AgTrials initiative to date, to analyze user interaction and usage data, and to survey users on their experiences. Our analysis includes information from the project website (www.agtrials.org) from mid-2011 to the end of 2016. We reviewed records from the sign-up information provided by users when registering on the website, including their motivation for participation. Statistics reports provided by Google Analytics were used to assess traffic on the website, along with the record of data downloads. The AgTrials website also includes data and statistics on the institutions providing data, the crops for which data is available, the location of trials and the participation of institutions involved in the initiative.\n\nThe project team administered a survey in August 2016 to evaluate the perspective of the AgTrials user base (survey results available from https://en.surveymonkey.net/results/SM-YLGDFPQG/) and survey data accompanies this article (Dataset 1; Hyman et al., 2017). As an incentive to participate in the survey, users were offered the possibility to enter a draw for a smartphone, an incentive reflected in the 44% response rate of users who received an invitation. 146 of 326 registered and active users took part in the survey; 19 of the survey replies were not fully complete. The survey covered topics on incentives and motivations for providing data, use of data, the potential for a global repository of trial information, and other topics. A copy of the survey questions can be found in Supplementary File 1.\n\n\nResults\n\nMore than 400 people have registered on the AgTrials website, providing their contact information and their motivation for participation (Figure 1). However, 326 of the registered users are considered active, having returned to the site after registration. Between the middle of 2011 and July 2016, the website had 25,648 visits, 64% of these being return visits. Most of the visitors were from Colombia, reflecting the program’s outreach from the International Center for Tropical Agriculture (CIAT), which hosts the initiative and is based in Cali, Colombia. Large numbers of visitors were also registered from the United States, Nigeria, Kenya and the Netherlands. Our data users downloaded 1,531 datasets during the analysis period.\n\nUser motivations. The stated motivations for joining the initiative varied across the more than 400 users from across the world (Figure 1), with the following being the most common reasons to look for data in AgTrials: for validating crop models; for studies on genotype by environment interaction; for acquiring daily weather or soil information; or for use in the context of studies on climate change. Many users were students working on a thesis project. The initiative came to the attention of at least two hackathons – events where coders and developers use API to bring data into their own applications. Some registered users of AgTrials were professionals working on aspects of open data initiatives, interested in data curation, metadata, and how the initiative was set up. Some of these were working with other networks that use agricultural evaluation data, such as the AgMIP (http://www.agmip.org/) and iPlant (rebranded as CyVerse, with the URL http://www.cyverse.org/) initiatives. A few research and development donors and their beneficiaries joined the initiative to share and verify evaluations resulting from their projects.\n\nTrial contributions by users. Users of the AgTrials platform contributed over 35,000 records of trial information from locations across the world (Figure 2). Approximately 85 percent of the trial records only include metadata, obligating those interested in the data to directly contact the information provider. The large majority of trials in the database were maize (29,461), followed by common bean (1881), cassava (1751), rice (411) and forages (405) to round out the top five. The countries with the largest number of trials in AgTrials were Mexico, India, Colombia, Guatemala and Ethiopia – making up over half the trials in the database. Cotaxtla and Tlaltizapan in Mexico, Palmira in Colombia and Las Vegas, Guatemala were among four trial sites that contributed more than 500 trials to the database. Other sites that were large contributors of trials include Agua Fria, Mexico, Hyderabad and Bangalore in India, Nioro in Senegal and Bako in Ethiopia. The top research centers contributing data were the International Maize and Wheat Improvement Center (CIMMYT), the International Center for Tropical Agriculture (CIAT), the International Institute of Tropical Agriculture (IITA) and the International Crop Research Institute for the Semiarid Tropics (ICRISAT); all centers of the CGIAR network. The database contains trials from 2,553 sites (Figure 3), most often national partners and collaborators of the CGIAR Centers, which generate most of the data.\n\nUser profiles. The AgTrials user survey is based on responses from 146 registered users of the platform who provided information. Nearly 40% of these were from CGIAR Centers, 20% from universities, 14% from government agencies, 6% from NGOs and 8% from the private sector. More than 43% of those surveyed had actually used the data in the context of their original motivation for registering for the platform. For these users, the data was useful for crop modeling and evaluation, genotype by environment interaction studies, phenological studies, as reference data, for data curation of their own databases, for checking weather data, for responding to data requests and for research on data repositories.\n\nAgTrials-relevant data in institutional repositories. The survey asked users how many trials their institution holds that can potentially be part of a global trial repository. 53% of respondents (equivalent to 77 people) indicated that their institution holds between zero and 100 trials that could potentially be part of a database. A total of 16 respondents (11%) suggested they could provide between 100 and 250 trials. Another 15 respondents (~10%) believed that their institutions can provide between 250 and 50,000 trials. Finally, four respondents indicated that their institutions could provide more than 50,000 trials. When asked to comment on the number of trials that might be part of a repository, there was a range of replies. Several users said that their organization did not have permission to publish trials of farmer partners. Many of the respondents simply did not know, primarily because they worked in large organizations and didn’t have numbers for trials carried out in other departments or other research locations.\n\nBarriers to contributing data to AgTrials. The survey also asked users about barriers that discouraged them from contributing data to the AgTrials platform (Table 1). Of the 112 respondents to this question, the most common answer given by ~34% of the respondents revolved around their belief that their data was not sufficiently organized for public sharing. 28% indicated that they needed more funding and resources to help them organize and upload data. 27% of the respondents believed that they or their institution simply did not have data to contribute. Another important reason for not wanting to contribute data, offered by 22% of respondents, was that it had not yet been published, and they did not want to share it until they had a chance to publish. Nearly 17% of respondents said that their data was published on another platform and that they did not want to duplicate efforts. Some respondents (13%) were discouraged from contributing data because they do not know how to upload or make it available. Over 12% of respondents considered that the policies or institutional culture of their institutions discouraged or forbade them from making data available. In about 10% of the responses, users said that donors or partners had asked that data not be made open access. Nearly 10% of the respondents indicated that they did not like the technical and design aspects of the AgTrials platform.\n\nIncentives to encourage contributions to AgTrials. The survey asked users what some incentives might be that would motivate them or their institution to contribute data to the platform (Table 2). Over 60 respondents, equivalent to 53%, indicated that the possibility that their data contribution could be cited and acknowledged would be a motivating factor for contributing to the platform. An equal number of users specified that they would be motivated by the possibility that their data could be combined with other datasets, dynamically linked to other platforms to facilitate meta-analyses or linked to larger studies. More than 44% of respondents cited the value of organizing their data in an application specifically designed for managing and sharing agricultural trial data. More than 43% of respondents suggested that they recognize the value of their data and they didn’t want it to be lost, but rather remain available and useful to others. Other incentives and motivating factors for contributing data included being able to comply with their organization or donor’s data policy (38%), if they were to receive funding to organize, document and upload the data (29%) and whether recognition of their data contribution could support their institutional or individual performance evaluation (25%).\n\nSurveyed users were asked whether they would be willing to provide both metadata and the full dataset, only metadata without the data, or neither data nor metadata. Almost 67% of respondents agreed that they were willing to provide both metadata and the full dataset. More than 27% would be willing to provide metadata only, giving their contact information in order to establish subsequent communication with interested users of their data. In total, 5% of respondents would be unwilling to provide either data or metadata.\n\nData sources for a global repository. The survey asked users what sources of data should be collected in a global repository. Users indicated that data collected from CGIAR centers (89% of those surveyed) and national agricultural researchers systems (86%) should be included. 81% of those surveyed suggested that trial data from farmer fields carried out through farmer participatory research should be included. Only 59% of those surveyed indicated that data from seed companies or agroindustry should be included in a global trial repository. Users also cited the inclusion of data from agronomic trials that test management practices, environmental and remotely sensed data related to trials and data from breeder trials focus on selecting varieties that perform well.\n\nApplications of a global agricultural repository. Respondents to the survey also provided information on key applications of an international database of agricultural trials. Several respondents mentioned the use of data for crop modeling, site similarity and other spatial analysis. These applications would evaluate the performance of crop varieties in different places and then estimate where else they might do well, suggesting where a variety release program might focus its activities. Applications of the dataset might examine crop pests and diseases and how their impacts vary with location. Other suggestions considered that the data would be useful in “big data” combinations and analyses of different datasets. For example, one respondent suggested that GPS coordinates from the trials could be used to acquire remote-sensing imagery for each plot, enabling the use of the acquired imagery for phenotyping. Respondents mentioned the possibility of using pedigree information to link genotype to phenotype in order to understand molecular level dynamics in different environmental settings. Several respondents mentioned the possibility of combining trial data with household surveys to evaluate possibilities for variety adoption. Another group of applications were suggested around priority setting and impact assessment, using crop performance data as a measure of the level of ex-ante impact.\n\nGeneral user reflections. Finally, respondents were asked to reflect on how this type of international trial database initiative could improve, and what direction it might take in the future. Several respondents mentioned the need for technical improvements in the structure of the database and the user interface. A few respondents said that it simply needed more data. Others mentioned the need for capacity building among users in order to be able to use the data more effectively. Many respondents indicated that the combination of this data with other existing datasets needed to be exploited to realize the full potential of the information resource. Several respondents said that the information resource leads to better integration of open data concepts, such as interoperability, metadata and data discovery and sharing. Many respondents mentioned the need for a stronger and more organized institutional arrangement to develop and use agricultural trial information.\n\n\nDiscussion\n\nOur experience to date in developing an agricultural trial database and the responses to our user survey suggest a number of key topics that need to be considered in the future development of this or similar initiatives. These topics concern the quality and quantity of data, improvements needed in the database and website, possible combinations of this information resource with other information initiatives, open data and data infrastructure issues and institutional arrangements to make this kind of effort succeed.\n\nOne interesting question surrounding this initiative is what its scope might be in terms of the quantity of data that might be included. Currently there are 35,000 trials in the database, mostly reflecting a large number of CIMMYT maize trials, plus a smaller collection of other crops. Respondents who said that their organization could potentially contribute more than 50,000 trials were from CGIAR centers. About seven of the CGIAR centers have major breeding programs, and if each of these could contribute 50,000 trials, the total would be 350,000 trials. Many of these CGIAR-reported trials are carried out by national agricultural research institutes and are simply part of larger CGIAR databases. There may be a number of other international organizations that participate in the initiative and could perhaps contribute more than 100,000 trials. There may be no way to know how many trials are carried out by agricultural colleges, universities, NGOs and others around the world. But overall we can speculate that the potential size of this database could be between 500,000 to 1 million agricultural trials. If a database initiative could reach those kinds of numbers, it is easy to see the potential of this information for all kinds of research and development.\n\nAnother set of issues related to agricultural trial data concerns the quality of the data and the database itself. It is notable that respondents to the survey said one of the greatest barriers to including data was that their information was not well organized and documented. Our experience has been that data curation efforts have lacked the resources needed to ensure data quality. Data curation is not one of the most exciting tasks, but it is critical for subsequent use of trial result data. Legacy data very often suffers from data quality problems. Therefore, data providers need to use best practices at the moment that data is recorded, as well as curate legacy data to overcome any deficiencies in the original collection of trial information.\n\nAny global trial data and information resource must pay considerable attention to the details of developing quality data that is well documented and an information system that facilitates ease of use in providing or acquiring data. For example, some AgTrials users pointed to the lack of detailed information on experimental design of the trials. Crop modelers typically want as much phenotypic data as possible, but also detailed data on the weather during the trial and the soil conditions of the trial site. For legacy data, it is perhaps unrealistic to expect huge numbers of trials with the full data that a crop modeler might want. But at the very least, data documentation and search mechanisms need to give users a clear picture of the data available in any given trial or set of trials. Therefore in the future of this or similar agricultural trial data resources, developers must make the needed investment to ensure that the data and the interface with the data meet minimum standards for documentation and ease of use.\n\nThe open data movement in agriculture is a trend that will surely affect the development of data and information resources like AgTrials. The CGIAR and many of its centers have recently developed new data policies oriented around data sharing and open access. These policies may motivate producers of trial data to participate in AgTrials or similar initiatives as a way to verify their work and share data with stakeholders. However, it will be necessary to emphasize the importance of standardized data initiatives that allow published data to be interoperable. For example, simply uploading data to a system, such as Dataverse, in which providers are not obligated to standardize data, would leave us with datasets that cannot be combined or studied together without a substantial effort by users. AgTrials uses the Crop Ontology initiative as the basis for standardizing data and making it interoperable, further development of which is crucial for supporting open trial data (Matteis et al., 2013). The open data movement’s emphasis on metadata is another development that could lead to better use of agricultural trial data. As the AgTrials data resource is adopting the CGIAR metadata schema, new opportunities for data discovery will likely become more apparent. Open data will also promote principles such as clear designation of intellectual property and digital identifiers for interoperability and proper citation, developments that could substantially improve the use of agricultural trial data.\n\nFinally, the survey and our experience in developing an agricultural trial database suggests the difficulties in developing a network of data providers and users. Private sector actors, such as large seed and agricultural input companies, have very well developed trial and phenotyping databases. They have a much more direct line between the data and its impact because understanding their trials and using the data directly affects their profits. As private sector companies tend to be vertically integrated hierarchical organizations, they can enforce discipline among their employees in developing and using agricultural trial data. Public sector efforts, on the other hand, depend on a networking model to organize themselves. They must all agree and be motivated to contribute to a data initiative. There are few negative consequences if they do not pursue an open data policy towards their trial data. Given the international agricultural research environment we are working in, public efforts to build agricultural trial information resources need a combination of carrots and sticks, incentives to participate and disincentives to go it alone. Developing these motivations for participation is particularly difficult considering the large number of stakeholders that would need to be brought together for a global trial data initiative.\n\n\nConclusions\n\nThis evaluation considered the development of a global agricultural trial database that can be established and used by a large number of stakeholders interested in crop improvement. Our experience in the initiative to date has shown that this type of effort has great potential for a number of applications. There is very likely a large number of agricultural trials that could be part of a global database. An initiative of this type requires development of well documented data and systems that facilitate ease-of-use. Barriers and incentives to participate could be addressed using a carrot and stick approach, where data providers and users work within an enabling environment to advance the initiative. Changes in practice are necessary for documenting and providing trial data as it is collected from the field or greenhouse. Data providers need to apply best practices and open data principles at the outset of their data collection programs. Legacy data will need increased curation, an effort that is not trivial and requires substantial resources.\n\nA future global agricultural trial data initiative will have to address the need for actors in the public sector to organize themselves around the goals of the effort. International and national institutions would need a strong commitment to participate. Donors to research and development projects would also need to commit themselves to requiring participation from the trial work they fund. The growing open data movement might provide one element of the enabling environment for such an initiative to be successful. The institutional and organizational barriers to creating a global trial information resource are much greater than any technical obstacles.\n\n\nEthics statement\n\nWe invited registered and active AgTrials users to participate in the survey reported in this paper, guaranteeing that their responses to the questionnaire will remain anonymous. Only the survey administrator was given access to the identity of the respondents, in the case that we wanted to follow up on certain questions. After reviewing survey responses, we determined that there was no need to follow up on questions.\n\n\nData availability\n\nThe survey data that was the basis of this paper is available on Survey Monkey at the following URL: https://en.surveymonkey.net/results/SM-YLGDFPQG/. Readers are also encouraged to visit the AgTrials website where metadata and data on agricultural trials can be accessed (www.agtrials.org).\n\nDataset 1: Anonymous individual responses and survey questions. doi, 10.5256/f1000research.11179.d155255 (Hyman et al., 2017).",
"appendix": "Author contributions\n\n\n\nGH, DA and LM conceived the survey and wrote the original questionnaire. HE, PC and GH prepared data tables and figures and managed the online survey. GH, MD, EA CP, KS and ST contributed to the writing of the manuscript. All authors interpreted and discussed the results and commented on the manuscript.\n\n\nCompeting interests\n\n\n\nAll, but one, of the authors works for the CGIAR, whose research programs and centers funded this research. Future funding for the authors could be influenced by the results of this research. The authors declare no direct competing interests. All authors work at non-profit public institutions dedicated to producing open access global public goods.\n\n\nGrant information\n\nThis research was commissioned by the CGIAR Systemwide Management Office. The CGIAR Fund (http://www.cgiar.org/who-we-are/cgiar-fund/) provided financial support for the study to the authors of four CGIAR centers – Bioversity International, CIAT, CIMMYT and ICRISAT. Additional support was provided by USAID (Project C-063-14 Joint CCAFS-AgMIP Activities G110).\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 AgTrials initiative has been primarily supported by the CGIAR Research Program on Climate Change and Agricultural Food Security (CCAFS). Additional support for the initiative and the development of the user survey was provided by the CGIAR System Management Office via the CGIAR Fund and the Bill and Melinda Gates Foundation. We thank Nora Casteneda for comments and suggestions on an earlier draft of this paper. We especially thank to those colleagues who responded to our survey on user perceptions and experience of the AgTrials information resource.\n\n\nSupplementary material\n\nSupplementary File 1: Survey questions.\n\nClick here to access the data.\n\n\nReferences\n\nAnnicchiarico P, Bellah F, Chiari T: Defining subregions and estimating benefits for a specific-adaptation strategy by breeding programs: a case study. Crop Sci. 2005; 45(5): 1741–1749. Publisher Full Text\n\nAnnicchiarico P, Bellah F, Chiari T: Repeatable genotype × location interactions and its exploitation by conventional and GIS-based cultivar recommendation for durum wheat in Algeria. Eur J Agron. 2006; 24(1): 70–81. Publisher Full Text\n\nBadu-Apraku B, Akinwale RO, Menkir A, et al.: Use of GGE biplot for targeting early maturing maize cultivars to mega-environments in West Africa. Afr Crop Sci J. 2011; 19(2): 79–96. Publisher Full Text\n\nGourdji SM, Mathews KL, Reynolds M, et al.: An assessment of wheat yield sensitivity and breeding gains in hot environments. Proc Biol Sci. 2012; 280(1752): 20122190. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGustafson DI, Jones JW, Porter CH, et al.: Climate adaptation imperatives: untapped global maize yield opportunities. International Journal of Agricultural Sustainability. 2014; 12(4): 471–486. Publisher Full Text\n\nHyman G, Barona E, Biradar C, et al.: Priority regions for research on dryland cereals and legumes [version 1; referees: 2 approved]. F1000Res. 2016; 5: 885. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHyman G, Espinosa H, Camargo P, et al.: Dataset 1 in: Improving agricultural knowledge management: The AgTrials experience. F1000Research. 2017. Data Source\n\nHyman G, Hodson D, Jones P: Spatial analysis to support geographic targeting of genotypes to environments. Front Physiol. 2013; 4: 40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHwang C, Correll MJ, Gezan SA, et al.: Next generation crop models: A modular approach to model early vegetative and reproductive development of the common bean (Phaseolus vulgaris L). Agric Syst. Available online 22 November 2016, ISSN 0308-521X. 2016. Publisher Full Text\n\nJarvis A, Ramírez-Villegas J, Nelson V, et al.: Farms of the Future: an innovative approach for strengthening adaptive capacity.Agricultural Innovation Systems in Africa (AISA), 29–31 May 2013, Nairobi, Kenya, 2014; 11P.\n\nJones PG, Diaz W, Cock JH: Homologue: A computer system for identifying similar environments throughout the tropical world.2005. Reference Source\n\nLampe M, Willenbockel D, Ahammad H, et al.: Why do global long-term scenarios for agriculture differ? An overview of the AgMIP Global Economic Model Intercomparison. Agr Econ. 2014; 45(1): 3–20. Publisher Full Text\n\nLobell DB, Bänziger M, Magorokosho C, et al.: Nonlinear heat effects on African maize as evidenced by historical yield trials. Nat Clim Chang. 2011; 1(1): 42–45. Publisher Full Text\n\nMatteis L, Chibon PY, Espinosa H, et al.: Crop ontology: vocabulary for crop-related concepts. Semantics for Biodiversity (S4BioDiv 2013). 2013; 37. Reference Source\n\nPorter CH, Villalobos C, Holzworth D, et al.: Harmonization and translation of crop modeling data to ensure interoperability. Environ Model Softw. 2014; 62: 495–508. Publisher Full Text\n\nRamírez-Villegas J, Lau C, Kohler AK, et al.: Climate analogues: finding tomorrow’s agriculture today. 2011. Reference Source\n\nRenkow M, Byerlee D: The impacts of CGIAR research: A review of recent evidence. Food Policy. 2010; 35(5): 391–402. Publisher Full Text\n\nRosenzweig C, Jones JW, Hatfield JL, et al.: The Agricultural Model Intercomparison and Improvement Project (AgMIP): Protocols and Pilot Studies. Agric For Meteorol. 2013; 170: 166–182. Publisher Full Text\n\nSetimela P, Chitalu Z, Jonazi J, et al.: Environmental classification of maize-testing sites in the SADC region and its implication for collaborative maize breeding strategies in the subcontinent. Euphytica. 2005; 145(1): 123–132. Publisher Full Text"
}
|
[
{
"id": "21255",
"date": "10 Apr 2017",
"name": "Anne-Françoise Adam-Blondon",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is appropriate to the content of the paper, that describes the result of a survey of the users of an open database for crop trials.\nThe aim of the survey was to understand globally the usefulness of such a database and precisely the problems encountered by users for contributing to it. The survey was performed with an online, easy tool and the questions are mostly targeted to understand the bottleneck for getting new data into the information system.\nThe introduction lacks a bit the strategy of the AgTrial initiative, its positioning in relation with other similar initiatives (at least BMS or others that are developed in the CGIARs): who are the targeted users more precisely. I understand between the lines that they are more agronomists than breeders, although this is not completely sure. The choices in terms of standardization should be also a bit more detailed, with its efforts and gaps: the crop dictionary is used for the traits description but what about the genetic material, which is also a very important object for linking genotypic information for breeders and geneticists?\nThe results of the survey are fully provided and interesting, pointing again the need to incentivize and support data curation and data management but also to a better recognition of data ownership to get more data in such central repositories. However, when looking more in detail to the last questions about the usefulness of AgTrial, the question of the links with other initiatives (e.g. BMS, CG Big data) is also strongly argued both to facilitate the submission or the harvest of properly formatted (and standardized?) data and to link data with other data. This should be more clearly discussed as I think this is also a critical issue for the users: when multiple initiative do exist, how do they complement or link between each others?\nThe bottlenecks might also be linked to the perception of the usefulness of the data once open in such database. This usefulness is very dependent on the type of standardization (again, the correct identification of both traits and genotypes is crucial for breeders before anything else, while modelers have other priorities) but also on the real accessibility to the data (which may depend on the community you belong to: for instance I was not able to access to the data I searched for).\nThe discussion of paper should better address the positioning of AgTrial regarding these two issues (link with other similar initiatives and future possible directions for improvement of the information systems that would enhance usefulness of AgTrial) in different scenarios in terms of users/submitters targeted: really open data for agronomists, really open data for breeders, CGIARs/community data more or less open to the rest of the world for agronomists, same for breeders. The priorities might be a bit different.",
"responses": [
{
"c_id": "3070",
"date": "02 Oct 2017",
"name": "Glenn Hyman",
"role": "Reader Comment",
"response": "This data initiative tried to take the approach of casting a wide net of potential users. Generally the users could be 1) agronomists or agricultural systems specialists looking to improve practices, 2) people working in crop improvement, especially breeders, 3) crop simulation modelers who are looking to calibrate their models. However, as with many \"big data\" initiatives, the users of many data sets are often unknown to the providers of the original data. This difference in communities of users led to some tensions among stakeholders of the project. Breeders, agronomists and crop modelers may not need the same types of data. It is important to note that the project didn't tried to restrict the use of this data to any particular target audience."
},
{
"c_id": "3071",
"date": "02 Oct 2017",
"name": "Glenn Hyman",
"role": "Reader Comment",
"response": "The reviewer asks about the links of the AgTrials initiative with other data initiatives that may be complementary or otherwise useful. The paper does mention the connection with the Crop Ontology initiative and the AgMIP projects. A revised version also mentions a few other possibilities. But the reviewer's point is well taken that there should be greater effort to find these useful connections."
},
{
"c_id": "3072",
"date": "02 Oct 2017",
"name": "Glenn Hyman",
"role": "Reader Comment",
"response": "The point that the reviewer makes about data accessibility is a valid point. We added some text to address this. Part of the program strategy was to allow data providers to control how their data is shared with others. In some cases this meant that a data provider only uploaded metadata, allowing potential users to know the details of the trial and what was measured. But if they want to get the actual data, they would need to contact the data provider. We felt like we had the balance the incentive of giving data providers control over how they share with the broader desire of data users to get the data directly without delay. We hope that in the end connections are made and collaborations are strengthened in the cases where the full data set was not provided."
}
]
},
{
"id": "22090",
"date": "22 May 2017",
"name": "Ruth Bastow",
"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 looks at the use of data and data submissions to the AgTrials initiative. To assess what was productive and effective in this initiative and what could be improved if the initiative was developed further. The survey of users, data collection, data analysis and conclusions are clearly described and understandable.\nIt would be helpful if the paper had provided a clearer introduction to the AgTrials project perhaps with some examples of data that AgTrials holds. It would also be useful to understand what similar projects or initiatives are being undertaken elsewhere in the world and how they have dealt with the issues raised in the paper. For example the paper notes that 17% of respondents published their data on a different platform. As funding bodies, donors and governments push for greater open access/open data the important issues raised in this paper (at both the technical and sociological levels) will need to be dealt with and most importantly coordinated at the international level to help ensure the effective use of agricultural trial data in improving agricultural and agricultural practices.\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/6-317
|
https://f1000research.com/articles/6-1531/v1
|
21 Aug 17
|
{
"type": "Research Article",
"title": "Renal impairment in stroke patients: A comparison between the haemorrhagic and ischemic variants",
"authors": [
"Pratyush Shrestha",
"Shalima Thapa",
"Shikher Shrestha",
"Subash Lohani",
"Suresh BK",
"Oscar MacCormac",
"Lekhjung Thapa",
"Upendra Prasad Devkota",
"Shalima Thapa",
"Shikher Shrestha",
"Subash Lohani",
"Suresh BK",
"Oscar MacCormac",
"Lekhjung Thapa",
"Upendra Prasad Devkota"
],
"abstract": "Background: Renal impairment is regularly seen in hospitalized stroke patients, affecting the outcome of patients, as well as causing difficulties in their management. A prospective cohort study was conducted to assess the trend of renal function in hospitalized ischemic and haemorrhagic stroke patients. The incidence of renal impairment in these subgroups, the contributing factors and the need for renal replacement in renal impaired patients was evaluated. Methods: Alternate day renal function testing was performed in hospitalized stroke patients. Estimated glomerular filtration rate (e-GFR) was calculated and the trend of renal function in the two stroke subgroups (haemorrhagic and ischemic) was assessed, with renal impairment defined as e-GFR < 60mL/ minute per 1.73m2. Results: Among 52 patients, 25 had haemorrhagic stroke (mean age 59.81 ± 14.67) and 27 had ischemic stroke (mean age 56.12 ± 13.08). The mean e-GFR (mL/minute per 1.732m2) at admission in the haemorrhagic stroke subgroup was 64.79 ± 25.85 compared to 86.04 ± 26.09 in the ischemic stroke subgroup (p=0.005). Sixteen out of 25 (64%) patients in the haemorrhagic stroke subgroup and 9 out of 27 (33.3%) patients in the ischemic subgroup developed renal impairment (p=0.27). The location of the bleed (p=0.8), volume of hematoma (p=0.966) and surgical intervention (p=0.4) did not predispose the patients to renal impairment. One out of 16 patients with haemorrhagic stroke (who eventually died), and 2 out of 9 patients with ischemic stroke required renal replacement. Conclusion: Renal impairment is commonly seen in stroke patients, more so in patients who suffered haemorrhagic strokes. The impairment, however, is transient and rarely requires renal replacement therapy.",
"keywords": [
"Estimated Glomerular Filtration Rate",
"Haemorrhagic",
"Ischemic",
"Renal Impairment",
"Stroke"
],
"content": "Introduction\n\nRenal dysfunction is commonly seen in hospitalized stroke patients. Ischemic stroke is frequently associated with renal dysfunction and nearly a third of patients hospitalized with intracerebral haemorrhage (ICH) have chronic kidney disease (CKD) (estimated glomerular filtration rate [e-GFR] < 60ml/minute per 1.73m2)1,2. The severity of the impairment and the requirement of renal replacement therapy for these patients in the course of their treatment are important management issues not currently well addressed by the literature. It has been established that good overall medical care can greatly influence the outcome of patients with stroke3. Cerebrovascular accident itself has a high burden of morbidity and mortality, and additionally, CKD is an independent predictor of poor clinical outcome and mortality after an initial stroke4,5. Co-existence of adverse conditions, such as anemia, oxidative stress, platelet dysfunction, electrolyte imbalance and hyperhomocysteinemia, in patients with CKD have been implicated as the reason why these patients have poorer outcomes compared to the normal population6. Moreover, even mild stages of CKD increases the risk of future ischemic and haemorrhagic strokes7. e-GFR decreased <40mL/min indicates that the risk of symptomatic stroke in the general population is increased by 3.1 times8. Hence proper and routine evaluation of renal function in hospitalized patients with cerebrovascular accident needs to be ensured to improve the outcome of these patients.\n\nSince there is inadequate knowledge concerning the trend of renal function in hospitalized stroke patients and a lack of clear guidelines regarding the need for renal replacement in these type of patients, we conducted an observational study of the trends in renal function in all stroke patients hospitalized at our centre.\n\n\nMethods\n\nA prospective study was conducted at the National Institute of Neurological and Allied Sciences, a 100-bed, tertiary care neuro-centre in Kathmandu, Nepal. Stroke patients admitted between December 2014 and April 2015 were recruited to the study. The Institutional Review Board of the National Institute of Neurological and Allied Sciences approved the study (6/2014). The review board confirmed that as this is a descriptive study with no active interventions performed on the patients and their personal identity was not disclosed, no informed consent from the patients was required.\n\nNon-probability, consecutive sampling was performed; all patients with haemorrhagic and ischemic stroke admitted during the study period were enrolled into the study. Patients admitted less than 5 days were excluded as trend analysis was not possible in them. The mean e-GFR at admission and at one week interval was calculated. Serum urea and creatinine were evaluated on alternate days throughout their hospital stay, and the e-GFR was calculated as per the Modification of Diet in Renal Disease Study group formula: e-GFR (mL/minute per 1.732m2) = 186 X [serum creatinine] - 1.154 X age - .203 X [.742 if female] X [1.21 if of African descent]9. Renal impairment was defined when the e-GFR was < 60mL/minute per 1/73m2.\n\nAll data were taken from patient hospital records. If data was not available, the information was sought from the patients’ relatives, which was obtained as part of routine care. The primary outcome measure was the occurrence of renal impairment during the hospital stay. The trend of renal function and the need for renal replacement in these patients were analyzed over the duration of admission in the hospital. Demographic, clinical profile and the trend in renal function of patients with haemorrhagic stroke were compared with those of ischemic stroke. The number of patients who had renal impairment according to our criteria was analyzed and any surgical interventions carried out on these patients were recorded. Of the patients that developed renal impairment, the ones that required renal replacement were strictly followed. Since renal replacement is not available at our centre, the patient were monitored when they were moved to nearby hospitals.\n\nData were entered in IBM SPSS (version 20; SPSS Inc., Chicago, IL, USA) and Pearson's chi square test, Fischer's exact test and independent variable t-test were applied. Line graphs were made to show the trends in the e-GFR in all the patients.\n\n\nResults\n\nA total of 55 patients were admitted during this period of five months to the intensive care unit and general wards; however, three patients had to be excluded as their hospital stay was less than 3 days. Hence the trend in renal function was followed up in 52 patients – 25 with haemorrhagic stroke and 27 with ischemic stroke. The mean hospital stay of the patients in the haemorrhagic stroke group was 14.58 ± 7.19 days and 9.86 ± 5.12 days in the ischemic stroke group; however, there were two patients from each group who were admitted for more than a month.\n\nThe mean age of patients in the haemorrhagic group was 59.81 ± 14.67 and that in the ischemic stroke group was 56.12 ± 13.08, which was comparable. In the haemorrhagic stroke group, 17 were men and 8 were women, while in the ischemic stroke group, 17 were men and 10 were women.\n\nThe mean systolic and diastolic blood pressures (mmHg) in the patients with haemorrhagic stroke at admission were 183.6 ± 30.4 and 105.6 ± 18.1, respectively. In the ischemic stroke group, the mean systolic and diastolic blood pressures at admission were 146.9 ± 27.1 and 90.9 ± 14.6, respectively. 23/25 patients with haemorrhagic stroke had hypertension (14 uncontrolled and 9 newly diagnosed); and only two were normotensive. 18/27 patients with ischemic stroke had hypertension (9 controlled, 8 uncontrolled and 1 newly diagnosed) and 9 were normotensive. 10/14 patients with uncontrolled hypertension and haemorrhagic stroke had renal impairment; however, 2/8 patients with uncontrolled hypertension and ischemic stroke had renal impairment. There were 9 patients with ischemic stroke who had controlled hypertension and 4 of them developed renal impairment. No significant association between hypertension and renal impairment was observed (p=0.993; Table 1). The mean arterial pressure at admission in patients who eventually had renal impairment was 124.35 ± 22.60, and in those who did not have renal impairment was 117.32 ± 19.03, which was not statistically significant (p=0.311).\n\nThe trend in the renal functions in the two groups was plotted over time (Figure 1). It is evident from this plot that the trend of e-GFR in the ischemic stroke patients is mostly more than 60mL/min per 1.73m2; whereas in the group of patients with haemorrhagic stroke, many of the patients have e-GFR values that demonstrate renal impairment. This was analysed on a two by two table and a Pearson Chi Square test was done to determine if this finding was statistically significant.\n\nTrends of e-GFR in patients with (A) hemorrhagic and (B) ischemic stroke. (C) Trend of average e-GFR.\n\nSixteen out of 25 (64%) patients with haemorrhagic stroke and 9 out of 27 (33.3%) patients with ischemic stroke had some grade of renal impairment during their hospital stay, which was statistically significant (Pearson's chi square test, p=0.027; Table 2).\n\nPatients with haemorrhagic stroke presented with an initial lower e-GFR compared to patients with ischemic stroke. The mean e-GFR (mL/minute per 1.732m2) on the day of admission in the haemorrhagic stroke group was 64.79 ± 25.85 compared to 86.04 ± 26.09 in the ischemic stroke group, which was statistically significant (independent sample t-test, p=0.005). However, by the end of a week, the difference was not statistically significant; 80.91 ± 30.74 for the haemorrhagic stroke group and 98.11 ± 31.92 for the ischemic stroke group (independent sample t-test, p=0.294).\n\nThe location of the intracerebral bleed in the haemorrhagic stroke group did not predispose the patient to renal impairment (p=0.80; Table 3). In the haemorrhagic stroke group, the mean volume of hematoma in the patients that developed renal impairment was 29.23 ± 24.23 mL and in the patients that did not develop renal impairment it was 28.61± 42.24 mL, which was not statistically significant (p=0.966). Hence, the volume of bleed did not influence the patient developing renal impairment.\n\nConsidering the management of the patients, in the haemorrhagic stroke subgroup, 6 patients underwent evacuation of hematoma, 5 patients underwent CSF drainage procedures and 14 patients were managed conservatively. In the ischemic stroke group, 6 patients underwent decompressive craniectomy, 19 were conservatively managed and 2 patients with cerebellar infarction underwent posterior fossa decompression and evacuation of the infarction.\n\nIn the haemorrhagic stroke group, 9 out of 11 patients who underwent surgical intervention developed renal impairment; yet, this finding was not statistically significant (p=0.40; Table 4) Patients requiring surgery were the ones that were more unwell and were likely to be at a higher risk of renal impairment; however, a significant association could not be established (Table 4).\n\nSixteen out of 25 patients (64%) with haemorrhagic stroke and nine out of 27 patients (33.3%) with ischemic stroke developed some degree of renal impairment. However, only three patients (one with haemorrhagic and two with ischemic stroke) required renal replacement therapy. In the vast majority of the patients, 22 out of 25 (88%), the renal impairment was transient and improved without hemodialysis.\n\nThe patient who underwent renal replacement therapy after haemorrhagic stroke, was a 45 year old, who had 75mLs of ganglionic bleed evacuated, and required hemodialysis from the 5th day of admission. This patient succumbed to multiple organ dysfunction syndrome on the 14th day. The two patients with ischemic stroke who required renal replacement on the 4th and 31st day of admission improved with hemodialysis.\n\n\nDiscussion\n\nRenal failure is a rare primary cause of death in patients following stroke; however, impaired renal function is a significant predictor of both short and long term mortality in these patients. The Oxford Community Stroke Project reported that stroke survivors tend to die 2.3 times more than the matched general population every year post-stroke, and non-stroke cardiovascular diseases is the most common cause of death following the first year of initial stroke10. Calculated e-GFR ≥ 51.27ml/min predicts a better long term survival in stroke patients after adjustment of confounders (age, neurological state, ischemic heart disease, hypertension, smoking and diuretic use). MacWalter et al. suggested the use of ACE inhibitors in this subgroup of patients, as they had higher risk of mortality11.\n\nHypertension is attributed to a large share of patients progressing to end stage renal failure12. In the present study, 23 out of 25 patients with haemorrhagic stroke had hypertension (14 uncontrolled and 9 newly diagnosed). However, 18 out of 27 patients with ischemic stroke had a normal blood pressure (9 without history of hypertension and 9 controlled). Even the patients with well-regulated blood pressure continued to have renal impairment, i.e. 5 out of 9 patients. Additionally, 4 out of 11 patients without hypertension, 4 out of 9 patients with controlled hypertension, 12 out of 22 patients with uncontrolled hypertension and 5 out of 10 patients with newly diagnosed hypertension developed renal impairment, which was not statistically significant (independent t-test, p=0.793). Therefore, in our patients, hypertension did not play a significant role in predisposing patients to renal impairment.\n\nIn our study, some degree of renal impairment was seen in 64% of patients with haemorrhagic stroke and 33.3% of patients with ischemic stroke. Ovbiagele et al. have reported renal impairment in nearly a third of patients with ICH; and Mishra and colleagues in 38.46% of such patients1,13. Use of mannitol could be a confounding variable in our study. Dziedzic et al. found that the use of mannitol decreased intra cranial pressure, caused transient elevation of urea and creatinine, but did not cause anuria or oliguria14. As ours is a tertiary care centre receiving referred cases from all over the country, selected severe patients could have been enrolled into our study. Of 614,454 patients admitted with ICH in hospitals in the USA between 2005 and 2011, 41694 (6.8%) had acute renal failure (ARF), with 700 patients (1.7%) requiring in-hospital dialysis. These patients with ARF had higher rates of moderate-to-severe disability and in-hospital mortality15.\n\nAfter excluding patients with a history of renal disease, dehydration, nephrotoxic drugs use or septicemia, transient renal impairment was found in 30 of 78 patients (38.46%) with acute ICH. Renal functions normalized in 2–4 weeks15. Hence, only a small percentage of patients with ICH who develop renal impairment, need renal replacement. Only one of the 25 patients with ICH and two of the 27 patients with ischemic stroke required renal replacement in our study. This finding is coherent with Saheed et al., who found that of only 700 (1.7%) of 41, 694 patients with some renal impairment requiring renal replacement in 614,454 patients with ICH admitted in the USA15. The Oxford Community Stroke Project further states that renal failure is a rare primary cause of death in post-stroke patients, but is a significant predictor of increased mortality in both the short and long term10,16.\n\nThe size of the hematoma did not predispose patients to renal impairment in our study, as the average hematoma volume in the group of patients that developed renal impairment was 29.23 ± 24.23mL and in the patients that did not develop renal impairment was 28.61± 42.24 (p=0.966). In a similar study by Cutting et al. (2004), there was no correlation between admission GFR and ICH volume (p=0.77), and patients with GFR <60 versus patients with GFR ≥ 60mL/min/1.73 m2 also had similar ICH volumes (median 10.8mL vs 11.4mL; p=0.54)17. However, Molshatzki and colleagues (2011) were of the opinion that patients with larger ICH were predisposed to having more severe renal impairment. In their no renal impairment group, mild impairment group and the moderate to severe impairment group, the median hematoma volumes were 15.3mL, 16.6mL and 50.2mL, respectively (p=0.009)18. In addition, patients with moderate/severe impairment exhibited a 2.3-fold higher hematoma volume than the other groups (p=0.04). In our study, the location of the hematoma did not have any association with the severity of the renal impairment. Molshatzki et al. concluded that patients with moderate to severe renal impairment had a > 6-fold higher odds of lobar location of the bleed (95% CI = 1.59-24.02) as compared to the patients with no impairment.\n\nThe mean e-GFR (mL/minute per 1.732m2) on the day of admission in the haemorrhagic stroke group was significantly lower (64.79 ± 25.85) compared to the ischemic stroke group (86.04 ± 26.09) in our study. In Hao et al.’s (2010) study, the mean e-GFR at admission in the haemorrhagic and ischemic stroke groups were comparable 77.57 ± 51.73 vs 77.07 ± 29.89 (p=0.285)19. Patients presented to our centre at various times following the index stoke; hence, the days mentioned are not the days after the stroke event, but the days after admission to the hospital. This is a limitation of our study as the values mentioned do not reflect the trend profile of renal parameters following stroke.\n\nSmall vessel disease is a generalized condition affecting the vascular beds in the brain, kidneys and retina. Makin and colleagues (2015) tried to study the relation of small vessel (lacunar) stroke with renal impairment with the hypothesis that lacunar infarction would be associated with more renal impairment compared to cortical stroke; however, there was no such difference20. We are not able to comment on this as we have not categorized ischemic stroke into these subgroups in this study.\n\n\nConclusions\n\nRenal impairment is commonly seen in stroke patients and is more common in the haemorrhagic variety, particularly in the initial days of the ictus. The impairment however is transient and rarely requires renal replacement therapy. The volume of the intracerebral bleed, its location and whether the patients were surgically or conservatively managed did not predispose them to renal impairment.\n\n\nData availability\n\nRaw data for renal impairment in stroke patients is available from http://dx.doi.org/10.7910/DVN/EILQSZ21.\n\nCoding schema for Excel spreadsheet (schema included in SPSS version): SN, Serial Number; age (1, <20; 2, 21–30; 3, 31–40; 4, 41–50; 5, 51–60; 6, 61–70; 7, 71–80; 8, 81–90); sex (0- female, 1-male); location, location of the bleed; IVE, intraventricular extension (1-yes, /- no); Renal_impair, patients that developed renal impairment (1-yes, 0-no); Renal_replace, patients that required renal replacement (1-yes, 0-no); Volume, volume of bleed in millilitres; htnhth, hypertension (0-absent, 1-controlled, 2-uncontrolled, 3-newly diagnosed), sbp, systolic blood pressure in mmHg at admission; dbp, diastolic blood pressure in mmHg at admission; MAP, mean arterial pressure at admission; operation (EVD, external ventricular drain; VP shunt, ventriculoperitoneal shunt; evacuation, evacuation of bleed or infarction); interv, intervention (1, conservative management (haemorrhagic); 2, evacuation of hematoma; 3, CSF drainage; 4, conservative (Infarction); 5, decompressive craniectomy; 6, decompression and evacuation of the infarction); egfr1, estimated glomerular filtration rate at day 1; Meaneffr, mean egfr of each patient; Meandrop, maximum drop in egfr during the hospital stay of the patient as compared to the day of admission.\n\nNOTE: The hospital stay can be determined by counting the number of egfrs recorded.",
"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\nOvbiagele B, Schwamm LH, Smith EE, et al.: Hospitalized hemorrhagic stroke patients with renal insufficiency: clinical characteristics, care patterns, and outcomes. J Stroke Cerebrovasc Dis. 2014; 23(9): 2265–73. PubMed Abstract | Publisher Full Text\n\nTsukamoto Y, Takahashi W, Takizawa S, et al.: Chronic kidney disease in patients with ischemic stroke. J Stroke Cerebrovasc Dis. 2012; 21(7): 547–50. PubMed Abstract | Publisher Full Text\n\nMorgenstern LB, Hemphill JC 3rd, Anderson C, et al.: Guidelines for the management of spontaneous intracerebral hemorrhage: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2010; 41(9): 2108–2129. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYahalom G, Schwartz R, Schwammenthal Y, et al.: Chronic kidney disease and clinical outcome in patients with acute stroke. Stroke. 2009; 40(4): 1296–303. PubMed Abstract | Publisher Full Text\n\nKim HJ, Kim JK, Oh MS, et al.: A low baseline glomerular filtration rate predicts poor clinical outcome at 3 months after acute ischemic stroke. J Clin Neurol. 2015; 11(1): 73–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchiffrin EL, Lipman ML, Mann JF: Chronic kidney disease: effects on the cardiovascular system. Circulation. 2007; 116(1): 85–97. PubMed Abstract | Publisher Full Text\n\nLee M, Ovbiagele B: Reno-cerebrovascular disease: linking the nephron and neuron. Expert Rev Neurother. 2011; 11(2): 241–9. PubMed Abstract | Publisher Full Text\n\nNakayama M, Metoki H, Terawaki H, et al.: Kidney dysfunction as a risk factor for first symptomatic stroke events in a general Japanese population--the Ohasama study. Nephrol Dial Transplant. 2007; 22(7): 1910–5. PubMed Abstract | Publisher Full Text\n\nNational Kidney Foundation: K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis. 2002; 39(2 Suppl 1): S1–S266. PubMed Abstract\n\nDennis MS, Burn JP, Sandercock PA, et al.: Long-term survival after first-ever stroke: the Oxfordshire Community Stroke Project. Stroke. 1993; 24(6): 796–800. PubMed Abstract | Publisher Full Text\n\nMacWalter RS, Wong SY, Wong KY, et al.: Does renal dysfunction predict mortality after acute stroke? A 7-year follow-up study. Stroke. 2002; 33(6): 1630–5. PubMed Abstract | Publisher Full Text\n\nWalker WG: Hypertension-related renal injury: a major contributor to end-stage renal disease. Am J Kidney Dis. 1993; 22(1): 164–73. PubMed Abstract | Publisher Full Text\n\nMisra UK, Kalita J, Srivastava M, et al.: Transient renal impairment in acute intracerebral haemorrhage. J Neurol. 1996; 243(5): 417–20. PubMed Abstract | Publisher Full Text\n\nDziedzic T, Szczudlik A, Klimkowicz A, et al.: Is mannitol safe for patients with intracerebral hemorrhages? Renal considerations. Clin Neurol Neurosurg. 2003; 105(2): 87–9. PubMed Abstract | Publisher Full Text\n\nSaeed F, Adil MM, Piracha BH, et al.: Acute renal failure worsens in-hospital outcomes in patients with intracerebral hemorrhage. J Stroke Cerebrovasc Dis. 2015; 24(4): 789–94. PubMed Abstract | Publisher Full Text\n\nRowat A, Graham C, Dennis M: Renal dysfunction in stroke patients: a hospital-based cohort study and systematic review. Int J Stroke. 2014; 9(5): 633–9. PubMed Abstract | Publisher Full Text\n\nCutting S, Castro C, Lee VH, et al.: Impaired renal function is not associated with increased volume of intracerebral hemorrhage. J Stroke Cerebrovasc Dis. 2014; 23(1): 86–90. PubMed Abstract | Publisher Full Text\n\nMolshatzki N, Orion D, Tsabari R, et al.: Chronic kidney disease in patients with acute intracerebral hemorrhage: association with large hematoma volume and poor outcome. Cerebrovasc Dis. 2011; 31(3): 271–7. PubMed Abstract | Publisher Full Text\n\nHao Z, Wu B, Lin S, et al.: Association between renal function and clinical outcome in patients with acute stroke. Eur Neurol. 2010; 63(4): 237–42. PubMed Abstract | Publisher Full Text\n\nMakin SD, Cook FA, Dennis MS, et al.: Cerebral small vessel disease and renal function: systematic review and meta-analysis. Cerebrovasc Dis. 2015; 39(1): 39–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPratyush S: Renal impairment in stroke patients-a comparison between haemorrhagic and ischemic variants. Harvard Dataverse, V3, UNF:6:sifkcIKrEyzWbC1CQS313g==, 2017. Data Source"
}
|
[
{
"id": "25747",
"date": "20 Sep 2017",
"name": "Ravi Dadlani",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very interesting study. I congratulate the authors on this unique analysis.\n\nI, however, have found some issues with the data. The hemorrhagic group appears to have more statistically significant renal impairment than the ischemic group.\n\nRather than comparing data with regards to blood clot volume, surgery etc., It would be more pertinent to look at the basic management for these patients in terms of intravenous fluids, mannitol, glycerol, types of maintenance IV fluids, insensible los (fever etc.,), Nephrotoxic drugs, antibiotics used etc.,\nIf the above mentioned analysis is added to the paper, it may have some value. Also, then, specific recommendations may be given regarding the overall management of such patients. I believe the differences in the two groups is artificial because the general management differs.\n\nI would not recommend the paper be published in it's present 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? 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": [
{
"c_id": "3082",
"date": "04 Oct 2017",
"name": "Pratyush Shrestha",
"role": "Author Response",
"response": "We would like to thank our referee for finding our study interesting, the analysis unique and most important of all, giving us his precious time.We agree to his suggestion that it would be more pertinent to look at the basic management for these patients in terms of IV fluids, mannitol, insensible loss, use of antibiotics and nephrotoxic drugs. We could probably continue to work on these issues in our future publications.We, however, disagree to his claim that the difference in this two groups is artificial and is due to the difference in the general management.We have calculated the mean e-GFR at admission for both the groups. The e-GFR in the haemorrhagic stroke group is significantly low compared to the ischemic stroke subgroup.\"The mean e-GFR (mL/minute per 1.732m2) at admission in the haemorrhagic stroke subgroup was 64.79 ± 25.85 compared to 86.04 ± 26.09 in the ischemic stroke subgroup (p=0.005)\" We agree that the management of haemorrhagic stroke and ischemic stroke differs. But we would like to stress that these subgroups were patients admitted in the same unit, over the same period of time;and, on the whole the baseline management in both the groups, in regards to the IV fluids used, the use of mannitol, insensible loss, use of antibiotics and nephrotoxic drugs was grossly indifferent.We have included this issue in our discussion and Dziedzic et. al. did find that mannitol caused transient elevation of urea and creatinine, but did not cause anuria or oliguria.Finally, we would like to thank our referee once again for his valuable suggestions."
}
]
},
{
"id": "25749",
"date": "22 Sep 2017",
"name": "Umit Eroglu",
"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 searched renal impairment rate among stroke patients. That’s a valuable study especially for ICU units of neurology and neurosurgery departments. Authors evaluated hematoma volume, surgical interventions and hematoma location. But ventricular hematoma can cause biochemical changes so this condition had to be evaluated separately. Cholesterol level, alcohol intake, smoking, seizures (status) and antiepileptic drugs could be the other parameters for haemorrhagic stroke. It is appropriate to index the article in this state.\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": "25450",
"date": "25 Sep 2017",
"name": "Dibya Singh Shah",
"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 we have very few literature and data in Nepalese populations in this field, this study is a good initiation. In this study eGFR at presentation was lower in hemorrhagic stroke group as compared to ischemic stroke. One explanation could be due to much higher blood pressure at presentation in this population as compared to ischemic stroke. As this is a small single center study larger multicentre studies are needed to draw further 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? 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/6-1531
|
https://f1000research.com/articles/6-1809/v1
|
06 Oct 17
|
{
"type": "Review",
"title": "A short review on the potential of coffee husk gasification for sustainable energy in Uganda",
"authors": [
"Gilbert John Miito",
"Noble Banadda",
"Noble Banadda"
],
"abstract": "Agricultural biomass is widely recognized as a clean and renewable energy source, with increasing potential to replace conventional fossil fuels in the energy market. Uganda, like other developing countries, has a high dependency (91%) on wood fuel, leading to environmental degradation. With a coffee production of 233 Metric Tonnes per annum, relating to 46.6 Mega Tonnes of coffee husks from processing, transforming these husks into syngas through gasification can contribute to resolving the existing energy challenges. The objective of this article is to briefly review the energy potential of coffee husks through gasification, and how the gasification process could increase energy recoveries for coffee farmers. Previous findings indicate that the 46.6 Mega Tonnes per year of coffee husks generated in Uganda, with a heating value of 18.34 MJ/kg, is capable of generating 24 GWh of energy. This will address a 0.7% portion of the energy situation in Uganda, while protecting the environment.",
"keywords": [
"Agricultural Biomass",
"Coffee Husks",
"Gasification",
"Wood Fuel",
"Energy Recovery"
],
"content": "Introduction\n\nEnergy demand is rising rapidly, mainly due to population increase and increasing industrial activities. This demand has led to an increase in prices of major energy sources, for instance fossil fuels1–4. Use of fossil fuels causes air pollution, which, over time, escalates climate change effects as emissions of greenhouse gases and other pollutants increases5–8. Due to the increase in energy demand and prices of fossil fuels, researchers are motivated to discover additional viable energy sources. A case of interest is Fernandes and Costa9, who noted that biomass, including wood agro residue, in Portugal has an annual energy potential of 160 Tera joules (TJ). In Mozambique, Vasco and Costa10 pointed out that agricultural biomass can cater for 32% of the country’s energy demand.\n\nMany sub-Saharan African (SSA) countries use agricultural biomass to cover most of their energy needs5, but the efficincies are low, and thus the raw material usage is high11. In Uganda, for example, woody biomass is used as wood fuel to provide for over 93% of the country’s energy needs12. This reliance on wood fuel significantly increases the rate at which the country’s forest cover is shrinking, and is associated with the adverse change in weather patterns and consequent climate variability experienced in the country13. With Uganda’s population growing at a rate of 3.2% per annum14, the pressure on the country’s forests is bound to increase if the country’s dependence on wood fuel continues. On the other hand, alternative biomass sources, like agricultural and saw mill waste, and technologies, like pyrolysis and gasification, remain unexplored. Coffee, which is Uganda’s top-earning export crop, is widely grown throughout the country, and yielded 233 mega tons (MT) in 201415. During the processing of the coffee, a substantial bulk of coffee husks are generated. In total, 46.6 MT of coffee husks were generated from the 233 MT of coffee produced, based on a 0.2 waste factor for coffee16. These husks are currently being used as beddings in poultry units, farmers also use coffee husks to replenish nutrients in pineapple and banana gardens across the country and to a smaller extent for use in briquetting. However, the current uses amount to underutilization of the coffee husks in monetary and energy terms, and there is thus a need to improve coffee waste handling alternatives that encompass maximum utilization of the energy component of the waste, while also addressing the challenges associated with the use of woody biomass for energy reliance. This won’t only address the energy crisis in the country, but also increase the economic value of the husks17.\n\nDirect use of coffee husks as an energy source is hindered by the low efficiency of the energy recovery systems used. It is essential to transform the husks to a form that improves energy recovery. One process that can be considered is gasification, which involves alteration of compact carbonaceous fuel, in this case being the husks, into ignitable gas by partial incineration at elevated temperatures and moderate heating rates11,18. Through this process, coffee husks can be thermally converted to producer gas. Producer gas is a blend of carbon monoxide, hydrogen, methane, carbon dioxide and nitrogen. Producer gas is multipurpose in its use, as opposed to the solid biomass, from which it is derived19. This article reviews the potential for coffee husks, as an alternative to wood as feedstock, for gas fuel production through gasification in Uganda.\n\n\nThe energy situation in Uganda\n\nAccording to Karekezi and Kithyoma20, Africa is the least electrified continent in the world, and, in East Africa, Uganda is amongst the least electrified countries (Table 1). Uganda presently has one of the lowest electricity consumption per capita in the world at 215 kWh per annum21. The typical value for SSA is 552 kWh per capita, while that for the world is 2,975 kWh per capita22. This low electricity coverage is attributed to electric tariffs and the fact that the electricity grid is concentrated in urban areas. Hence, people have continued to use wood fuel for their energy needs.\n\nAn examination of the power grid in East Africa indicates that the spread of electricity is mainly limited to main town areas, while rural regions have no access23 (Figure 1). In addition, some homesteads that are close to the network lines cannot afford electricity connections and user fees. Indeed access to electricity at a national level in Uganda is about 15%, which is lower than other SSA24, with a rural coverage of 1%5. As a result, many Ugandans depend on firewood and charcoal as their main energy source for lighting and cooking25–27. According to the Rural Electrification Agency, the total woody biomass consumption in Uganda in 2013 was 31.7 million tons. The total energy consumption per year in Uganda is approximately 45.1 million tonnes of wood, 1.2 million m3 of oil products, with a hydropower installed capacity of about 691.5 MW and 100 MW of thermal power28.\n\nAdapted from 32.\n\nA small portion (7%) of the energy demand in Uganda is met by fossil fuel29, which is primarily used for automobiles and generators, and, to a smaller extent, in form of natural gas. Although fossil fuels contribute a small portion of the energy demand, data shows that there is increase in the volume of petroleum products imported into the country. In 2013, there was an increase of 4.6 and 4.3% in the import volume of kerosene and diesel, respectively (Figure 2)15. This increment implies an increased reliance on an energy source that is unsustainable. From Figure 2, it is also observable that there was a greater increase in the amounts of kerosene imported compared with other energy sources. This is highly attributed to the higher demand for kerosene, which the majority of the rural population uses for lighting30.\n\nUganda is an energy deprived nation with restricted access to electricity and heavy dependence on wood fuel to cater for the energy requirements. There has been a general increase in the wood consumption for fuel in the country (Figure 3)33. This heavy consumption of wood fuel has contributed to high rates of deforestation, further leading to unreliable rainfall and rampant soil erosion34.\n\nCharcoal is largely used in metropolitan areas, whereas firewood, agricultural biomass and wood chippings are commonly utilized in rural areas35. The sugar industry is the sole industry in Uganda that exploits agricultural biomass in the form of bagasse for cogeneration, but the technology should be adopted even for energy production in homesteads.\n\nThe promotion of renewable energy has been recognized as a potential method of addressing power shortages in the East African region20. The renewable energy policy of Uganda states that the increasing cost of fossil-based fuels makes them costly for developing countries, and fossil fuels have an unreliable future. Uganda has extensive renewable energy resources for production of energy and the delivery of energy services, such as biomass, geothermal, large scale hydro, hydro, wind and solar energy, yet these remain un-tapped, primarily due to the apparent technical and financial incapacitations. With the exclusion of biomass, whose energy contribution is significant, the other sources contribute ~5% of the country’s total energy consumption. This hinders the scope and productivity of economic activities that can be undertaken in any part of the country. Biomass is an extremely available resource, and agricultural waste, such as coffee husks, is a large quota of the biomass yield. Therefore, it is advisable that the use of these readily abundant resources should be increased.\n\nGasification technology is not extensively used nor known in Uganda11, but in Kenya, a neighboring country of Uganda, gasification of sawmill dust has been implemented with a power output of 76 GWh36. Although previous investigations showed that small-scale wood gasifiers could be economically and socially feasible energy systems to generate electricity in rural areas37, it is not a widely implemented technology in SSA. Obua et al.38 noted that gasification is not widely applied in Uganda and reported only two established gasification units: one at Muziizi Tea Estate for electricity generation using wood feedstock and the other at Paramount Cheese Diaries for industrial heat production, which uses papyrus reeds. The gasification unit at Muziizi Tea Estate was stated to be 87 kW average power output, although rated at 200 kW39, indicating a low operating efficiency. Buckholz et al.37 described the economics of a 10 kW wood gasification unit in Mukono, Uganda, and concluded that the technology is proven, efficient and economically viable. The existing gasification units in Uganda rely on firewood, which is a factor in deforestation, and yet the literature supports use of coffee husks. Previous studies in Sweden40 and Portugal41,42 have reported successful gasification of coffee husks for various applications, including fuel, electrical generation and soil conditioning, but the possibility of the technologies in less developed countries is still abstract.\n\n\nDeforestation situation\n\nUganda has an expanse of 241,550.7 square kilometers, including water bodies. Forests are one of the chief land uses although there has been a significant decline in forest area over the past two decades5,43. Between 1990 and 2010 total forest cover dropped from 24 to 12.7% of the total land area, implying an average decline of close to 2% per year (Figure 4)28. Although agriculture is the major cause of deforestation, wood extraction for energy production, which has been steadily rising over the years, has been noted as one of the direct reasons for deforestation38. The forest area reduction contributes to environmental hazards and unreliable rainfall manifested in the country. Currently, approximately 90,000 ha of forest and vegetation are destroyed yearly, leading to fuel wood shortage in rural areas and increasing prices of charcoal and fuel wood15.\n\n\nCoffee production in Uganda\n\nUganda is ranked second to Ethiopia in regards to coffee production in Africa44, and there is a current government intervention of coffee replanting, which is bound to boost coffee production in Uganda45. Uganda produces two types of coffee: Robusta coffee and Arabica coffee (also termed as Mountain coffee). Over the years, Robusta coffee has been produced in larger quantities and accounts for 80% of production compared with Arabica coffee. In 2013, Uganda produced a sum of 232,561 tons of coffee (Figure 5) from a producing area of about 310,000 hectares, of which 75% was Robusta15. Currently, the (Figure 5 and Figure 6)15.\n\nThe major coffee growing regions include the Western Highlands, Bugisu around Mt. Elgon and areas around Lake Victoria basin (Figure 7). According to Lora and Andrade16, coffee has a waste factor of 0.2 by weight, and as such this yields a coffee husk production of approximately 46.6 MT per year46. These husks are used as soil conditioners, briquetting, poultry bedding, and some are burnt as biomass energy sources, but with low efficiency. Significant to note is that the production surpasses the use of the husks24.\n\nPermission to use this figure was granted by Kyagalanyi Coffee Ltd (http://kyagalanyi.co.ug/sustainability/sustainable-coffee-schemes/).\n\n\nGasification potential of coffee husks\n\nAccording to Acharya et al.47, processing of coffee yields about 22% of its weight as coffee husk. The annual generation of coffee husks in Uganda is estimated to be 46.6 M tons per annum, rising from 172 M tons per annum reported in 200448. According to Mhilu40, coffee has an energy value of 18.34 MJ/kg. At a maximum this would harness efficiencies of 855 TJ with 46.6 M tons of coffee husks. At a conversion efficiency of 65%46, the generated coffee husks have a potential of 24 GWh energy production. This would translate to better efficiencies and environmental protection, yet reducing the reliance on wood fuel.\n\nA study was carried out by Pereira et al.49 concerning the development of gasification stoves. They found that the gasification process yields emissions to the environment, but the amounts are small compared with fossil fuels and direct ignition of the biomass. Through gasification, the coffee husks can be put to an environmentally sound use, as the emissions associated with open burning are reduced50. The technology has been shown to be viable for a variety of feedstock, including waste from paper mills, mixed plastics, forest industry waste and agricultural residues28. The coffee husk gasification technology has the potential to better livelihoods and contribute to local progress by providing electricity access to societies in rural Uganda30.\n\n\nConclusions\n\nThis short review critically focusses on the fact that gasification of coffee husks can address a portion the energy demand problem in Uganda, while enhancing cleaner production. The review of the literature shows that coffee husks have the potential for conversion to clean gas fuels for energy generation on an industrial scale through gasification. If the renewable energy policy of Uganda emphasizes the adoption of highly efficient renewable technologies, the heavy reliance of wood for fuel will be curbed. The Government of Uganda should effectively implement the coffee replanting scheme. This will increase the coffee yield, which in turn will increase the amounts of coffee husks produced. These coffee husks, if properly harnessed, can be a clean energy alternative. Uganda has a massive potential to produce energy from coffee husks, as coffee is a major cash crop from the country. If utilized in a viable manner, coffee husks could contribute to 24 GWh of energy, while decreasing deforestation and environmental degradation in the country, which are associated with the current energy sources. The technology adoption will further improve the livelihood of the residents, as well as lessen the pressure on wood fuel. However, the possibility of sustainable energy derivation from coffee husks is attached to the increasing production of coffee by allocating more unutilized land to coffee growing and also growing of improved and high yielding coffee varieties. Nevertheless, emphasis should be also put on food crops to avoid food insecurity.",
"appendix": "Author contributions\n\n\n\nBoth authors made substantial contributions to conception and design, and acquisition of data, and analysis and interpretation of data. They also participated in drafting the article and revising it. The second author also gave final approval of the version submitted.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWolfram C, Shelef O, Gertler P: How will energy demand develop in the developing world? J Econ Perspect. 2012; 26(1): 119–138. Publisher Full Text\n\nOmer AM: Energy, environment and sustainable development. Renew Sustainable Energy Rev. 2008; 12(9): 2265–2300. Publisher Full Text\n\nAsif M, Muneer T: Energy supply, its demand and security issues for developed and emerging economies. Renew Sustainable Energy Rev. 2007; 11(7): 1388–1413. 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Open Journal of Renewable Energy and Sustainable Development. 2014; 1(1): 1–13. Publisher Full Text\n\nMenéndez JA, Domínguez A, Fernández Y, et al.: Evidence of self-gasification during the microwave-induced pyrolysis of coffee hulls. Energ Fuel. 2007; 21(1): 373–378. Publisher Full Text\n\nCouto N, Silva V, Monteiro E, et al.: Experimental and numerical analysis of coffee husks biomass gasification in a fluidized bed reactor. Energy Procedia. 2013; 36: 591–595. Publisher Full Text\n\nOmeja PA, Chapman CA, Obuaa J, et al.: Intensive tree planting facilitates tropical forest biodiversity and biomass accumulation in Kibale National Park, Uganda. Forest Ecol Manag.2011; 261(3): 703–709. Publisher Full Text\n\nHammond S: Uganda Coffee Annual Uganda Coffee Annual Report. 2012. Reference Source\n\nBenin S, You L: Benefit-Cost Analysis of Uganda's Clonal Coffee Replanting Program, An Ex-Ante Analysis. Intl Food Policy Res Inst. 2007; 744. Reference Source\n\nYin XL, Wu CZ, Zheng SP, et al.: Design and operation of a CFB gasification and power generation system for rice husk. Biomass Bioenergy. 2002; 23(3): 181–187. Publisher Full Text\n\nAcharya B, Dutta A, Basu P: An investigation into steam gasification of biomass for hydrogen enriched gas production in presence of CaO. Int J Hydrogen Energ. 2010; 35(4): 1582–1589. Publisher Full Text\n\nWang J, Hu Z, Xu X, et al.: Emissions of ammonia and greenhouse gases during combined pre-composting and vermicomposting of duck manure. Waste Manag. 2014; 34(8): 1546–1552. PubMed Abstract | Publisher Full Text\n\nPereira EG, de Silva JN, da Oliveira JL, et al.: Sustainable energy: A review of gasification technologies. Renewable and Sustainable Energy Reviews. 2012; 16: 4753–4762. Publisher Full Text\n\nHan J, Mol AP, Lu Y, et al.: Small-scale bioenergy projects in rural China: lessons to be learnt. Energy Policy. 2008; 36(6): 2154–2162. Publisher Full Text"
}
|
[
{
"id": "27378",
"date": "02 Nov 2017",
"name": "Jamidu H.Y. Katima",
"expertise": [
"Reviewer Expertise Renewable energy"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 review is relevant considering the current energy situation i.e. dwindling of fossil fuel resources and the impact of the same to climate change. Use of sustainable renewable resource which are considered to be carbon neutral is very important.\n\nHowever, the current generation of coffee husks (only meeting 0.7% of energy demand) does not provide comfort for sustainable energy supply. Although the paper discusses the intention of Uganda to increase acreage of coffee plantation, it does not discuss to what extent this will be done to meet even 20% of Uganda’s energy supply.\n\nSpecific Comments Abstract\nLine 4, the Authors are stating that Uganda is producing 233 Metric Tonnes of coffee, which produce 46.6 Mega Tonnes. These numbers do not make sense. Unless 233 are in Mega Tonnes Line 5, insert “per year” between “husks” and “from” Line 13, 0.7% of total energy from coffee husks do not tally with the use of “sustainable energy” – probably instead the Authors should use contribute to energy mix\n\nIntroduction\nLine 5, the Authors imply other pollutants contribute to greenhouse gases, they should be specific by mentioning which ones, otherwise they should change the statement Second paragraph, second line, last word “efficiencies” is misspelled\n\nThe Energy Situation in Uganda Fossil Fuels\nLine 3, the Authors are making reference to use of Natural Gas. My understanding is that Uganda is not using NG, probably they wanted to mean “Liquefied Petroleum Gas (LPG)”\n\nBiomass Energy on page 4\nSecond paragraph, line 5, Authors should change “Should” to “could”\n\nStatus of gasification in Uganda\nLine 21 “ replace the word “electrical” with “electricity”\n\nGasification potential of coffee husks (page 5)\nLine 2 the Authors states that the coffee yield 22% of its weight as coffee husks. When you consider that Uganda produces 233 Mega Tonnes, then the coffee husks do not compute to 46.6 Mega Tonnes The statement ” The annual generation of coffee husks in Uganda is estimated to be 46.6 M tons per annum, rising from 172 M tons per annum reported in 2004” does not seem correct. 172 Mega Tonnes is larger than 46.6 Mega Tonnes\nConclusion\nLine 8, use of statement “the heavy reliance of wood for fuel will be curbed” is a heavy statement without qualification particularly when you consider that the coffee husks contribute only 0.7% of energy in Uganda Line 12, insert the word “source” between “a” and “clean”\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? Partly",
"responses": []
},
{
"id": "26767",
"date": "04 Dec 2017",
"name": "Innocent Nhapi",
"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 giving me this opportunity to review this article. The review is well written and focused and gives a good overview of the subject and possibility of utilising coffee husk gasification for sustainable energy in Uganda. I only made a few notable observations which are outlined below:\nThere are some sections with bold and factual statements which needs to be backed by literature. An example is on page 2, paragraph 2, where uses of rice husks in Uganda are discussed. Another example is this sentence from page 4: With the exclusion of biomass, whose energy contribution is significant, the other sources contribute ~5% of the country’s total energy consumption. Some factual statements are referenced but the authors can go on and find some statistics on coverage - an example is page 4, second paragraph.\nThis sentence on page 5 is not complete: Currently, the (Figure 5 and Figure 6)15\nMake this sentence more clearer in meaning: The annual generation of coffee husks in Uganda is estimated to be 46.6 M tons per annum, rising from 172 M tons per annum reported in 2004 - what is rising here?\nThe energy needs to be solved are distributed around Uganda and so also is the cultivation of coffee. The authors need to consider the logistical and economic implications of their proposal and recommend how the gasification plants and energy produced will be distributed for the benefit of the rural peasants who are currently using firewood as source of fuel.\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": []
}
] | 1
|
https://f1000research.com/articles/6-1809
|
https://f1000research.com/articles/6-1805/v1
|
05 Oct 17
|
{
"type": "Review",
"title": "Illustrative examples of probable transfer of resistance determinants from food animals to humans: Streptothricins, glycopeptides, and colistin",
"authors": [
"Hattie E. Webb",
"Frederick J. Angulo",
"Sophie A. Granier",
"H. Morgan Scott",
"Guy H. Loneragan",
"Frederick J. Angulo",
"Sophie A. Granier",
"H. Morgan Scott",
"Guy H. Loneragan"
],
"abstract": "Use, overuse, and misuse of antimicrobials contributes to selection and dissemination of bacterial resistance determinants that may be transferred to humans and constitute a global public health concern. Because of the continued emergence and expansion of antimicrobial resistance, combined with the lack of novel antimicrobial agents, efforts are underway to preserve the efficacy of current available life-saving antimicrobials in humans. As a result, uses of medically important antimicrobials in food animal production have generated debate and led to calls to reduce both antimicrobial use and the need for use. This manuscript, commissioned by the World Health Organization (WHO) to help inform the development of the WHO guidelines on the use of medically important antimicrobials in food animals, includes three illustrations of antimicrobial use in food animal production that has contributed to the selection—and subsequent transfer—of resistance determinants from food animals to humans. Herein, antimicrobial use and the epidemiology of bacterial resistance are described for streptothricins, glycopeptides, and colistin. Taken together, these historical and current narratives reinforce the need for actions that will preserve the efficacy of antimicrobials.",
"keywords": [
"olistin",
"glycopeptides",
"streptothricins",
"antimicrobial resistance",
"Critically Important Antimicrobials"
],
"content": "Context\n\nApart from a few molecules, many antimicrobial agents, such as antibiotics, either occur in nature or are derived from natural compounds. Likewise, their corresponding resistance determinants have occurred naturally for millennia. Mounting evidence, however, informs us that decades of global, anthropomorphic antimicrobial overuse has resulted—and is resulting—in the selection and spread of antimicrobial resistant bacteria and their determinants. Much of this antimicrobial use is occurring in food animal production; while some over-selection from this use does not extend to distinctly human pathogens, zoonotic bacteria that can be transmitted from food animals to humans through the food supply and environment may pose an increased risk to humans due to adverse consequences of antimicrobial resistance such as treatment failure. Human deaths attributed to all bacterial resistance are currently estimated to be 700,000 annually1, and—unless action is taken—this estimate is projected by economists to exceed 10 million by 2050, thereby surpassing cancer2.\n\nMany organizations have begun to engage in efforts to reduce the potential public-health impact of bacterial resistance associated with the use of antimicrobials in food animals. In particular, the World Health Organization (WHO) has established an Advisory Group on Integrated Surveillance of Antimicrobial Resistance (AGISAR). This group has been key in producing guidelines on the use of antimicrobials in food-producing animals (hereafter, termed the “guidelines”), the integrated surveillance of antimicrobial resistance, regularly revised lists of critically important antimicrobials for human medicine (CIA List), and supporting capacity building and infrastructure development efforts in the developing world. This review was commissioned in the context of informing the development of the WHO guidelines on use of medically important antimicrobials used in food animals to be published in October, 2017. Therefore, our objective is to provide three specific examples that illustrate selection and subsequent transfer of resistant determinants from food animals to humans. These illustrative examples are streptothricins, glycopeptides, and colistin.\n\n\nLimitations\n\nOur general knowledge on antimicrobial resistance among bacteria is ever evolving; in particular, the story of colistin resistance is rapidly unfolding. This review was prepared for the WHO AGISAR meeting of October, 2016 (Raleigh, North Carolina, US) at which time the WHO guidelines was being drafted; therefore, the cited literature is considered up to date as of September 15, 2016. The scope of this review paper was centered on the evidence of antimicrobial use (and amount of use) in food animals and the epidemiology of common resistance mechanisms. Routes of antimicrobial administration were not evaluated or discussed within this report, but likely also play a role in antimicrobial resistance determinant selection. Further, dissemination of bacteria and resistance genes are frequently not unidirectional events. As such, we do not discount the importance of other directional routes of transfer (e.g. direct or indirect transfer from human to animal populations); however, the scope of this review was limited to transfer from food-producing animals to humans. The selection and dissemination of antimicrobial resistance is a complex, multifactorial phenomenon. Unfortunately, there is no ‘perfect’ experiment or controlled environment to demonstrate selection, dissemination, and the subsequent risks imposed through the sharing of resistance determinants among bacterial and host populations, and we acknowledge up front that there remain data gaps.\n\n\nStreptothricins\n\nStreptothricins are a distinct group of antibiotic compounds isolated from the genus Streptomyces3,4. The first streptothricin compound (F) was described in 19425. Antibiotic agents of the streptothricin group are composed of varying combinations and proportions of the streptothricin compounds (A, B, C, D, E, F, and X)6. More than 70 mixtures of streptothricin compounds have been described and subsequently named, including: streptolin, racemomycin, geomycin, grisein, pleocidin, and nourseothricin; however, the amount of detail available regarding the chemical structure and antibacterial activity of each of the streptothricin antibiotic agents varies greatly. Nonetheless, the streptothricin antibiotic agents are known to be effective against pathogenic fungi and have both bacteriostatic and bactericidal effects on Gram-negative and Gram-positive bacteria through the inhibition of protein synthesis and misreading of genetic information7–9.\n\nNephrotoxicity associated with streptothricin antibiotic agents has prevented clinical use of these agents in human medicine10,11. As a result, use of the streptothricin antibiotic agents has been largely limited to plant production and animal husbandry in a select few countries, particularly China and the former German Democratic Republic (GDR; East Germany)12,13. The most detailed accounts of streptothricin use and the apparent subsequent dissemination of resistance are available from the GDR. Between 1981 and 1989, nourseothricin—a mixture of streptothricin D and F—was used in the GDR for in-feed growth promotion in the swine industry5,14. No data are available about the amounts of streptothricins or nourseothricin produced, distributed, or used in the swine industry during this time. Nourseothricin was not used in animals in the GDR prior to the introduction of its use in swine, and nourseothricin use in the GDR was limited to the swine industry15. Furthermore, no use of other streptothricin antibiotic agents in animals or humans has ever been reported in the GDR.\n\nIt has been reported that, prior to utilization in the GDR swine industry in 1981, acquired nourseothricin resistance in Enterobacteriaceae among animal and human isolates was rare and believed to be solely associated with chromosomal mutations12,16,17. Furthermore, when phenotypic resistance was reported, it was never found to be a mobilizable resistance, although the extent of antimicrobial surveillance or screening is not cited and is unknown for that period of time. In 1981, less than one year after the initial use of nourseothricin in the swine industry, a streptothricin-streptomycin-spectinomycin resistance phenotype was observed in Escherichia coli isolated from rectal swabs from pigs on multiple farms, “sewage”, and from the feces of those in direct contact with the pigs (i.e. farm personnel)17. This resistance was found to be mediated by streptothricin-acetyltransferase (sat) genes coding for a nourseothricin-inactivating enzyme, which is carried on a transposon, designated Tn182517.\n\nFrom 1981 to 1983, plasmid-mediated streptothricin resistance was documented in E. coli isolated from rectal swabs of pigs being treated with nourseothricin and slurry from their farms in multiple geographical locations within the GDR14. Hummel and colleagues also identified streptothricin-resistant E. coli in piglets being treated with nourseothricin, the gut flora of persons with direct contact with the pigs (i.e. farm personnel), the gut flora of persons with in-direct contact with the pigs, who had no other connection to the livestock industry (i.e. farm personnel’s family members), and among the gut flora of outpatients living in the same region that had no apparent contact with pigs17,18. Remarkably, the authors did not observe streptothricin resistance in samples from piglets or humans in regions where nourseothricin was not being used. Further, the prevalence of streptothricin resistance was highest in E. coli isolated from piglets (33% of 306) and declined in the following order: isolates from farm personnel (18% of 377), isolates from farm personnel’s family members (17% of 334), isolates from outpatients in the region (16% of 266) and isolates from urinary tract infections in outpatients in the region (1% of 28).\n\nDespite discontinuation of nourseothricin use in the GDR swine in 1988, the identification of streptothricin resistance and associated resistance determinants continued and broadened. Streptothricin resistance has now been associated with the sat, stat, and nat genes19. In 1992, the first report of streptothricin-resistance Campylobacter isolated from pig slurry was published20,21. Integrons harboring the gene sequence of these resistance determinants have also been observed in other bacteria (clinical isolates, animal environments, and food-producing animals), including Salmonella enterica, Enterococcus faecium, Acinetobacter baumannii, Burkholderia cenocepacia, Vibrio cholerae, Shigella sonnei, and S. flexneri12,22–27.\n\nInterestingly, the spread of the streptothricin resistance gene to these other ecological niches and bacterial populations has occurred without direct selection pressure (i.e. use of streptothricins in animals or human medicine)12. Importantly, the streptothricin resistance genes are often harbored in integrons with resistance determinants present to other antimicrobial agents, namely determinants coding for resistance to streptomycin, spectinomycin, trimethoprim, or kanamycin22–25. It is possible that such co-resistance may have contributed to the early dissemination of streptothricin resistance, but the early epidemiological studies did not report information on use of other antimicrobial agents. Little to no information is provided about the animals and humans from which the isolates were collected. Furthermore, because there were few studies that searched for streptothricin resistance prior to the 1980s, it is not known if streptothricin resistance determinants were present in bacteria before this time. Nonetheless, this illustrative example outlines the published account of the likely emergence and dissemination of plasmid-borne resistance from swine to humans.\n\nNourseothricin, a streptothricin antimicrobial agent, was widely used as a growth promoter in the swine industry in the former German Democratic Republic from 1981–1988. In contrast, toxicity prevented use of streptothricin antimicrobial agents in humans. Less than one year after the introduction of nourseothricin in swine, a plasmid-borne streptothricin resistance (sat) seemingly emerged in E. coli isolated from swine administered nourseothricin. Subsequently, plasmid-borne streptothricin resistance was detected in the gut flora of humans with direct, indirect, and no contact to pig farms, but living in the same regions. Following reports of the plasmid-mediated streptothricin resistance demonstrates an illustrative example of the detection—and apparent emergence—of streptothricin-resistant bacteria in swine as a result of antimicrobial use, and the dissemination of the resistant bacteria and mobile genetic elements conferring resistance to humans.\n\n\nGlycopeptides\n\nGlycopeptides are a broad-spectrum antimicrobial class, including vancomycin, and its derivatives teicoplanin, telavancin, dalbavancin, oritavancin, and avoparcin28. Glycopeptides block cell wall assembly in Gram-positive bacteria by inhibiting peptidoglycan synthesis28. Therefore, the clinical importance of the glycopeptide class has been the treatment of infections caused by Gram-positive pathogens. For a large part of the 1980s and 1990s glycopeptides were the drugs of last-resort for multidrug-resistant Gram-positive infections in humans29.\n\nVancomycin, the first antibiotic of the glycopeptide class, was first described in 1955 and was subsequently approved for human use by the United States (US) Food and Drug Administration (FDA) in 195829–31. The dates of approval and beginnings of human use in European countries are unknown. Renal toxicity and ototoxicity (largely due to impurities in the drug) limited vancomycin use in humans until the early 1980s when multidrug-resistant Gram-positive bacteria began to emerge and purified formulations of vancomycin became available32,33. Annual vancomycin usage in humans in the US climbed from 2,000 kg in 1984 to 11,460 kg in 199433. In Europe and Australia, human vancomycin use was more limited33; for example, in Australia, an average of 193 kg of vancomycin was used in humans annually between 1991 and 199334,35. France reported 200 kg of vancomycin was used in humans in 1984, increasing to only 1,151 kg in 199433. Annual vancomycin usage in humans in Germany, Italy, United Kingdom (UK), the Netherlands, and Denmark each ranged between 24 to 408 kg in 199433,36. Human use of vancomycin began to decline after 1994 following efforts to promote vancomycin conservation, an attempted to limit dissemination of glycopeptide-resistant bacteria.\n\nAlthough vancomycin use in humans in Europe was very limited in the 1990s, avoparcin, a glycopeptide antimicrobial, was heavily used in many European countries and Australia as an antimicrobial growth promoter in livestock34. Avoparcin use for growth promotion is documented in Europe as early as 1975 and products containing avoparcin have been registered in Australia since 197837–39; while data supporting heavy use of avoparcin in many European countries are limited, data from Denmark indicate 24,000 kg of active avoparcin were used in swine and broilers in 199436. Austria reported an average of 62,642 kg of avoparcin for animal production use were imported per year from 1992 to 199640. Australia used an annual average of 125,000 kg of avoparcin between 1991 and 199334,35. Avoparcin has never been licensed for use in animals in the US41. Following the isolation of glycopeptide-resistant bacteria from food animal products at the retail level, attempts to mitigate the risk of human exposure to glycopeptide-resistant enterococci (GRE) through the food chain led to the ban of avoparcin for growth promotion use in Denmark and Norway in 1995, Germany in 1996, followed by the remaining European Union member states in 1997, and withdraw of avoparcin from the Australian market in 200020,38,39,42–46.\n\nTransferable glycopeptide resistance in enterococci was first reported in human patients in both France and the UK in 1986, and then in the US in 198747–49. However, it wasn’t until the 1990s that considerable attention turned to the evaluation of glycopeptide use and resistance due to differing epidemiological trends between GRE in the US and Europe. In the US in the 1990s, GRE emerged as a significant cause of healthcare-associated infection and colonization in many hospitals—frequently associated with the high use of vancomycin in those hospitals50–52. Hospital-associated GRE infections rose at an endemic rate; with the proportion of vancomycin resistant enterococcal blood isolates climbing from little to no resistance in 1989 to 25.9% in 200039,53. In the 1990s in Europe, prevalence rates of GRE in hospitals remained low; however there were reports of GRE in healthy human carriers in the community (e.g. people with no association to a hospital) and sporadic hospital outbreaks54–56.\n\nMonitoring of antimicrobial resistance to growth promoters was not common practice prior to the mid-1990s57. Perhaps as a result, the first detection of GRE isolated from sewage, animals, and healthy humans in the community (i.e. outside of hospitals) were reported in the mid-1990s39,42,45,56,58–66. Notably, an association was made between use of avoparcin and the occurrence of GRE in livestock and their environments in Belgium, Denmark, Finland, France, Germany, UK, and the Netherlands—directing a spotlight to food animal production42,50,51,57,60,61,65,67–74.\n\nThe differing epidemiological trends in GRE between the US and Europe led to considerable interest to compare GRE from European farm animals fed avoparcin, hospitalized humans, and non-human sources using various molecular methods32. Such investigations provided a great deal of insight about the epidemiology of acquired resistance genotypes associated with glycopeptide resistance, particularly the most globally widespread and prevalent glycopeptide resistance in enterococci, vanA resistance. vanA is an inducible resistance to vancomycin and often teicoplanin mediated by a complex cluster of resistance genes (ORF1, ORF2, vanR, vanS, vanH, vanA, vanX, vanY, and vanZ) often carried on a 10,851 bp transposon designated Tn154667,75–77.\n\nAnalysis of GRE with vanA resistance revealed a certain level of host-association51,78–80. Reports using deoxyribonucleic acid (DNA) sequence typing and phylogenetic analysis for genotyping clustered vanA Enterococcus faecium isolates from varying ecological backgrounds into distinct genogroups. Strains collected from pigs and healthy people often clustered together forming a single genotype or cluster. In contrast, isolates collected from poultry and their farmers, veal calves and their farmers, and hospitalized patients from epidemics worldwide each form genetically distinct clusters78–80. One of the first insights of genetic relatedness was the observation of a single base change (G8234T) in the vanX of Tn1546, which was first described by Jensen et al.50,51 The G-variant was associated with isolates collected from poultry and poultry farmers in multiple countries51,57,68,81. The T-variant, on the other hand, was predominantly observed in swine isolates from differing countries51,80. Interestingly, both G- and T-variants were associated with isolates likely of human origin51. In fact, it was observed that all human samples from a Muslim country—a population that likely eats little or no pork—belong to the G-variant associated with poultry, thus further suggesting GRE transmission may occur between food animals and humans51.\n\nFurther investigation of vanA mechanism by Willems et al.80, revealed amplified fragment length polymorphism (ALFP) genotyping clustered a bank of 255 E. faecium isolates from various ecological niches and geographic locations into four genogroups (designated A–D). All isolates collected from pigs and 76% of isolates collected from healthy people clustered to form Genogroup A. Almost all isolates collected from poultry (95%) and 50% of isolates from poultry farmers clustered to form Genogroup B, and Genogroup D contained 70% of isolates collected from veal calves and their farmers. Further, 84% of isolates collected from hospitalized patients from epidemics in the UK, US, and Australia formed a genetically distinct cluster from the healthy humans and animal genogroups, which the authors designated Genogroup C80. Similar findings have been demonstrated using various other genotypic methods39,72,78,79,82–84.\n\nThe VanA gene cluster is now one of many described genotypic determinants encoding glycopeptide resistance, and the early genotypic studies described herein only evidence the likely dissemination of a single glycopeptide resistance determinant from animals to healthy people. Further, the differing epidemiological trends between the US and Europe detail two situations that consequently led to the selection of glycopeptide resistance determinants in distinct ecological niches—one in hospitalized patients and the other in healthy humans and animals. Nonetheless, the genetic characterization of the VanA gene cluster provides an illustrative example of the dissemination of glycopeptide resistance from animals to humans following selection, due to use of avoparcin for growth promotion.\n\nAvoparcin appears to have been widely used in food animals, particularly in chickens and pigs, in parts of Europe, since before the mid 1970s. Vancomycin use in humans, in contrast, was very limited in Europe until the late 1990s. It appears likely that the use of avoparcin in food animals selected for the emergence and dissemination of a resistance gene cluster (VanA), which was increasingly identified in animals and healthy people. Molecular subtyping of the VanA gene cluster has identified variants that are more likely to be associated with certain food animal species. Subsequently, GRE were transmitted and found to colonize healthy humans, presumably via the food chain. Therefore, evaluation of the VanA gene cluster variants provides an illustrative example of the emergence and selection of a genetic resistance determinant as a consequence of antimicrobial use in food animals, and subsequent dissemination of the resistant bacteria to humans.\n\n\nColistin\n\nPolymyxin E (herein simply referred to as colistin) is a cationic, multicomponent lipopeptide antimicrobial agent of the polymyxin family that was first discovered in 1949 and isolated in 195085. Polymyxins are effective against Gram-negative bacilli through their affinity to bind to the negatively charged lipopolysaccharide (LPS) of the cell outer membrane86. This binding, more specifically to the anionic lipid A of the LPS, leads to disruption of the cell membrane integrity, ultimately leading to leakage induced cell death86–88. Two forms of the colistin compound are available for clinical use: colistin sulfate (colistin S) and the pro-drug, colistimethate sodium (colistin methanesulfonate sodium, colistin sulfomethate sodium, colistin M).\n\nThe US FDA first approved colistin for human use in 1962—in the form of colistin sulfate; this first approval was for ear drops89. The FDA subsequently approved a product for injection—in the form of colistimethate sodium—for human use in 197090. No US data are available on the quantities of colistin used in humans, although use in the US is thought to have been very low as parenteral use in human medicine quickly fell out of favor due to initial reports of nephro- and neurotoxicity90–96. More recently, colistin has reemerged as an antimicrobial of interest as a last-resort treatment option for life threatening human infections of multidrug-resistant Gram-negative bacteria, particularly Pseudomonas aeruginosa, Acinetobacter baumannii strains, and carbapenem-resistant Enterobacteriaceae97–102. Approval dates for human use of colistin products in member states of the EU are not clear; however, it is believed that human use began in the 1960s. More recent estimates of polymyxin consumption in humans are available in the EU/European Economic Area103. A sum of 0.8 tonnes of active polymyxin ingredients—including colistin and polymyxin B—were consumed by humans in 22 European countries in 2012104. In 2014, polymyxin consumption in humans in Europe was 0.012 defined daily doses (DDD) per 1,000 inhabitants—a 50% increase since the 0.008 DDD per 1,000 inhabitants was reported in 2010105. Countries reporting highest use of polymyxin in humans include Greece, Italy, and Slovakia (0.095, 0.025, and 0.025 defined daily doses per 1,000 inhabitants, respectively)105.\n\nIn animals, the extent of colistin sales and use is largely unknown outside of the EU106–109. In the US, one colistin product, in the form of an injectable colistimethate sodium, was approved for use in chickens in 1998110; however its marketing status is unclear. In Canada, colistin is not approved for veterinary medicine; however, a loophole in regulation leaves opportunity for “own-use importation,” meaning farmers may import—and use—unlicensed, non-prescription antimicrobials in their animals111. As such, use in swine production has been explored under the veterinarian’s liability (dose, withdrawal period)112,113. In the EU, colistin-containing products for use in animals are authorized114, though marketing authorization is on a national level and little historical information is available. It is believed that colistin has been used in food animals in the EU since the 1950s103. Colistin is chiefly administered as an oral group treatment in food-producing species to alleviate and prevent Gram-negative infections of the gastrointestinal tract107. Such use is predominantly reported in pigs, poultry, cattle, sheep, goats, and rabbits; however, colistin is also used in laying hens and milk-producing cattle, sheep, and goats106,107. To date, no data are available that would allow comparison among uses in differing animal species on a European level.\n\nColistin is also reported to be used in food animal production in Asia, although publically available data are scarce. In China, approximately 90% of the 17.5 million tonnes of colistin produced in 2014 were reportedly consumed by the domestic agriculture industry108. If so, China likely represents the largest colistin producer and consumer in the world. In comparison, a sum of 545.2 tonnes of active polymyxin ingredients—including colistin and polymyxin B—were consumed by food-producing animals, primarily in poultry and swine, in 22 European countries in 2012104. In 2013, polymyxins were estimated to be the fifth most commonly sold antimicrobial class (7%) for food-producing animals across the EU107. Reported consumption of colistin in animals varied greatly, ranging from <0.2 tonnes in Slovenia, Sweden, Ireland, and Luxembourg to >100 tonnes in Germany, Italy, and Spain104. In another report, annual colistin use in animals in Europe ranged between 0 mg (Finland, Iceland, and Norway) to more than 20 mg (Italy and Spain) per kg of animal biomass115.\n\nUse of colistin for growth promotion in China was banned effective November 1, 2016—which was expected to decrease colistin use in food animal production in China by an estimated 8,000 tonnes116. In March 2015, the European Commission adopted a Decision restricting indications, target species, duration of treatment, and added prudent use warnings to products administered orally to animals that contain colistin as the sole active ingredient117. Evidently, such conversations have continued, as the European Commission recently implemented a Directive to withdraw marketing authorizations for all veterinary medicinal products containing colistin in combination with other antimicrobial substances to be administered orally118. The European Medicines Agency issued a recommendation advising colistin to be used solely as a second line treatment in animals and for sales to be minimized EU-wide103. In Canada, the “own-use importation” loophole has been acknowledged and regulation changes have been proposed that would prohibit such practices119.\n\nDespite widespread and continuous veterinary use, data gaps persist around colistin resistance. Lack of agreement on standardized in vitro screening methods and interpretation criteria has complicated and hindered phenotypic surveillance efforts86,120–123. This dilemma is largely a consequence of two important colistin characteristics: a large molecule size—which reduces its rate of diffusion into media—and its affinity to adhere to plastics—which are commonly used in phenotypic methods86,123. Until recently, colistin resistance was believed to be extremely rare; however, surveillance efforts were minimal. In fact, mandatory EU monitoring for colistin resistance in Salmonella and E. coli only began in 2014124,125. Even so, many member states have reported technical difficulties in using the only recommended screening method (i.e., broth dilution)103.\n\nBefore November 2015, described phenotypic colistin resistance was associated with chromosomal mutations, which, at least in theory, would be limited to vertical (clonal) dissemination86,126. However, this previous belief was proven too narrow by the description of a novel, conjugable plasmid-mediated gene conferring colistin resistance108. The gene, designated mobile colistin resistance, or mcr-1, was described in E. coli and Klebsiella pneumoniae isolated from human clinical isolates, retail meat, and food animals in China, between 2011 and 2014108. The discovery prompted an immediate worldwide response with screening via genomic data mining exercises or else a combination of phenotypic and polymerase chain reaction (PCR)-based methods127–133. It has now been retrospectively identified with 100% homology in other members of the Enterobacteriaceae family isolated from human, animal, food, and environmental samples and from multiple continents133–140.\n\nIn humans, the earliest identified mcr-1 was found in a Shigella sonnei isolate arising from a hospitalized child with diarrhea in Vietnam in 2008141. Bacteria harboring mcr-1 have also been reported in isolates from humans (both infected patients and asymptomatic human carriers) in Canada142,143, China108,136,144–154, Denmark106,140, Ecuador155, Egypt156, France130,157, Germany137,158,159, Hong Kong159,160, India161,162, Italy159,163–165, Laos130, Malaysia159,166, Netherlands131,167–170, Norway171, Poland159,172, Portugal173, Russia159, Saudi Arabia174, Singapore175,176, South Africa177,178, Spain159,179, Sweden180,181, Switzerland182–185, Taiwan186, Thailand130,187, United Arab Emirates174, UK188, US159,189–191, Venezuela192, and Vietnam141,193. Bacteria harboring the mcr-1 gene sequence have likewise been documented from food samples on multiple continents108,129,136,138,140,142,168,173,185,186,188,194–199, suggesting this may be an important route of dissemination from animals to humans.\n\nTo date, the earliest identified mcr-1-positive isolates are three E. coli isolates collected from chickens in China during the 1980s200. Interestingly, mcr-1 has not been detected in isolates arising during the two subsequent decades; however, the reported proportion of mcr-1-positive isolates in China begins increasing in 2009200. Furthermore, in Europe the earliest mcr-1-positive isolate was identified as an E. coli originating from a diarrheic veal calf in France in 2005132. Observations of mcr-1 in bacteria isolated from food-producing animals, their products, or environments now includes: pigs (Belgium128,201, Brazil202, China203,204, France127, Germany137,205,206, Japan133,207, Laos130, Malaysia136,138,166, Spain208, Taiwan186, Venezuela192, Vietnam209,210, UK211, US212), poultry (Algeria130,213, Brazil202,214,215, China216–218, Denmark199, Egypt218, France127, Germany205, Italy106,219, Malaysia136,138,166, Netherlands169, South Africa220,221, Spain208, Taiwan186, Tunisia185, Vietnam193,209), and cattle (Belgium128, Denmark199, Egypt222, France127,132,223, Germany205, Japan133, Netherlands169).\n\nWidespread reports of mcr-1 shortly after its initial characterization indicate the gene was likely being disseminated in an uncharacterized state, and thereby undetected rather than not being present, for a long period of time. The gene has evidently been widely disseminated geographically, as well as across multiple bacterial species of differing origins. Thus far, mcr-1 has mostly been reported in E. coli, although mcr-1-positive Citrobacter224,225, Klebsiella108,157,175,224, Shigella141, Enterobacter144,160,176,224, and Salmonella129,135,136,173,188,195,208,211,217 spp. have also been documented. Furthermore, mcr-1 has been observed in bacteria from wild animals and water samples, indicating the resistance determinant has also disseminated into the environment194,224,226–230.\n\nRetrospective screening for colistin-resistant bacteria may be limited by the availability of historical isolates and their genomic data. Further, lack of standardized phenotypic screening methods and the delay in genotypic description have likely lead to the underestimation of colistin resistance; nonetheless, the identification and description of the gene has opened the door for screening via genotypic methods. Nevertheless, resistance is still believed to be rare, particularly in humans and in some regions of the world. The initial paper reported the mcr-1 gene sequence in 1.4% of 902 E. coli and 0.7% of 420 Klebsiella pneumoniae clinical isolates in China; however, prevalence among E. coli isolates originating from pigs and retail meats in China were surprisingly higher: 20.6% of 804 isolates from pigs at slaughter collected between 2012–14 and 14.9% of 523 isolates from retail meats (chicken and pork) collected between 2011–2014108. Still, in the US the mcr-1 gene sequence is rare. It was detected in one E. coli isolate out of 949 animal intestine samples screened and was not detected in more than 44,000 Salmonella and 9,000 E. coli and Shigella isolates from the National Antimicrobial Resistance Monitoring System (NARMS) and National Center for Biotechnology Information (NCBI) genomic database212. In many reports to date, phenotypic screening is frequently performed prior to genotypic screening. For example, in France, Perrin-Guyomard and colleagues report mcr-1 in 0.3% of 590 isolates from healthy pigs in 2011–13, 1.8% of 227 isolates from broilers in 2014, and 5.9% of 239 isolates from turkeys in 2014127; importantly, screening for mcr-1 was performed only on isolates with a colistin minimum inhibitory concentration > 2 mg/L. Some limitations are inevitable with this approach, as it implies a level of dependency on the much-debated breakpoints and phenotypic methods.\n\nThe technological response afforded by genomics-based methods is also not without limitations, especially by not detecting variants of mcr-1. In fact, on July 7, 2016, the first account of mcr-2, a seemingly distinct gene also conferring colistin resistance was described in E. coli isolated from calves and piglets in Belgium231. This mcr-2 appeared to be more prevalent than mcr-1 among colistin-resistant E. coli of porcine origin231. Then, on July 11, 2016, the first functional variant of mcr-1, designated mcr-1.2, was reported in K. pneumoniae isolated from a surveillance rectal swab of a child in Italy232. Since this report was prepared, a number of other mcr variants have been reported, some of which also appear to have been disseminating globally prior to characterization233–235. While some of these genes may also contribute towards evidencing selection—and subsequent dissemination—of the colistin resistance determinant from food animals to humans, the focus of this report was the initial epidemiology of colistin resistance (i.e. mcr-1). Very likely, there remain additional yet-to-be-characterized mechanisms of colistin resistance. Much more work is needed to explore other mechanisms of resistance and to fully comprehend the overall prevalence of colistin resistance determinants and their phenotypic characteristics.\n\nColistin has been widely used in food animals—particularly poultry and swine—in areas of Europe and Asia for decades, perhaps since the early 1980s or earlier. In contrast, colistin use in humans has been extremely limited, at least until recently. It appears highly probable that the use of colistin in food animals has selected for a novel resistance gene (mcr-1), identified as far back as the mid-1980s in chickens in China, which has become increasingly identified in isolates from food animals in many regions of the world since its discovery in 2015. This novel resistance gene has more recently been identified among isolates from humans; however, to date mcr-1 has been more frequently associated with food animal and meat isolates compared to human isolates. Prevalence of mcr-1 in animal samples—and to some degree in human samples—appears to be proportional to its use in animals. These chains of events, despite the data gaps, provide an illustrative example of the emergence, selection, and widespread dissemination of a resistance gene as a consequence of antimicrobial use in food animals, and subsequent transfer of bacteria harboring that resistant gene to humans.\n\n\nConclusions\n\nIn this review, we have focused on three illustrative examples (i.e. streptothricins, glycopeptides, and colistin) of selection—and subsequent transfer of antimicrobial resistance determinants from food animals to humans. The use of antimicrobials in food animal production contributes to the selection and dissemination of antimicrobial resistance determinants that may reach human populations. However, this review is only part of the picture if taken in a One Health perspective. Its objectives do not encompass the impact of other industries (i.e. environment, human, companion animals, etc.) that also contribute to selection of antimicrobial resistance and it’s consequences on each health sector. To tackle the problem of selection and dissemination of antimicrobial resistance in a true One Health perspective, there is need to fully investigate the role of each of those industries. Nevertheless, the three examples we have described serve to illustrate that use of antimicrobials in food animals can result in antimicrobial resistance that can be transmitted to humans. Therefore, these illustrative examples support the need for actions, such as the proposed WHO Guidelines on use of medically important antimicrobials in food animals, to mitigate the risk of adverse human health consequences resulting from the use of antimicrobial agents in food animals.",
"appendix": "Competing interests\n\n\n\nGL has accepted consulting fees, honoraria, and reimbursement of travel expenses from Zoetis, Elanco Animal Health, Merck Animal Health, and Bayer.\n\n\nGrant information\n\nThis work was commissioned by the WHO. The authors have been given permission to publish this article.\n\n\nAcknowledgements\n\nWe appreciate Yves Millemann and Gérard Moulin for providing their expertise concerning antimicrobials and their use. Also, we would like to thank John M. Conly, Peter Collignon, and Scott McEwen for their critical eye and feedback during the preparation of this review. We would like to acknowledge the WHO Secretariat, Yuki Minato, and the Coordinator of the WHO AGISAR, Awa Aidara-Kane.\n\n\nReferences\n\nO’Neill J: Review on AMR. Antimicrobial resistance: tackling a crisis for the health and wealth of nations. London: HM Government and the Wellcome Trust, 2014. 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PubMed Abstract | Publisher Full Text\n\nLi A, Yang Y, Miao M, et al.: Complete Sequences of mcr-1-Harboring Plasmids from Extended-Spectrum-β-Lactamase- and Carbapenemase-Producing Enterobacteriaceae. Antimicrob Agents Chemother. 2016; 60(7): 4351–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang R, Huang Y, Chan EW, et al.: Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis. 2016; 16(3): 291–2. PubMed Abstract | Publisher Full Text\n\nZhang XF, Doi Y, Huang X, et al.: Possible Transmission of mcr-1-Harboring Escherichia coli between Companion Animals and Human. Emerg Infect Dis. 2016; 22(9): 1679–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZheng B, Dong H, Xu H, et al.: Coexistence of MCR-1 and NDM-1 in Clinical Escherichia coli Isolates. Clin Infect Dis. 2016; 63(10): 1393–5. PubMed Abstract | Publisher Full Text\n\nYe H, Li Y, Li Z, et al.: Diversified mcr-1-Harbouring Plasmid Reservoirs Confer Resistance to Colistin in Human Gut Microbiota. mBio. 2016; 7(2): e00177–16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRuppé E, Le Chatelier E, Pons N, et al.: Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis. 2016; 16(3): 290–1. PubMed Abstract | Publisher Full Text\n\nHe QW, Xu XH, Lan FJ, et al.: Molecular characteristic of mcr-1 producing Escherichia coli in a Chinese university hospital. Ann Clin Microbiol Antimicrob. 2017; 16(1): 32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDu H, Chen L, Tang YW, et al.: Emergence of the mcr-1 colistin resistance gene in carbapenem-resistant Enterobacteriaceae. Lancet Infect Dis. 2016; 16(3): 287–8. PubMed Abstract | Publisher Full Text\n\nYu H, Qu F, Shan B, et al.: Detection of the mcr-1 Colistin Resistance Gene in Carbapenem-Resistant Enterobacteriaceae from Different Hospitals in China. Antimicrob Agents Chemother. 2016; 60(8): 5033–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrtega-Paredes D, Barba P, Zurita J: Colistin-resistant Escherichia coli clinical isolate harbouring the mcr-1 gene in Ecuador. Epidemiol Infect. 2016; 144(14): 2967–2970. PubMed Abstract | Publisher Full Text\n\nElnahriry SS, Khalifa HO, Soliman AM, et al.: Emergence of Plasmid-Mediated Colistin Resistance Gene mcr-1 in a Clinical Escherichia coli Isolate from Egypt. Antimicrob Agents Chemother. 2016; 60(5): 3249–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCaspar Y, Maillet M, Pavese P, et al.: mcr-1 Colistin Resistance in ESBL-Producing Klebsiella pneumoniae, France. Emerg Infect Dis. 2017; 23(5): 874–876. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFritzenwanker M, Imirzalioglu C, Gentil K, et al.: Incidental detection of a urinary Escherichia coli isolate harbouring mcr-1 of a patient with no history of colistin treatment. Clin Microbiol Infect. 2016; 22(11): 954–955. PubMed Abstract | Publisher Full Text\n\nCastanheira M, Griffin MA, Deshpande LM, et al.: Detection of mcr-1 among Escherichia coli Clinical Isolates Collected Worldwide as Part of the SENTRY Antimicrobial Surveillance Program in 2014 and 2015. Antimicrob Agents Chemother. 2016; 60(9): 5623–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWong SC, Tse H, Chen JH, et al.: Colistin-Resistant Enterobacteriaceae Carrying the mcr-1 Gene among Patients in Hong Kong. Emerg Infect Dis. 2016; 22(9): 1667–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumar M, Saha S, Subudhi E: More Furious Than Ever: Escherichia coli-Acquired Co-resistance Toward Colistin and Carbapenems. Clin Infect Dis. 2016; 63(9): 1267–68. PubMed Abstract | Publisher Full Text\n\nBernasconi OJ, Kuenzli E, Pires J, et al.: Travelers can import colistin-resistant Enterobacteriaceae, including those possessing the plasmid-mediated mcr-1 gene. Antimicrob Agents Chemother. 2016; 60(8): 5080–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGiufrè M, Monaco M, Accogli M, et al.: Emergence of the colistin resistance mcr-1 determinant in commensal Escherichia coli from residents of long-term-care facilities in Italy. J Antimicrob Chemother. 2016; 71(8): 2329–31. PubMed Abstract | Publisher Full Text\n\nCannatelli A, Giani T, Antonelli A, et al.: First detection of the mcr-1 colistin resistance gene in Escherichia coli in Italy. Antimicrob Agents Chemother. 2016; 60(5): 3257–8. 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"id": "27473",
"date": "03 Nov 2017",
"name": "Wolfgang Witte",
"expertise": [
"Reviewer Expertise Molecular epidemiology of antibiotic resistance",
"studies on transmission of antibiotic resistant bacteria and their resistance genes at the interface between livestock and humans"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nManuscript “Illustrative examples of probable transfer of resistance determinants from food animals to humans: Streptothricins, glycopeptides, and colistin” describes evidence for antibiotic resistance gene transfer from bacteria colonizing / infecting livestock to bacteria colonizing / infecting humans by means of three examples: streptothricine-resistance and glycopeptide resistance as historical examples, and mcr mediated resistance to colistin as an important recent example. Altogether this review is well written and based on a very careful literature research together with a balanced selection of the really relevant publications. Above all the comprehensive and condensed presentation of studies on colistin resistance is impressing.\nA few points will need attention:\nA short discussion on the consequences of the ban of avoparcin as growth promoter would be of interest (e.g. significant reduction of gastrointestinal colonization of healthy humans in the community, Klare et al., 1999). It should also be mentioned that despite this ban several European countries faced an increase of VRE among isolates from blood cultures (EARSnet) since 2004. The question whether the ban was of little significance for the VRE situation in human medicine or whether it could have come much more worse in case of a continuing existing van gene pool in the community cannot be answered in retrospect.\n\nFor the chapter on colistin resistance I suggest to mention that the extent to which mcr contributes to colistin resistance in clinical isolates cannot exactly be assessed so far, and that there are cases of acquisition of mcr by carbapenemase producing K.pneumoniae (Newton-Food et al., 2017). Furthermore, the emergence of E.coli harbouring mcr-1 and blaKPC-2 after meropenem and colistin therapy is of interest (Tacao et al., 2017).\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": "27472",
"date": "14 Nov 2017",
"name": "Séamus Fanning",
"expertise": [
"Reviewer Expertise Molecular mechanism of antimicrobial resistance",
"dissemination of resistance determinants",
"WGS characterisation of AMR genotypes"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Illustrative examples of probable transfer of resistance determinants from food animals to humans: strepothricins, glycopeptides and coilstin, these authors provide a timely update on this important topic of interest to veterinary public health. The three selected antimicrobial agents highlighted are described so as to provide the reader with a background on their uses; the emergence of resistance to these compounds and possible routes of dissemination. Two of the three examples provided (streptothricin- and glycopeptide-resistance) are historical in nature, whilst the colistin-resistant chapter is more recent. The selection of these topics clearly demonstrated the consequences of antimicrobial compound usage and the subsequent response from bacteria of importance to animal- and human-health.\nThe manuscript is well designed and clearly written, as presented. It is scientifically sound. The topic is of immediate interest and would serve as a very useful and comprehensive review of our current understanding of this subject. In places the authors highlight some of the limitations in current approaches to the study of some of these resistance mechanisms, particularly in regard to colistin.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1805
|
https://f1000research.com/articles/6-1802/v1
|
04 Oct 17
|
{
"type": "Research Article",
"title": "Risk perception of medicinal marijuana in medical students from northeast Mexico",
"authors": [
"Sandra Castillo-Guzmán",
"Dionicio Palacios-Ríos",
"Teresa A. Nava-Obregón",
"Julio C. Arredondo-Mendoza",
"Olga V. Alcalá-Alvarado",
"Sofía A. Alonso-Bracho",
"Daniela A. Becerril-Gaitán",
"Omar González-Santiago",
"Sandra Castillo-Guzmán",
"Dionicio Palacios-Ríos",
"Teresa A. Nava-Obregón",
"Julio C. Arredondo-Mendoza",
"Olga V. Alcalá-Alvarado",
"Sofía A. Alonso-Bracho",
"Daniela A. Becerril-Gaitán"
],
"abstract": "Background. Several studies have shown support from the public toward the use of medicinal marijuana. In this cross-sectional study, we assess the risk perception to medicinal marijuana in a sample of medical students. Methods. To estimate risk perception, a visual scale that ranges from 0 cm (without risk) to 10 cm (totally risky) was used. Risk perception was expressed as the median of the cm marked over the scale. Differences among groups was tested with the Mann-Whitney and Kruskal-Wallis tests, as appropriate. Results. 283 students participated in the study. Risk perception to medicinal marijuana was 4.22, paracetamol 1.56 and sedatives 5.0. A significant difference in risk perception was observed in those that self-reported to smoke and consume alcohol. Conclusions. Risk perception of medicinal marijuana is 4.22 in medical students of northeast of Mexico. Students may underestimate its adverse effects. More studies with respect to this are needed.",
"keywords": [
"Medicinal marijuana",
"medical students",
"Mexico",
"Risk perception",
"Drugs"
],
"content": "Introduction\n\nSeveral studies have showed the potential benefits of medicinal marijuana (MM) for the treatment of some illnesses, such as pain in cancer, multiple sclerosis, Alzheimer’s disease, post-traumatic stress disorder, epilepsy, Crohn’s disease, and glaucoma1,2. However, like other drugs, MM is not exempt from serious adverse effects, such as psychiatric disorders, euphoria, disorientation, confusion, somnolence, and fatigue; therefore, it should be used with extreme caution3. Legally, physicians cannot prescribe MM; however, in some countries, they can recommend its use when they consider it necessary. Some recommendations on how to prescribe marijuana for the care of patients have been published previously4.\n\nOn the other hand, risk perceptions towards medicinal drugs have been reported that influence in the prescribing behavior and the willingness of physicians to report adverse drug reactions. Knowing the risk perceived by medical students to drug prescription is important, since it establishes the requirement of education regarding certain medicines, like MM. In this respect, there are no studies regarding the risk perception toward MM in the general population and among health professionals. The existing literature has focused mainly on attitudes and support toward its legalization. Across countries, the factors associated with support for its legalization are political tolerance, ideology, and the views toward government5.\n\nIn Mexico, as in others countries, the use of MM was prohibited; however, in December 2016, the Senate approved the legalization of MM and sent the bill to the Chamber of Representatives for ratification, which occurred on April 28, 2017. The next steps for its appropriate use include the publication of laws, regulations, and guidelines by the Health Ministry, medical schools, and medical associations. While this continues, it is important that health professionals be updated on this topic, so that they can rationally recommend the use of MM. To achieve this, a first approach would be to know the attitudes and willingness to recommend the use of MM, and the risk perceived by physicians. In this study, we evaluate the risk perception of MM in medical students from northeast Mexico and determine associated factors.\n\n\nMethods\n\nA cross-sectional study that uses a visual scale to estimate risk perception of MM. The survey was applied during July and August 2017 in a public university from northeast of Mexico.\n\nThe inclusion criteria were as follow: Medical students of Universidad Autónoma de Nuevo León, both genders, any semester of study, and ages older than 18 years. Participants with incomplete surveys of second section were excluded. The medical students were contacted personally in the study areas and halls of the Faculty of Medicine. After obtaining verbal consent the survey was applied. This was self-administered with supervision. This study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Faculty of Medicine at Universidad Autónoma de Nuevo León (registration number PI17-00134). Only verbal consent was requested from participants because the study is of low risk. This type of consent was approved by the Ethics Committee.\n\nThe survey used was composed of two sections, the first section, which was optional, collected demographic information, such as age, gender, semester of study, self-reported alcohol and smoking status (undefined level, only yes or no), and currently self-reported disease (unspecific, only yes or no). The second section evaluates the risk perception toward the use of MM. For this, a visual scale of 10 cm, from 0 cm (without risk) to 10 cm (completely risky), was used. Participants marked over the scale the risk that they perceived when MM is used. This section also contained two additional scales for paracetamol and sedatives, which act as relatively safe (negative control) and risky (positive control) drugs, respectively. The order of scales, including MM, was randomly allocated. The use of this visual 10-cm scales has been used previously in studies that assess the risk perception to other medicinal drugs6–8. The complete survey is available as Supplementary File 1.\n\nDescriptive statistics is reported for demographic data. Risk perception was reported as the median of centimeters marked over the scale (from 0 to the mark). The results of risk perception were grouped by three age groups (18–20, 21–23 and >23 years), gender, semester of study (1–3, 4–7 and >7 semester), and self-reported alcohol and smoking status. Differences among groups were evaluated with the Mann-Whitney and Kruskal-Wallis tests, as appropriate. The statistical package NCSS version 11 was used for all analysis. The level of significance was p < 0.05.\n\n\nResults\n\nOverall, the rate of response was 97%. In total 283 students were interviewed, of which 50.8% were men; 61% had an age range of 21–23 years, and 62.8% were enrolled in semesters 8 or higher. In addition, 18.7% self-reported smoking, 48.1% consuming alcohol and 14.1% self-reported having a (unspecified) disease (Table 1).\n\n* Some data are missing due to participants that did not respond to the questions.\n\nOverall, the risk perception of MM was 4.22 cm, while for paracetamol and sedatives it was 1.56 and 5.00 cm, respectively (Figure 1). The observed differences among the three drugs were statistically significant (P < 0.05). This pattern was similar according to gender, age, semester of study, smoking and alcohol consumption and having some (unspecified) disease.\n\nTwo additional scales for paracetamol and sedatives are included, which act as relatively safe (negative control) and risky (positive control) drugs.\n\nThe analysis of individual drugs did not show a significant difference between gender, smoking and alcoholism status, having a disease, or among age groups and the semester of study in the risk perception of paracetamol and sedatives (Table 2). However, a significant difference was observed between self-reported smoking and alcohol consumption in the risk perception of MM. Those that self-reported smoking or alcoholism had a lower risk perception of MM.\n\n\nDiscussion\n\nIn this study, we assessed the risk perceived by medical students toward the use of MM. Previous reports have analyzed the risk perception to several drug prescriptions; however, this is the first study that analyzes the risk perception to MM using the same instrument that these studies used.\n\nAlthough several studies in the general population and among health professionals have shown a support for the legal use of MM9–12, physicians undoubtedly require solid knowledge of both its benefits and adverse effects if they want recommend it appropriately. Our results show that the risk perceived to MM (4.2 cm) is higher than paracetamol but lower than sedatives in Mexican medical students. Compared with other studies, the risk perceived is similar to the risk perceived to antibiotics, hypocholesterolemia drugs, and antihypertensives (median range 3–5 cm), but lower than that to nonsteroidal anti-inflammatory drugs (NSAIDs), antidepressants, and anticoagulants (median > 6 cm)6–8,13. Although the objective was not to establish whether perceived risk was adequate, we speculate that observed outcome is low. We think that a reasonable risk perception of MM should be more than 6, close to other drugs as sedatives, antidepressants or anticoagulants.This due to the difficulty of dosing, specifically if it is smoked, and the frequency of adverse effects of MM. We speculate that students with risk perception values of MM lower than 5 could underestimate its adverse effects and probably could recommend it indiscriminately during their practice. More studies concerning this are needed.\n\nOn the other hand, the risk perception of paracetamol was low and is similar to that previously reported14. The implications of this finding could be the same as with MM; students could underestimate its adverse effects, which is mainly liver damage15. It is worth remembering that lack of awareness of potential harm from taking or administering paracetamol improperly in adults and adolescents is a cause of emergency department visits16.\n\nAn interesting result was the significant difference in risk perception between those that self-reported smoking and alcohol consumption. The users of these recreational drugs had a lower risk perception of MM. In this sense, it has been proven in previous studies that tobacco and alcohol consumption are risk factors for the use of recreational marijuana17,18. In addition, the consumption of these drugs has been associated with support for legalization of recreational and MM19,20.\n\nThe risk perception observed could be due in part to attention from mass media regarding its potential uses. Previous studies have found an association between public support for MM and its coverage in media21,22. Another factor that could impact risk perception values, is the lack of formal courses regarding MM in the syllabus of students surveyed23. In this sense, previous studies have proven that formal courses of pharmacology increase risk perception toward common drugs like NSAIDs7. The design and implementation of formal courses regarding MM may have the same impact.\n\nFinally, we consider that our results should be interpreted with caution, as it is possible that our findings may not be generalized to other countries, due to differences in teaching methods. Replication in others countries, especially where the use of MM has been recently made legal, is needed. Although the risk perception of drugs has been studied with visual scales, the development of other instruments could improve the assessment.\n\n\nConclusions\n\nThe risk perception of MM was 4.22 in medical students of the northeast of Mexico. With the legalization of MM in Mexico, formal courses regarding dosing, and adverse and beneficial effects of MM will be needed in medical schools.\n\n\nEthical statement\n\nThis study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Faculty of Medicine (registration number, PI17-00134).\n\n\nData availability\n\nDataset 1: Raw data of risk perception to medicinal marijuana in medical students. doi, 10.5256/f1000research.12638.d17906924",
"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\nTo all medical students that participated in this study. To Dr Sergio Lozano for his help in the edition of manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: Survey in English and Spanish.\n\nClick here to access the data.\n\n\nReferences\n\nWhiting PF, Wolff RF, Deshpande S, et al.: Cannabinoids for Medical Use: A Systematic Review and Meta-analysis. JAMA. 2015; 313(24): 2456–2473. PubMed Abstract | Publisher Full Text\n\nWilkie G, Sakr B, Rizack T: Medical Marijuana Use in Oncology: A Review. JAMA Oncol. 2016; 2(5): 670–675. PubMed Abstract | Publisher Full Text\n\nCiccone CD: Medical Marijuana: Just the Beginning of a Long, Strange Trip? Phys Ther. 2017; 97(2): 239–248. PubMed Abstract | Publisher Full Text\n\nChaudhry HJ, Hengerer AS, Snyder GB: Medical Board Expectations for Physicians Recommending Marijuana. JAMA. 2016; 316(6): 577–578. PubMed Abstract | Publisher Full Text\n\nCruz JM, Queirolo R, Boidi MF: Determinants of public support for marijuana legalization in uruguay, the united states, and el salvador. J Drug Issues. 2016; 46(4): 308–325. Publisher Full Text\n\nBongard V, Ménard-Taché S, Bagheri H, et al.: Perception of the risk of adverse drug reactions: differences between health professionals and non health professionals. Br J Clin Pharmacol. 2002; 54(4): 433–436. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDurrieu G, Hurault C, Bongard V, et al.: Perception of risk of adverse drug reactions by medical students: influence of a 1 year pharmacological course. Br J Clin Pharmacol. 2007; 64(2): 233–236. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuendia JA, Zuluaga AF: [Physicians´ insight about adverse drug reaction to frequently used medication groups in Bogotá (Colombia)]. Biomedica. 2014; 34(3): 403–8. PubMed Abstract\n\nSupport increases for marijuana legalization | Pew Research Center [Internet]. [cited 2017 May 5]. Reference Source\n\nMillhorn M, Monaghan M, Montero D, et al.: North americans’ attitudes toward illegal drugs. J Hum Behav Soc Environ. 2009; 19(2): 125–141. Publisher Full Text\n\nChan MH, Knoepke CE, Cole ML, et al.: Colorado Medical Students' Attitudes and Beliefs About Marijuana. J Gen Intern Med. 2017; 32(4): 458–463. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKondrad E, Reid A: Colorado family physicians’ attitudes toward medical marijuana. J Am Board Fam Med. 2013; 26(1): 52–60. PubMed Abstract | Publisher Full Text\n\nCullen G, Kelly E, Murray FE: Patients’ knowledge of adverse reactions to current medications. Br J Clin Pharmacol. 2006; 62(2): 232–236. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCastillo-Guzman S, González-Santiago O, Delgado-Leal IA, et al.: Perception of the risk of adverse reactions to analgesics: differences between medical students and residents. PeerJ. 2016; 4: e2255. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTittarelli R, Pellegrini M, Scarpellini MG, et al.: Hepatotoxicity of paracetamol and related fatalities. Eur Rev Med Pharmacol Sci. 2017; 21(1 Suppl): 95–101. PubMed Abstract\n\nBudnitz DS, Lovegrove MC, Crosby AE: Emergency department visits for overdoses of acetaminophen-containing products. Am J Prev Med. 2011; 40(6): 585–592. PubMed Abstract | Publisher Full Text\n\nvon Sydow K, Lieb R, Pfister H, et al.: What predicts incident use of cannabis and progression to abuse and dependence? A 4-year prospective examination of risk factors in a community sample of adolescents and young adults. Drug Alcohol Depend. 2002; 68(1): 49–64. PubMed Abstract | Publisher Full Text\n\nIglesias V, Cavada G, Silva C, et al.: [Early tobacco and alcohol consumption as modifying risk factors on marijuana use]. Rev Saude Publica. 2007; 41(4): 517–522. PubMed Abstract | Publisher Full Text\n\nSznitman SR, Bretteville-Jensen AL: Public opinion and medical cannabis policies: examining the role of underlying beliefs and national medical cannabis policies. Harm Reduct J. 2015; 12: 46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan der Sar R, Brouwers EPM, van de Goor LAM, et al.: The opinion on Dutch cannabis policy measures: A cross-sectional survey. Drugs Education Prevention and Policy. 2011; 18(3): 161–171. Publisher Full Text\n\nSznitman SR, Lewis N: Is cannabis an illicit drug or a medicine? A quantitative framing analysis of Israeli newspaper coverage. Int J Drug Policy. 2015; 26(5): 446–452. PubMed Abstract | Publisher Full Text\n\nSantos MT, Camacho I: El tratamiento del cannabis en la prensa española. Cuadernos info. 2017; 153–171. Publisher Full Text\n\nPrograma Académico | Universidad Autónoma de Nuevo León. Facultad de Medicina [Internet]. [cited 2017 Sep 10]. Reference Source\n\nCastillo-Guzmán S, Palacios-Ríos D, Nava-Obregón TA, et al.: Dataset 1 in: Risk perception of medicinal marijuana in medical students from northeast Mexico. F1000Research. 2017. Data Source"
}
|
[
{
"id": "27228",
"date": "07 Nov 2017",
"name": "Nicholas Chadi",
"expertise": [
"Reviewer Expertise Addiction 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\nOverarching comments:\n\nThis article describes the results of a cross-sectional study that used a self-administered survey to explore the perception of riskiness of medical marijuana in comparison to paracetamol and sedatives among a sample of medical students in a single medical school in northeast Mexico. The study addresses an important and timely topic given the current movement towards legalization of medicinal marijuana in North America. Also, provider perception of risk associated with medicinal marijuana has not previously been reported.\n\nHowever, in its current state, the manuscript is unfit for publication. The introduction is insufficiently supported by evidence, and contains several grammatical and structural mistakes that make it very difficult to read. The aims and objectives of the study are not clearly stated. The methodology has several gaps and generally lacks in rigor. Specifically, the question asked to study participants is unclear (“What is the level of risk associated with medicinal marijuana”) as participants are not told what type of risk we are concerned about. The discussion section also has several grammatical mistakes, includes several statements that are not supported by evidence, and does not adequately explain the significance of the findings. Also, the authors fail to underline key limitations in their study. Finally, the conclusions are not appropriately related to study data and lack in clinical relevance.\n\nAll in all, the authors have attempted to research an important issue, but the level of rigor in their investigation is insufficient. The survey designed by the authors fails to ask a clear question to study participants and the very high number of grammatical mistakes in the manuscript severely impacts readability. We recognize the effort that was put into this study, and congratulate the authors for selecting an important and timely topic, but cannot recommend publication of this manuscript.\n\nDetailed comments:\nConsider changing the term medicinal marijuana to medical marijuana (this term is more commonly used in North America).\n\nAbstract:\n\nIn the results section, the authors should clearly state that students who used alcohol or cigarettes had a lower risk perception of marijuana. Also, authors need to specify the units of the risk perception scale of put it into context. The conclusions need to be rephrased. A score of 4.22 on a visual analogue scale does not have any clinical or practical significance in and of itself.\n\nIntroduction:\nThe text has several grammatical and syntactic mistakes that make it difficult to read. Several statements in the Introduction are not supported by a reference. This needs to be addressed.\n\nThe second paragraph is very difficult to read and needs to be rewritten. In the third paragraph, the authors appear to support the recommendation of medical marijuana for patients (“…so that they can rationally recommend the use of MM…”). Authors refrain from making recommendations based on personal opinion or rewrite this sentence so that it appears neutral. The aims and objectives of the study are not clearly stated and should be clearly described.\n\nMethods:\nThe first sentence is grammatically incorrect and needs to be revised. Participants: The reason for exclusion of participants with incomplete data is unclear (this data needs to be reported). The sentence where this is stated is ambiguous and grammatically incorrect. Authors need to detail who obtained consent and who was providing supervision while participants were filling in their surveys as this could be a significant source of bias. Also, was privacy respected? This is a critical point. Were surveys administered anonymously? This needs to be stated explicitly in the manuscript. It is unclear why the authors refer to the Declaration of Helsinki, this should be removed. Instrument: It is unclear why the demographic section was optional. This highly impacts the results of the study. It is unclear why the authors asked participants about having a disease. Authors should say more explicitly how they asked the question on perceived riskiness of medical marijuana (this should appear in the methodology section).\n\nResults:\nIn Table 1, authors need to be more specific about how much data is missing as this could introduce a source of bias It is unclear what the response rate represents. Is it referring to the proportion of students who were offered to participate or is it the proportion of medical students at the medical school? In Figure 1, authors state that paracetamol is safe and that sedatives are risky. This should be supported by evidence. Table 2 would benefit from more detail in the row headers as they are hard to interpret. Also, authors should include interquartile ranges in Table 2. Finally, it is not clear what the p-values for the rows in Table 2 are comparing – highest to lowest values would mean comparing paracetamol to sedatives – presumably that is not what authors mean. The term “control” is inadequate in this context. While there is no statistically significant difference between semester of study, it looks like there could be a difference between students early in their studies (1-3) vs >4 if the data were dichotomized in this way. That could be interesting because it might suggest that as students learn more in school they become more aware of the risks of marijuana use.\n\nDiscussion:\nThe first sentence of the discussion should summarize the authors’ research findings. Authors should acknowledge that there is a high level of controversy around the use of medical marijuana. The authors compare the results of their study with results from other studies. This comparison needs to be detailed. Were all the studies asking the exact same question? The comment in the discussion that the “risk is too low and should be closer to 6” seems to have no justification, and should be removed (if the manuscript is to be edited). This is strictly the authors’ speculation about a scale that has even been validated for this type of experiment (or has it? If it has been this needs to be mentioned in the manuscript) – in other words we don’t even know what a 6/10 means, nor is there any evidence that the scale is anything more than ordinal. If the authors have more data about the scale and its validity they should include it. The authors fail to recognize several methodological limitations in their study (ie: missing data, mode of recruitment, one single medical school...). The authors’ explanation of why the fact that students who use alcohol or cigarettes have lower risk perception is an interesting finding is incoherent. The authors appropriately recognize opportunities for education but could make better use of study data to support their point. Ex: mention that the risk perception of MM does not change with the number of school semester, suggesting a potential missed opportunity in medical education, as students should become more aware of the risks of recommending MM if taught about those risks. The authors appropriately recognized that the study findings might not be generalizable to other countries, but should also acknowledge that they have surveyed students and not healthcare providers. In general, the discussion lacks in depth and clinical relevance. Finally there is no sense of the relevance of this data. Are higher risk perceptions associated with less frequent recommendations for other substances or are they associated with some other meaningful impact on provider behavior? Without this, the data is decontextualized and not easy to meaningfully interpret.\nConclusions:\nConclusions lack clinical relevance and are not appropriately supported by the research data.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "3159",
"date": "08 Nov 2017",
"name": "Omar González-Santiago",
"role": "Author Response",
"response": "Thank you very much to Dr Chadi and Dr Levy for your comments regarding our manuscript. All your comments are important for us. I would like to clarify some points.Grammatic will be revised by an profesional translator.METHODSParticipants: The reason for exclusion of participants with incomplete data is unclear (this data needs to be reported). The sentence where this is stated is ambiguous and grammatically incorrect.Response. At this respect we consider to exclude from the analysis the responses of participants with incomplete surveys of the second section. This is due to that the section correspond to risk perception of MM. Fortunately, all participants completed the second section so we will delete this sentence.Authors should say more explicitly how they asked the question on perceived riskiness of medical marijuana (this should appear in the methodology section).Response. The specific question will be written in the method section. However, you can find it in the survey, which is provided as a supplementary file. RESULTSIn Figure 1, authors state that paracetamol is safe and that sedatives are risky. This should be supported by evidence.Response. In Figure 1 we do not affirm that paracetamol and sedative are completely safe and risky respectively. We state that two additional scales were included for paracetamol and sedatives which act “RELATIVELY” safe and risky control. We agree with the term control is inadequate in this context and will be removed.While there is no statistically significant difference between semester of study, it looks like there could be a difference between students early in their studies (1-3) vs >4 if the data were dichotomized in this way.Response. Thank you very much for your observations. We will consider dichotomies as you recommend. DISCUSSIONThe comment in the discussion that the “risk is too low and should be closer to 6” seems to have no justification, and should be removed (if the manuscript is to be edited). This is strictly the authors’ speculation about a scale that has even been validated for this type of experiment (or has it? If it has been this needs to be mentioned in the manuscript) – in other words we don’t even know what a 6/10 means, nor is there any evidence that the scale is anything more than ordinal. If the authors have more data about the scale and its validity they should include itResponse. Thank you very much, we have clearly written in the paragraph that “we speculate” at this respect and expose our reasons. In addition, we recommend more studies at this respect in the last sentence.Finally there is no sense of the relevance of this data. Are higher risk perceptions associated with less frequent recommendations for other substances or are they associated with some other meaningful impact on provider behavior? Without this, the data is decontextualized and not easy to meaningfully interpret.Response. With respect to MM there is no data. However, we comment the association of risk perception of medicinal drugs with behaviors of physicians in the second paragraph of introduction (it will be more referenced). These paragraph will be placed in discussion section for improve the sense of relevance."
}
]
},
{
"id": "46028",
"date": "08 Apr 2019",
"name": "Sultan Alnohair",
"expertise": [
"Reviewer Expertise -chronic disease as Diabetes",
"Hypertension"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 discussion of relevant epidemiological data conducted in Mexico and other countries could be included. Correlates such as socio-demographic, culture-related factors of Mexico and other countries pertaining to adolescent smoking could also be discussed in the Introduction Section.\nMethods - The information below is missing in the paper but should be included:\nThe authors did not tell us how many students participated in the study and how many of them was included and excluded? The criteria for selection of the participating medical students should be listed. What is the percentage of those who participated compared to the total medical students? Is it daily interview or once per week from Jun up to August? What were the inclusion/exclusion criteria of the participants? Regarding the assessment, the authors need to let the readers know more about the variables which were included in the questionnaire. The authors should state the scales that have been used to measure the variables, the reliability etc. All of those were missing in the Methods Section. Since the authors outlined some information about northeast Mexico, it would be better if the authors could devote a section to discuss northeast Mexico in the Introduction part so as to let the readers understand what are the existing cultural factors affecting adolescents in that area. The authors also need to state that the rights and confidentiality of the participants have been communicated to them. I suggest to make correlation between smoking status and their perception. Also, the survey should mention about the family history of smoking, using marijuana in the home. The study was done on medical students but actually no clear question about the effect of medical schools on their perception. How many lectures per semester talk about drug abuse.\nThe study has implications on policy and how adolescents are educated to resist peer pressure when it comes to smoking. In general, a good study but needs more clarification about the comments above and how the medical school will increase the awareness of medical students about the harmful of MM.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1802
|
https://f1000research.com/articles/6-1801/v1
|
04 Oct 17
|
{
"type": "Case Report",
"title": "Case Report: Managing a giant, high-grade carotid body tumor in a resource-limited setting",
"authors": [
"Sunil Munakomi",
"Samrita Chaudhary",
"Iype Cherian",
"Samrita Chaudhary",
"Iype Cherian"
],
"abstract": "Herein we report the management of a giant, high-grade and vascular carotid body tumor in a young woman. She presented with slowly progressive neck swelling. Vascular imaging revealed a left-sided, high-grade giant carotid body tumor (> 8cm). The tumor was completely excised by caudocranial subadventitial dissection. Histology of the tumor revealed a characteristic Zellballen pattern of the lesion, suggestive of a paraganglioma. The patient made an uneventful recovery. We also discuss newer insights regarding the management of such highly vascular lesions.",
"keywords": [
"giant",
"carotid body",
"tumor"
],
"content": "Introduction\n\nGiant carotid body tumors are rare. Management of such highly vascular lesions, which completely encase the major carotid artery along with its branches, is even more challenging. We report the management strategy for a similar case, where caudocranial subadventitial dissection completely excised this tumor, one of the largest reported in the literature1,2.\n\n\nCase report\n\nA 22-year-old woman presented to the Neurosurgery clinic in Nobel Teaching Hospital with a history of slow progressive but painless swelling over her left neck, ongoing for the last 6 months. She was also experiencing some difficulty while swallowing. She denied history of trauma, any episodes suggestive of transient ischemic attacks or paroxysmal episodes of severe headache, flushing or chest pain. She did not have major surgery or medical illnesses in the past, or any significant family history.\n\nLocal examination revealed a pulsatile swelling on her left neck. There was no audible bruit over the swelling. The patient then underwent a CT angiography, which revealed a well-defined large (> 8 cm) heterogeneous and hyperdense soft tissue lesion; showing intense arterial enhancement at the level of carotid bifurcation (Figure 1). It was causing significant compression and displacement of a long segment of the internal carotid artery (ICA) and external carotid artery (ECA), encasing a broad area of the ICA and ECA ( Shamblin Grade 3) (Figure 2). The lesion was getting vascular supply from both the ICA and ECA branches (Figure 3). These findings were all highly suggestive of a carotid body tumor.\n\nUltrasound imaging of the abdomen to assess adrenal glands was normal. 24-hour urine vanillylmandelic acid (VMA) and plasma metanephrines were within normal range.\n\nThe patient was thoroughly counseled regarding her condition and the need for operative management. The risks associated with the surgery, including intra-operative uncontrolled bleeding, lower cranial nerve palsy, ischemic stroke and even death, were detailed. Anaesthetic precautionary measures were implemented to reduce the risk of hypertensive crisis (during tumor manipulation) and hypotensive episodes (following tumor removal), by administering antihypertensive drugs and fluid support, respectively. After ensuring temporary carotid control with vascular loops, sub-adventitial dissection of the lesion was carried out starting from the common carotid caudally and then progressing cranially towards the bifurcation and its branches (Figure 4). The major vascular supplies were sequentially isolated, ligated and divided. A venous graft from the long saphenous vein was prepared for repair in case of inadvertent tears within the carotid or any of its branches. The internal jugular vein and the vagus nerve within the carotid sheath, and the hypoglossal nerve were all selectively isolated and well preserved. The lesion was completely excised and sent for histopathological analysis. There was only one instance of temporary bradycardia throughout the procedure. Patient made an uneventful recovery with no lower cranial nerve deficits or any vascular insults. The histology from the lesion revealed a characteristic Zellballen pattern, highly suggestive of a Paraganglioma (Figure 5). The patient has been for regular follow-ups in the last 4 months, with complete resolution of her previous symptoms. She has been advised for lifelong periodic visits.\n\n\nDiscussion\n\nCarotid body tumors, first described by Von Haller in 1743, are of neuro-ectodermal origin3. They are the most common tumors of this origin to occur in the head and neck region4. Since the majority of cases seem to occur in people residing at high altitudes, chronic hypoxemia has been postulated to be a cause for such tumors5. Most patients present with features owing to compression of the lower cranial nerves by the lesion6 In few cases, paroxysmal symptoms resulting from excessive circulating catecholamine may be evident7,8.\n\nExamination should begin by assessing the pulsatility of the tumor. It has a characteristic mobility in the horizontal direction whereas it has restricted mobility in the vertical direction. This aspect is also referred to as the ‘Fontaine sign’9.\n\nDuplex ultrasound study of the carotid vessels is the first line diagnostic imaging modality10. It helps diagnose the lesion, determine its extent and the involvement of the major vessels. It also aids in simultaneous assessment of both the carotid vessels, to rule out bilateral involvement and carotid artery disease, especially in aged patient groups. In high-risk patients with positive family history or in active catecholamine secreting lesions, this type of imaging can help rule out multiple infections by evaluating the adrenal glands. Shamblin et al classified these highly vascular lesions depending on how they are placed in relation to the carotid vessel, which helps plan their management strategy11. Most high-grade lesions require an adjuvant protocol, either in the form of preoperative embolisation or intra-operative temporary or permanent interposition vascular grafts.Needle biopsy is contraindicated due to the risk of haemorrhage, thrombosis or pseudo-aneurysm12. It is also advisable to rule out synchronous neural crest lesions by imaging the tympanic cavity and the adrenal glands, especially in patients with positive family history13. Although rare, active lesions (seen in around 5% of cases) should be ruled out by assessing the plasma catecholamine or the urinary VMA levels14.\n\nThe risk of intra-operative and post-operative neurological and the vascular complications increases with the grade of the lesions. The size, vascularity and thereby intra-operative blood loss is minimized with the use of embolisation15. However, there is an inadvertent risk of stroke and increased difficulty in excising the tumor, when it is glued with embolized particles intra-operatively16.\n\nThe first successful excision of a carotid body tumor was performed by Albert J Van Der Kogel in 188917. Surgical excision is the management modality of choice, with embolisation and radiotherapy used as adjuncts in a few selected cases10. Cerebral protection from hypotensive insults during vascular clamping or bypass and management of arrhythmias (following vagal nerve stimulation) during tumor manipulation are some of the challenges faced during the intra-operative period18,19. Surgery may be carried out under local, regional or general anesthesia. Local or cervical plexus block offers the advantage of a continuous neurological assessment to rule out cerebral hypo-perfusion20,21. Blood loss can be minimized with meticulous sub-adventitial dissection of the lesion from the vessel walls. Intra-operative vascular control can be achieved by temporary carotid ligation, use of temporary arterial bypass or by use of vascular inter-position graft in cases of inadvertent tear within the vessels22. Hypotensive anesthesia is another valid option to maintain a bloodless field23. A temporary clamp of less than 10 minutes is considered safe13. The IJV, vagus nerve and hypoglossal nerve should always be isolated and well protected throughout the procedure. The higher the grade of the lesion and the need for repair or reconstruction, the higher the risk of stroke24,25.\n\nHuge carotid body paragangliomas continue to cause a high incidence of pre- and postoperative complications17,26, including peri-operative stroke and persistent nerve palsy27.\n\nCranial nerve palsy is seen in as high as 40% of cases28. The hypoglossal nerve is the most common nerve to be affected in the post-operative period, thereby undue stretching should be avoided. Mortality has been seen in 3% of cases, with tumors greater than 5 cm29. In cases of transmural involvement of the vessel by the tumor, excision of the main vessel along with the tumor is justified. Internal carotid artery ligation with reconstruction requirements as well as permanent cranial nerve deficits have been observed in 23% of cases, all belonging to Shambling grade 3 tumors30.\n\nA characteristic Zellballen pattern in the histology and positive staining with neuron-specific enolase (NSE) for Paraganglioma and S100 for sustentacular cells is definitive in diagnosing the tumor31.\n\nThere are no defined pathological criteria to differentiate benign tumors from their malignant counterpart. Distant metastasis and the involvement of the lymph nodes are the only definitive markers to determine its malignant behavior32.\n\nMalignant transformation and local or distant metastasis is estimated to affect around 10% of cases33. Follow-up with doppler and duplex ultrasound of the carotid vessels is currently advocated17.\n\nLifelong follow-ups are required34. Succinate dehydrogenase (SDH) mutations are commonly seen in young patients, and testing for these may provide us with clues when assessing patients at high risk35.\n\n\nConclusions\n\nLarge and complicated vascular tumors can be managed with proper planning and execution36. With judicious anesthetic care and meticulous subadventitial dissection, such lesions can be managed even in a rural setup37.\n\n\nConsent\n\nWritten informed consent for publication of clinical data 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\nSpinelli F, Massara M, La Spada M, et al.: A simple technique to achieve bloodless excision of carotid body tumors. J Vasc Surg. 2014; 59(5): 1462–1464. PubMed Abstract | Publisher Full Text\n\nHinojosa CA, Ortiz-Lopez LJ, Anaya-Ayala JE, et al.: Comparison of retrocarotid and caudocranial dissection techniques for the surgical treatment of carotid body tumors. J Vasc Surg. 2015; 62(4): 958–964. PubMed Abstract | Publisher Full Text\n\nVon Haller Cited by Gratiot JH. Carotid tumors: A collective review. Abstr Surg. 1943; 7: 117–186.\n\nMathew BK, Bandgar T, Menon PS, et al.: Carotid body tumours: three case reports. Singapore Med J. 2009; 50(2): e58–e60. PubMed Abstract\n\nYildiz BS, Sasmazel A, Baysal A, et al.: Assessment of carotid body tumor and its association with tetralogy of fallot: effect of the chronic hypoxia. Heart Views. 2014; 15(3): 86–88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPadberg FT Jr, Cady B, Persson AV: Carotid body tumor: The Lahey Clinic experience. Am J Surg. 1983; 145(4): 526–528. PubMed Abstract | Publisher Full Text\n\nWright DJ, Pandya A, Noel F: Anesthesia for carotid body tumour resection. Anaesthesia. 1979; 34: 806–808. Publisher Full Text\n\nKnight TT Jr, Gonzalez JA, Rary JM, et al.: Current concepts for the surgical management of carotid body tumor. Am J Surg. 2006; 191(1): 104–10. PubMed Abstract | Publisher Full Text\n\nWang SH, Chiu KM, Cheng PW: Bilateral carotid body paragangliomas. CMAJ. 2011; 183(9): E606. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTong Y: Role of duplex ultrasound in the diagnosis and assessment of carotid body tumour: A literature review. Intractable Rare Dis Res. 2012; 1(3): 129–133. PubMed Abstract | Free Full Text\n\nShamblin WR, ReMine WH, Sheps SG, et al.: Carotid body tumor (chemodectoma). Clinicopathologic analysis of ninety cases. Am J Surg. 1971; 122(6): 732–39. PubMed Abstract | Publisher Full Text\n\nStaats EF, Brown RL, Smith RR: Carotid body tumors, benign and malignant. Laryngoscope. 1966; 76(5): 907–916. Publisher Full Text\n\nPatetsios P, Gable DR, Garrett WV, et al.: Management of carotid body paragangliomas and review of a 30-year experience. Ann Vasc Surg. 2002; 16(3): 331–8. PubMed Abstract | Publisher Full Text\n\nPellitteri PK, Rinaldo A, Myssiorek D, et al.: Paragangliomas of the head and neck. Oral Oncol. 2004; 40(6): 563–75. PubMed Abstract | Publisher Full Text\n\nThakkar R, Qazi U, Kim Y, et al.: Technique and Role of Embolization using Ethylene Vinyl-Alcohol Copolymer before Carotid Body Tumor Resection. Clinics and Practice. 2014; 4(3): 661. Publisher Full Text\n\nKrishnamoorthy T, Gupta AK, Rajan JE, et al.: Stroke from delayed embolization of polymerized glue following percutaneous direct injection of a carotid body tumor. Korean J Radiol. 2007; 8(3): 249–253. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSajid MS, Hamilton G, Baker DM, et al.: A multicenter review of carotid body tumour management. Eur J Vasc Endovasc Surg. 2007; 34(2): 127–130. PubMed Abstract | Publisher Full Text\n\nKarigar SL, Kunakeri S, Shetti AN: Anesthetic management of carotid body tumor excision: A case report and brief review. Anesth Essays Res. 2014; 8(2): 259–262. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKavakli AS, Ozturk NK: Anesthetic approaches in carotid body tumor surgery. North Clin Istanb. 2016; 3(2): 97–103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nToktaş F, Yümün G, Gücü A, et al.: Protective Surgical Procedures for Carotid Body Tumors:A Case Series. Erciyes Med J. 2014; 36(3): 133–5. Publisher Full Text\n\nJones HG, Stoneham MD: Continuous cervical plexus block for carotid body tumour excision in a patient with Eisenmenger's syndrome. Anaesthesia. 2006; 61(12): 1214–8. PubMed Abstract | Publisher Full Text\n\nZeng G, Zhao J, Ma Y, et al.: Use of an intraoperative shunt for easy resection of complicated carotid body tumors. Head Neck. 2013; 35(1): 61–4. PubMed Abstract | Publisher Full Text\n\nO’Neill S, O'Donnell M, Harkin D, et al.: A 22-year Northern Irish experience of carotid body tumours. Ulster Med J. 2011; 80(3): 133–140. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGwon JG, Kwon TW, Kim H, et al.: Risk factors for stroke during surgery for carotid body tumors. World J Surg. 2011; 35(9): 2154–8. PubMed Abstract | Publisher Full Text\n\nKakkos SK, Reddy DJ, Shepard AD, et al.: Contemporary presentation and evolution of management of neck paragangliomas. J Vasc Surg. 2009; 49(6): 1365–73.e2. PubMed Abstract | Publisher Full Text\n\nElsharawy MA, Alsaif H, Elsaid A, et al.: Management of sizeable carotid body tumor: Case report and review of literature. Avicenna J Med. 2013; 3(4): 106–108. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSen I, Stephen E, Malepathi K, et al.: Neurological complications in carotid body tumors: a 6-year single-center experience. J Vasc Surg. 2013; 57(2 Suppl): 64–8S. PubMed Abstract | Publisher Full Text\n\nPor YC, Lim DT, Teoh MK, et al.: Surgical management and outcome of carotid body tumours. Ann Acad Med Singapore. 2002; 31(2): 141–4. PubMed Abstract\n\nLuna-Ortiz K, Rascon-Ortiz M, Villavicencio-Valencia V, et al.: Does Shamblin's classification predict postoperative morbidity in carotid body tumors? A proposal to modify Shamblin's classification. Eur Arch Otorhinolaryngol. 2006; 263(2): 171–175. PubMed Abstract | Publisher Full Text\n\nLim JY, Kim J, Kim SH, et al.: Surgical treatment of carotid body paragangliomas: outcomes and complications according to the shamblin classification. Clin Exp Otorhinolaryngol. 2010; 3(2): 91–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWieneke JA, Smith A: Paraganglioma: carotid body tumor. Head Neck Pathol. 2009; 3(4): 303–306. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMendenhall W, Werning J, Peister D: Treatment of head and neck cancer (Paragangliomas). In: Devia V, Lawrence T, Rosenberg S, eds. Cancer: Principles and Practice of Oncology, 9th ed. Philadelphia: Lippincott, 2011; 722–3.\n\nWang SJ, Wang MB, Barauskas TM, et al.: Surgical management of carotid body tumors. Otolaryngol Head Neck Surg. 2000; 123(3): 202–206. PubMed Abstract | Publisher Full Text\n\nKollert M, Minovi AA, Draf W, et al.: Cervical paragangliomas-tumor control and long-term functional results after surgery. Skull Base. 2006; 16(4): 185–191. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavila VJ, Chang JM, Stone WM, et al.: Current surgical management of carotid body tumors. J Vasc Surg. 2016; 64(6): 1703–1710. PubMed Abstract | Publisher Full Text\n\nGalyfos G, Stamatatos I, Kerasidis S, et al.: Multidisciplinary Management of Carotid Body Tumors in a Tertiary Urban Institution. Int J Vasc Med. 2015; 2015: 969372. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWani B, Agni N, Rathod V, et al.: Rural centre based management of the carotid body tumour. Indian J Otolaryngol Head Neck Surg. 2011; 63(Suppl 1): 107–109. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "26639",
"date": "13 Nov 2017",
"name": "Arif Hussian Sarmast",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a very beautifully written case report, however I would like to clarify from the authors how a giant carotid body tumour is to be addressed in a normal setting, resource limitation does not change the management in a big chunk of cases like that. It needs a change in headline.\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": "27906",
"date": "24 Nov 2017",
"name": "Yi Guo",
"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 case report about how to manage a Shamblin Grade 3 carotid body tumor (CBT). Though surgery is first-line treatment of CBT, it remains a challenge because it’s highly vascular and adherent to the vagus nerve in high-grade CBT. The authors discussed the diagnosis and treatment of CBT in detail. In large tumors like this case, cerebral collateral circulation should be evaluated in case of vessel transplantation or ligation performed. HyperForm might be used for intra-operative distal vascular control if tumor is very large to reach the skull base. However, in a resource-limited setting it is difficult for detailed pre-operative evaluation and intra-operative vascular control.\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": "28506",
"date": "01 Dec 2017",
"name": "Luis Rafael Moscote-Salazar",
"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\nParagangliomas are a type of infrequent head and neck tumor originating in the paraganglionic brachymeric tissue involved in the development of the branchial arches. Five families of paragangliomas have been described:\n\nBrachymeric, intravagal, sympathetic, viscero-autonomic and adrenal. At the histopathological level, this type of tumors has an identical configuration regardless of its origin. Sporadic cases of this class of tumors are more frequent in females. A high frequency of this class of tumors has been established in patients who live at high altitudes and those suffering from the chronic obstructive disease. It has been suggested that chronic hypoxemia plays a role in the genesis of this class of tumors\n\nMunakomi et al. presented a very well documented case and with successful resolution managed in a resource-limited setting.\n\nThe therapeutic choice depends fundamentally on the location of this kind of tumors, the size of the lesion, age, health status of the patient plus the experience of the surgeons. The use of various resources as angiography and preoperative embolization suggests that this type of tumors should be ideally managed in third-level care centers. The case managed by Munakomi et al. testifies that the preparation and expertise of the surgeon are fundamental for the successful resolution of this class of injuries.\n\nFinally, it is important to mention that this type of tumors has a significant risk of urinary complications and several postoperative complications have been described, including cranial nerve lesions, cerebral infarction, hemorrhages among others.\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": "28491",
"date": "11 Dec 2017",
"name": "Guru Dutta Satyarthee",
"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\nParagangliomas are neoplasms arising from extra-adrenal chromaffin tissue. It frequently cause symptoms by overproduction of catecholamines and carry risk of hypertension during surgery and post –excision, patient may develop hemodynamic insatiability [1]. Carotid body tumor is a slow-growing neoplasm, usually localized at the bifurcation of the common carotid artery represent most commonly encountered are head and neck paragangliomas, accounting for 60–78% [2]. It is usually benign and rarely malignant and accounts for about 0.5% of all head and neck tumors [2].\nIn 1971, Shamblin et al. classified carotid body tumor into three classes: in Class I tumor is small and surgically resectable, Class II includes adherent tumor, which surrounding the carotid vessels partially, resection is considered as technically more difficult, and class III, the size of the tumor is large ie more than 5 cm and usually completely encases the carotid vessels, resection is considered very difficult, and always carry risk of the cerebral circulation interruption always [3].\nDSA is considered as important imaging modality of choice for diagnosis as clearly defines, epicentre of tumor, location, size and encasement of vessel branches in tumor and aid in aiding transarterial embolization therapy which reduces intraoperative blood loss. However, it carry limitation poor visualization of adherence of the tumor mass to the peripheral tissues, cerebral embolism causing ischemic stroke and vascular injury are also reported and even risk cardiac asystole with carotid body hypersensitivity. However DSA of cerebral vessels including cross circulation is essential if needed of cerebral vessel revascularization surgery is required [4]. So, currently, computed tomography scan, CT angiography and MRI are preffered routinely .\n\nSurgery is preferred treatment modality and chief concern s include highly vascular mass, dense adherence to the vagus nerve. Hua et al observed microneurosurgical technique is said in dissection. And prevent vascular and cranial nerve injury [1]. Radiotherapy is an alternative management option for patients with co-morbid illness or who may not tolerate the surgery, presence of metastasis subsequent to surgery complications include otitis externa, otitis media, osteoradionecrosis, cranial nerve lesion and brain injury [5]. Munakomi and neuroanesthesia team should be commended for successful surgery, perioperative hemodynamic adequate control with good 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": []
}
] | 1
|
https://f1000research.com/articles/6-1801
|
https://f1000research.com/articles/6-1798/v1
|
03 Oct 17
|
{
"type": "Research Article",
"title": "Identification of high-risk patients for early death or unplanned readmission using the LACE index in an older Portuguese population",
"authors": [
"João Fonseca",
"Flávio Costa",
"José Mateus",
"Diana Ferreira",
"Hugo Clemente",
"Manuel Veríssimo",
"Armando Caravalho",
"Flávio Costa",
"José Mateus",
"Diana Ferreira",
"Hugo Clemente",
"Manuel Veríssimo",
"Armando Caravalho"
],
"abstract": "Background: Unplanned readmissions are frequent, associated with high costs and potentially preventable. Pre-discharge risk screening is a crucial step to prevent hospital readmissions. This study evaluates the LACE index as a tool capable of identifying patients with high risk of early readmission or death in an older Portuguese population. Methods: We performed a retrospective study in a tertiary care hospital in Portugal. All acute patients, aged ≥ 65 years, discharged from the Internal Medicine Service between 1 January and 30 June 2014 were included. Data was collected from hospital records. The LACE index was calculated for each patient. A comparative analysis was performed based on a cutoff of 10 (≥10 indicates a high-risk population) for the LACE score. Results: 1407 patients were evaluated, with a mean age of 81.7±7.6 years; 41.2% were male, 52.2% were dependent for ≥1 activities of daily living, the average Charlson comorbidity index was 3.54±2.8. There were 236 (16.8%) readmissions, 132 (9.4%) deaths and 307 (21.8%) patients were dead and/or readmitted within 30 days of discharge. At 90 days, 523 (37.2%) patients were dead and/or readmitted. The LACE score was higher in patients who died or were readmitted within 30 days compared with those who were not (13.2±2.7 versus 11.5±3.0, p <0.0001). Patients with LACE score ≥10 had significantly higher mortality and readmission rates compared to those with LACE score <10: at 30 days, 25.5% versus 9.3% (OR 3.34, 95% CI 2.24-4.98, p <0.0001); at 90 days, 43.4% versus 16.2% (OR 3.98, 95% CI 2.89-5.49, p <0.0001). However, the discriminative capacity of LACE index assessed by C-statistic was relatively poor: 0.663 (95% CI 0.630-0.696) and 0.676 (95% CI 0.648-0.704), respectively. Conclusions: This study shows that the LACE index should be used with reservations for predicting 30 and 90-day readmission or death in complex elderly patients.",
"keywords": [
"hospital readmissions",
"elderly",
"LACE index"
],
"content": "Introduction\n\nUnplanned hospital readmissions are frequent1,2, particularly in elderly patients with comorbidities3,4. These are associated with high costs and a poor global prognosis1–3. Portugal is no different from other Western countries, in which there is a rapid ageing of the population. As a consequence, the number of complex elderly patients with multiple diseases requiring hospitalisation has increased in the last two decades, similarly to the readmission rate5.\n\nThe problem of hospital readmissions has gained great interest from the medical community and the governments of most developed countries1,2. Several studies suggest that rehospitalizations are potentially preventable6,7, and the ratio of readmissions is now seen as a quality benchmark for health care systems8–11. There have been a large number of publications in recent years on this subject, and some countries have made efforts to cultivate programs to reduce the impact of this major problem2,12,13.\n\nA pre-discharge risk screening is the first crucial step in any model that attempts to prevent unplanned hospital readmissions. Several readmission screening tools have been created14,15, such as the LACE index16, PARR-3017, 8Ps18, and HOSPITAL score19. However, none prove to be completely adequate14,15.\n\nThe LACE index was first presented in 2010, by Van Walraven et al. This is a simple, quick and inexpensive tool that includes length of stay, acuity of the admission (emergency), comorbidities (measured with the Charlson comorbidity index [CCI]) and previous emergency department visits before readmission. It was designed to predict early death or unplanned readmission after discharge from hospital to the home setting16 (Table 1). However, subsequent studies have failed to obtain consistent results20–24.\n\nNote: The LACE score is calculated by summing the points attributed to the 4 variables.\n\nThe original LACE index was based on a middle-aged population living in Canada, with good functional status and few comorbidities16. This population is not representative of the kind of patient usually observed in acute care facilities in Portugal. Further research is essential to clarify whether the LACE index is a useful tool in predicting the risk of readmission in elderly patients with multiple diseases.\n\nThe aim of this study was to assess the ability of the LACE index to predict the risk of readmission or death within 30 and 90 days after discharge in elderly patients at a Portuguese tertiary hospital.\n\n\nMethods\n\nWe performed a retrospective study in Coimbra Hospital and Universitary Centre, a tertiary care hospital in Portugal. The study included all acute patients aged ≥ 65 years, who were discharged from the Internal Medicine Service between 1 January and 30 June 2014 (n=1619). The first hospitalisation during the study period was established as the index admission, and we counted no more than one readmission for each patient. Exclusion criteria were planned admission, discharge to rehabilitation or continuing care facility, transfer to another acute care hospital, leaving the hospital against medical advice, or death. A total of 1407 patients were eligible for analysis (Figure 1).\n\nMedical and demographic data were collected from hospital records, including age, gender, residence, functional status, comorbidities (CCI), reason for admission, medications, length of stay and healthcare utilisation in the previous 6 months (emergency department visits and number of hospital admissions). Readmissions and mortality at 30 and 90 days since the indexed admission were also obtained.\n\nThe LACE index was calculated for each patient. The LACE score ranges from 0 to 19 - higher scores are related to increased risk of readmission or death after discharge16. Previous investigations have failed to reach an optimal cut-off for defining high-risk populations. Therefore, we decided to establish 10 as the cutoff, considering that it has already been used in some publications20–22. For a more accurate interpretation of the data obtained, we also found it useful to divide patients into three risk groups: low (0–7), moderate (8–13), and high (≥14) risk.\n\nWe compared the characteristics of patients with or without 30-day readmission or death using Chi-square for categorical variables and Student’s t-test for continuous variables. The area under the receiver operating characteristic (ROC) curve C-statistic (ROC area) was estimated to assess the discriminating ability of the LACE index to predict: (1) readmission, (2) death, and (3) readmission or death within 30 and 90 days. Statistical significance was set at p <0.05. Descriptive and statistical analysis were performed in SPSS v.23.0 (IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard deviation.\n\n\nResults\n\nA total of 1407 patients were evaluated. Their demographic and clinical baseline characteristics are shown in Table 2, as well as the comparison between patients with and without readmission or death after 30 days of discharge. The age of patients ranged from 65 to 104 years (mean 81.7±7.6); 41.2% were male, 52.2% were dependent on one or more activities of daily living (ADL), the average CCI score was 3.54±2.8, and 23.2% of patients had a hospital admission in the previous 6 months. Admissions were predominantly emergencies (99.6%), and the most common reasons included pneumonia, urinary tract infection, chronic obstructive pulmonary disease, and heart failure. The patients were hospitalised for 9.5±8.9 days, discharged with 7.53±3.4 different medications, and the average LACE index score was 11.8±3.0. The median time to complete the LACE index was 44 seconds for each patient.\n\nIn the overall cohort, there were 236 (16.8%) readmissions, 132 (9.4%) deaths and 307 (21.8%) patients were dead and/or readmitted within 30 days of discharge. At 90 days there were 404 (28.7%) readmissions, 255 (18.1%) deaths, and 523 (37.2%) patients were dead and/or readmitted (Table 3).\n\nThe group of patients with 30-day events (death or readmission) were slightly older and had a higher percentage of men compared to non-event patients (Table 2). In the comparison of these two groups, dependence for one or more ADL, CCI, hospital admission in the previous 6 months, length of initial hospital stay, and number of medications at discharge showed statistically significant differences (Table 2). The LACE score was also significantly higher in patients with death or readmission within 30 days compared with non-event patients (13.2±2.7 versus 11.5±3.0, p <0.0001).\n\nLACE scores ranged between 4 and 19. There were no patients with a LACE score of ≤3. As seen in Figure 2 and Figure 3, those with a higher LACE score presented with an increased risk of readmission or death. The 30-day death or readmission ranged from 0.0% for a LACE score of 4–5 to 30.0–44.4% for a LACE score of 18–19. The same pattern was found at 90 days, with the risk of death or readmission ranging between 0.0% for a LACE score of 4, and 60.0–67.7% for a LACE score of 17–19.\n\nPatients with a LACE score ≥10 represented 77.1% (1085) of the sample. In this group, we observed significantly higher mortality and readmission rates compared to patients with LACE score <10 (Table 3): 30-day readmission, 19.8% versus 6.5% (OR 3.54, 95% CI 2.22-5.65, p <0.0001); 30-day death, 11.1% versus 3.7% (OR 3.21, 95% CI 1.75-5.89, p <0.0001); 30-day death or readmission, 25.5% versus 9.3% (OR 3.34, 95% CI 2.24-4.98, p <0.0001); 90-day readmission, 33.7% versus 11.8% (OR 3.80, 95% CI 2.65-5.46, p <0.0001); 90-day death, 21.5% versus 6.8% (OR 3.73, 95% CI 2.36-5.89, p <0.0001); 90-day death or readmission, 43.4% versus 16.2% (OR 3.98, 95% CI 2.89-5.49, p <0.0001).\n\nThe ROC curves of the LACE index as a predictor of readmission, death, and readmission or death within 30-days are displayed in Figure 4. The C-statistics associated with the LACE index were 0.652 (95% CI 0.615-0.689), 0.672 (95% CI 0.625-0.718), and 0.663 (95% CI 0.630-0.696), respectively (Table 3). However, the highest C-statistic achieved was relative to mortality at 90 days (0.678, 95% CI 0.644-0.713). The discriminative capacity of the LACE index as assessed by C-statistic was relatively poor in all outcomes evaluated, and tended to be a better predictor for evaluating death than for readmissions. These results are consistent with those found in other works21,22.\n\nNote: ROC curves for the LACE index as a predictor of readmission (left), death (middle) and readmission or death (right) within 30 days.\n\nWe performed an additional descriptive analysis, dividing patients into three risk groups: 122 patients (8.7%) were at low risk (LACE 0-7); 873 (62.0%) were at moderate risk (LACE 8-13), and 412 (29.3%) were at high risk (LACE ≥14). As we see in Figure 5, in the high-risk group, a third (33.7%) of patients were readmitted or died 30 days after discharge, and more than a half (54%) had an event at 90 days. In contrast, individuals defined as low and moderate risk had a much lower event rate (5.7% and 18.4% at 30 days, 13.1% and 32.8% at 90 days, respectively).\n\n\nDiscussion\n\nIt is now clear that unplanned readmissions are associated with high costs and avoidable health risks for patients1,2. This major problem is even greater in countries such as Portugal, where most of patients are elderly with complex and multiple comorbidities. Available data confirm that this is an increasing problem in Portuguese hospitals5, but the annual cost of readmissions is unknown.\n\nIn the US, about 17% (US$17.4 billion) of total hospital payments by Medicare in 2004 were related to readmissions1. Previous studies have shown that a significant portion of early readmissions can be prevented6,7. The implementation of the Affordable Care Act's Hospital Readmission and Reduction Program reinforced this idea. This program, which economically penalises US hospitals with high readmission rates, is associated with a significant decline in the number of rehospitalizations13.\n\nThe development of readmission prevention programs should be a priority for hospital and health system leaders. Unplanned hospital readmissions and early death after discharge can be taken as markers of health care quality8–10. However, considering these events as a direct consequence of inadequate care or premature discharge is simplistic and reductive11,12. Hospital readmissions are still difficult to predict, resulting from the complex relationship between multiple factors.\n\nPrevention strategies and the implementation of a post-discharge plan requires the identification of individuals at a high risk of readmission, which depends on prediction tools12. There are not predictors of hospital readmission validated for the Portuguese population. For this study, the LACE index was chosen as it is a simple, quick and easy instrument created to predict the risk of readmission and early death at 30 days16. The predictive capability of LACE has shown tremendous inconsistency in different populations20–24. However, attempts to improve the LACE index through the incorporation of other variables have resulted in more complex and time-consuming models without showing any significant advantage24–27.\n\nThis single-center retrospective study was based on a population of elderly people with significant comorbidity, polypharmacy, and usually compromised functional status. These baseline characteristics are representative of the general reality of the Portuguese Internal Medicine wards. This is in contrast to the much younger, independent and “relatively healthy” Canadian population used to derive the original LACE index16.\n\nAs expected, the hospital readmission rate and mortality were elevated, with 21.8% of patients experiencing readmission or death at 30 days, and 37.2% at 90 days after discharge. Most patients had a high LACE score (77.1% had LACE score ≥10), due to high CCI, prolonged hospitalisations, and almost all admissions were emergencies.\n\nIn the study population, those with a LACE score ≥10 had a 3-fold increased risk of being readmitted or dead at 30 days, and this risk was even higher at 90 days - almost 4 times compared to the group of patients with a LACE score <10. However, the LACE index had a relative poor discriminative ability in predicting 30-day readmissions alone (c-statistic 0.652), and only a slightly better performance in 30-day mortality prediction (c-statistic 0.672). A similar pattern was obtained at 90 days. This performance was poorer than the findings in the original study by Van Walraven et al.16, but very consistent with other studies conducted in similar populations (UK21 and Singapore22).\n\nDespite these results, we found it interesting to classify patients into three risk groups. With this approach, we obtained interesting results. In the high-risk group (LACE score 14–19), more than half of the patients were readmitted or died at 90 days; therefore, making this a hypothetical perfect target to integrate a structured post-discharge preventive plan. This strategy may be more advantageous in clinical practice than defining low- and high-risk patients based on a single cutoff. Further research is required to determine if a specific post-discharge intervention in this group (LACE score 14–19) may decrease the number of early deaths and readmissions.\n\nOur study has some limitations. First, this single-center study was based on a relatively small sample of patients. Second, as a retrospective study, only the variables usually collected were evaluated. Characteristics such as economic status and frailty were not assessed. Third, the causes of unplanned readmission and death were not analysed. Fourth, patients readmitted in hospitals other than ours were not included. This may have led to an underestimated readmission rate in this study. Fifth, the outcomes related to 90 days after discharge were never validated and were mentioned in a very few studies, which limits the comparative analysis of our findings. The same is true for the risk stratification in three groups.\n\n\nConclusions\n\nThis study shows that the LACE index should be used with reservations for predicting 30 and 90-day readmission or death in complex elderly patients. Further research is needed to determine an effective way to stratify patients at risk of readmission according to the LACE index and to clarify the real impact of post-discharge intervention in these patients.\n\n\nData availability\n\nDataset 1: Demographic and clinical data for all 1407 patients. The data file contains the main medical and demographic variables collected for all 1407 patients, including: age, gender, residence, functional status, number of medications at discharge, length of stay, CCI score, hospital admission in the previous 6 months, LACE index score, readmissions and mortality at 30/90 days, and risk group categorization. doi, 10.5256/f1000research.11315.d17675528\n\n\nEthical approval\n\nThis study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Ethics Committee of Coimbra Hospital and Universitary Centre. The need for written informed consent from the participants was waived by the committee due to the retrospective nature of the study based only on data from medical records.",
"appendix": "Author contributions\n\n\n\nAll authors have contributed equally to the preparation of the paper and study design; JF and FC collected the majority of data. MV and AC were responsible for the critical revision of the article and the final approval of the version to be published.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nJencks SF, Williams MV, Coleman EA: Rehospitalizations among patients in the Medicare fee-for-service program. N Engl J Med. 2009; 360(14): 1418–28. PubMed Abstract | Publisher Full Text\n\nJoynt KE, Jha AK: Thirty-day readmissions--truth and consequences. N Engl J Med. 2012; 366(15): 1366–9. PubMed Abstract | Publisher Full Text\n\nGooding J, Jette AM: Hospital readmissions among the elderly. J Am Geriatr Soc. 1985; 33(9): 595–601. PubMed Abstract | Publisher Full Text\n\nRaval AD, Zhou S, Wei W, et al.: 30-Day Readmission Among Elderly Medicare Beneficiaries with Type 2 Diabetes. Popul Health Manag. 2015; 18(4): 256–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSousa-Pinto B, Gomes AR, Oliveira A, et al.: [Hospital readmissions in Portugal over the last decade]. Acta Med Port. 2013; 26(6): 711–20. PubMed Abstract\n\nJack BW, Chetty VK, Anthony D, et al.: A reengineered hospital discharge program to decrease rehospitalization: a randomized trial. Ann Intern Med. 2009; 150(3): 178–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Walraven C, Bennett C, Jennings A, et al.: Proportion of hospital readmissions deemed avoidable: a systematic review. CMAJ. 2011; 183(7): E391–402. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAshton CM, Kuykendall DH, Johnson ML, et al.: The association between the quality of inpatient care and early readmission. Ann Intern Med. 1995; 122(6): 415–21. PubMed Abstract | Publisher Full Text\n\nBalla U, Malnick S, Schattner A: Early readmissions to the department of medicine as a screening tool for monitoring quality of care problems. Medicine (Baltimore). 2008; 87(5): 294–300. PubMed Abstract | Publisher Full Text\n\nStefan MS, Pekow PS, Nsa W, et al.: Hospital performance measures and 30-day readmission rates. J Gen Intern Med. 2013; 28(3): 377–85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWish JB: The role of 30-day readmission as a measure of quality. Clin J Am Soc Nephrol. 2014; 9(3): 440–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKripalani S, Theobald CN, Anctil B, et al.: Reducing hospital readmission rates: current strategies and future directions. Annu Rev Med. 2014; 65: 471–485. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZuckerman RB, Sheingold SH, Orav EJ, et al.: Readmissions, Observation, and the Hospital Readmissions Reduction Program. N Engl J Med. 2016; 374(16): 1543–51. PubMed Abstract | Publisher Full Text\n\nKansagara D, Englander H, Salanitro A, et al.: Risk prediction models for hospital readmission: a systematic review. JAMA. 2011; 306(15): 1688–98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou H, Della PR, Roberts P, et al.: Utility of models to predict 28-day or 30-day unplanned hospital readmissions: an updated systematic review. BMJ Open. 2016; 6(6): e011060. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Walraven C, Dhalla IA, Bell C, et al.: Derivation and validation of an index to predict early death or unplanned readmission after discharge from hospital to the community. CMAJ. 2010; 182(6): 551–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBillings J, Blunt I, Steventon A, et al.: Development of a predictive model to identify inpatients at risk of re-admission within 30 days of discharge (PARR-30). BMJ Open. 2012; 2: pii: e001667. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim CS, Flanders SA: In the Clinic. Transitions of care. Ann Intern Med. 2013; 158(5 Pt 1): ITC3–1. PubMed Abstract | Publisher Full Text\n\nDonzé JD, Williams MV, Robinson EJ, et al.: International Validity of the HOSPITAL Score to Predict 30-Day Potentially Avoidable Hospital Readmissions. JAMA Intern Med. 2016; 176(4): 496–502. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGruneir A, Dhalla IA, van Walraven C, et al.: Unplanned readmissions after hospital discharge among patients identified as being at high risk for readmission using a validated predictive algorithm. Open Med. 2011; 5(2): e104–e111. PubMed Abstract | Free Full Text\n\nCotter PE, Bhalla VK, Wallis SJ, et al.: Predicting readmissions: poor performance of the LACE index in an older UK population. Age Ageing. 2012; 41(6): 784–9. PubMed Abstract | Publisher Full Text\n\nTan SY, Low LL, Yang Y, et al.: Applicability of a previously validated readmission predictive index in medical patients in Singapore: a retrospective study. BMC Health Serv Res. 2013; 13: 366. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang H, Robinson RD, Johnson C, et al.: Using the LACE index to predict hospital readmissions in congestive heart failure patients. BMC Cardiovasc Disord. 2014; 14(1): 97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLow LL, Lee KH, Hock Ong ME, et al.: Predicting 30-Day Readmissions: Performance of the LACE Index Compared with a Regression Model among General Medicine Patients in Singapore. Biomed Res Int. 2015; 2015: 169870. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Walraven C, Wong J, Forster AJ: LACE+ index: extension of a validated index to predict early death or urgent readmission after hospital discharge using administrative data. Open Med. 2012; 6(3): e80–90. PubMed Abstract | Free Full Text\n\nSpiva L, Hand M, VanBrackle L, et al.: Validation of a Predictive Model to Identify Patients at High Risk for Hospital Readmission. J Healthc Qual. 2016; 38(1): 34–41. PubMed Abstract | Publisher Full Text\n\nLow LL, Liu N, Wang S, et al.: Predicting 30-Day Readmissions in an Asian Population: Building a Predictive Model by Incorporating Markers of Hospitalization Severity. Steyerberg EW ed. PLoS One. 2016; 11(12): e0167413. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFonseca J, Costa F, Mateus J, et al.: Dataset 1 in: Identification of high-risk patients for early death or unplanned readmission using the LACE index in an older Portuguese population. F1000Research. 2017. Data Source"
}
|
[
{
"id": "26621",
"date": "09 Oct 2017",
"name": "Nan Liu",
"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 the opportunity to review. In this manuscript, the authors validated the LACE index on identifying high risk elderly patients in a selected Portuguese population. 1407 patients were retrospectively recruited to evaluate the performance of LACE on its associations with the risk of readmission or death in 30 days and 90 days. The results were consistent with several similar international studies and the authors suggested that the LACE index should be used with reservations.\nThe study is well designed and the manuscript is clearly written. The methods are reported in a standard approach and the results are sufficient in supporting the conclusions. One suggestion: since 90-day outcome is studied, the authors are suggested to add in the results on 90-day readmission or death into Table 2 and Figure 4.\nIn Methods (study population subsection, first paragraph second sentence), the authors mentioned that they included all “acute” patients, so please define “acute” and explain why these patients from the Internal Medicine Service were studied (for example, are they are subset of all internal medical patients?). As shown in the results that “admissions were predominantly emergencies (99.6%)”, the authors need to clarify the actual inclusion criteria.\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": "40074",
"date": "05 Nov 2018",
"name": "Robert Robinson",
"expertise": [
"Reviewer Expertise Hospital readmissions"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 well written and explores the utility of the LACE index in a different setting than other published studies - A tertiary care center in Portugal.\nThe background is well written, citing relevant articles and explaining the limitations in the ability to identify patients at highest risk of hospital readmission with various systems. Differences in the study population and the derivation and validation studies for the LACE index is discussed. Consider including the references listed below in the introduction to include literature since the publication of the first version of this paper.\nMethods are conventional. Inclusion of a Brier score and a Hosmer-Lemeshow goodness of fit score for each of the three outcomes (mortality, 30 day readmission, and 90 day readmission) is essential for a better understanding of the significance of the AUC values for the ROC curves and will allow a more detailed understanding of the value of the scores in this population.\n\nThe Brier score will evaluate the accuracy of the LACE index in predicting outcomes in this study, and the Hosmer-Lemeshow test will evaluate test calibration in the patient population.\n\nThe full inclusion and exclusion criteria (including definitions) for this study should be described in detail, in addition to the figure showing the reasons for patient exclusion from analysis.\nThe percentages in axis labels for Figures 2 and 3 do not need numbers to the right of the decimal point (100% instead of 100.00%).\nDiscussion and conclusions are well written, framing the results of this study in the current knowledge at the time of initial release of this paper regarding the LACE index as a predictor of mortality and readmission. Additional publications on this topic since the release date would not change the conclusions drawn by the authors. The inclusion of the Brier score and Hosmer-Lemeshow test results in the discussion and contrasting the results against other studies would be appropriate.\nThese methodological concerns limit the rating of this interesting and potentially useful article to “Approved with Reservations.”\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": []
}
] | 1
|
https://f1000research.com/articles/6-1798
|
https://f1000research.com/articles/6-1795/v1
|
03 Oct 17
|
{
"type": "Software Tool Article",
"title": "Tools for annotation and comparison of structural variation",
"authors": [
"Fritz J. Sedlazeck",
"Andi Dhroso",
"Dale L. Bodian",
"Justin Paschall",
"Farrah Hermes",
"Justin M. Zook",
"Andi Dhroso",
"Dale L. Bodian",
"Justin Paschall",
"Farrah Hermes",
"Justin M. Zook"
],
"abstract": "The impact of structural variants (SVs) on a variety of organisms and diseases like cancer has become increasingly evident. Methods for SV detection when studying genomic differences across cells, individuals or populations are being actively developed. Currently, just a few methods are available to compare different SVs callsets, and no specialized methods are available to annotate SVs that account for the unique characteristics of these variant types. Here, we introduce SURVIVOR_ant, a tool that compares types and breakpoints for candidate SVs from different callsets and enables fast comparison of SVs to genomic features such as genes and repetitive regions, as well as to previously established SV datasets such as from the 1000 Genomes Project. As proof of concept we compared 16 SV callsets generated by different SV calling methods on a single genome, the Genome in a Bottle sample HG002 (Ashkenazi son), and annotated the SVs with gene annotations, 1000 Genomes Project SV calls, and four different types of repetitive regions. Computation time to annotate 134,528 SVs with 33,954 of annotations was 22 seconds on a laptop.",
"keywords": [
"structural variants",
"whole genome sequencing",
"bioinformatics",
"NGS",
"annotation"
],
"content": "Introduction\n\nThe advent of high throughput sequencing (HTS) facilitates the investigation of genomic differences among and within organisms, populations, and even diseases such as cancer. While the identification of single nucleotide polymorphisms (SNPs) is currently well established, structural variant (SV) calling remains challenging and little is known about the sensitivity (correctly inferring SVs) and false discovery rate (FDR) (falsely inferring SVs) of structural variation detection (Guan & Sung, 2016). Recent SV discovery methods, such as LUMPY (Layer et al., 2014) and PBHoney (English et al., 2014), focus on one callset per technology, but SV detection and call evaluation would benefit from comparison of the data from multiple technologies. However, many challenges exist in comparing and merging SV calls due to uncertainty in breakpoints, sequencing errors, and multiple possible representations of SVs in repetitive regions (Wittler et al., 2015). In addition, Sudmant et al. (Sudmant et al., 2015) as well as Jeffares et al. (Jeffares et al., 2017) mention that the methods often lack sensitivity and suffer from an inestimable FDR. Jeffares et al. coped with this problem by merging SV calls generated by multiple callers to reduce the FDR, but this approach also slightly reduced sensitivity (Jeffares et al., 2017).\n\nTo enable comparison and evaluation of SV callsets generated by different algorithms, we developed methods to compare and annotate SV calls, represented in variant call format (VCF), with other SVs as well as other genomic features. Genomic features can include gene annotations, mappability tracks, and any feature that can be represented as a region in BED or GFF format. SNPs can also be used for annotation by representing them as regions of 1bp.\n\nAs a proof of concept, we apply these novel methods to the Genome in a Bottle (GiaB) data generated on the Ashkenazi son (NIST Reference Material 8391, aka HG002 and NA24385) to explore SV type and breakpoint concordance of SV calling algorithms. GiaB provides SV calls generated using five different technologies (including Illumina short read sequencing, Complete Genomics nanoball sequencing, Pacific Biosciences long read sequencing, 10X Genomics linked reads, and BioNano optical mapping) and 16 different SV calling algorithms on the same genome. We used SURVIVOR (Jeffares et al., 2017) to merge SV calls and our novel method (SURVIVOR_ant) to annotate and predict more precise breakpoints. All data sets (including merged SV and annotated SV) and methods used in this manuscript are available at https://github.com/NCBI-Hackathons/svcompare.\n\n\nMethods\n\nSURVIVOR_ant. To enable annotation and comparison of the SV callsets, we implemented a new extension of SURVIVOR that aims to assign genomic features, including previously known/established SVs, to merged SV callsets produced by SURVIVOR. SURVIVOR_ant takes any VCF file (list of SVs) as an input (-i) as well as annotation sets specified as a list of BED files (--bed), GFF files (--gff) and additional VCF files (--vcf). Each of the three file types are optional and the user can specify multiple files for each type, separated by commas. SURVIVOR_ant reads in the original VCF file to be annotated and constructs a self-balancing interval tree originally taken from SURVIVOR (Jeffares et al., 2017). Next, it reads in any annotations in VCF files (e.g. from the 1000 Genomes Project) and compares these to the original VCF entries in the interval tree. The comparison is based on the individual breakpoints, given a maximum distance parameter (by default 1kb). Subsequently, SURVIVOR_ant runs through the BED files and GFF files and parses the provided intervals and identifiers. In the case of a GFF file, SURVIVOR parses the first name in the 9th column: gene=. For BED files, SURVIVOR_ant uses the fourth column as the name for each entry (or the file name, if the BED file does not include a four columns).\n\nEach entry of a BED or GFF file is assigned to deletions and duplications in the SURVIVOR_ant VCF if they overlap the SV +/- a user-defined distance/wobble parameter (by default 1kb). For translocations, insertions and inversions, SURVIVOR_ant only takes the breakpoints into account and assigns genomic features within a user-defined distance/wobble parameter (by default 1kb). Figure 1 shows the schematic based on three genes. The distance/wobble parameter is necessary to account for differences in accuracy of the technology, mapping, or the SV calling algorithm. Often breakpoints are positioned in repeated regions which makes it hard to place the breakpoints accurately.\n\nBy default SURVIVOR_ant takes 1kbp surrounding the start and stop coordinates into account. Furthermore, for deletions and duplications we take the overlapping regions into account.\n\nAfter all files are read in and compared to the original VCF file, SURVIVOR_ant prints the original VCF file and extends the INFO field with information on how many VCF files have supportive information (“overlapped_VCF=”) as well as how many genomic features within the VCF files could be assigned per original SV (“total_Annotations=”). If SURVIVOR_ant found overlapping genomic features the names associated to these are printed out in a comma separated list (“overlapped_Annotations=”). SURVIVOR_ant is maintained at https://github.com/NCBI-Hackathons/svcompare.\n\nSummary statistics scripts. These statistics were generated with code available at https://github.com/NCBI-Hackathons/svcompare, which analyzes the data as structured by data_structures.pl. The R script stats_plots_v2.R can be used to generate a variety of figures like those in this paper.\n\nSV analyses. Several statistics were computed by event, by call set, and by variant type:\n\n\n\n1. The number of callers supporting an event is the count of the number of callers for which SURVIVOR identified a variant call at the same location for an event, subject to the 1 kb wobble parameter and independent of the variant type.\n\n2. For the total number of variant calls per callset by variant type, the SVs of each type were counted separately. For events in which a single caller made more than one call (sub-calls), each sub-call is counted separately (e.g., if a callset has two different deletions that SURVIVOR merged into a single VCF row, it is counted as two deletions).\n\n3. For analysis of the SURVIVOR_ant annotations, the number of events with at least one annotation of the selected type were counted.\n\n4. Breakpoints were compared for events supported by at least four callers, and for which all calls were of the same type. The type was examined for all calls supporting that event, including multiple calls from any one caller. First, the median start and end positions for were calculated for all calls for that event. Second, the distance of each call’s start from the median start, and each call’s end from the median end were computed. For calls with multiple sub-calls from a single caller, the minimum start position and maximum end position were used as the start and end, respectively.\n\nSURVIVOR_ant is based on C++ and does not require any preinstalled packages or libraries. The analysis scripts are using BioPerl (http://bioperl.org/).\n\nGenomic data. We used 16 candidate-SV callsets from the GiaB Ashkenazi son data set available at https://github.com/genome-in-a-bottle. We reformatted the files if they did not correspond to the VCF 4.1 standard then merged the SVs in the 16 files into one multi-sample VCF using SURVIVOR (Jeffares et al., 2017) with 1 kb as the distance parameter and without requiring type specificity. Next we downloaded three BED files defining repetitive regions from the GA4GH Benchmarking Team at https://github.com/ga4gh/benchmarking-tools/tree/master/resources/stratification-bed-files. Furthermore, we downloaded the gene annotations for GRCh37 from ensembl. Population genomic data was downloaded for the 1000 Genomes Project from dbVar (estd219) (Sudmant et al., 2015) and filtered to produce a unique set of variant sites. SURVIVOR_ant (Version 0.0.1) was used to annotate the merged SVs with all the annotation data sets. The merged SVs are referred to as “events.”\n\n\nResults\n\nWe merged the output of 16 different callers containing variants >19 bp (Table 1) that were run on the Ashkenazi son data (GiaB) using SURVIVOR (Jeffares et al., 2017). The resulting VCF file contained 134,528 SV events and was annotated by our novel method SURVIVOR_ant. We annotated the SVs with genes from hg19 (GFF), the 1000 Genomes project (dbVar) population-based structural variant calls, and repetitive regions from the GA4GH Benchmarking Team (3 bed files). SURVIVOR_ant compared the five files to the merged SV calls for the Ashkenazi son data within 22 seconds. It identified 4,506 overlapping SVs between the 1000 Genomes Project and our data set (Table 2). Furthermore, SURVIVOR_ant identified genomic features in the 3 BED files and the GFF gene list overlapping 66,166 SVs out of the total merged 134,528 VCF entries.\n\nThe SURVIVOR_ant output is also useful for comparing the output of callers. Each caller assigns an SV type (e.g., insertion, deletion, translocation, etc.) and breakpoints for each SV call. Overall we identified 125,909 (93.6%) SVs that were supported by fewer than four callsets. Figure 2 depicts the widely varying number of candidate SV calls of different types across callsets, which contributes to the large fraction of calls that are supported by fewer than four callsets. Of 134,528 calls from the Ashkenazi son data from the callers in Table 1, 11,474 (8.5%) had more than one SV type discovered by different callers in the same region, and 6,280 (4.7%) had more than one SV type discovered by the same caller in the same region. It is possible either that these different types are due to errors in the calls or that there is a true complex SV consisting of multiple nearby SV types. In addition, duplications of a large region in tandem could be described as an insertion by some callers and as a duplication by other callers. These results illustrate the disagreement of multiple callers over the same data set, as well as the complexity of integrating calls from different methods.\n\nFor characterization of the consistency of breakpoint prediction of the different callers, we analyzed the 5,386 SV events with support from at least four callsets, and for which all calls are of the same type, so that a useful median start and end position could be calculated. Figure 3 depicts example histograms of distance to the median start position for two callsets, one from long reads and one from short reads. In general, more of the short read caller’s start positions are closer to the median breakpoint, but this could be due to a variety of factors, including lower error rates in short reads, easier less repetitive sites detected by short reads, filtering rules, etc. Note that since the number of callers per technology varies and calls supported by more callsets are likely to be easier to detect, this likely introduces a bias in the variants assessed. We calculated these statistics as an example of using our methods, not as a generalizable estimate of breakpoint accuracy.\n\nOnly sites with calls from at least 4 different callsets were included in order to calculate a useful median value at each site.\n\n\nConclusions and future work\n\nIn this paper, we introduced SURVIVOR_ant, an annotation and comparison tool especially designed for comparing SVs and genomic features (e.g. genes). SURVIVOR_ant is novel in that it enables a type-specific comparison to multiple genomic annotations and other features of interest. The resulting VCF file can be loaded in existing methods such as IGV or bedtools for further manual inspections. SURVIVOR_ant and all resources used here are available at https://github.com/NCBI-Hackathons/svcompare. This tool is an important first step to enable the comparison of SVs to each other, to known SVs, and to genomic features. Here, we defined genomic features as being information about the properties of the underlying genome sequence (e.g., repetitive regions), as well as annotations such as genes or even chromatin assays. Furthermore, we have made available scripts to calculate a variety of statistics that characterize the similarity and differences between many callsets from a single genome, including the number of callsets supporting similar calls in a region and concordance between their breakpoints.\n\nFuture work will include estimating the underlying breakpoints for each SV, potentially based on machine learning methods that utilize information gained from the GiaB consortium on the accuracy of different technologies for different SVs types and sizes. In addition, future work will involve comparing predicted SVs in repetitive regions, since these can often be represented in multiple ways in multiple locations in the genome.\n\nIn summary, we present a method (SURVIVOR_ant) for fast annotation of SVs and represents a first step in understanding type and breakpoint concordance for any type of SV, as well as the potential impact of SVs on genes.\n\n\nData and software availability\n\nAll the datasets used in this study are available at https://github.com/NCBI-Hackathons/svcompare.gi. Additional raw data can be obtained at https://github.com/genome-in-a-bottle.\n\nArchived source code of the software used as at the time of publication is available at: http://doi.org/10.5281/zenodo.898078 (dbodian et al., 2017)",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the National Science Foundation awards (DBI-1350041) and National Institutes of Health awards (R01-HG006677 and UM1-HG008898), and by the Inova Health System.\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 Timothy Hefferon for advice on dbVar datasets. Furthermore, we thank Ben Busby and the NCBI Hackathon Team of August 2016 for helpful discussions. We thank Lisa Federer, NIH Library, for editing assistance. We thank members of the Genome in a Bottle Consortium for generating the data and SV calls used in this study. Certain commercial equipment, instruments, or materials are identified in this paper only to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the NIST, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.\n\n\nReferences\n\nAbyzov A, Urban AE, Snyder M, et al.: CNVnator: an approach to discover, genotype, and characterize typical and atypical CNVs from family and population genome sequencing. Genome Res. 2011; 21(6): 974–984. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarnevali P, Baccash J, Halpern AL, et al.: Computational techniques for human genome resequencing using mated gapped reads. J Comput Biol. 2012; 19(3): 279–292. PubMed Abstract | Publisher Full Text\n\nChaisson MJ, Huddleston J, Dennis MY, et al.: Resolving the complexity of the human genome using single-molecule sequencing. Nature. 2015; 517(7536): 608–611. PubMed Abstract | Publisher Full Text | Free Full Text\n\ndbodian, adhroso, Sedlazeck F, et al.: NCBI-Hackathons/svcompare: Initial release. Zenodo. 2017. Data Source\n\nEnglish AC, Salerno WJ, Hampton OA, et al.: Assessing structural variation in a personal genome-towards a human reference diploid genome. BMC Genomics. 2015; 16(1): 286. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEnglish AC, Salerno WJ, Reid JG: PBHoney: identifying genomic variants via long-read discordance and interrupted mapping. BMC Bioinformatics. 2014; 15: 180. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarrison E, Marth G: Haplotype-based variant detection from short-read sequencing. 2012. Reference Source\n\nGuan P, Sung WK: Structural variation detection using next-generation sequencing data: A comparative technical review. Methods. 2016; 102: 36–49. PubMed Abstract | Publisher Full Text\n\nHénaff E, Zapata L, Casacuberta JM, et al.: Jitterbug: somatic and germline transposon insertion detection at single-nucleotide resolution. BMC Genomics. 2015; 16: 768. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJeffares DC, Jolly C, Hoti M, et al.: Transient structural variations have strong effects on quantitative traits and reproductive isolation in fission yeast. Nat Commun. 2017; 8: 14061. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLayer RM, Chiang C, Quinlan AR, et al.: LUMPY: a probabilistic framework for structural variant discovery. Genome Biol. 2014; 15(6): R84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H: FermiKit: assembly-based variant calling for Illumina resequencing data. Bioinformatics. 2015; 31(22): 3694–3696. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMak AC, Lai YY, Lam ET, et al.: Genome-Wide Structural Variation Detection by Genome Mapping on Nanochannel Arrays. Genetics. 2016; 202(1): 351–362. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcKenna A, Hanna M, Banks E, et al.: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20(9): 1297–1303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMohiyuddin M, Mu JC, Li J, et al.: MetaSV: an accurate and integrative structural-variant caller for next generation sequencing. Bioinformatics. 2015; 31(16): 2741–2744. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNattestad M, Schatz MC: Assemblytics: a web analytics tool for the detection of variants from an assembly. Bioinformatics. 2016; 32(19): 3021–3023. PubMed Abstract | Publisher Full Text\n\nRitz A, Bashir A, Sindi S, et al.: Characterization of structural variants with single molecule and hybrid sequencing approaches. Bioinformatics. 2014; 30(24): 3458–3466. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSudmant PH, Rausch T, Gardner EJ, et al.: An integrated map of structural variation in 2,504 human genomes. Nature. 2015; 526(7571): 75–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWittler R, Marschall T, Schönhuth A, et al.: Repeat- and error-aware comparison of deletions. Bioinformatics. 2015; 31(18): 2947–2954. PubMed Abstract | Publisher Full Text\n\nZhao M, Wang Q, Wang Q, et al.: Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives. BMC Bioinformatics. 2013; 14 Suppl 11: S1. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "26608",
"date": "09 Oct 2017",
"name": "Mitchell A. Bekritsky",
"expertise": [
"Reviewer Expertise Genomics",
"copy number variants",
"short read sequencing analysis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, Sedlazeck et al. describe a tool, SURVIVOR_ant, that uses interval trees to intersect multiple SV sets and annotations to produce a single VCF that can be used to compare SV callsets to each other, to known SV datasets, or to other reference annotations. The use of a 'wobble' parameter described in the manuscript enables them to overlap features that are nearby, but may not directly overlap. This is particularly important because SV representations can be pretty variable, although I wouldn't mind seeing a little more direct evidence from the authors motivating the need for this parameter.\nThe authors demonstrate an analysis possible with SURVIVOR_ant on 16 HG002 SV callsets, along with the 1000 Genomes Project Phase 3 SV calls, and several reference annotation datasets. While they demonstrate that their analysis produces interesting results, some of their observations might benefit from a little more analysis to round them out. In addition, one of the more interesting points that is apparent in one of the figures is not mentioned in the text, which might be worth bringing up. While I realize this manuscript is not intended to be a thorough analysis of the HG002 SV callsets, it is a very good opportunity for the authors to give their readers some interesting insights into these datasets, and could also help point SURVIVOR_ant users in the direction of some useful questions they can ask once they've run this tool.\nAlthough no direct analog to SURVIVOR_ant currently exists, some of its functionality overlaps with bedtools intersect. It seems as though their analysis pipeline might be similar to extending the input SV callset by the wobble parameter on either side, translating it into a BED file, then intersecting it with the other datasets. I think SURVIVOR_ant is distinct enough in its focus on SV in VCFs without further intermediate steps that it's a meaningfully distinct tool.\nSoftware The code for SURVIVOR_ant is written in C++. Some of it could be organized a little better (for instance, the SVS_Node class was in Parser.h, which I found a little confusing). When I tried to compile the code on my Mac, it did not compile because I didn't have a compiler configured with OpenMPI. While that's not a dealbreaker, I'm not sure why there's an OpenMPI requirement in a program with no indication of any parallelization. Perhaps it's from an external library?\nAnother concern that I have is the lack of any testing for the interval tree structure implemented by the authors, especially because it can be a somewhat complex data structure to implement. From the header, it looks like this was written a while ago and might have been taken from another package, where perhaps there was more testing done. If that's the case, it might still be good to include the tests here so that any new bugs might be uncovered by future users of this tool. If not, perhaps some inspiration can be taken from bedtools's tests?\nMissing wobble parameter implementation? I was able to clone and compile the code on a different machine and run it without any arguments, so the code seems sound. However, I did not attempt to rerun the authors' analysis since the wobble parameter did not appear to be implemented. With this option missing, I did not believe the software provided in the repo linked to the manuscript would allow me to replicate the analysis described by the authors. Not finding any 'wobble' parameter implementation in the C++ code or in the compiled executable, which seems to be a feature that's emphasized in the manuscript, is my primary concern about this manuscript. When I downloaded the source code as at the time of publication, it appeared that the SURVIVOR_ant directory was actually empty. Perhaps the authors neglected to update the code in the repo they refer to in the text to a later version?\n\nWobble intervals The motivation for the wobble parameter could be backed up by a little more data (although I don't doubt its need). Perhaps one of these two plots might be helpful:\nThe length of an event on one axis and the distance to the nearest overlapping event on the other (0 for when the events overlap) might make it clear that lots of events throughout the SV size spectrum that are nearby do not overlap, which would provide more motivation for this parameter. Similarly, for SV calls that are grouped into a single region by SURVIVOR_ant using the wobble parameter, a plot of the reciprocal overlaps for the variants would show that calls that are grouped in a region typically have no overlap, and therefore the wobble parameter is needed.\nAdditionally, I wonder if a fixed wobble size (defined as 1 kb in the manuscript) is the right choice. My concerns are motivated by 2 factors:\nFor small events, let's say, on the order of 100 bp, extending the interval by 1kb on either side might be a bit liberal, assuming that the size of the SV isn't grossly underestimated and that the breakpoints are in unique regions. In this case, something that's 1kb away from that event might not be a good overlap candidate. For very large events, on the order of 10s to 100s of kb or more, extending the SV on either side by 1kb might conversely be too conservative. Particularly if breakpoints are uncertain, one could imagine the ends of the event moving by 5kb+.\nAdditional SV analyses The analyses proposed by the authors are very interesting, but might perhaps be trivially extended to provide additional useful information. One option that might be especially interesting to see is an extension of their 4th analysis proposal (breakpoint variation) to genotype variation. Namely, if 4+ callers have made calls of the same type, it would be good to know if they all have consistent genotypes (e.g. single or double null at a site where a deletion has been called).\nOne thing that was unclear to me from the current list of SV analyses is whether there's any per-locus report of the type diversity. It is obviously discussed later in the results section, so I clearly misunderstood one of the analyses described. My money's on 2 or 3. Perhaps the descriptions of these analyses could be made clearer?\nSVs overlapping with HG002 The authors report that 4,506 1000 Genomes SVs overlap with their merged SV set for HG002 out of a total of 134,528 total events. Some context would be helpful for these 2 numbers. In the case of SNPs, Figure 3 from the 1000 Genomes project1 suggests that if the 1000 Genomes project had comprehensively identified all SVs in their study cohort, HG002 should have a significantly higher overlap with previously characterized common SVs. Instead, the data here suggests that just 3% of the SVs identified in HG002 were previously described in the Phase 3 SV release from the 1000 Genomes Project. This is a bit lower than I would expect. There are, of course, possible explanations for this lower overlap that the authors could explore:\nVariants of any type in HG002 are not generally found in the 1000 Genomes Project Either due to the methods used or the sequencing data available, the 1000 Genomes project SV dataset is far from complete, and the authors are describing novel SVs. It remains possible that many of the novel SVs described in the merged HG002 SV set are false positives (not SURVIVOR_ant's problem, that's a callset problem)\nWhile the point of this paper isn't to thoroughly analyze extant HG002 callsets, this number is a significant enough departure from expectation that it might be worth addressing.\nBreakpoint prediction consistency In the authors' explanation of why short read caller's start positions are closer to the median breakpoint, they posit that the sites detected by short read callers may be easier and less repetitive. Given that the authors have overlapped their calls with the GA4GH repetitive region BED files, they are in a position to substantiate that claim if it were true, or invalidate if it were false. A breakdown of repetitive overlap status for shared calls, calls unique to short read technologies, and calls unique to long read technologies should be done to dig into this claim a little bit more if the authors are going to make it.\nAdditionally, the authors that the number of callers per technology varies, but by my count, there are 7 Illumina callers and 7 PacBio callers. At least within the subset of calls detected by either of these 2 technologies, the number of callers is equal, so perhaps it is not a bias is introduced simply due to the number of callers for these 2 technologies.\nFigures\nFigure 2 is a little difficult to read -- perhaps log-scale the y-axis and color / sort the bars by platform / technology?\nThis plot also makes it apparent that many of the calls in this analysis come from a single dataset and a single variant type -- deletions from MetaSV on Illumina data. This is at least worth noting in the manuscript, and it may be worth providing numbers for the analyses done with and without this dataset since it is such a significant outlier. Again, perhaps not the point of the paper, but maybe something the authors could elaborate on a bit.\nMisc A few small suggested edits as well:\nPage 3, Column 2, Paragraph 1 (end of paragraph): if the BED file does not include a four columns -> if the BED file does not include four columns Page 4, Column 1, Paragraph 3: The analysis scripts are using BioPerl -> The analysis scripts use BioPerl Page 5, Column 2, Paragraph 1: including lower error rates in short reads, easier less repetitive sites detected by short reads, filtering rules, etc. -> including lower error rates in short reads; easier, less repetitive sites detected by short reads; filtering rules, etc. OR including lower error rates in short reads, easier and less repetitive sites detected by short reads, filtering rules, etc.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "26611",
"date": "16 Oct 2017",
"name": "Andrew Carroll",
"expertise": [
"Reviewer Expertise Structural variant calling",
"short-read analysis",
"long-read analysis",
"benchmarking methods for NGS tools"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 deals with the methods to compare and overlap structural variant events and presents an analysis of these methods against one of the best studied single genome for structural variant calling - the Genome in a Bottle HG002 - and the extensive population-level structural variant callset from the 1000 Genomes Project. Although in SNP and Indels the problem of determining whether two representations of a call are the same event, this problem is significantly more difficult more structural variant events. In the current state of the field, this is an open and challenging problem.\nThe presented analyses are valid. Any review of this work can point out an almost infinite number of parameters for the analysis that can be argued over, such as:\nWhat are reasonable size ranges to merge events? What are proper criteria for filtering events? Is it appropriate to include calls from so many methods in merging - should we identify some as unreliable? Should certain callers be weighted more highly than other based on reliability? How do we consider orthogonal support from different methods - are 3 Illumina methods based on different signals (coverage, insert size) as good as 2 Pacbio? Are the differences in how we discover events types (INS/TRA/DEL) significant enough that we need new comparison methods for some? What are we missing, but we're not aware we're missing. Do the current methods have any major blindspots?\nEtc... This list could be nearly infinite, mostly because so many questions in this field are unsettled.\nThe most important contribution of this work is as a foundation that frames these discussion points. The ability to point to data around a strategy for overlapping and analysis is an important step in progressing beyond discussion of all the items that can be considered.\nThe results may be valuable to these tool developers to identify structural variant types, sizes, and genomic contexts which their methods perform poorly on. In addition, the annotation ability may be useful in the annotation and interpretation of structural variant events (one could imagine making BED files for genome regions identified in ExAC or gnomAD as important for and scanning samples or structural variant events that look unusual or damaging in an individual or which impact genes associated with phenotypes of interest).\nThe number of possible future directions for this work is unusually large (the questions listed above are a good start).\nThere are a number of decisions about the approach that I disagree with and would do differently, but to determine which of those opinions/approaches are valid would require work to the scope of being one (or many) additional publications.\n\nI look forward to community efforts which continue to refine the methods to compare and annotate structural variant calls building on the concepts outlined here.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "26610",
"date": "01 Nov 2017",
"name": "Aaron R. Quinlan",
"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\nDetecting variation in genome structure is notoriously difficult with existing sequencing technologies and algorithms. Consequently, researchers typically utilize multiple SV detection algorithms, and in some cases, multiple sequencing technologies to maximize accuracy.\nSedlazeck and colleagues introduce SURVIVOR_ant as a new tool in the SURVIVOR package for annotating a SV callset in VCF format with SV predictions from other tools as well as genome features in BED or GFF format. While the motivation, performance and implementation are sound, I think the authors need to compare their software to both bedtools intersect and especially vcfanno given that vcfanno already provides all or nearly all of the functionality described here. In particular, vcfanno was designed to properly annotate SV events, as it takes the confidence interval about an SV's predicted breakpoints into account. I suggest that the authors extend the manuscript to provide a paragraph comparing the functionality and speed of SURVIVOR_ant to that of vcfanno and bedtools intersect, as this will help readers to better understand its strengths and weaknesses with respect to existing solutions.\nSecondly, I think the authors should state up front exactly what types of events they consider to be SVs, as some call sets listed in Table one restricted to SNPs and short INDELs, which are typically driven by different mutational mechanisms and typically not referred to as SVs.\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": []
}
] | 1
|
https://f1000research.com/articles/6-1795
|
https://f1000research.com/articles/6-1018/v1
|
28 Jun 17
|
{
"type": "Review",
"title": "MicroRNAs in the development and neoplasia of the mammary gland",
"authors": [
"Manoj Kumar Jena"
],
"abstract": "Study on the role of microRNAs (miRs) as regulators of gene expression through posttranscriptional gene silencing is currently gaining much interest,due to their wide involvement in different physiological processes. Understanding mammary gland development, lactation, and neoplasia in relation to miRs is essential. miR expression profiling of the mammary gland from different species in various developmental stages shows their role as critical regulators of development. miRs such as miR-126, miR-150, and miR-145 have been shown to be involved in lipid metabolism during lactation. In addition, lactogenic hormones influence miR expression as evidenced by overexpression of miR-148a in cow mammary epithelial cells, leading to enhanced lactation. Similarly, the miR-29 family modulates lactation-related gene expression by regulating DNA methylation of their promoters. Besides their role in development, lactation and involution, miRs are responsible for breast cancer development. Perturbed estrogen (E2) signaling is one of the major causes of breast cancer. Increased E2 levels cause altered expression of ERα, and ERα-miR cross-talk promotes tumour progression. miRs, such as miR-206, miR-34a, miR-17-5p, and miR-125 a/b are found to be tumour suppressors; whereas miR-21, miR-10B, and miR-155 are oncogenes.Studies using an ACI rat model showed similar findings of miR dysregulation due to excess E2, and a natural phenol antioxidant ellagic acid showed therapeutic properties by reversing the miR dysregulation. This review focuses on the recent findings concerning the role of miRs in developmental stages of the mammary gland (mainly lactation and involution stages) and their involvement in breast cancer progression. Further studies in this area will help us understand the molecular details of mammary gland biology,as well as miRs that could be therapeutic targets of breast cancer.",
"keywords": [
"Mammary gland",
"miRNA",
"development",
"lactation",
"neoplasia"
],
"content": "Introduction\n\nMicroRNAs (miRs) are small, endogenous noncoding RNAs that regulate gene expression post-transcription, by degrading target mRNA or repressing translation. They are hardy in nature, being resistant to RNAse degradation, acidic pH, and repeated freezing and thawing, and are stable at room temperature1. In addition to their involvement in many other physiological processes, they play crucial roles in mammary gland development and lactation, and their deregulation can lead to breast cancer2. This review provides an overview on the recent findings concerning the role of miRs in the developmental stages of the mammary gland and breast cancer progression.\n\n\nmiRs in mammary glands\n\nThe study of miRs in mammary gland development is essential, as miRs in milk are derived from the mammary gland and can be a biomarker of a healthy lactating gland, as well as protecting infants and promoting their development3. The role of miRs in milk is still being debated. For example, whether they are only simple nutrients or if they have some regulatory functions in milk recipients after entering systemic circulation is still disputed4.\n\nComparative analysis of the miRNome of bovine milk, mammary tissue, milk fat, and whey revealed 188 miRs in common, with some novel miRs discovered5. The miR signature of milk fat was similar to that of mammary gland tissue miRs. Functional studies of highly expressed miRs in those fractions indicated their role as regulators of mammary gland functions contributing to healthy milk production5.\n\n\nmiRs controlling lactation\n\nIt was observed that miR-126 plays a role in mammary epithelial cells (MECs) by modulating lipid synthesis6. FASN (fatty acid synthase) gene expression is increased when miR-126-3p is inhibited, suggesting its involvement in lipid metabolism in the mammary gland. In addition, estradiol and progesterone enhanced lipid synthesis by downregulating the levels of miR-126-3p6. Similarly, miR-150 hampers lipogenesis in MECs and reduces secretory activity7, while miR-145 facilitates milk fat synthesis in lactating goats by targeting the gene INSIG1 (insulin induced gene 1)8.\n\nThe role that miRs play as regulators of lipogenesis was confirmed by overexpression studies, which showed that there was more synthesis of fat droplets, accumulating triacylglycerols, and a higher proportion of unsaturated fatty acids in lactating MECs. For instance, miR-24 was found to be expressed at a much higher level during peak lactation in goats and affects triacylglycerol content, unsaturated fatty acid concentration, and expression of target genes, such as FASN, SREBF1 (sterol regulatory element binding transcription factor1), SCD (stearoyl-CoA desaturase), GPAM (glycerol-3-phosphate acyltransferase; mitochondrial), and ACACA (acetyl-CoA carboxylase)9.\n\nmiR expression in MECs is regulated by lactogenic hormones (dexamethasone, insulin, and prolactin), as evidenced by an increased miR-148a level in bovine MECs, which is probably associated with increased milk production during lactation in cows10. The miR-29 family affects lactation by regulating the DNA methylation of target genes DNMT3A and DNMT3B (DNA (cytosine-5)-methyltransferase 3A and 3B) in MECs of dairy cows11. Moreover, inhibition of the miR-29 family resulted in hypermethylation of promoters of lactation–related genes, leading to decreased secretion of triglycerides, proteins, and lactose by the epithelial cells. miR-486 facilitates lactation by downregulating PTEN (phosphatase and tensin homolog) target gene. Downregulation of this gene affects the expression of downstream genes, such as AKT, mTOR and β-casein, which have crucial roles in mammary gland development and lactation12. Additionally, diet restriction has been shown to affect the miRNome of lactating mammary glands, indicating the role of miRs in regulating milk composition13.\n\n\nmiRs controlling involution and breast cancer\n\nIdentification and characterization of miRs involved in breast cancer will facilitate targeting miRs for possible therapy. Improper involution possibly contributes towards tumour development14. miR-424(322)/503 regulates mammary involution in humans by targeting BCL-2 (B-cell lymphoma 2; anti-apoptotic) and IGF1R (insulin like growth factor-1-receptor) genes. The loss of this miR leads to malignancy and nonresponse to chemotherapy, demonstrating its role as a tumour suppressor14. By contrast, some miRs, such as miR-660-5p, promote tumour development and metastasis, and the level of miR-660-5p was found to be increased in MCF7 breast cancer cell lines15. This miR’s tumour promoting activity was confirmed by observation of reduced invasion of MCF7 cells at reduced miR-660-5p levels.\n\nOne of the major causes of breast cancer is disturbed estrogen signaling, where the altered expression of estrogen receptor α (ERα) and its cross-talk with the related miR culminates in neoplasia16. Studies on the effect of E2 on the expression pattern of miRs in MCF7 and ZR75 cell lines revealed 172 miRs that were up or downregulated. Notable miRs are miR-206, miR-34a, miR-17-5p, and miR-125 a/b, which act as tumour suppressors, and miR-21, miR-10B, and miR-155, which act as oncogenes17. Another study using an ACI rat model for the effect of E2 on the miR signature showed 33 dysregulated miRs18. Additionally, the use of ellagic acid (natural phenol antioxidant) reversed the dysregulation of miR-206, miR-182, miR-375, miR-127, miR-183, and miR-122, subsequently modulating the target proteins ERα, RASD1, cyclin D1, FoxO1, FoxO3a, Bcl-w, Bcl-2 and cyclin G11818. Furthermore, overexpression of the tumour suppressing miR-133a in MCF-7 and MDA-MB-231 cells suppressed phosphorylated Akt (p-Akt) protein and inhibited p-Akt nuclear translocation, and this miR also regulates the cell cycle of cancerous cells by targeting the EGFR (epidermal growth factor receptor) gene19.\n\nmiR-206 is found to play a crucial role in BRCA1 (Breast CAncer susceptibility gene; tumour suppressor) depleted mouse mammary gland20. Overexpression of miR-206 showed no effect on lactation, but did have a role in tissue remodeling through increasing fat tissue and reducing branching morphogenesis. There may be a possibility of increased miR-206 levels due to BRCA1 loss, culminating in mammary gland remodeling and tumour development20. miR-184 is found to be a tumour suppressor by regulating the number of genes in the PI3K/AKT/mTOR pathway, as observed by miR profiling of the pubertal mouse mammary gland21. This pathway is important in mammary gland development and lactation22. miR-184 is only expressed in epithelial cells, and the level increases during differentiation of cells from terminal end bud into ductal epithelial cells21.\n\n\nConclusions\n\nmiRs have been shown to be one of the major regulators of mammary gland development and neoplasia. miRNome studies of mammary gland in different developmental stages and cancerous tissues will elucidate biomarkers for early cancer diagnosis, and may be used as therapeutic targets. Future studies focusing on the cross-talk between miRs and target genes with the signaling pathways involved in development and neoplasia of mammary gland will open the door to understand mammary gland biology and oncogenesis in molecular detail.",
"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\nGigli I, Maizon DO: microRNAs and the mammary gland: A new understanding of gene expression. Genet Mol Biol. 2013; 36(4): 465–474. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang CD, Long K, Jin L, et al.: Identification of conserved microRNAs in peripheral blood from giant panda: expression of mammary gland-related microRNAs during late pregnancy and early lactation. Genet Mol Res. 2015; 14(4): 14216–14228. PubMed Abstract | Publisher Full Text\n\nAlsaweed M, Lai CT, Hartmann PE, et al.: Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk. Sci Rep. 2016; 6: 20680. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMelnik BC, Kakulas F, Geddes DT, et al.: Milk miRNAs: simple nutrients or systemic functional regulators? NutrMetab (Lond). 2016; 13: 42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi R, Dudemaine PL, Zhao X, et al.: Comparative Analysis of the miRNome of Bovine Milk Fat, Whey and Cells. PLoS One. 2016; 11(4): e0154129. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChu M, Zhao Y, Feng Y, et al.: MicroRNA-126 participates in lipid metabolism in mammary epithelial cells. Mol Cell Endocrinol. 2017; pii: S0303-7207(17)30309-X, In press. PubMed Abstract | Publisher Full Text\n\nHeinz RE, Rudolph MC, Ramanathan P, et al.: Constitutive expression of microRNA-150 in mammary epithelium suppresses secretory activation and impairs de novo lipogenesis. Development. 2016; 143(22): 4236–4248. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang H, Shi H, Luo J, et al.: MiR-145 Regulates Lipogenesis in Goat Mammary Cells Via Targeting INSIG1 and Epigenetic Regulation of Lipid-Related Genes. J Cell Physiol. 2017; 232(5): 1030–1040. PubMed Abstract | Publisher Full Text\n\nWang H, Luo J, Chen Z, et al.: MicroRNA-24 can control triacylglycerol synthesis in goat mammary epithelial cells by targeting the fatty acid synthase gene. J Dairy Sci. 2015; 98(12): 9001–14. PubMed Abstract | Publisher Full Text\n\nMuroya S, Hagi T, Kimura A, et al.: Lactogenic hormones alter cellular and extracellular microRNA expression in bovine mammary epithelial cell culture. J Anim Sci Biotechnol. 2016; 7: 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBian Y, Lei Y, Wang C, et al.: Epigenetic Regulation of miR-29s Affects the Lactation Activity of Dairy Cow Mammary Epithelial Cells. J Cell Physiol. 2015; 230(9): 2152–63. PubMed Abstract | Publisher Full Text\n\nLi D, Xie X, Wang J, et al.: MiR-486 Regulates Lactation and Targets the PTEN Gene in Cow Mammary Glands. PLoS One. 2015; 10(3): e0118284. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMobuchon L, Marthey S, Le Guillou S, et al.: Food Deprivation Affects the miRNome in the Lactating Goat Mammary Gland. PLoS One. 2015; 10(10): e0140111. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRodriguez-Barrueco R, Nekritz EA, Bertucci F, et al.: miR-424(322)/503 is a breast cancer tumor suppressor whose loss promotes resistance to chemotherapy. Genes Dev. 2017; 31(6): 553–566. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShen Y, Ye YF, Ruan LW, et al.: Inhibition of miR-660-5p expression suppresses tumor development and metastasis in human breast cancer. Genet Mol Res. 2017; 16(1): gmr16019479. PubMed Abstract | Publisher Full Text\n\nManavathi B, Dey O, Gajulapalli VN, et al.: Derailed estrogen signaling and breast cancer: an authentic couple. Endocr Rev. 2013; 34(1): 1–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFerraro L, Ravo M, Nassa G, et al.: Effects of oestrogen on microRNA expression in hormone-responsive breast cancer cells. Horm Cancer. 2012; 3(3): 65–78. PubMed Abstract | Publisher Full Text\n\nMunagala R, Aqil F, Vadhanam MV, et al.: MicroRNA 'signature' during estrogen-mediated mammary carcinogenesis and its reversal by ellagic acid intervention. Cancer Lett. 2013; 339(2): 175–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCui W, Zhang S, Shan C, et al.: MicroRNA-133a regulates the cell cycle and proliferation of breast cancer cells by targeting epidermal growth factor receptor through the EGFR/Akt signaling pathway. FEBS J. 2013; 280(16): 3962–74. PubMed Abstract | Publisher Full Text\n\nWronski A, Sandhu GK, Milevskiy MJ, et al.: MicroRNA-206 is differentially expressed in Brca1-deficient mice and regulates epithelial and stromal cell compartments of the mouse mammary gland. Oncogenesis. 2016; 5: e218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPhua YW, Nguyen A, Roden DL, et al.: MicroRNA profiling of the pubertal mouse mammary gland identifies miR-184 as a candidate breast tumour suppressor gene. Breast Cancer Res. 2015; 17: 83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Z, Hou X, Qu B, et al.: Pten Regulates Development and Lactation in the Mammary Glands of Dairy Cows. PLoS One. 2014; 9(7): e102118. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "24612",
"date": "07 Aug 2017",
"name": "Mohammed Alsaweed",
"expertise": [
"Reviewer Expertise microRNA",
"gene expression regulation",
"lactation",
"mammary gland",
"milk",
"human milk"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 review entitled \"MicroRNAs in the development and neoplasia of the mammary gland\" has briefly presented recent studies in the relevant studies in mammary gland microRNAs field. This kind of review is very good for scientists to update their knowledge without reading informative reviews. However, minor comments are below to make the review suitable for indexing:\n\nIntroduction:\nMore information is needed to understand the mechanism of microRNA roles in the mammary gland.\n\nmiRs in mammary gland:\nAlong with Alsaweed et al. study, Modepalli et al. (2014, BMC Genomics) has provided evidence that mammary gland is the main source of microRNAs in milk. Therefore, milk microRNAs are not considered as nutrients as stated, so better to not use \"whether they are only simple nutrients\". Examples about the milk microRNAs are important as the mammary gland is synthesized and secreted milk.\n\nmiRs controlling lactation:\nHighly expressed microRNAs in milk/mammary gland have different roles in synthesize various milk nutrient contents such as triacylglycerols and lactose, so the role of highly expressed microRNAs must be linked to related functions (Alsaweed et al 2016, PLoS ONE and IJMS). Li et al. (2012, BMC Genomics) has presented very important aspects of the microRNAs and lactation performance.\n\nmiRs controlling involution and breast cancer:\nZhang et al. (2013, Nature Cell Biology) has provided breast cancer inhibits progression by microRNAs expression roles, so it must be considered in this review.\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": "2943",
"date": "09 Aug 2017",
"name": "Manoj Jena",
"role": "Author Response",
"response": "IntroductionMechanism of action to be incorporated: Matured miRs integrate into the RNA - induced silencing complex (RISC) and direct it to target mRNAs through partial sequence complementarity. miRs down regulate the target gene expression by translation repression (48 % through RISC complex), or mRNA degradation (29 %), or by both phenomena (23 %). (Jin and Xiao, 2015).miRs in mammary glands To be removed: The role of miRs........still disputed.To be incorporated: miRs in milk during lactation are putative markers of activities in mammary gland and act as functional signals for proper growth and development of the young. The highly abundant miRs in milk during lactation cycle are miR-191, 184, 181, 148, 375, and miRs of let-7 family (7f, 7a and 7i) as evidenced from studies in the marsupial tammar wallaby (Modepalli et al., 2014). miRs controlling lactationTo be incorporated: Lin et al (2013) observed group of miRs (such as miR-23a, 27b, 103, and 200a) affect milk fat synthesis with synergistic action. miR profiling of lactating and nonlactating bovine mammary gland revealed miR-125b, 181a, and 199b expression level reduced in non-lactation period; whereas miR-141, 484, and 500 expression level was higher in lactation period. (Li et al., 2012). Highly expressed miRs such as let-7f-5p is found to target many genes involved in protein, carbohydrate, and triglyceride synthesis. The human milk enriched miR such as miR-22-3P regulates development and differentiation of T-lymphocytes. The miR-181a-5p and miR-182-5p have crucial role in immune cell differentiation, miR-375 is required for glucose homeostasis, and miR-148a-3p is a tumour suppressor and involved in liver development (Alsaweed et al., 2016).miRs controlling involution and breast cancer To be incorporated: The miR 126/126* pair is observed to repress the recruitment of mesenchymal stem cells and inflammatory monocytes into the tumour stroma, thus inhibiting breast cancer metastasis (Zhang et al., 2013)."
}
]
},
{
"id": "25931",
"date": "19 Sep 2017",
"name": "Shantibhusan Senapati",
"expertise": [
"Reviewer Expertise Tumor microenvironment",
"animal models",
"stroma and cancer cell crosstalk"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWith this review, the author provide a concise literature summary of microRNAs in the development and neoplasia of the mammary gland. For the most part the review is complete and informative. To further improve the quality, the author is advised to address these minor points:\nExosomes secreted by different cells of mammary gland carry microRNA. It has been demonstrated that cancer-associated fibroblasts release exosomal microRNA that induce aggressiveness in breast cancer cells (e.g. Donnarumma et al. (2017)1). At the same time, the microRNA present in the exosomes secreted by the cancer cells also play a critical role in the pathogenesis of breast cancer (e.g. Melo et al. (2014)2). Hence, the authors might consider to include these information.\n\nThe beginning sentence under “miRs in mammary gland” heading needs to be restructured. The presence of miRNA in milk and the role of miRNA in mammary development should be mentioned separately.\n\nIn the abstract, the sentence “Further studies in this area will help us understand ……” should be “Further studies in this area will help us to understand ……”).\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": "3049",
"date": "25 Sep 2017",
"name": "Manoj Jena",
"role": "Author Response",
"response": "To be incorporated in the heading \"miRs controlling involution and breast cancer\": Exosomes are cell - derived small vesicles (40 - 100 nm) containing mRNA, protein, miR etc. They are crucial mediator of inter cellular signaling during cancer development. Exosomes derived from cancer associated fibroblasts in breast cancer have 3 miRs such as miR-21, -143, and -378e which promote stemness, epithelial-mesenchymal transition, and anchorage-independent cell growth, thus accelerating oncogenic signaling in breast cancer cells (Donnarumma et al., 2017). The exosomes released from cancer cells of breast cancer patients induce the non-tumorigenic epithelial cells to form tumors with the help of dicer endonuclease (Melo et al., 2014).The beginning sentence under “miRs in mammary gland” will be restructured. The presence of miRNA in milk and the role of miRNA in mammary development will be separated.In the abstract, the sentence “Further studies in this area will help us understand ……” will be rectified as “Further studies in this area will help us to understand ……”."
}
]
},
{
"id": "24742",
"date": "20 Sep 2017",
"name": "Bhudev C. Das",
"expertise": [
"Reviewer Expertise Molecular oncologist"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview article on “MicroRNAs in the development and neoplasia of the mammary gland” submitted by M.K. Jena deals with how various microRNAs that regulate gene expression through post transcriptional gene silencing are important during development and progression as well as prognosis of cancer of the mammary gland in women. Though I understand it is a mini-review but it has not been written in a very focused and systematic way. There are huge numbers of publications in this field, particularly the role of mRNA in breast carcinogenesis. However, the author cites only 22 references in this article which is highly inadequate to justify the review on the role of miRNA in mammary gland development and carcinogenesis. Author must consult recently published literatures including a recent paper by Thakur et al. (2016)1 and many of the references therein. There are a lot of sweeping comments have been made which indicates that the author has nothing much to do with cancer specialty. The comments in abstract say ‘miRs are responsible for breast cancer development’. It is not straight forward and clear that miRNA are responsible of breast cancer development. They may be associated with but not directly correlated with cancer development. Similar sweeping comments are many in the text and these need to be addressed properly and the manuscript needs to be elaborative and focused. The presentation of the review article should be made with proper sub-headings on mammary gland development, lactation and carcinogenesis.\n\nIn summary, I suggest that the article can be considered for indexing only after satisfactory revision.\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? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly",
"responses": [
{
"c_id": "3077",
"date": "03 Oct 2017",
"name": "Manoj Jena",
"role": "Author Response",
"response": "All the suggestions made by the referee has been complied in the second version of this article."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1018
|
https://f1000research.com/articles/6-1790/v1
|
02 Oct 17
|
{
"type": "Research Article",
"title": "Genetic diversity and multiplicity of Plasmodium falciparum merozoite surface protein 2 in field isolates from Sudan",
"authors": [
"Shaza O. Mustafa",
"Muzamil M. Abdel Hamid",
"Mariam A. Aboud",
"Mutaz Amin",
"Mohamed S. Muneer",
"Kyakonye Yasin",
"Nouh S. Mahgoub",
"Nabiela M. El Bagir",
"Shaza O. Mustafa",
"Mariam A. Aboud",
"Mutaz Amin",
"Mohamed S. Muneer",
"Kyakonye Yasin",
"Nouh S. Mahgoub",
"Nabiela M. El Bagir"
],
"abstract": "Background: Malaria is a major health problem, with over one third of worldwide populations currently at risk. Determining the genetic diversity of plasmodium parasites is essential for assessing the efficacy of antimalarial drugs and for future vaccine development. This study investigated the genetic diversity of P. falciparum merozoite surface protein 2 (MSP2), and multiplicity of infection (MOI) in different geographic regions in Sudan. Methods: A total of 271 patients with uncomplicated malaria were recruited from four ecological sites during malaria transmission season, 2011-2013. P. falciparum was confirmed using species specific primers targeting the rDNA gene. All P. falciparum positive samples were genotyped for the major MSP2 allelic families (IC1/3D7 and FC27 MSP2 allele) using nested PCR. Multiplicity of infection and allele frequencies were determined. Results: A total of 241 samples (88.9%) were confirmed positive for P. falciparum. The number of different MSP2 alleles were 14, 15, 13 and 12 in Khartoum, Gezira, River Nile and Red Sea states, respectively. The 3D7 allelic family was more prevalent in the states of Khartoum, Gezira, River Nile and Red Sea compared to the FC27 allelic family. Multiclonal infections were observed in 25.8% of patients, with a mean multiplicity of infection (MOI) of 1.45. MOIs were highest in the age group over 40, with an average of 2 and 1.68 in Khartoum and Gezira states, respectively, however MOIs in River Nile and Red Sea states were higher in age groups below 18, with an average of 1.37 and 1.33, respectively. Conclusions: MSP2 allelic genotyping revealed MOI and diversity of the Sudanese P. falciparum isolates. The results of our study are expected to influence current and future malaria control strategies, since the MOI predicts development of clinical malaria and subsequent efficacy of antimalarial treatment.",
"keywords": [
"malaria",
"Plasmodium",
"falciparum",
"multiplicity of infection",
"allele",
"msp2",
"Sudan"
],
"content": "Introduction\n\nMalaria was and still is a major health problem, with over one third of worldwide populations being at risk (World Health Organization, 2015). The burden of this disease falls the heaviest on Sub-Saharan African countries (World Health Organization, 2015). In Sudan, almost 90% of the population live under high malaria transmission levels, as recently reported by the WHO (“WHO | Sudan,” 2016; World Health Organization, 2015). Malaria in Sudan is transmitted mainly by Anopheles arabiensis and the majority of cases are due to P. falciparum (Ageep et al., 2009). The merozoite surface protein-2 (MSP2) is a highly polymorphic plasmodium membrane protein and the most expressed in the surface of the merozoite (Gerold et al., 1996). It has been long studied in vaccine trials for being accessible by the immune system (McCarthy et al., 2011). MSP2 protein is highly polymorphic, which gives an opportunity for molecular epidemiologists to differentiate between recurrent and new infections (Mwingira et al., 2011). Genetic diversity of MSP2 alleles is due to mutations and proliferation of highly polymorphic central repeats (Ferreira & Hartl, 2007). The allelic diversity of MSP2 proteins can be used to calculate multiplicity of infection (MOI), which is the number of different strains of P. falciparum co-infecting the same host and is a valuable for estimating malaria transmission level (Vafa et al., 2008). Sudan experiences a range of climate variations, from arid desert in the north, to tropical areas in the south and a short rainy season (Noor et al., 2012). The current study aimed to determine the level of genetic diversity of the P. falciparum MSP2 gene, using field isolates across different geo-ecological regions in Sudan. The study also aimed to determine the relationship between genetic diversity and the characteristics between different patients. Little has been documented regarding the molecular diversity of P. falciparum in hypo and meso-endemic regions of Sudan, in particular after adoption of ACT as antimalarial treatment.\n\n\nMethods\n\nThis study was conducted across the two geographical zones of Sudan: the semi-desert regions of the Red Sea (Northeast) and the savannah regions of Khartoum (Central), Gezira and River Nile states. It took place between August 2011 and December 2013 – peak malaria transmission is between July and December (rainy season) in the poor and rich savannah regions, but in the semi desert regions of the Red Sea, peak malaria transmission occurs between November and January, during the winter season.\n\nEthical clearance was obtained from the scientific and research ethics committee, Institute of Endemic Diseases, University of Khartoum. Informed consent was obtained from patients, or guardians if the patient was a minor, for participation in this study. Anonymity and confidentiality of patient information were maintained throughout.\n\nA total of 271 symptomatic malaria patients from all age groups regardless of gender were recruited from selected health facilities (Omdurman teaching hospital and Omdoom and Mubarak Zaroog health centers in Khartoum, Wad madani teaching hospital in Gezira, Zeedab, Kaboushia and Elbouga hospitals in River Nile and Unity hospital in Red Sea) during peak malaria transmission season. Patients were passively recruited from the outpatient clinics of these hospitals during the study period. Patients were diagnosed with malaria using blood film microscopy. Parasite density and hemoglobin level were measured on each sample upon collection. Thick and thin blood smears were stained with Giemsa stain. Parasite density was determined by counting the number of asexual parasites per 200 white blood cells using the formula described by Cook (Cook, 1990). The data was grouped based on the level of parasitemia, into low = 1–5,000 parasites/µl, intermediate = 5,001–10,000 parasites/µl and high = >10,000 parasites/µl.\n\nParasite DNA was extracted from dried blood samples spotted on filter papers using a method described by Musapa et al. (Musapa et al., 2013). Plasmodium species were identified by 18S rDNA based nested PCR, using genus and species specific primers manufactured by Macrogen, Korea, as described by Snounou et al. [15]. Msp2 alleles were further amplified with slight modification of the standardized nested-PCR protocols described previously by Snounou et al. (Snounou et al., 1993)\n\nThe amplified PCR products were run on 2% Agarose gel (Caisson, Utah, USA) stained with 4µl ethidium bromide at 100V and 30A for 60 minutes. DNA fragments were estimated using 100 bp DNA ladder marker (Vivantis, Selangor DarulEhsan, Malaysia) and the bands were viewed under UV light using trans-illuminator (BioDoc-It UVP, Cambridge, UK).\n\nThe multiplicity of infection (MOI) was calculated by dividing the total number of fragments observed in MSP2 by the number of positive samples. Isolates with a single genotype were considered monoclonal infections while isolates with more than one genotype was considered multiclonal infections.\n\nData was analyzed using SPSS version 20 (SPSS, Inc., Chicago, IL, USA). Proportions were compared for significance using the χ2-test. The association between MOIs, parasite densities and age groups were computed using Spearman’s rank correlation coefficient. Statistical significance among MOIs and mean hemoglobin levels for the different age groups were calculated using the Kruskal-Wallis H-test at a P-value of ≤ 0.05.\n\n\nResults\n\nP. falciparum rDNA was detected in a total of 241 (88.9%) malaria patients using nested PCR (Figure 1). 158 (65.6%) were male, while 83 (34.4%) were female. The distribution of patients across the four studied states: Khartoum, Gezira, River Nile and the Red Sea was 81 (33.6%), 58 (24.1%), 47 (19.5%) and 55 (22.8%), respectively. The age groups ranged between 3–90 years, with a mean of 31.39 ±14.9 years. The mean parasite density was higher in the River Nile state (15869 parasites/µl) compared to Khartoum, Gezira, and Red Sea states (14563, 11266, and 14603 parasites/µl, respectively), and this difference was statistically significant (p-value = 0.02). The mean hemoglobin level was lowest in Khartoum (5.23 g/dl) compared to the other regions; Gezira, River Nile, and Red Sea (7.94, 9.26, and 7.74 g/dl, respectively), and the difference was statistically significant (p-value = 0.02) (Table 1).\n\nAmplification of the P. falciparum ribosomal DNA gene using genus and species specific primers by nested PCR, yielding amplicons of 220 bp in size.) Molecular weight marker (MW) is 100 bp (iNTRON, Inc.). Lanes 2, 3, 4, and 6 are positive for samples of P. falciparum; 1 and 5 are negative for samples of P. falciparum; lane 7 is the negative control.\n\nAllele genotyping revealed the highly polymorphic nature of Sudanese P. falciparum isolates with respect to the MSP2 gene. Both IC1/3D7 and FC27 MSP2 allele types were identified (Figure 2). The total number of different sized alleles detected in this study was 42. Among them IC1/3D7 (160–400 bp) and FC27 (140–400 bp) allele families were noted. Frequencies of different MSP2 alleles and their combinations and multiplicity of infection across the study sites are shown in Table 2. The frequency of samples with only IC1/3D7 and FC27 were 56.4% (136/241), and 17% (41/241), respectively and IC1/3D7/FC27 combinations were found in 26.6% (64/241) of samples. The prevalence of IC1/3D7 and FC27 allelic types was 82.9% (200/241) and 43.5% (105/241), respectively. Multiple clones were detected in all study sites. Multiplicity of infection (MOI) was highest in P. falciparum infected patients from Gezira state (1.67 genotypes per infection) and lowest in those from Red Sea state (1.20 genotypes per infection) and it was statistically significant, (p-value = 0.001) (Table 2). The difference in distribution of allelic polymorphism of MSP2 was only significant for total MSP2 and FC27 (p-value = 0.002 and p-value = 0.042, respectively). However, for IC1/3D7 the result was not statistically significant (p-value = 0.89). The estimated mean MOI of all studied areas was 1.46 genotypes per infection (Table 2). There was a negative correlation between age and parasite density (Spearman rank coefficient = 0.03; p-value = 0.65), though it was not significant. The 18–40 age group had the highest mean parasite density (16354 parasites/µl, Table 3).\n\nKey:\n\n*: Fragment size by base pair\n\nPCR typing of MSP2 multiple clones (FC27) band size range (250–400 bp) and (IC1/3D7) band size range (500–750 bp). MW: 100 bp ladder (iNTRON, Biotechology). Lanes 1, 3 and 7 are positive for IC1/3D7, lane 9 is negative for the IC1/3D7 allelic family. Lanes 2, 4, and 6 are positive for the FC27 allelic family, 8 is negative for FC27 and lane 11 is a negative control. Lane 5 is positive for IC1/3D7, showing multiple clones (500 and 600 bp).\n\nThe distributions of P. falciparum MSP2 block 3 allelic types across different age groups are shown in Table 3. The prevalence of IC1/3D7 and FC27 is shown in the same table.\n\nThere is no significant correlation between multiplicity of infection and the age group the patient is in (Spearman rank coefficient = 0.01; P-value= 0.50). The distribution of IC1/3D7 and FC27 according to different levels of parasitaemia is shown in Table 4. Neither the individual distribution of IC1/3D7and FC27 nor the multiclonal isolates were significantly different among different parasitaemic groups (p-value = 0.7 and p-value = 0.957, respectively).\n\n\nDiscussion\n\nIncreasing our knowledge of the genetic diversity of P. falciparum induced malaria will certainly help us understand its pathogenesis, acquired immunity and drug resistance. 14, 15, 13 and 12 different alleles of MSP2 were identified in Khartoum, Gezira, River Nile and Red Sea states, respectively. This data is consistent with previous studies in low and unstable malaria transmission regions in eastern Sudan (A-Elbasit et al., 2007), central Sudan (Hamid et al., 2013), and other areas of seasonal unstable malaria transmission (Elmahdi et al., 2012). 3D7 alleles were more prevalent in the four states (Khartoum, Gezira, River Nile and Red Sea) compared to FC27 alleles. This finding differs from those of previous studies in central and eastern Sudan, where FC27 was the most predominant allelic family (A-Elbasit et al., 2007; Babiker et al.,1997; Hamid et al., 2013). In our study, 25% of studied samples were found to have mixed infections with multiple parasite clones; a result similarly reported in eastern Sudan (Babiker, 1998).\n\nIn our study, multiplicity of infection was highest in the >40 age group (with an average MOI of 2 and 1.68 in Khartoum and Gezira states, respectively), whereas the MOI was highest in the <18 age group (with an average of 1.37 and 1.33 in River Nile and Red Sea states, respectively), although the difference was not statistically significant. Recent studies on the variation of MOI with age have suggested that the influence of age on MOI is highly affected by the endemicity of malaria (Pinkevych et al., 2015). this is consistent with studies that have shown an age-dependent MOI in villages with intense perennial malaria transmission (Smith et al., 1999) and some areas with hypo-meso-endemic malaria transmission like Ghana (Agyeman-Budu et al., 2013),\n\nThe relatively higher number of alleles detected at high parasite densities might also mean that more diverse parasite populations are present in such infections. This suggestion is partly supported by the finding that all the different allelic types were more often concurrently detected in the high-density samples (Färnert et al., 2001). The present study reported that the increasing level of multiplicity was seen with increased parasite density of the samples. These findings are compatible with a previous study that has shown a clear trend of increasing parasite density with increased multiplicity for genetic markers on both genes, MSP1 and MSP2 (Peyerl-Hoffmann et al., 2001). The results of our study will likely influence current and future malaria control strategies since MOI can predict antimalarial treatment response (Kyabayinze et al., 2008).\n\n\nConclusions\n\nAllele genotyping revealed the highly polymorphic nature of Sudanese P. falciparum isolates with respect to the MSP2 gene. The results of our study are expected to have an influence on the current and future malaria control strategies, since MOI predicts development of clinical malaria and subsequent efficacy of antimalarial treatment.\n\n\nAbbreviations\n\nMSP2: merozoite surface protein 2, PCR: polymerase chain reaction, MOI: multiplicity of infection, ANOVA: analysis of variance, WHO: World Health Organization, rDNA: ribosomal DNA, ACT: artemisinin based combination therapy.\n\n\nEthical statement\n\nThe study received approval from the scientific and research ethics committee of the Institute of Endemic Diseases; University of Khartoum, Sudan. Informed consent was obtained from all participants (or from the parents/legal guardians when the participant was a minor), prior to their enrolment. All malaria patients approached in the study were treated using the standard treatment of WHO protocol for malaria (Olumese, 2017).\n\n\nData availability\n\nDataset 1: Raw data supporting the findings presented in this study. The dataset is available both in XLSX and SPPS format. DOI, 10.5256/f1000research.12585.d179181 (Mustafa et al., 2017).",
"appendix": "Competing interests\n\n\n\nAll authors have declared that they have no conflict of interest.\n\n\nGrant information\n\nThis study was partially funded by the National Malaria Program Administration, Ministry of Health, the University of River Valley, Sudan and Third World Academy of Science (TWAS), Trieste, Italy [project no. 13-145 RG/BIO/AF/AC_G].\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 all malaria patients who participated in the study. The authors would also like to thank Prof. Muntaser E. Ibrahim, director of the Dr. Douglas Barker Molecular Biology Lab, Institute of Endemic Diseases, University of Khartoum, for hosting the molecular work and data analysis.\n\n\nReferences\n\nA-Elbasit IE, ElGhazali G, A-Elgadir TM, et al.: Allelic polymorphism of MSP2 gene in severe P. falciparum malaria in an area of low and seasonal transmission. Parasitol Res. 2007; 102(1): 29–34. PubMed Abstract | Publisher Full Text\n\nAgeep TB, Cox J, Hassan MM, et al.: Spatial and temporal distribution of the malaria mosquito Anopheles arabiensis in northern Sudan: influence of environmental factors and implications for vector control. Malar J. 2009; 8: 123. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAgyeman-Budu A, Brown C, Adjei G, et al.: Trends in multiplicity of Plasmodium falciparum infections among asymptomatic residents in the middle belt of Ghana. Malar J. 2013; 12: 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBabiker HA: Unstable malaria in Sudan: the influence of the dry season. Plasmodium falciparum population in the unstable malaria area of eastern Sudan is stable and genetically complex. Trans R Soc Trop Med Hyg. 1998; 92(6): 585–9. PubMed Abstract | Publisher Full Text\n\nBabiker HA, Lines J, Hill WG, et al.: Population structure of Plasmodium falciparum in villages with different malaria endemicity in east Africa. Am J Trop Med Hyg. 1997; 56(2): 141–7. PubMed Abstract | Publisher Full Text\n\nCook GC: Parasitic Disease in Clinical Practice. London: Springer London, 1990. Publisher Full Text\n\nElmahdi ZA, Nugud AA, Elhassan IM: Estimation of malaria transmission intensity in Sennar state, central Sudan. East Mediterr Health J. 2012; 18(9): 951–6. PubMed Abstract\n\nFärnert A, Arez AP, Babiker HA, et al.: Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study. Trans R Soc Trop Med Hyg. 2001; 95(2): 225–32. PubMed Abstract | Publisher Full Text\n\nFerreira MU, Hartl DL: Plasmodium falciparum: Worldwide sequence diversity and evolution of the malaria vaccine candidate merozoite surface protein-2 (MSP-2). Exp Parasitol. 2007; 115(1): 32–40. PubMed Abstract | Publisher Full Text\n\nGerold P, Schofield L, Blackman MJ, et al.: Structural analysis of the glycosyl-phosphatidylinositol membrane anchor of the merozoite surface proteins-1 and -2 of Plasmodium falciparum. Mol Biochem Parasitol. 1996; 75(2): 131–43. PubMed Abstract | Publisher Full Text\n\nHamid MM, Mohammed SB, El Hassan IM: Genetic Diversity of Plasmodium falciparum Field Isolates in Central Sudan Inferred by PCR Genotyping of Merozoite Surface Protein 1 and 2. N Am J Med Sci. 2013; 5(2): 95–101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKyabayinze DJ, Karamagi C, Kiggundu M, et al.: Multiplicity of Plasmodium falciparum infection predicts antimalarial treatment outcome in Ugandan children. Afr Health Sci. 2008; 8(4): 200–5. PubMed Abstract | Free Full Text\n\nMcCarthy JS, Marjason J, Elliott S, et al.: A phase 1 trial of MSP2-C1, a blood-stage malaria vaccine containing 2 isoforms of MSP2 formulated with Montanide® ISA 720. PLoS One. 2011; 6(9): e24413. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMusapa M, Kumwenda T, Mkulama M, et al.: A simple Chelex protocol for DNA extraction from Anopheles spp. J Vis Exp. 2013; 71: pii: 3281. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMustafa SO, Abdel Hamid MM, Aboud MA, et al.: Dataset 1 in: Genetic diversity and multiplicity of Plasmodium falciparum merozoite surface protein 2 in field isolates from Sudan. F1000Research. 2017. Data Source\n\nMwingira F, Nkwengulila G, Schoepflin S, et al.: Plasmodium falciparum msp1, msp2 and glurp allele frequency and diversity in sub-Saharan Africa. Malar J. 2011; 10(1): 79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNoor AM, ElMardi KA, Abdelgader TM, et al.: Malaria risk mapping for control in the republic of Sudan. Am J Trop Med Hyg. 2012; 87(6): 1012–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOlumese P: (n.d.). FOR THE TREATMENT OF MALARIA GUIDELINES WHO Library Cataloguing-in-Publication Data. 2017. Reference Source\n\nPeyerl-Hoffmann G, Jelinek T, Kilian A, et al.: Genetic diversity of Plasmodium falciparum and its relationship to parasite density in an area with different malaria endemicities in West Uganda. Trop Med Int Health. 2001; 6(8): 607–13. PubMed Abstract | Publisher Full Text\n\nPinkevych M, Petravic J, Bereczky S, et al.: Understanding the relationship between Plasmodium falciparum growth rate and multiplicity of infection. J Infect Dis. 2015; 211(7): 1121–7. PubMed Abstract | Publisher Full Text\n\nSmith T, Beck HP, Kitua A, et al.: Age dependence of the multiplicity of Plasmodium falciparum infections and of other malariological indices in an area of high endemicity. Trans R Soc Trop Med Hyg. 1999; 93 Suppl 1: 15–20. PubMed Abstract | Publisher Full Text\n\nSnounou G, Viriyakosol S, Jarra W, et al.: Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol. 1993; 58(2): 283–92. PubMed Abstract | Publisher Full Text\n\nVafa M, Troye-Blomberg M, Anchang J, et al.: Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal: relation to transmission, age and erythrocyte variants. Malar J. 2008; 7(1): 17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO | Sudan: 2016; Retrieved December 28, 2016. Reference Source\n\nWorld Health Organization: World Malaria Report (2008–2015). 2015; Retrieved August 8, 2016. Reference Source"
}
|
[
{
"id": "37789",
"date": "05 Sep 2018",
"name": "Maria Isabel Veiga",
"expertise": [
"Reviewer Expertise Plasmodium falciparum drug resistance"
],
"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 article as it stands, although on an important topic specific for malaria research, needs further improvement to achieve sufficient scientific relevance for indexing. The work describes the diversity of a very polymorphic gene, pfmsp2 in P. falciparum in various sites around Sudan adjudicating the multiplicity of infection. They compare the genetic diversity between different regions where samples were collected as well as with patients’ characteristics.\nConsidering the Figure 2, that should represent the results for all the samples, the results are very difficult to extract, questioning the true genotyping and consequently the MOI of the samples. The nested PCR method used is very delicate and demands a good quality of agarose gel pictures, that is not the case of Figure 2. Unless all samples were run in the same gel (which is not the case), the bands from the molecular marker has to be evident to be able to evaluate the band sizes of the samples. The families IC1 and FC27 can differ of just 50 bp difference, making the resolution of the Figure 2 an impossible task to achieve. This technical issues have to be resolved for the accuracy of the data.\nIn the article should also be described the malaria epidemics in the regions where the samples were collected to understand if the number of samples herein analyzed are representative.\nThe method to identify plasmodium species is lacking the reference and is not really understood if other species were identified.\nSome of the authors of this article have published1 before submission of this manuscript a similar study on samples collected in one of the regions herein studied in the same year and the same genotyping of the msp2. They don’t mention this publication not even in the discussion section. Are some of the samples overlapping this study?\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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
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https://f1000research.com/articles/6-1790
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https://f1000research.com/articles/6-1786/v1
|
02 Oct 17
|
{
"type": "Research Article",
"title": "Could night-guards be used as a simple method to detect leached-elements from dental restorations intra-orally? A study on amalgam restorations",
"authors": [
"Rasha Mohamed Abdelraouf"
],
"abstract": "Background: Detection of leached-elements from dental restorations intra-orally has been a subject of prime importance in dental research. However, this is challenging as most of the present techniques have some limitations. In this study, a new simple method was proposed via using night-guards. Thus, the aim of the study was to verify if night-guards could detect leached-elements from restorations as dental amalgam. Methods: Ten upper custom-made night-guards were fabricated for patients suffering from bruxism, who had amalgam-restorations in their upper molars. The night-guards were delivered to the patients and they were instructed to wear the night-guards during when they were asleep. After six months, the night-guards were taken from the patients to be analyzed. A new unused night-guard was fabricated from the same material to be used as a control. In the used night-guards, two areas were studied: the fitting surfaces contacting the amalgam restorations and the fitting surfaces not contacting amalgam restorations. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Analysis (EDXA) were used to examine the structural and elemental changes in the night-guards.\n\nResults: SEM of the unused night-guard revealed a homogenous structure, and the composition was carbon and oxygen, as shown using EDXA (C=88.9wt% and O=11.1wt%). By contrast, the fitting surfaces of the night-guards contacting amalgam restorations showed numerous lustrous particles. Elemental analysis of these areas showed the presence of mercury and sulfur, in addition to carbon and oxygen (Hg=21.2wt%, S=2.5wt%, C=67.1wt% and O=9.2wt%). The night-guards’ fitting surfaces not contacting amalgam restorations showed slight cracking, and the composition was carbon and oxygen (C=88.3wt% and O=11.7 wt%). Conclusions: Analyzing fitting surfaces of night-guards contacting dental restorations, such as amalgam, could aid in understanding the nature of leached-elements from these restorations intra-orally. However, further studies about its application upon dental-restorations other than amalgam are recommended.",
"keywords": [
"Night-guard",
"Amalgam",
"Mercury",
"Elemental Analysis",
"leached Elements"
],
"content": "Introduction\n\nDetermination of leached-elements from dental restorations has been investigated as a result of exposure to oral conditions1. Several methods have been performed both in vitro and in vivo. Most of the in vitro techniques relied on storing specimens in surrounding media, such as distilled water or artificial saliva1–3. Various analytical means had been used to analyze the leached-elements, including inductively-coupled-plasma, atomic-absorption, Fourier-transform-Infrared spectroscopies and chromatographic methods1,2,4.\n\nOn the other hand, in vivo methods have been used to assess the actual situation intra-orally5. In vivo techniques have been performed either in animals or humans. Cavities are prepared in the teeth of animals, such as monkeys6, pigs7,8 or rats9, then filled by dental-restorations. The released-elements are either detected after sacrificing the animals7 or by analyzing secretory products, such as urine9 or fecal samples6.\n\nYet, measuring the released elements in humans may be best. Several studies quantified the excretion of mercury in urine, hair and nails of patients with dental amalgam-restorations and found a positive correlation10,11. In contrast, others did not find a correlation between mercury content in hair and amalgam-restoration, but mercury in hair has been correlated with fish consumption12. Thus, assessing the leached-elements in the oral-cavity may be more specific to eliminate other possible systemic sources. Thus, several studies examined released ions in patients’ saliva13,14. However, these ions may be affected by salivary secretions or washed out by swallowing. In addition, saliva collection and storage may be technique sensitive15. Thus, a new simple method was proposed in this study through using night-guards (polymeric occlusal-splints used to decrease bruxism and clenching teeth) in gathering leached-elements from dental-restorations.\n\n\nMethods\n\nTen upper dental alginate impressions (Tropicalgin, Zhermack, Italy) were taken as part of routine treatment at the Dental Family Clinic (Cairo, Egypt), between February and December 2016, for ten patients suffering from bruxism, and who also had amalgam-restorations in their upper molars. This was performed after all the patients signed written informed consent forms agreeing to participate in the study. The participants were six females and four males with the following inclusion criteria: adult (>18 years), non-periodontal-affected patients suffering from bruxism, and had amalgam restoration. Medically-compromised patients were excluded. Custom-made night-guards (example shown in Figure 1) were fabricated for each patient from 2mm soft polymeric sheets (Easy-Vac-Gasket, 3A-Medes, Korea), using a vacuum forming machine (Pro-Form, Keystone, Germany). The night-guards were delivered to the patients in the clinic after one week from first visit and they were instructed to wear the night-guards when they were asleep (≈7 hours±2). Recall visits were given to the patients in the regular check-up schedule after six months. In this visit, the night-guards were taken from the patients to be analyzed. A new unused night-guard was fabricated from the same material to be used as a control.\n\nScanning-Electron-Microscopy (SEM) and Energy-Dispersive-X-ray-Analysis (EDXA) were used to examine structural and elemental changes in the used night-guards, and compared to the unused one. In the used night-guards, two areas were studied: the fitting surfaces contacting amalgam-restorations and the fitting surfaces not contacting amalgam-restorations. The patients’ records were used to identify teeth that had restorations. Parts from these areas of interest were cut using a scissor (Singer, Germany), mounted on coded brass stubs and sputter coated with 10Å gold platinum and observed at 20000× magnification. The SEM and EDXA (Supra40, Carl-Zeiss-NTS-GmbH, Germany) were used with an accelerating voltage of 20.0–30.0kV.\n\n\nResults\n\nThe SEM of the unused night-guard revealed a homogenous structure (Figure 2), with its composition consisting of carbon and oxygen (C=88.9wt%±0.3 and O=11.1wt%±0.4; Figure 3). By contrast, the night guard surfaces contacting amalgam-restorations showed numerous lustrous particles (Figure 4). Elemental analysis of these areas showed the presence of mercury and sulfur, in addition to carbon and oxygen (Hg=21.2wt%±0.6, S=2.5wt%±0.5, C=67.1wt%±0.3 and O=9.2wt%±0.5; Figure 5). The surfaces not contacting amalgam-restorations under SEM showed slight cracking, but were still homogenous without lustrous particles (Figure 6). Their composition was carbon and oxygen (C=88.3wt%±0.6 and O=11.7wt%±0.6; Figure 7).\n\nMagnification: 20000×.\n\nMagnification: 20000×. Image representative of 10 night guards.\n\nImage representative of 10 night guards.\n\n\nDiscussion\n\nThe oral-cavity is an aggressive environment, which may affect the integrity of dental restorations16–18. It is essential to examine the resultant leached-elements to assess the degradation products of the filling materials in the oral-cavity and investigate if these materials could provoke an adverse systemic effect19.\n\nSeveral studies have examined released elements by in vitro testing through fabricating specimens and soaking them in medium1,2,20. However, simulating the oral-cavity is difficult due to its complex nature with multifactorial variables17. Thus, some studies have analyzed patients’ saliva21–23. However, saliva is composed of various components and its analysis has shown variation in methodology. In addition, contamination of saliva may occur15.\n\nAccordingly, in this study, a new technique was introduced via examining night guards contacting dental restorations. Night guards are polymeric materials designed to fit the occlusal-surfaces of patients to decrease signs and symptoms of bruxism and teeth clenching. Consequently, the study was performed by providing patients who suffered from bruxism with custom-made night guards. An additional inclusion criterion was the presence of amalgam fillings in their upper molars. Amalgam restorations were selected rather than any other dental restoration, as it has been well proven in the literature that mercury is released from such material19. Therefore, utilizing night guards in gathering leached-elements from amalgam-restorations intra-orally was evaluated in this study.\n\nBoth the unused night guard and the used surfaces not contacting amalgam-restorations were homogenous, yet the latter showed cracking, which may be due to bruxism. This consistent structure may be attributed to the following: these areas were not contacting dental restorations and no leached elements were deposited. This was confirmed by the EDXA results, which were only carbon and oxygen in both. The night-guards are polymeric materials, with their basic structure consisting of carbon, oxygen and hydrogen24. However, hydrogen was not detected as it did not have core electron, only one valence electron that enters in chemical bonding25. Analysis relied on excitation of electrons in lower shells, not the valence electron, which was shared in covalent bonding in the case of hydrogen25.\n\nThe night guard surfaces contacting amalgam restorations showed numerous lustrous particles, which were identified by EDXA as mercury. This is in agreement with numerous studies that have detected mercury released from amalgam restorations19,26. Sulfur was also detected, which may be due to tarnish and corrosion of the amalgam27.\n\nSince leached elements detected by night guards matched with that reported in the literature, night-guards could be used as a simple method to detect released elements from dental restorations such as amalgam intra-orally.\n\n\nData availability\n\nDataset 1. SEM images for night guard surfaces contacting and not contacting amalgam restorations. 10.5256/f1000research.12311.d17738328\n\nDataset 2. Raw EDXA data for night guard surfaces contacting and not contacting amalgam restorations. 10.5256/f1000research.12311.d17738429",
"appendix": "Author contributions\n\n\n\nIt should be noted that all the work, including the clinical steps, was performed by the author.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nZhou M, Drummond JL, Hanley L: Barium and strontium leaching from aged glass particle/resin matrix dental composites. Dent Mater. 2005; 21(2): 145–155. PubMed Abstract | Publisher Full Text\n\nLee SY, Greener EH, Menis DL: Detection of leached moieties from dental composites in fluids simulating food and saliva. Dent Mater. 1995; 11(6): 348–353. PubMed Abstract | Publisher Full Text\n\nGjorgievska E, Nicholson JW, Gjorgovski I, et al.: Aluminium and fluoride release into artificial saliva from dental restoratives placed in teeth. J Mater Sci Mater Med. 2008; 19(10): 3163–3167. PubMed Abstract | Publisher Full Text\n\nWataha JC, Craig RG, Hanks CT: The effects of cleaning on the kinetics of in vitro metal release from dental casting alloys. J Dent Res. 1992; 71(7): 1417–1422. PubMed Abstract | Publisher Full Text\n\nMarshall Protocol Knowledge Base: Differences between in vitro, in vivo, and in silico studies (MPKB). Autoimmun Res Found. 2014. Reference Source\n\nSummers AO, Wireman J, Vimy MJ, et al.: Mercury released from dental “silver” fillings provokes an increase in mercury- and antibiotic-resistant bacteria in oral and intestinal floras of primates. Antimicrob Agents Chemother. 1993; 37(4): 825–834. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHörsted-Bindslev P, Danscher G, Hansen JC: Dentinal and pulpal uptake of mercury from lined and unlined amalgam restorations in minipigs. Eur J Oral Sci. 1997; 105(4): 338–343. PubMed Abstract | Publisher Full Text\n\nAkyüz S, Caglar E: Pulpal uptake of mercury from lined amalgam restorations in guinea pigs. Eur J Oral Sci. 2002; 110(6): 460–463. PubMed Abstract | Publisher Full Text\n\nGalić N, Prpić-Mehiić G, Prester LJ, et al.: Elimination of mercury from amalgam in rats. J Trace Elem Med Biol. 2001; 15(1): 1–4. PubMed Abstract | Publisher Full Text\n\nGul N, Khan S, Khan A, et al.: Quantification of Hg excretion and distribution in biological samples of mercury-dental-amalgam users and its correlation with biological variables. Environ Sci Pollut Res Int. 2016; 23(20): 20580–20590. PubMed Abstract | Publisher Full Text\n\nRichardson GM, Wilson R, Allard D, et al.: Mercury exposure and risks from dental amalgam in the US population, post-2000. Sci Total Environ. 2011; 409(20): 4257–4268. PubMed Abstract | Publisher Full Text\n\nKruzikova K, Modra H, Kensova R, et al.: Mercury in human hair as an indicator of the fish consumption. Neuroendocrinol Lett. 2008; 29(5): 675–679. PubMed Abstract\n\nZheng P, Li M, Jurevic R, et al.: A gold nanohole array based surface-enhanced Raman scattering biosensor for detection of silver(i) and mercury(ii) in human saliva. Nanoscale. 2015; 7(25): 11005–11012. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFakour H, Esmaili-Sari A, Zayeri F: Scalp hair and saliva as biomarkers in determination of mercury levels in Iranian women: amalgam as a determinant of exposure. J Hazard Mater. 2010; 177(1–3): 109–113. PubMed Abstract | Publisher Full Text\n\nNunes LA, Mussavira S, Bindhu OS: Clinical and diagnostic utility of saliva as a non-invasive diagnostic fluid: a systematic review. Biochem Med (Zagreb). 2015; 25(2): 177–192. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErdemir U, Yildiz E, Eren MM, et al.: Surface hardness evaluation of different composite resin materials: influence of sports and energy drinks immersion after a short-term period. J Appl Oral Sci. 2013; 21(2): 124–131. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHengtrakool C, Kukiattrakoon B, Kedjarune-Leggat U: Effect of naturally acidic agents on microhardness and surface micromorphology of restorative materials. Eur J Dent. 2011; 5(1): 89–100. PubMed Abstract | Free Full Text\n\nOkada K, Tosaki S, Hirota K, et al.: Surface hardness change of restorative filling materials stored in saliva. Dent Mater. 2001; 17(1): 34–39. PubMed Abstract | Publisher Full Text\n\nRoberts HW, Charlton DG: The release of mercury from amalgam restorations and its health effects: a review. Oper Dent. 2009; 34(5): 605–614. PubMed Abstract | Publisher Full Text\n\nAl-Salehi SK: Effects of bleaching on mercury ion release from dental amalgam. J Dent Res. 2009; 88(3): 239–243. PubMed Abstract | Publisher Full Text\n\nMortazavi SM, Daiee E, Yazdi A, et al.: Mercury release from dental amalgam restorations after magnetic resonance imaging and following mobile phone use. Pak J Biol Sci. 2008; 11(8): 1142–1146. PubMed Abstract | Publisher Full Text\n\nGarhammer P, Hiller KA, Reitinger T, et al.: Metal content of saliva of patients with and without metal restorations. Clin Oral Investig. 2004; 8(4): 238–242. PubMed Abstract | Publisher Full Text\n\nPizzichini M, Fonzi M, Sugherini L, et al.: Release of mercury from dental amalgam and its influence on salivary antioxidant activity. Sci Total Environ. 2002; 284(1–3): 19–25. PubMed Abstract | Publisher Full Text\n\nTezuka Y, Oike H: Topological polymer chemistry. Prog Polym Sci. 2002; 27(6): 1069–1122. Publisher Full Text\n\nStojilovic N: Why can’t we see hydrogen in X-ray photoelectron spectroscopy? J Chem Educ. 2012; 89(10): 1331–1332. Publisher Full Text\n\nBengtsson UG, Hylander LD: Increased mercury emissions from modern dental amalgams. Biometals. 2017; 30(2): 277–283. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFathi Phd M, Mortazavidds V: A Review on Dental Amalgam Corrosion and Its Consequences. J Res Med Sci. 2004; 1: 42–51. Reference Source\n\nAbdelraouf R: Dataset 1 in: Could night-guards be used as a simple method to detect leached-elements from dental restorations intra-orally? A study on amalgam restorations. F1000Research. 2017. Data Source\n\nAbdelraouf R: Dataset 2 in: Could night-guards be used as a simple method to detect leached-elements from dental restorations intra-orally? A study on amalgam restorations. F1000Research. 2017. Data Source"
}
|
[
{
"id": "26560",
"date": "11 Oct 2017",
"name": "Sabry A. El-Korashy",
"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\nDifferent methods are used in determination of the leached-elements from dental restorations intra-orally are reported. Some of these methods have been performed both in vitro and vivo, using different analytical techniques. They were carried out via surrounding media, such as distilled water or artificial saliva. However, these methods still having some reservations and almost take a great effort. In the present article, the author used very simple and new technique for measuring the leached elements from amalgam restorations via fabricated polymeric night guards for the tested patients suffering from bruxism. The concentrations of the leached elements after removing the night guards were measured using SEM with EDAX spectroscopy.\n\nIn my opinion, the article is accepted for publication after answering the following questions:\nThe results of EDAX (Fig. 5) have detected the Hg, O, S and Ca elements. The author did not address the sources of sulfur and calcium where they came from?\n\nIt is known that the amalgam contains some other metals such as copper and silver and yet none of them appeared in the analysis. Why?\n\nIt is well known that hydrogen has no spectrum in analysis by using EDAX technique and hence the paragraph in the discussion section (page 6) begins with\" However, hydrogen was not detected……..….. case of hydrogen 25\" must be deleted.\n\nIn the future, the author must do the same study on patients who do not have bruxism for comparison.\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": "28198",
"date": "07 Dec 2017",
"name": "Brian W. Darvell",
"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\nOn the face of it, the concept has some attraction, but it is predicated on something unstated: that the mouthguard material will adsorb any ions arising from the amalgam. Here we have the first problem: dissolution is not leaching, so the premise is faulty. Secondly, there is no evidence that such adsorption could occur - certainly it is unlikely but it is, more to the point, untested. Where is the positive control? However, what is actually detected, it would seem, are particulates (apparently HgS, but not specifically demonstrated). These are either the result of mechanical action, which seems likely, embedding them in the polymer surface, or the result of reaction with mercury ions forming a precipitate on the polymer. The latter would be very serious in its implications, and for which I know of no precedent. The fact that the chemistry is not considered in any way is major deficiency. (The naïvety of the remarks in para.4 of the Discussion suggests a lack of such comprehension.)\nAt the very least, elemental mapping should have been used, identifying the restoration borders carefully and looking for migration beyond them. Particulates should have been studied in more detail, but the experiment fails to achieve what it sets out to do, but it seems that there is no comprehension of this or why. For example, was the sulphur already on the amalgam surface as sulphide (which is likely) and simply transferred? I can think of two ways of testing this - both very simple. But, without this information, we have no idea what is going on. Indeed, why is there no silver?\n\nThe English is pretty poor, with failures of logic and sense in many places. At the very least it should have been copy-edited by a native English speaker versed in technical writing before submission. It is in the best interests of all authors that this be done where English is not the first language. It would save referees an awful lot of trouble, and authors a lot of time.\nScientifically, this paper is below the standard required in several respects. Division of the content into the proper sections is required. However, the ‘review’ section is of no value, and the discussion completely empty except for repetition of introductory material and other material of no value.\nWith some sensible direction, something useful might have been achieved with the basic idea, but this has not occurred.\n\nSome few comments. A comprehensive list would take too long.\nAbstract\nverify if >> verify whether could detect : logically wrong - night-guards cannot detect anything leached-elements >> leached elements restorations as >> restorations such as during when >> while analyzed >> analysed (the z is always illiterate) homogenous >> homogeneous the composition was carbon and oxygen : no ... wt% >> mass%\n\nIntroduction\nSeveral studies ... : there is no trace here of a critical review so the value of the various reports cannot be assessed or the present attempt actually justified.\n\nMethods\npolymeric sheets : what material? instructed to wear the night-guards when they were asleep : ... while ... 10Å >> SI equivalent, please, and with a space between value and symbol: but does this not obscure the test surface and prevent elemental detections? homogenous >> homogeneous\n\nDiscussion\npara 1: >> introduction para 2: repetition, delete. para 3: repetition! para 4: This was confirmed ... : no, this is the only evidence you have, the assertion does not count. The remainder of this paragraph is padding. para 5: results, delete. para 6: is the totality of the discussion? Actually, a conclusion, but very weak.\n\nFig. 2 and 6 are near pointless - the contrast is bad. There is no point in Figures 3, 5, and 7 - this is raw data.\nFig. 4 : elemental maps for Hg and S (as well as Ag, Sn, Cu, if ever detected locally) would make more sense. “Lustrous” is quite inappropriate for a description from an SEM image - this character is not detectable.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1786
|
https://f1000research.com/articles/5-2532/v1
|
18 Oct 16
|
{
"type": "Case Report",
"title": "Case Report: Whole exome sequencing identifies variation c.2308G>A p.E770K in RAG1 associated with B- T- NK+ severe combined immunodeficiency",
"authors": [
"Geeta Madathil Govindaraj",
"Shamsudheen Karuthedath Vellarikkal",
"Rijith Jayarajan",
"Rowmika Ravi",
"Ankit Verma",
"Krishnan Chakkiyar",
"Machinari Puthenpurayil Jayakrishnan",
"Riyaz Arakkal",
"Revathi Raj",
"Sridhar Sivasubbu",
"Vinod Scaria",
"Geeta Madathil Govindaraj",
"Shamsudheen Karuthedath Vellarikkal",
"Rijith Jayarajan",
"Rowmika Ravi",
"Ankit Verma",
"Krishnan Chakkiyar",
"Machinari Puthenpurayil Jayakrishnan",
"Riyaz Arakkal",
"Revathi Raj"
],
"abstract": "Severe combined immunodeficiency is a large clinically heterogeneous group of disorders caused by a defect in the development of humoral or cellular immune responses. At least 13 genes are known to be involved in the pathophysiology of the disease and the mutation spectrum in SCID have been well documented. The widespread application of whole-exome sequencing based on next-generation sequencing has offered a new opportunity to systematically screen these genes in clinical scales. In this report, we describe the application of whole exome sequencing for arriving at a molecular diagnosis in a child suffering from B- T- NK+ severe combined immunodeficiency. Apart from making the accurate molecular diagnosis, we also add a genetic variation c.2308G>A p.E770K to the compendium of variations associated with the disease.",
"keywords": [
"Severe Combined Immunodeficiency",
"B- T- NK+ SCID",
"Whole Exome Sequencing",
"RAG1"
],
"content": "Introduction\n\nSevere combined immunodeficiency (SCID) encompasses a constellation of clinically and genetically heterogeneous diseases resulting in defects of the humoral and/or cellular immune defence mechanism1. Accurate molecular diagnosis of the disease is of prime importance, not only in offering appropriate genetic counselling, but also in understanding the exact molecular defect and would potentially enable prenatal screening2. The accurate molecular diagnosis would provide a fresh opportunity to enable cost-effective screening of family members and offer appropriate genetic counselling especially for populations where there is a high degree of consanguinity3,4. So far, arriving at a precise molecular diagnosis has been quite cumbersome, technically challenging and expensive as over a dozen genes are known to be implicated in the disease, which would require systematic targeted sequencing of each of the gene5,6. The advent of next generation sequencing, especially whole exome and sometime whole genome sequencing has significantly enabled the rapid identification of the causative genetic variations in clinical settings6.\n\nIn the present report, we describe the application of whole exome sequencing for the accurate molecular diagnosis of a case of B- T- NK+ SCID. Our report also adds a genetic variation c.2308G>A p.E770K to the compendium of variations associated with the disease.\n\n\nCase report\n\nHere we report a case of a seven-month-old boy, born out of a third degree consanguineous marriage, with a history of recurrent episodes of pneumonia, acute otitis media, diarrhea and oral thrush since two months of age. The child was pale, emaciated, febrile, and had respiratory distress with lower chest retractions. He was in compensated shock. There was no clubbing, cyanosis or lymphadenopathy. There was no facial dysmorphism and skin and hair were normal. He weighed 5.4 kg; measured 64 cm in length and head circumference was 39.5 cm. Examination of the chest showed evidence of bronchopneumonia while there was no evidence of congenital heart disease or neurological deficits. There was mild hepatomegaly with a liver span of 6.5 cm.\n\nThe baby was normal in the perinatal and postnatal period. His birth weight was normal (3.04 kg) and was asymptomatic until 2 months of age. There was a history of admission to PICU and artificial ventilation for severe pneumonia at the age of 2 months. He was admitted for 22 days during that episode. The child had gross motor developmental delay and no adverse events following immunization. He had a male sibling who expired at seven months of age due to persistent pneumonia and two unaffected female siblings, apart from a half-brother and half-sister both of whom were asymptomatic (Figure 1A).\n\n(a) Pedigree of the family (b) domain mapping of the RAG1 p.E770K on RAG1 protein showing the variation lies on RAG1 domain highlighted with red triangle and (c) capillary sequencing of the locus in the proband and family members. The homozygous variation c.2308G>A in the proband is marked with an asterisk.\n\nOn investigation, the child was found to have hypochromic microcytic anemia, lymphocytopenia with absolute lymphocyte counts less than 1000/cu.mm and a normal platelet count. Liver and renal function tests were normal. The ionized calcium was 1.1 mg%. Blood culture was positive for Enterobacter species. The baby was negative for HIV infection and there was no evidence of tuberculosis. His chest X-Ray showed absence of the thymus shadow apart from evidence of bronchopneumonia.\n\nA close workup of the immunoglobulin profile revealed hypogammaglobulinemia-IgA 23 mg%, IgG 44 mg% and IgM 26 mg%. IgE was 1 IU/L. The absolute CD3 count was 464 cells/ul (normal range 1,460–5,440 cells/ul), absolute CD19 lymphocyte count was 12 cells/ul (normal 430–3,300 cells/ul) and absolute NK cell count was 1,328 cells/ul (normal 80–340 cells/ul). Flow cytometry suggested absent B and markedly reduced T cell populations suggestive of B- T- NK+ SCID.\n\nThe child was treated with piperacillin (80mg/kg/dose Q8H), vancomycin (15 mg/kg/dose Q6H), dopamine (10 mic/kg/min), IVIG and other supportive measures and was put on cotrimoxazole (6 mg/kg/day OD) prophylaxis. He was treated with ganciclovir for CMV infection and for staphylococcal pneumonia.\n\nThe clinical diagnosis of SCID and family history of sibling death prompted us to investigate the molecular genetic correlates of the disease. Since over 13 genes are implicated in SCID and regular molecular testing was not readily available for the genes, we resorted to whole exome sequencing. After obtaining informed consent from the parents, blood was drawn after venipuncture under aseptic precautions. DNA was isolated from whole blood using salting out method7. Exome capture was performed on DNA using the Illumina Nextera rapid capture expanded exome kit using standard protocols (Illumina Inc USA). We generated 47.95 million paired end reads and an average on target coverage of over 25x on Illumina HiSeq 2500 (Illumina Inc. USA). Alignment was performed using BWA (v0.7.12-r1039)8 and Stampy (v1.0.20)9 and variants were called using Platypus (v0.8.1)10. For the prioritisation of variants, we filtered all homozygous variants, further filtered by an allele frequency of <1% in the 1000 Genome and Exac. Variants in the 13 genes were prioritised and annotated for their deleteriousness using SIFT, Polyphen and Mutation Taster annotations obtained from annovar11.\n\nAnalysis revealed a homozygous missense variation (c.2308G>A) in exon number 2 of Recombination activating gene 1 (RAG1). The variant was predicted to be highly deleterious by SIFT (score 0.000), PolyPhen2 (0.991) and Mutation Taster (1.00). The variation causes an amino acid change p.E770K, which lies on RAG1 domain of the protein (Figure 1b). The present variation was not found in the 1000 Genome (http://browser.1000genomes.org/index.html), Exac (http://exac.broadinstitute.org/) or internal control database of over 150 exomes from South East Asian ancestry. Incidentally the mutation was previously reported and analysis suggested a significantly reduced recombination activity12.\n\nThe variation was further confirmed using targeted PCR amplification around the locus and confirmed by capillary sequencing. The variant was found to be heterozygous in both the parents as well as the surviving siblings (Figure 1c). The status of the variation could not be ascertained in the sibling who died because no sample was archived and primary immune deficiency was not suspected at the time.\n\n\nDiscussion\n\nMutations in RAG1 gene cause various degrees of severe combined immunodeficiency syndrome. RAG1 is involved in the V(D)J recombination1,13. The child was suspected to have a primary immune deficiency disorder since he had unusually frequent and severe infections and in addition had lost a male sibling due to similar illness. Further, he was born to third degree consanguineous parents. The early onset of symptoms by 2 months of life with increased susceptibility to both bacterial and fungal infections was a pointer to a T cell defect or a phagocytic defect rather than to an antibody deficiency like X linked agammaglobulinemia, which usually presents by 5 to 6 months of age, when maternal antibodies are on the wane14. The immunoglobulin profile showed that there was also a B cell defect. The low absolute lymphocyte counts coupled with radiological evidence of an absent thymus shadow was proof of a T cell defect as well. Thus, a provisional diagnosis of a severe combined immunodeficiency was made even before the flow cytometry results became available and helped confirm the diagnosis.\n\nThe possibility of Omenn syndrome was not considered since there was no history of a rash and there was no lymphadenopathy or hepatosplenomegaly, nor was there eosinophilia in the peripheral smear. XR SCID is characterized by an elevated percentage of B cells and in the absence of B cells in the child ruled this out. Jak3 deficiency was also not thought of for the same reason. ADA deficient SCID is characterized by bony abnormalities including rib cage defects, which were absent.\n\nRAG 1 or RAG 2 deficiencies are associated with a lack of both B cells and T cells and NK cells are predominant in the circulation13. With this possibility in mind, and with a view to offer genetic counselling to the family, whole exome sequencing was considered. The child was referred for a bone marrow transplant, since SCID is not compatible with life beyond infancy. The patient underwent a matched sibling donor bone marrow transplant at the age of 1 year and 3 months. Post-transplant, he developed hypertension and later developed septic shock, which were managed successfully. He is now one year three months post-transplant and off all medications including immunosuppressive therapy.\n\n\nEthics approval\n\nThe whole exome sequencing was approved by the Institutional Ethical Committee of CSIR - Institute of Genomics and Integrative Biology (IHECC proposal number 8).\n\n\nConsent\n\nWritten informed consent for publication of the patients’ details and/or their images was obtained from the patients/parents of the patient.\n\n\nData availability\n\nAll the raw sequencing data are available at the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra), accession number SRR4088561.",
"appendix": "Author contributions\n\n\n\nGG, CK, MPJ, RA and RR clinically evaluated and characterised the patient. SKV, RJ, RR and AV performed the whole exome sequencing, computational analysis, data interpretation and validation experiments. SS and VS oversaw all the experiments and data interpretation. VS, SKV, GG and SS contributed towards writing the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nSS and VS acknowledge funding from the Council of Scientific and Industrial Research (CSIR) India through Grant BSC0212.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgment\n\nAuthors acknowledge support from the GUaRDIAN consortium.\n\n\nReferences\n\nFischer A: Severe combined immunodeficiencies (SCID). Clin Exp Immunol. 2000; 122(2): 143–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmeratunga R, Woon ST, Neas K, et al.: The clinical utility of molecular diagnostic testing for primary immune deficiency disorders: a case based review. Allergy Asthma Clin Immunol. 2010; 6(1): 12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKingsmore S: Comprehensive carrier screening and molecular diagnostic testing for recessive childhood diseases. PLoS Curr. 2012; 4: e4f9877ab8ffa9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKingsmore SF, Lantos JD, Dinwiddie DL, et al.: Next-generation community genetics for low- and middle-income countries. Genome Med. 2012; 4(3): 25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang W, Cui H, Wong LJ: Application of next generation sequencing to molecular diagnosis of inherited diseases. Top Curr Chem. 2014; 336: 19–45. PubMed Abstract | Publisher Full Text\n\nSawyer SL, Hartley T, Dyment DA, et al.: Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care. Clin Genet. 2016; 89(3): 275–84. PubMed Abstract | Publisher Full Text\n\nMiller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988; 16(3): 1215. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14): 1754–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLunter G, Goodson M: Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 2011; 21(6): 936–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRimmer A, Phan H, Mathieson I, et al.: Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications. Nat Genet. 2014; 46(8): 912–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang K, Li M, Hakonarson H: ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010; 38(16): e164. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAsai E, Wada T, Sakakibara Y, et al.: Analysis of mutations and recombination activity in RAG-deficient patients. Clin Immunol. 2011; 138(2): 172–7. PubMed Abstract | Publisher Full Text\n\nBuckley RH: Molecular defects in human severe combined immunodeficiency and approaches to immune reconstitution. Annu Rev Immunol. 2004; 22: 625–55. PubMed Abstract | Publisher Full Text\n\nShearer WT, Dunn E, Notarangelo LD, et al.: Establishing diagnostic criteria for severe combined immunodeficiency disease (SCID), leaky SCID, and Omenn syndrome: the Primary Immune Deficiency Treatment Consortium experience. J Allergy Clin Immunol. 2014; 133(4): 1092–8. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "17720",
"date": "16 Nov 2016",
"name": "Paola Itliani",
"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 “Whole exome sequencing identifies variation c.2308G>A p.E770K in RAG1 associated with B- T- NK+ severe combined immunodeficiency” is the description of a case report, that is a child as a supposed case of SCID. Exactly, the authors found a homozygous variation c.2308G>A by whole exome sequencing and they hypothesized that this variant is associated with the disease. All the sections of the manuscript are well presented and written (title, abstract, content and discussion).\nHowever, there are two points that I would like to comment:\nIn the introduction the authors wrote that there are over a dozen genes known to be implicated in the disease. So, what about these genes in this case report? By performing a whole exome sequencing, did they also observed the variants of these genes? if yes or not, please comment!\n\nIn order to explain the functional consequences of the mutated protein, the authors write “The variation causes an amino acid change p.E770K, which lies on RAG1 domain of the protein……Incidentally the mutation was previously reported and analysis suggested a significantly reduced recombination activity” referring to work of Asai E et al. Clin Immunol. 2011. In Asai’s work the nucleotide mutation is referred as 2420 G>A with a E770K effect. The position of nucleotide between the two works is different, but the authors of the manuscript declare the same position for amino acid change and the same reduced recombination activity. Please, specify/insert the RS ID number of the homozygous variation.",
"responses": [
{
"c_id": "2915",
"date": "02 Oct 2017",
"name": "Vinod Scaria",
"role": "Author Response",
"response": "The manuscript “Whole exome sequencing identifies variation c.2308G>A p.E770K in RAG1 associated with B- T- NK+ severe combined immunodeficiency” is the description of a case report, that is a child as a supposed case of SCID. Exactly, the authors found a homozygous variation c.2308G>A by whole exome sequencing and they hypothesized that this variant is associated with the disease. All the sections of the manuscript are well presented and written (title, abstract, content and discussion).However, there are two points that I would like to comment:1. In the introduction the authors wrote that there are over a dozen genes known to be implicated in the disease. So, what about these genes in this case report? By performing a whole exome sequencing, did they also observed the variants of these genes? if yes or not, please commentClarification 1: We have observed the variations in the genes, which are previously reported with primary immunodeficiency. The prioritized variations are shown in table uploaded in answer to reviewer’s comments. (Sent to editor while uploading revised version). Among the variations, the RAG1:c.2308G>A:p.E770K variation was prioritised due to pathogenic and rarity of variation. 2. In order to explain the functional consequences of the mutated protein, the authors write “The variation causes an amino acid change p.E770K, which lies on RAG1 domain of the protein……Incidentally the mutation was previously reported and analysis suggested a significantly reduced recombination activity” referring to work of Asai E et al. Clin Immunol. 2011. In Asai’s work the nucleotide mutation is referred as 2420 G>A with a E770K effect. The position of nucleotide between the two works is different, but the authors of the manuscript declare the same position for amino acid change and the same reduced recombination activity. Please, specify/insert the RS ID number of the homozygous variation.Clarification 2: In the original publication by Asai et al. 2011, the nucleotide position is mentioned as c.2420G>A. While the position of amino acid is clear from the publication, we are not sure if nucleotide position denotes the position in cDNA or other. In our case, we mentioned the cDNA change as c.2308G>A and amino acid change as p.E770K. We clearly indicated the change in cDNA and it is also the reported nomenclature from database.For reference, Cosmic mutation ID: COSM3447065.The rsID for mutation is rs768260595."
}
]
},
{
"id": "19588",
"date": "31 Jan 2017",
"name": "David K. Buchbinder",
"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 provide a case report of a patient with severe combined immunodeficiency secondary to RAG deficiency. Whole exome sequencing was utilized to ensure a rapid and accurate diagnosis. Overall, the report is well written. Despite this there are some minor changes that should be considered. I also do not appreciate the novelty of this report and what it adds to the existing literature. The clinical diagnosis of severe combined immunodeficiency is clear. The routine use of next generation sequencing approaches are widely used to provide accurate and rapid diagnoses.\n\nIntroduction\nPage 3: I would consider focusing the introduction on the topic of RAG deficiency.\n\nPage 3: I would also note that an accurate and timely diagnosis is vital to the provision of life-saving therapy.\n\nPage 3: At the end of the first paragraph the word “sometime” is missing an “s”.\n\nPage 3: In the last paragraph of the introduction you talk about a genetic variation E770K. A genetic variation in what gene? Please state RAG1.\n\nCase Report\nPage 3: When talking about the patient weight, height, and head circumference I would add in percentiles.\n\nPage 3: Please define the abbreviation “PICU”.\n\nPage 3: I would review the units on measurements. I would consider using SI units. For example, please review the use of “mg%”. I would address this issue throughout the manuscript.\n\nPage 3: How was HIV and tuberculosis excluded?\n\nPage 3: Is chest X-Ray correct?\n\nPage 3: Was there any evidence of eosinophilia?\n\nPage 4: For the immunological evaluation - were antibody responses assessed? Were RA/RO populations assessed? Were T cell responses to mitogens assessed? Was TCR diversity assessed? Was maternal engraftment assessed?\n\nPage 4: Please define the abbreviation “IVIG”.\n\nPage 4: How was the CMV infection identified? Where was the “infection”? How was staph pneumonia identified?\n\nPage 4: I would consider adding a “Methods” section where you talk about the whole exome techniques used and confirmatory sequencing. I don’t really like how it is right in the middle of the case report.\n\nPage 4: In the 4th paragraph I would italicize “RAG1”. This should be addressed throughout the manuscript.\n\nPage 4: You mention that the mutation was previously reported. This detracts a bit from the novelty. I would also consider noting the RAG activity that was assessed in-vitro from the citation.\n\nDiscussion\nPage 4: You fail to use the abbreviation “SCID” in the first paragraph of the discussion which you defined earlier.\n\nPage 4: Please define the abbreviations “XR, Jak3, and ADA”.\n\nPage 4: I would include the portion about the transplant course in the case report section and not at the end of the discussion. I would add in additional details as well - what type of conditioning was used? Graft-versus-host-disease prophylaxis? What was the etiology of the hypertension? PRES? What was the organism isolated during sepsis? Any other post-transplant issues such as GVHD, VOD, etc.? Was engraftment assessed (i.e. lineage specific chimerism)?",
"responses": [
{
"c_id": "2914",
"date": "02 Oct 2017",
"name": "Vinod Scaria",
"role": "Author Response",
"response": "The authors provide a case report of a patient with severe combined immunodeficiency secondary to RAG deficiency. Whole exome sequencing was utilized to ensure a rapid and accurate diagnosis. Overall, the report is well written. Despite this there are some minor changes that should be considered. I also do not appreciate the novelty of this report and what it adds to the existing literature. The clinical diagnosis of severe combined immunodeficiency is clear. The routine use of next generation sequencing approaches are widely used to provide accurate and rapid diagnoses.Introduction Page 3: I would consider focusing the introduction on the topic of RAG deficiency. Clarification: Suggestions have been incorporated. Introduction is added with focus on RAG1 deficiency. Page 3: I would also note that an accurate and timely diagnosis is vital to the provision of life-saving therapy. Clarification: Suggestions have been incorporated. Page 3: At the end of the first paragraph the word “sometime” is missing an “s”. Clarification: Corrections has been incorporated. Page 3: In the last paragraph of the introduction you talk about a genetic variation E770K. A genetic variation in what gene? Please state RAG1. Clarification: Suggestions have been incorporated.Case Report Page 3: When talking about the patient weight, height, and head circumference I would add in percentiles. Clarification: His weight, length and head circumference were below the 3rd centile as per WHO Child Growth Standards. This is updated in the manuscript. Page 3: Please define the abbreviation “PICU”. Clarification: Suggestions have been incorporated. Page 3: I would review the units on measurements. I would consider using SI units. For example, please review the use of “mg%”. I would address this issue throughout the manuscript. Clarification: Ionized calcium was 0.28 mmol/L. Page 3: How was HIV and tuberculosis excluded? Clarification: The child’s mother was HIV ELISA negative and the child had a negative Mantoux test and negative gastric acid AFB stain. Page 3: Is chest X-Ray correct? Clarification: Lateral view of the Chest X Ray. Page 3: Was there any evidence of eosinophilia? Clarification: There was no evidence of eosinophilia Page 4: For the immunological evaluation - were antibody responses assessed? Were RA/RO populations assessed? Were T cell responses to mitogens assessed? Was TCR diversity assessed? Was maternal engraftment assessed? Clarification: Antibody responses, RA/RO populations, responses to mitogens, TCR diversity and maternal engraftment were not assessed. Page 4: Please define the abbreviation “IVIG”. Clarification: Suggestions have been incorporated. Page 4: How was the CMV infection identified? Where was the “infection”? How was staph pneumonia identified? Clarification: CMV infection was identified by DNA PCR. The child had disseminated CMV infection. Staph. pneumonia was identified by characteristic radiological findings and positive blood culture. Page 4: I would consider adding a “Methods” section where you talk about the whole exome techniques used and confirmatory sequencing. I don’t really like how it is right in the middle of the case report. Clarification: Suggestions have been incorporated. Page 4: In the 4th paragraph I would italicize “RAG1”. This should be addressed throughout the manuscript. Clarification: Suggestions have been incorporated. Page 4: You mention that the mutation was previously reported. This detracts a bit from the novelty. I would also consider noting the RAG activity that was assessed in-vitro from the citation. Clarification: We are not claiming that the mutation is novel. We are pointing that the variation is so rare that it is reported only once. In-vitro assay for RAG activity from citation is noted down in manuscript.Discussion Page 4: You fail to use the abbreviation “SCID” in the first paragraph of the discussion which you defined earlier. Clarification: Suggestions have been incorporated. Page 4: Please define the abbreviations “XR, Jak3, and ADA”. Clarification: Suggestions have been incorporated. Page 4: I would include the portion about the transplant course in the case report section and not at the end of the discussion. I would add in additional details as well - what type of conditioning was used? Graft-versus-host-disease prophylaxis? What was the etiology of the hypertension? PRES? What was the organism isolated during sepsis? Any other post-transplant issues such as GVHD, VOD, etc.? Was engraftment assessed (i.e. lineage specific chimerism). Clarification: Suggestion have been incorporated. The hypertension is an inadvertent typographical error. It should read ‘hypotension’. There was no issue of GVHD or VOD."
}
]
},
{
"id": "20355",
"date": "20 Feb 2017",
"name": "Lennart Hammarström",
"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\nGovindaraj et al. report a typical SCID patient with a known mutation in Rag1 (p.E770K) with a previously known defective recombination activity and thus, there is very little novel information in this manuscript. In fact, the manuscript is already in itself published in F1000 (October 18th, 2016) and available on the web. The mutation should simply be added to existing databases.\nAdditional comments:\nThe figure should be improved – providing the different domains. The pedigree (inheritance pattern is incomplete) - or simply state that the missing individuals were not sampled. Introduction should be more focussed on Rag1. Immunization schedule (if any) for live vaccines could be of interest for the readers (but not necessary). In the methods section, uniformity should apply (cc, mm ul etc). The number of NK cells appear to be too high (higher than the total lymphocyte count). Gene names should be in italics and transcript IDs should be given. Abbreviations should be correctly used and introduced at the first time of presentation.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-2532
|
https://f1000research.com/articles/5-2690/v1
|
17 Nov 16
|
{
"type": "Research Article",
"title": "Monitoring the compliance of the academic enterprise with the Fair Labor Standards Act",
"authors": [
"Adriana Bankston",
"Gary S. McDowell",
"Adriana Bankston"
],
"abstract": "Background: On December 1 2016, the Fair Labor Standards Act (FLSA) will be updated by the U.S. Department of Labor. The key changes are an increase in the salary threshold for exemption from overtime for working more than 40 hours per week, and indexing the salary level so that it is updated automatically every 3 years. This update is predicted to have a profound effect on the academic enterprise as a large proportion of the postdoctoral researcher population is currently paid at a salary below the new threshold for exemption. Here we review the key changes to the FLSA, how they came about, and how the postdoctoral population is affected by the ruling. Methods: We describe recent data collection efforts (checking university websites and contacting HR departments) to uncover what institutions in the 2014 NSF Survey of Graduate Students and Postdoctorates in Science and Engineering are doing to comply with the FLSA ruling for postdocs. Results: Our data show that 41% of the estimated postdoctoral workforce in STEM and 57% of institutions checked have not decided or have no public decision yet available one month prior to implementation, and only 35.5% of institutions are planning to raise salaries to the new minimum. Conclusions: Our data show the uncertainty of postdoc salaries in the U.S. one month prior to implementation of the FLSA ruling. This implementation also gives rise to various issues that have arisen in an already strained research enterprise, including short-, medium- and long-term effects on academe.",
"keywords": [
"FLSA ruling",
"postdoctoral salary",
"research enterprise",
"science policy",
"biomedical research",
"research funding",
"academic labor",
"institutional policy"
],
"content": "Background\n\nThe Fair Labor Standards Act (FLSA) (https://www.dol.gov/WHD/overtime/final2016/) establishes standards such as minimum wage and overtime pay for employees in both the public and private sectors in the United States. Through the FLSA a minimum wage and overtime pay (for working more than 40 hours per week) at 1.5 times the employee's regular rate are guaranteed (United States Department of Labor, 2016a).\n\nOn December 1 2016, the FLSA will be updated by the U.S. Department of Labor (DOL). One key change is an increase in the salary threshold for exemption from overtime pay from the 2004 level of $23,660 to $47,476. The other key change is indexing the salary level so that it is updated automatically every 3 years pegged to the 40th percentile of full-time salaried workers in the lowest-wage Census region, estimated by the DOL to be $51,168 in 2020. We first describe the timeline of how these updates were decided, and how they affect the postdoctoral researcher population.\n\nOn March 13 2014, a memorandum (https://www.whitehouse.gov/the-press-office/2014/03/13/presidential-memorandum-updating-and-modernizing-overtime-regulations) was issued by the White House from U.S. President Barack Obama to Secretary of Labor Thomas Perez, instructing the Department of Labor to investigate updating and modernizing current overtime regulations:\n\n“I hereby direct you to propose revisions to modernize and streamline the existing overtime regulations. In doing so, you shall consider how the regulations could be revised to update existing protections consistent with the intent of the Act; address the changing nature of the workplace; and simplify the regulations to make them easier for both workers and businesses to understand and apply (Obama, 2014).”\n\nOn July 6 2015, the Department of Labor issued a “Notice of Proposed Rulemaking,” soliciting feedback by September 4 2015. The notice proposed increasing the current exemption salary of $23,660, set in 2004, to $50,440 in 2016, with automatic updates to the level every 3 years (Obama, 2015; United States Department of Labor, 2016b).\n\nOn May 18 2016, the Secretary of Labor, Thomas Perez, gave notice of the final decision on the updates to overtime regulations in the FLSA. The exemption salary would be set at $47,476 (lower than the $50,440 originally proposed) with updates every 3 years determined by future wage growth (United States Department of Labor, 2016f). The date for implementation was set as December 1 2016. Therefore, 2 years after the first indication of a change to overtime regulations, and just under a year from the indication of what those changes were likely to be, an additional 6 months allowance was made to prepare for compliance with the new rule.\n\nAs of Nov 1 2016, ongoing efforts to delay implementation of the new rule included H.R.6094, the Regulatory Relief for Small Businesses, Schools, and Nonprofits Act, which passed the U.S. House of Representatives on September 28 2016 by 246 votes to 177. It was then passed to the Senate on September 29 2016 (Congress of the United States of America, 2016). An emergency motion for preliminary injunction was also filed by 21 States (Anon, 2016).\n\nThe changes to the FLSA proposed on July 6 2015 stood to make a large impact on higher education. The large rise in the salary threshold for exemption had the potential to affect a wide range of workers in academe. As stated by the College and University Professional Association for Human Resources (CUPA-HR), affected employees could include: “librarians; advisers; counselors; residence hall managers; admissions counselors; financial aid counselors’ student activities officers; human resources professionals and trainers; accountants; head cashiers; textbook managers; ticket managers; alumni relations; fundraising professionals; head of mail services; farm managers; information technology professionals; research and clinical professionals (including many with advanced degrees and those engaged in advanced training such as postdocs); managers in food service, security and building and grounds. Many of these jobs have always been and are well suited to exempt status (CUPA-HR, 2015a).”\n\nA concerted effort was therefore made to reduce the potential impact of FLSA changes on higher education. A letter to the Department of Labor was coordinated by CUPA-HR on behalf of 18 higher education organizations (CUPA-HR, 2015a; CUPA-HR, 2015b). Key recommendations made in the letter were: 1) the Department of Labor providing a longer time to adjust to the changes; 2) proposed lower salary level options of: $29,172, $30,004 or $40,352; and 3) rephrasing language to specifically exempt postdocs based on their “trainee” status in a similar manner to medical residents.\n\nSimilarly, the Association of American Medical Colleges (AAMC) submitted a letter supporting this position and adding:\n\n“Any increase in the salary threshold for exemption should be graduated and incremental. AAMC recommends an initial threshold that does not exceed the National Institutes of Health (NIH) guidelines for postdoctoral stipends, currently set at $42,840 for new trainees in [fiscal year] 2015. In addition, postdoctoral scientists should be considered salaried, FLSA-exempt “learned professionals,” similar to medical residents (AAMC, 2015).”\n\nOn the other hand, in addition to postdocs themselves commenting on the ruling (Wexler, 2016), on May 10 2016 four unions representing postdocs or higher education employees (American Federation of State, County and Municipal Employees; Service Employees International Union; the United Auto Workers and the National Education Association) met with the Department of Labor to argue against institutional calls for exemption for postdoctoral researchers (Penn, 2016).\n\nAttempts to exempt postdocs and to push for an exemption threshold below current postdoctoral salary levels were unsuccessful. With the announcement of updates to the FLSA, there was a simultaneous announcement that postdocs would not be exempt and would in fact be targeted by the ruling, discussed in the article co-authored by Director of the NIH Francis Collins and the Secretary of Labor Thomas Perez, “Fair Pay for Postdocs: Why We Support New Federal Overtime Rules (Collins & Perez, 2016).”\n\nEmployees must meet a series of tests in order to be exempt from overtime payments. First, they must be paid on a salary basis and not an hourly basis, by the “salary basis test.” Second, their salary must meet the minimum salary threshold of $913 per week or $47,476 annually, by the “salary level test” (which does not apply to doctors, lawyers or teachers). Finally, the employee’s primary job duty must pass the “standard duties test”. The duties test is either an executive exemption (e.g. managing a department), an administrative exemption (e.g. being in a primarily clerical role), or a professional exemption, such as that of a postdoc. Unless all 3 tests are passed, the employee is eligible for overtime payment.\n\nThe Department of Labor issued a summary of the impact that updates to the FLSA have on higher education (United States Department of Labor, 2016e) and guidance for higher education on compliance with the FLSA ruling (United States Department of Labor, 2016c). Limits to the impact of this ruling include exemptions for those who are in primarily teaching roles (such as adjunct faculty) and students (including undergraduate and graduate students) earning degrees. However, technical staff who are primarily carrying out benchwork and not clerical work are likely affected by the new ruling.\n\nFrom this point, we will focus particularly on postdoctoral researchers in Science, Engineering and Health, as this population has been the focus of our data collection efforts. However all postdocs (in Science, Technology, Engineering and Math (STEM) disciplines, as well as in humanities and social sciences) primarily engaged in research (and not teaching) at an U.S. institution regardless of visa status and salary source are affected by this ruling as follows:\n\n“Postdoctoral fellows are employees who conduct research at a higher education institution after the completion of their doctoral studies. Postdoctoral fellows are not considered students because they are not working towards a degree...Postdoctoral fellows often meet the duties test for the “learned professional” exemption but must also satisfy the salary basis and salary level tests to qualify for this exemption.” (United States Department of Labor, 2016c).\n\nRaising postdoctoral salaries in the U.S. under the FLSA. Recommendations have been made across a wide swathe of academe, as summarized by Pickett et al. (Pickett et al., 2015). The American Academy of Arts and Sciences (American Academy of Arts and Sciences, 2014), the National Academies (Committee to Review the State of Postdoctoral Experience in Scientists and Engineers et al., 2014), senior biomedical researchers (Alberts et al., 2014), junior scientists (McDowell et al., 2014), organizations representing postdocs (National Postdoctoral Association, 2016) and advisory groups to the NIH (Biomedical Research Workforce Working Group, 2012) have all recommended increases to postdoc salaries in the years prior to the FLSA update, often to the level of at least $50,000, which is higher than the current required level of $47,476 for overtime exemption.\n\nThe Department of Labor has issued the following statement in its guidance to higher education about current postdoctoral salaries:\n\n“Under the 2016 National Institutes of Health (NIH) salary guidelines for postdoctoral research fellows, some fellows earn more than the revised salary level. Other postdoctoral research fellows earn less, although it is the Department’s understanding that many postdoctoral research fellow salaries are close to the new salary level, and that any differences are not more than a few thousand dollars a year (United States Department of Labor, 2016c).”\n\nThere is an assumption that postdoctoral salaries are, on average, around $45,000 per year for a full-time postdoc (Collins & Perez, 2016) and that most institutions follow the NIH National Research Service Award (NRSA) stipend levels. In theory, therefore, the salary changes expected for postdocs in many cases should approximate the changes in the new NRSA levels (National Institutes of Health, 2016) as shown in Figure 1.\n\nThe current salary for postdocs on NRSA grants (blue) in 2016 increases from a minimum of $43,692 at a linear rate with increasing years experience. The FLSA ruling (red) will raise the minimum salary in 2017 to $47,484, which will remain relatively constant until 2 years experience and increase linearly therein (data source: NIH NOT-OD-16-134, (National Institutes of Health, 2016)).\n\nThe minimum salary should therefore rise from $43,692 for new postdocs to $47,484, which constitutes an increase of 8.7%. Thus, postdocs with more than 3 years of postdoctoral experience are, in theory, already exempt from this rule. However, it is difficult to gauge exactly how postdoctoral salaries are changing across the U.S. Transparent salary information for postdoctoral positions is very hard to find and the administration of postdocs (McDowell, 2016b) means that many may be on lower salaries than expected. It is not currently possible for all institutions in the United States to identify and obtain information on the salaries of all of their postdocs with certainty.\n\nAnother problem with the NRSA assumption is that not all institutions peg their salaries to NRSA levels, and institutional salary policies may indicate more realistic effects of the FLSA on salaries. In 2014, 89% of institutions had a minimum salary policy, where 51% of those institutions set their postdoctoral salary scales to the NRSA scale but ~30% set their minima lower and 7% did not enforce these policies (data in Figure 21, (Ferguson et al., 2014); for a visual description of this see also (McDowell, 2016)). It is also unclear how many postdocs are employed at each of these institutions (populations potentially from 1 to 5761, according to 2014 data from the National Science Foundation (NSF, (National Science Foundation, n.d.))) and therefore the number of U.S. postdocs at different points on this salary range is also unclear. If 11% of U.S. institutions haven’t set a minimum salary level, it is therefore possible (and legal) that there may be full-time postdocs currently earning as little as $23,660, and these salaries will need to double if they are to meet the FLSA exemption ruling.\n\nImplementing the salary change. Institutions have the choice to either increase the minimum salary for postdocs to $47,476, or to classify postdocs as hourly workers.\n\nThe first option is difficult because there is no extra money for this ruling, and PIs may have to pay postdocs from research grants such as R01 grants. Salaries of postdocs on training grants/fellowships (NRSA, HHMI and possibly NSF) are likely to be increased (National Institutes of Health, 2016; National Science Foundation, 2016).\n\nThe second option, however, may cost more. It would involve implementing a system for keeping track of the hours that postdocs spend in the lab. Many postdocs work, and are expected to work, in excess of a standard 40-hour week. One calculation posits that a postdoc earning the NRSA minimum of $43,692 working 50 hours a week under the new rule would have an increase in salary to $60,076 (Polka, 2016).\n\n\nMethods\n\nIn an effort to make salary information as transparently available as possible, we have been gathering information at the FLSA and postdocs resource at Future of Research (Future of Research, 2016b). This data-gathering has involved checking university websites and contacting HR departments at institutions for information on complying with the FLSA ruling for postdocs. We made it clear that this information is to be made publicly available, using as our guide data from the 2014 NSF Survey of Graduate Students and Postdoctorates in Science and Engineering (National Science Foundation, n.d.) using the total number of Science, Engineering and Health postdocs as our postdoc population (as all postdocs are affected). We are in a position, with one month prior to implementation, to describe the current landscape of publicly available information on changes to the administration of postdocs in compliance with the FLSA.\n\nThe only other datasource that is publicly available on this is a survey by the Council on Governmental Relations conducted of their membership in August (Council on Governmental Relations, 2016). Of 190 member institutions, 109 responded, 68 of which had medical schools. Out of these, 79 were public institutions and 30 were private.\n\nIn August, 63% claimed to have made a decision, 19% said the decision would be made in September, and 15% said their decision would be made in October. Therefore 97% surveyed by now, with a month before implementation, should have made a decision. Based on decisions that institutions made or were leaning towards, the survey reported 75% of institutions would raise salaries, and 25% would allow the tracking of hours, 55% of which leave the decision up to the individual PI. Also, 96 institutions have reported on salary levels with ⅔ reporting at least 50% of salaries, and ¼ reporting at least 75% of salaries were below the new threshold, and 2% reported that 90% of salaries exceeded the new threshold.\n\n\nResults\n\nIn a recent blogpost for Addgene, we reported data that had been collected so far (McDowell, 2016a). In this analysis, we looked at both the percentage of the postdoctoral workforce at institutions implementing various plans for the FLSA, as well as provided data on the percentage of institutions implementing various FLSA plans, as of October 21 2016.\n\nRepeating this analysis a month before compliance is required, we are now able to discuss data for institutions that we have checked or contacted covering 97.5% of the estimated postdoctoral workforce, or every U.S. higher education institution listed in the NSF dataset with > 35 postdocs in 2014 (Dataset 1).\n\nOut of these, 51% of the estimated postdoctoral workforce comes from institutions that have stated they are raising salaries,1.5% from institutions focused on tracking hours, and 4% from institutions allowing the tracking of hours while promoting (but not mandating) salary raising. However, still 41% of postdocs come from institutions that have either reported to us that they have not decided, have no information available, and/or have not yet responded to a request for information (Figure 2A).\n\nPie charts show the percentage of the postdoctoral population at institutions implementing various plans for FLSA (A) and the percentage of institutions implementing various plans for FLSA (includes institutions so far researched for the FLSA and postdocs resource) (B).\n\nOne month away from Dec 1st, we have now checked 56% of institutions, and of those checked, 35.5% are planning to raise salaries, and 57% have no public decision yet available (Figure 2B).\n\nTo illustrate the point of what postdocs may know at this point in time, with one month prior to implementation, we take Boston postdocs as an example. The Boston Postdoctoral Association has been taking an active role in gathering institutional information and preparing resources on the FLSA for its members (Boston Postdoctoral Association, 2016). There are 14 academic postdoctoral associations in the Boston Postdoctoral Association (http://bostonpostdocs.org/) and we list them here along with the numbers of postdocs from the NSF dataset we used for our analysis (where known: “Harvard” and “MIT” are each listed as a single institution) and current estimates of 9,000 postdocs in Boston (Table 1).\n\n*Unknown number out of 5,761 at “Harvard” and **Unknown number out of 1,516 at “MIT”\n\nOut of 9,000 postdocs in Boston, data about postdoc numbers and salaries are known for only 740. Whitehead Institute for Biomedical Research and the Department of Brain and Cognitive Sciences cannot be more than a small proportion of the 1,516 at MIT, while Boston Children’s, Dana Farber, Harvard Chan School of Public Health and Harvard University may be a larger number out of 5,761 but likely not the majority. It could be that as many as two thirds of postdocs in Boston - a very well organized group of postdocs already gathering information - are not aware of what their status will be in a month, either because they have not been told, or their institution has not yet made a decision.\n\n\nConclusions\n\nWe are assuming that all institutions will make information transparently and publicly available, whereas of course there is bound to be a great variation in the institutions that will publish postdoc salary information on the web or respond to queries for FLSA compliance information, and this does not seem to be linked to the size of the institution.\n\nSome institutions (such as “Harvard” and “MIT”) are listed as a single institution in the NSF dataset, and as we have so little data for each (as discussed above) we count them as not having yet made decisions. Combined with previous discussions that the NSF dataset may only approximate postdoc numbers, and this is 2014 data (not up to date), the numbers likely have some degree of variation. Also, in contrast to the Council on Governmental Relations report of August, we see a very striking difference in what has been publicly declared compared with private data. Decisions that have been made this time were predicted to be at 97% whereas we have found only 47% have publicly done so. The ratio of institutions planning to raise salaries to those planning to track hours was 3:1 in the report whereas our data show the ratio closer to 9:1.\n\nWe have not yet done a thorough analysis comparing salary reporting by region, or comparing between public and private institutions, however we hope to carry out this analysis as data continues to be collected through December 1st.\n\nAre postdocs on fellowships FLSA exempt? Both Brown and Brandeis universities have suggested that postdocs paid on training grant/fellowship stipends may be FLSA exempt, and so will not be mandating raises in the salaries of postdocs on stipends that are below the exemption level. At the time of writing this manuscript, we are still awaiting responses to requests for further information from institutions. Nevertheless, the position of Brown University is stated explicitly as follows:\n\n“Postdoctoral fellows are defined as non-employees, paid by stipend rather than salary, and are thus not covered by the FLSA (Brown University, 2016).”\n\nThe position of Brandeis University is stated publicly on their website as follows (with info that salaries need to be raised for postdoctoral associates, but with no mention of fellows):\n\n“Postdoctoral Fellows come to Brandeis to further their scholarly competence, with fellowship aid through sources other than the NRSA. These sources may be federal or non-federal. Appointments are usually for one semester or more and are renewable, based upon the terms and conditions of the individual award. Postdoctoral Fellows are trainees and do not provide services to the University, and are not considered to be employees. A Postdoctoral Fellow is eligible to be appointed as a Postdoctoral Associate after the term of the Postdoctoral Fellowship has ended (Brandeis University, 2016).”\n\nPostdoctoral fellows are not considered employees by institutions, as they are not paid by the institution. However, reading the directions from the Department of Labor, that is not the same as being recognized as exempt from the FLSA. A postdoc is federally recognized as both a trainee and an employee (Code of Federal Regulations, Title 2, part 200.400(f) (U.S. Government, 2014)) and unless they are in a primarily teaching role, all postdocs come under the FLSA (see Overtime Final Rule and Higher Education (United States Department of Labor, 2016c)). In addition, the Department of Labor defines what “employ’ means in the context of the FLSA:\n\n“The FLSA defines the term “employ” to include the words “suffer or permit to work”. Suffer or permit to work means that if an employer requires or allows employees to work, the time spent is generally hours worked (United States Department of Labor, n.d.).”\n\nThe “employer” is the institution that “suffers [someone] to work,” so institutions are the ones responsible for ensuring FLSA compliance. Postdoc fellows are “permitted” to work at the institution. In combination with further guidance from the Department of Labor on independent contractors, the closest possible analogy to postdoctoral fellows (United States Department of Labor, 2014), it is our understanding that if fellowships do not pay full FLSA rates, the employer is still responsible for making sure employees get supplemented up to federal standards. What matters is not where the money comes from, but what a person is doing at an institution that “suffers or permits them to work” there. Guidance in the latest update to the NRSA stipends and applying for supplements (NOT-OD-17-002) makes clear that NIH is taking no position on the status of workers at institutions (National Institutes of Health, 2016).\n\nThis may become part of the larger conflict in the debate over whether postdocs are trainees or employees, and what services (including intellectual property) they do or do not provide (Haak, 2002a; Haak, 2002b; Haak, 2002c). This may aggravate existing issues with postdoctoral fellowships, as recipients already face losing benefits (Gaval, 2014), or dealing with tax complications such as imputed income tax (National Postdoctoral Association, n.d.; University of California San Francisco 2015). Elsewhere we have discussed changes that may be taking place to postdoctoral benefits, such as reductions in fringe rates, as a similar but separate effect of the FLSA implementation at institutions (Future of Research, 2016a).\n\nIf violations to the FLSA ruling occur, will they be reported? One question that has occupied some discussion about the FLSA is whether violations, such as directing employees to give false reports on timesheets, will actually be reported. The Department of Labor gives advice on how to report these violations (United States Department of Labor, 2016d) and points out that there is a three year statute of limitations, reporting is completely confidential until the point of allegation being pursued and the person then deals with the Department of Labor and not the institution, in reporting violations. In addition, it is illegal for employers to take action against employees based on reporting of violations, and immigration status will not be investigated.\n\nA comparison is often made about the perceived lack of reporting hours’ violations during medical residency, a similar role in the medical system to the postdoc in academia, and so we use this comparison here to illustrate why reporting could be more common in academic science. First, medical residents are exempt from FLSA so a different system of reporting exists to begin with. Medical residents can have up to an additional ~$250,000 debt for tuition, compared to the relatively lower (undergraduate) student debts in the academic path, as well as the cultural eschewing of financial gain, and perhaps have more “skin in the game”. The bottleneck in the medical system is often getting into residency, from where job certainty is much higher than in academia, whereas most postdocs end up leaving academia despite a high interest in staying in it (Sauermann & Roach, 2016). In addition, medical residents may consider that reporting hours may actually harm their own training and the training of others, whereas whether many postdocs actually receive training is of great concern in academe (Pickett et al., 2015) and perhaps the perceived harm to that training may be seen as minimally impacting them. All of this, combined with the 3-year statute of limitations, makes “burning bridges” a much stronger possibility for postdocs.\n\nIf a violation of the FLSA is reported, it seems that the burden of proof is on the institution to counter the evidence from the complaint (discussed in (American Council on Education, 2016)). There are many common false assumptions being discussed in academia about how federal labor law can be implemented (Reynolds & Rudnick, 2010), which makes it very interesting that a number of institutions will let departments, or even PIs, decide on how to administer postdocs.\n\nMoving towards greater transparency in postdoc salaries. Our goal in presenting these data is to increase transparency about the postdoc position, in terms of the administration and benefits, in a similar manner to a call for transparency in career outcomes (Polka et al., 2015). We have presented our impression of the information currently available to postdocs about how their roles may be changing in one month, and aim to continue collecting and further analyzing data as we go forward until December 1, 2016.\n\nPossible effects of the FLSA ruling. Tracking hours runs counter to academic culture, and the notion of the postdoc as a “trainee”, someone in a mentored environment developing independence. It is also in contrast to behaviors learned in graduate school and the working culture of the faculty positions to which postdocs are meant to be directed. Analyzing data, writing and reading papers and carrying out other job related duties of the postdoc are often performed during nights and weekends, while away from the lab. How will those hours be tracked to everyone’s satisfaction? Conflicts arising could force postdocs to work fewer hours and thus make themselves less competitive compared to postdocs exempted by salary level or under/over-report their hours, risking reporting violations to the Department of Labor. An institution that tracks hours could also put its postdocs and PIs at a competitive disadvantage compared to other institutions raising salaries. At the moment, the relatively small numbers of institutions tracking hours suggest that the larger implications of the FLSA may be as a result of the raise in postdoctoral salaries.\n\nIt is unclear what the effects of the FLSA ruling may be on academe. How will this affect smaller institutions? Will there be a drop in new postdoc hires as they become more expensive? How many postdocs are about to lose their jobs? How many will have to shorten their current positions? Will junior faculty bear the brunt, will mid-career researchers be most strained, or will tenured professors be more likely to cut postdocs loose? Will institutions look to increase admission of graduate students, to keep up the labor at the bench? Will there be a shift to more postdocs on particular training mechanisms or fellowships, where the salary is provided, and less on other types of fellowships or research grants?\n\nPerhaps most importantly, will we as a research enterprise start acting on recommendations in a more timely fashion? The academic system will survive this modernization, and in the long term the likely decrease of the postdoctoral population may be a necessary cap on the expanding “trainee” population. This will make the research enterprise more sustainable by limiting the number of postdocs in the system that can be supported. The FLSA ruling change could have been handled far better by the system in favor of postdocs and hopefully is a call to action in terms of how to deal with other issues affecting the research enterprise in the future. The FLSA ruling imposes changes on the system, but these changes had been recommended for some time and could have been implemented more gradually, and with less pain inflicted on postdoctoral researchers (and not only), than they are now. How to correctly deal with the administration of postdocs at institutions has been in discussion over decades (Curtis, 1969), and raising postdoc salaries has been advocated for as we also describe above. Will other recommended changes within the research enterprise be made by the deliberate action of scientists and administrators, or will they have to be imposed by federal statutes? We hope that the abrupt nature of the FLSA revision serves as a call to redouble efforts for academia to become the driver, rather than the subject, of change.\n\n\nData availability\n\nF1000Research: Dataset 1. FLSA status of postdocs as of 10/31/16, http://dx.doi.org/10.5256/f1000research.10086.d142615 (Bankston & McDowell, 2016)",
"appendix": "Author contributions\n\n\n\nGS conceived the study and designed the experiments. AB and GS carried out the research. AB collected a large portion of the data. GS made the figures and wrote the manuscript with extensive editing input from AB. The final version was approved by both AB and GS.\n\n\nCompeting interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThis work was supported by a grant from the Open Philanthropy Project to Future of Research, awarded in April 2016.\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\nMany thanks to Jessica Polka, David Riglar and Rodoniki Athanasiadou for comments on the manuscript, and Chris Pickett for discussions. Future of Research is supported by a grant from the Open Philanthropy Project (http://www.openphilanthropy.org/focus/scientific-research/miscellaneous/future-research-general-support).\n\n\nReferences\n\nAAMC: AAMC submits comments to Department of Labor on Overtime Rule. 2015. Reference Source\n\nAlberts B, Kirschner MW, Tilghman S, et al.: Rescuing US biomedical research from its systemic flaws. Proc Natl Acad Sci U S A. 2014; 111(16): 5773–5777. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmerican Academy of Arts and Sciences: Restoring the Foundation: The Vital Role of Research in Preserving the American Dream. 2014. Reference Source\n\nAmerican Council on Education: New Federal Overtime Regulation and Higher Education. [Accessed: 3 October 2016], 2016. Reference Source\n\nAnon: State of Nevada et al v. United States Department of Labor et al, Docket No. 4: 16-cv-00731. [Accessed: 2 November 2016], 2016. Reference Source\n\nBankston A, McDowell G: Dataset 1 in: Monitoring the compliance of the academic enterprise with the Fair Labor Standards Act. F1000Research. 2016. Data Source\n\nBiomedical Research Workforce Working Group: Biomedical Research Workforce Working Group Report. (Report to the Advisory Committee to the Director), 2012. Reference Source\n\nBoston Postdoctoral Association: FLSA Overtime Pay Rule revision and Postdoc salaries. 2016. Reference Source\n\nBrandeis University: Brandeis Postdoc Policy, Effective 12/1/2016. [Accessed: 2 November 2016], 2016. Reference Source\n\nBrown University: Fair Labor Standards Act – 2016 Important Update. [Accessed: 2 November 2016], 2016. Reference Source\n\nCollins FS, Perez TE: Fair Pay for Postdocs: Why We Support New Federal Overtime Rules. Huffington Post. 2016. Reference Source\n\nCommittee to Review the State of Postdoctoral Experience in Scientists and Engineers, Committee on Science, Engineering, and Public Policy, Policy and Global Affairs, National Academy of Sciences, National Academy of Engineering and Institute of Medicine: The postdoctoral experience revisited. Washington (DC): National Academies Press (US); 2014. Reference Source\n\nCongress of the United States of America: H.R.6094 - 114th Congress (2015–2016): Regulatory Relief for Small Businesses, Schools, and Nonprofits Act. [Accessed: 2 November 2016], 2016. Reference Source\n\nCouncil on Governmental Relations: Fair Labor Standards Act (FLSA) Postdoctoral Researcher Survey Results. 2016. Reference Source\n\nCUPA-HR: DOL’s Proposed Overtime Rule: Higher Education’s Comments & Concerns. 2015a. Reference Source\n\nCUPA-HR: Re: Notice of Proposed Rulemaking; Defining and Delimiting the Exemption for Executive, Administrative, Professional, Outside Sales and Computer Employees (80 Fed. Reg. 38515, July 6, 2015) (RIN 1235-AA11). 2015b. Reference Source\n\nCurtis RB: Invisible University: Postdoctoral Education in the United States. Report of a Study Conducted Under the Auspices of the National Research Council. Washington, D.C.: National Academies Press; 1969. Reference Source\n\nFerguson K, Huang B, Beckman L, et al.: National Postdoctoral Association Institutional Policy Report 2014: Supporting and Developing Postdoctoral Scholars. National Postdoctoral Association. 2014. Reference Source\n\nFuture of Research: Changes to employee benefits for scientists after FLSA. [Accessed: 2 November 2016], 2016a. Reference Source\n\nFuture of Research: FLSA and Postdocs. [Accessed: 3 October 2016], 2016b. Reference Source\n\nGaval M: Stuff you should know but no one tells you: Employee benefits of postdoctoral appointments. [Accessed: 2 November 2016], 2014. Reference Source\n\nHaak L: Postdocs and the Law, Part 3--Are Postdocs Employees? [Accessed: 2 November 2016], 2002a. Reference Source\n\nHaak L: Postdocs and the Law, Part II: Principal Investigator Versus Individual Grants. [Accessed: 2 November 2016], 2002b. Reference Source\n\nHaak L: Postdocs and the Law: Meet the IRS. [Accessed: 2 November 2016], 2002c. Reference Source\n\nMcDowell GS: Addgene Blog. [Accessed: 2 November 2016], 2016a. Reference Source\n\nMcDowell GS: ASBMB Today. [Accessed: 9 July 2016], 2016b. Reference Source\n\nMcDowell GS: Future of Research and the Fair Labor Standards Act. F1000Res. 2016. Reference Source\n\nMcDowell GS, Gunsalus KT, MacKellar DC, et al.: Shaping the Future of Research: a perspective from junior scientists [version 2; referees: 2 approved]. F1000Res. 2014; 3: 291. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNational Institutes of Health: NOT-OD-16-134: Revised: Projected FY 2017 Stipend Levels for Postdoctoral Trainees and Fellows on Ruth L. Kirschstein National Research Service Awards (NRSA). [Accessed: 2 November 2016], 2016. Reference Source\n\nNational Postdoctoral Association: Overview of Tax Issues for Postdocs. [Accessed: 2 November 2016]. Reference Source\n\nNational Postdoctoral Association: Statement from the NPA on the Fair Labor Standards Act. [Accessed: 2 November 2016], 2016. Reference Source\n\nNational Science Foundation: Frequently Asked Questions (FAQs) on the National Science Foundation’s Implementation of the Department of Labor’s Fair Labor Standards Act (FLSA) Final Overtime Rule. 2016. Reference Source\n\nNational Science Foundation: Survey of Graduate Students and Postdoctorates in Science and Engineering. [Accessed: 6 January 2016]. Reference Source\n\nObama B: A Hard Day’s Work Deserves a Fair Day’s Pay. Huffington Post. 2015. Reference Source\n\nObama B: Presidential Memorandum -- Updating and Modernizing Overtime Regulations. [Accessed: 3 October 2016], 2014. Reference Source\n\nPenn B: Employers to White House: Give Us More Time on Overtime Rule. Bloomberg. 2016. Reference Source\n\nPickett CL, Corb BW, Matthews CR, et al.: Toward a sustainable biomedical research enterprise: Finding consensus and implementing recommendations. Proc Natl Acad Sci U S A. 2015; 112(35): 10832–10836. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPolka J: Fair Labor Standards Act—What It Means for Postdocs. [Accessed: 2 November 2016], 2016. Reference Source\n\nPolka JK, Krukenberg KA, McDowell GS: A call for transparency in tracking student and postdoc career outcomes. Mol Biol Cell. 2015; 26(8): 1413–1415. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReynolds PA, Rudnick B: So You Think You Know About the FLSA? Ten Common Assumptions About the FLSA That Can Land You in Hot Water. Bloomberg Law Reports. 2010; 4(10). Reference Source\n\nSauermann H, Roach M: SCIENTIFIC WORKFORCE. Why pursue the postdoc path? Science. 2016; 352(6286): 663–664. PubMed Abstract | Publisher Full Text\n\nUnited States Department of Labor: Compliance Assistance - Wages and the Fair Labor Standards Act (FLSA) - Wage and Hour Division (WHD). [Accessed: 3 October 2016], 2016a. Reference Source\n\nUnited States Department of Labor: Defining and Delimiting the Exemptions for Executive, Administrative, Professional, Outside Sales and Computer Employees. [Accessed: 3 October 2016], 2016b; 80(128): 38515–38612. Reference Source\n\nUnited States Department of Labor: Fact Sheet #13: Am I an Employee?: Employment Relationship Under the Fair Labor Standards Act (FLSA). 2014. Reference Source\n\nUnited States Department of Labor: FLSA hours worked advisor. [Accessed: 2 November 2016]. Reference Source\n\nUnited States Department of Labor: Guidance for Higher Education Institutions on Paying Overtime under the Fair Labor Standards Act. 2016c. Reference Source\n\nUnited States Department of Labor: How to File a Complaint - We Can Help. [Accessed: 3 October 2016], 2016d. Reference Source\n\nUnited States Department of Labor: Overtime Final Rule and Higher Education. 2016e. Reference Source\n\nUnited States Department of Labor: The Overtime Rule. [Accessed: 3 October 2016], 2016f. Reference Source\n\nUniversity of California San Francisco: Tax Issues Associated with Reporting Fellowships. 2015. Reference Source\n\nU.S. Government: Code of Federal Regulations. Title 2, part 200.400(f). U.S. Government Publishing Office. 2014. Reference Source\n\nWexler E: What does the Department of Labor’s overtime rule mean for higher education? Inside Higher Ed. 2016. Reference Source"
}
|
[
{
"id": "17777",
"date": "18 Nov 2016",
"name": "Nathan L. Vanderford",
"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\nBankston and McDowell provide a comprehensive and well-written report on the status and impact of the Fair Labor Standards Act (FLSA) on the US postdoc population. This article is very timely given the “go-live” date for implementing this Act is less than a month away and given that there has been great consternation among academic institutions in terms of how the mandates will be implemented and what impacts it will have on institutions, postdocs, and science in general.\nWhile acceptable for publication now, there are minor changes that could improve the article.\nThe non-yes (i.e, the “X” and “-“) annotations in dataset 1 should be defined.\n\nThe y-axis of Figure 1 should be labeled.\n\nAt the top of page 7, a deeper analysis comparing salary by region, public vs. private institutions, etc. is mentioned as a future direction. This is a critical aspect of understanding how the FLSA will differentially impact institutions and postdocs across the country. I believe it is a very important point that, for example, the cost of living in the middle of the country is not comparable to the cost of living on the east and west coast, which begs the question of whether such differences should be considered within the FLSA. Will this analysis be added to this current article as more data is collected or will this form the basis of another article? It is my opinion that this analysis would greatly strengthen and compliment the current article’s argument and impact.\n\nIt may be interesting (and impactful) to add a current snapshot of what postdocs think of the FLSA implementation at their institutions. At my institution, postdocs have been very concerned about the short- and long-term impacts on their current positions and how any potential changes (eg, losing their position) may impact their career progression. It would be interesting to hear from postdocs representing institutions across the country (not just on the coasts).\n\nAt the bottom of page 8 the authors begin to question how the FLSA will impact the biomedical enterprise. It would be more powerful if the authors were more definitive and concrete with their own opinions as to what impact FLSA will have on science. Some have argued that the FLSA will shrink the postdoc pool thus shrink the pipeline of future researchers thus set back the momentum of new science discoveries. The article could be more impactful if the authors wrote specific speculations as to the threats and opportunities that the FLSA will have on the biomedical enterprise.\n\nSimilar to point #4, could the authors provide some specific recommendations to institutions, postdocs, the government, etc.?\n\nWhat implications will/could a new political party and leader have on FLSA?\n\nIn summary, this is an important article that is acceptable for publication now although there are several areas that could be improved upon as noted above. As such, I look forward to reviewing any revised version of the article.",
"responses": []
},
{
"id": "17778",
"date": "22 Nov 2016",
"name": "Christopher L. Pickett",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research article “Monitoring the compliance of the academic enterprise with the Fair Labor Standards Act” by Bankston & McDowell is a review of academic compliance with changes to the FLSA overtime rules as of Nov. 1. The authors give a good primer on the history of the FLSA overtime rule change and how it is expected to affect academic research starting Dec. 1. They then discuss their extensive outreach to institutions with postdocs, and their efforts to determine how the institutions are adjusting to the new overtime rule with regard to postdoc salaries.\n\nThis paper and the results of the authors’ outreach are important for the postdoc and larger academic communities to understand how institutions are dealing with the increase to postdoc pay.\n\nMajor concerns:\nThe authors should devote some space to a discussion of international postdocs and how they are affected by the FLSA overtime rule change. Anecdotally, international postdocs may not be paid on the same scale as American postdocs, even at institutions with well-defined salary minima. Since the nation of origin does not affect whether someone is FLSA exempt, just how widespread the disparities in U.S. versus international postdoc pay are could have significant repercussions in the relative populations of each in the enterprise moving forward.\n\nThe authors should take the time to explain exactly what is going on in Boston. There seems to be an assumption that the reader will know all of the institutions that are classified under “Harvard” or “MIT”. The assumption comes across in phrases such as institutions “…cannot be more than a small proportion…” As someone who has never done research in Boston, it is not clear to me why this phrase must be true. The authors should take the time to indicate, both in the text and Table 1, which institutions are part of “Harvard” for example, which have released decisions about postdoc pay, and why this may be confusing for postdocs employed there.\n\nThe conclusions section opens with a meandering and confusing sentence and the first section is about the difficulties of data collection. I agree these are real concerns, but the authors should state their overall and most important conclusions at the beginning of this section as a way to frame the rest of the discussion.\n\nMinor concerns\nBackground\nThe sentence beginning, “The other key change is indexing…” is not clear. Recommend breaking the sentence into two with the second sentence starting something like, “This means the OT threshold will be $51,168 in 2020.”\n\nIt would be helpful to know the current status of H.R. 6094. Was it vetoed or does it await action?\n\nOn page 4, in the paragraph about the test that must be passed to be eligible for overtime pay, the authors should consider a short example at the end of the paragraph. For example, “A first year postdoc in 2015 earning a salary of $XX,XXX would pass the salary basis test, would fail the salary level test and pass the standard duties test. Hence the focus on the overtime pay threshold.”\n\nIn the paragraph starting “The minimum salary should therefore…”: for consistency, recommend the authors indicate years experience as they do in Fig. 1 (Year 0, 1, etc.) as opposed to calling them “new postdocs”.\n\nThe two problems with the assumption of the NRSA minimum as the actual minimum pay for postdocs, as I understand them are, (1) transparent salary information does not exist and (2) institutional salary minima are unknown or unenforced. These seem very similar problems to me and I don’t understand the distinction the authors are trying to make. I recommend the authors draw a starker division between the two problems they see or discuss them as one.\n\nDue to the date-sensitive nature of the information, I recommend the authors add the date to the titles of all figures and tables.\n\nIn the discussion, the comparison between medical residents and postdocs:\nThe authors should explicitly state why, after each condition they site, why postdocs would be more likely to report violations. For example, “…debts in the academic path, meaning postdocs are significantly less dependent on academic employers and may be more inclined to report violations.” “…postdocs end up leaving academia meaning postdocs are less likely to be concerned about how academic employers will view their willingness to report violations.”\n\n“…compared to the relatively lower (undergraduate) student debts…” It is not clear why the authors specify these as undergraduate debts. Graduate students can also secure student loans. I recommend removing the parenthetical.",
"responses": []
},
{
"id": "18035",
"date": "28 Nov 2016",
"name": "Paula E Stephan",
"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 Bankston & McDowell article is highly informative on two counts. First, it highlights the extreme difficulty encountered in collecting information regarding postdocs. The Boston data are but one case in point. Second, it contributes information concerning the percent of postdocs which, at the time Bankston & McDowell collected their data, were likely to be affected by the FLSA ruling. This constitutes a significant contribution to our understanding of how the law may affect actual salaries.\n\nFor my taste, however, the article could go a bit further. First, the authors present no information regarding who will pay for the increase on campuses that intend to raise salaries sufficiently so that postdocs will be exempt. The authors speculate that the funds will come out of grants. And in the long run, they are probably right. But, in the short run the evidence is that universities and departments will pick up some of the increases. (See Council on Governmental Relations 2016 Survey). Second, they do not speculate on how the increase will likely affect the number of postdocs employed. To the extent the demand for postdocs is sensitive to salary, one would predict that in the long run the regulation will lead to a decrease in the number of postdocs working at universities.\n\nThe authors also imply that it is not clear as to whether the regulation applies to postdocs on fellowships. It is my understanding that a decision by the Department of Labor means that those on fellowships are to be treated the same as are postdocs supported in other ways.\nThe authors also make no effort to explain why their results seem so at odds with the results found by the Council on Governmental Relations Survey, which suggests that something like 75% of campuses surveyed were choosing to raise salaries so that postdocs would be exempt from overtime pay.\n\nFinally, there is the issue that the authors could not have foreseen that a Federal Judge would issue an injunction November 22, 2015 which preserves the status quo. While the injunction is temporary, many believe that the judge’s language “indicated he was likely to strike down the regulation.” http://www.nytimes.com/2016/11/22/business/obama-rule-to-expand-overtime-eligibility-is-suspended-by-judge.html.\nTo the extent the implications of the regulation for postdoc pay were difficult to understand before this ruling, they are even more so today. Assuming the rule is found to be invalid, campuses will find themselves in the awkward position of having to decide if they will take back a promised increase. Or, take back an increase that has already taken effect.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-2690
|
https://f1000research.com/articles/6-227/v1
|
07 Mar 17
|
{
"type": "Software Tool Article",
"title": "Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality",
"authors": [
"Scott Gigante"
],
"abstract": "Oxford Nanopore Technologies' (ONT) MinION and PromethION long-read sequencing technologies are emerging as genuine alternatives to established Next-Generation Sequencing technologies. A combination of the highly redundant file format and a rapid increase in data generation have created a significant problem both for immediate data storage on MinION-capable laptops, and for long-term storage on lab data servers. We developed Picopore, a software suite offering three methods of compression. Picopore's lossless and deep lossless methods provide a 25% and 44% average reduction in size, respectively, without removing any data from the files. Picopore's raw method provides an 88% average reduction in size, while retaining biologically relevant data for the end-user. All methods have the capacity to run in real-time in parallel to a sequencing run, reducing demand for both immediate and long-term storage space.",
"keywords": [
"DNA Sequencing",
"Genome Informatics",
"Nanopore Sequencing",
"Compression",
"Data Storage"
],
"content": "Introduction\n\nOxford Nanopore Technologies’ (ONT) nanopore sequencing technology MinION provides a high-throughput, low-cost alternative to traditional Next-Generation Sequencing (NGS) technologies1. The sequencing device itself is handheld and connects by USB to a laptop computer. Together with all equipment and reagents required for DNA library preparation, the equipment required to use MinION is minimal; entire laboratories have even been transported overseas in a suitcase, allowing a versatile and agile approach towards DNA and RNA sequencing2.\n\nOver the course of ONT’ Early Access Program, several improvements in software and chemistry have led to a rapid increase in yield, through an increase in average read length, an improvement in basecalling accuracy rates and an increase in total number of reads. In October 2015, the MinION Analysis and Reference Consortium (MARC), using R7.3 flow cells and SQK-MAP005 (2D) chemistry, reported a median of 60,600 reads with a median yield of 650,000 events across 20 MinION experiments3. In contrast, ONT claim to have obtained a total base yield of 17 gigabases using an R9.4 flowcell on the latest version of their MinKNOW software (https://nanoporetech.com/about-us/news/minion-software-minknow-upgraded-enable-increased-data-yield-other-benefits). The drastic increase in data generation from a single experiment is rapidly becoming the limiting factor in uptake of the technology.\n\nThe concerns over data storage extend beyond the data generation capabilities of a single flowcell. Recent attempts to perform de novo assembly of eukaryotic genomes have combined the data generated by multiple flowcells in order to gain sufficient coverage of the genome4. To this end, ONT have begun the pre-commercial release of the PromethION, a benchtop nanopore sequencing device with 48 flowcells. In addition, each of these flowcells contains 3000 channels, as opposed to the 512 channels in a single MinION flowcell, with data generation projected at 6 terabases per flowcell per day5.\n\nNumerous methods have been developed for the efficient analysis of the increasingly large nanopore datasets. However, current methods to reduce the data storage footprint are extremely limited. Nanopore runs uploaded to online repositories, such as the European Nucleotide Archive, are bundled into a tarball, a process which facilitates upload as a single file, but does not decrease file size. Moreover, ONT runs bundled into a tarball (which could then be compressed using traditional means) are not able to be read by any existing nanopore analysis tools. Moreover, traditional compression technologies are poorly adapted to the needs of the individual user, many of whom have no need for a large portion of the data stored by ONT’s basecallers. Therefore, we developed Picopore, a tool for reducing the storage footprint of the ONT runs without preventing users from using their preferred analysis tools. Picopore uses a combination of storage reduction techniques, including built-in dynamic compression in the HDF5 file format, reduction of data duplication, efficient allocation of memory within the file, and the removal of intermediate data generated during basecalling, which is deemed unnecessary by the end-user.\n\n\nMethods\n\nPicopore is developed using the Python h5py module (http://www.h5py.org/), an interface to the HDF5 file format (http://www.hdfgroup.org/HDF5), used by ONT under the FAST5 file extension. Picopore implements a number of different compression methods, a selection of which are applied according to user preferences, before using HDF5’s h5repack to rebuild the file according to the reduced file size requirements.\n\nBuilt-in GZIP compression. The HDF5 file format allows for both files and datasets within files to be written using a number of different compression filters, the most universally implemented being GZIP. GZIP applies traditional compression to the data stored in the HDF5 file with choices of compression level between 1 and 9. ONT’s default compression uses GZIP at level 1; Picopore increases this to level 9 in order to decrease disk space usage.\n\nDynamic memory allocation for variables. Data stored in the HDF5 file format uses fixed-size file formats provided by NumPy, which provides a vast array of options for storing integers, decimals and strings within high-dimensional datasets6. ONT’s native data is written using the largest data types provided by NumPy: 64-bit integers, 64-bit decimals, and \"variable-size\" strings. Picopore vastly reduces disk space usage by analyzing each dataset to determine the minimum number of bytes required by a given variable in the file, changing the data type accordingly.\n\nCollapsing of file structure. The advantage of the HDF5 file format is that it provides a file directory-like storage format for datasets and properties, making reading and writing to the files straightforward and easy to understand. However, the inherent nature of the highly-structured file format requires HDF5 to allocate slots of memory to \"groups\", which represent the internal directory structure of the file. Picopore reduces the disk space used by this file metadata by collapsing the directory structure, while retaining the option for users to reverse this action when tools that only recognize the original file format are required.\n\nIndexing of duplicated data. ONT’s most widely used basecalling software, the cloud-based Metrichor service (https://metrichor.com/s/), performs feature recognition (or \"event detection\"). This segments the electrical signal representing each nanopore read into events, each of which represents a period of time when the DNA was stationary in the nanopore. These events are then converted into basecalled data, which provides a single k-mer (at present a 5-mer) of DNA representing the bases in the nanopore contributing to the signal at that time. Each event corresponds to a single row in the basecalled dataset, and both the event detection and basecalled datasets thus store the mean signal, standard deviation, start time and length of the event. Picopore reduces disk space usage by indexing the basecalled dataset to the event detection dataset, removing the duplicated data while retaining the option for users to reverse this action when tools that require access to this data are required.\n\nRemoval of intermediate data. The primary function of all basecalling software is to generate a FASTQ file containing the genomic sequence and associated quality scores representing the read stored in each FAST5 file. While some software tools, such as nanopolish7 and nanoraw8, do make use of the signal, event detection and basecalled datasets, the large majority of analyses, including alignment, assembly and variant calling, simply require access to the FASTQ data. Picopore allows users to remove the intermediate data generated during the process of converting raw signal to FASTQ, while retaining the signal data, should they ever want to re-basecall the run to attain improved FASTQ data or to access this intermediate data at a later stage.\n\nRequirements. Picopore is built in Python 2.7 (www.python.org) and runs on Windows, Mac OS and Linux. It requires the following Python packages:\n\nh5py 2.2.0 or later\n\nwatchdog 0.8.3 or later\n\nIn addition, Picopore requires HDF5 1.8.4 or newer, with development headers (libhdf5-dev or similar), including the binary utility h5repack, which is included therein.\n\nInstallation. The latest stable version of Picopore is available on PyPi and bioconda (see Software availability). It can be installed according to the following commands:\n\nLinux and Mac OS: pip install picopore\n\nWindows: conda install picopore -c bioconda -c conda-forge\n\nPicopore can also be installed from source (see Software availability) using the command python setup.py install.\n\nExecution. Picopore is run from the command-line as a binary executable.\n\nPicopore accepts both folders and FAST5 files as input. If a folder is provided, it will be searched recursively for FAST5 files, and all files found will be considered as input.\n\nThere are three modes of compression available, each of which performs a selection of the techniques described above.\n\nlossless: performs built-in GZIP compression and dynamic memory allocation for variables. This mode is both fast and allows continued analysis of data by any existing software.\n\ndeep-lossless: performs lossless compression, as well as collapsing of file structure and indexing of duplicated data. This mode obtains the best compression results without removing any data, but comes at the cost of requiring reversion before most software tools can analyse the data.\n\nraw: performs lossless compression, as well as removal of intermediate data, partially reverting files to the \"raw\" pre-basecalled file format. This mode is fast, obtains the best filesize reduction, and allows continued analysis by tools that extract FASTQ and related data, but comes at the cost of removing intermediate basecalling data required for some niche applications, such as nanopolish, which cannot be retrieved by Picopore (but can be regenerated using basecalling software.)\n\nOptional arguments include:\n\n–revert: reverts lossless compressed files to their original state to allow high-speed access at the cost of disk usage;\n\n–realtime: watches for file creation in the given input folder(s) and performs the selected mode of compression on new files in real time to reduce the footprint of an ongoing MinION run;\n\n–prefix: allows the user to specify a filename prefix to prevent in-place overwriting of files;\n\n–group: allows the user to select only one of the analysis groups on files that have been processed by multiple basecallers;\n\n–threads: allows the user to specify the number of files to be processed in parallel.\n\n\nResults\n\nTo demonstrate the effectiveness of Picopore’s compression, we ran all three modes of compression on four toy datasets of 40 FAST5 files run using the R9 SQK-RAD001 (R9_1D), R9 SQK-NSK007 (R9_2D), R9.4 SQK-RAD002 (R9.4_1D) and R9.4 SQK-LSK208 (R9.4_2D) protocols. The files for the toy datasets were chosen randomly from the pass folder of four MinION datasets generated at the Australian Genome Research Facility (see Software and data availability). For the R9_1D dataset, DNA was extracted from the lung of a juvenile 129/Sv mouse using the DNeasy Blood and Tissue kit (Qiagen). For the R9_2D, R9.4_1D and R9.4_2D datasets, DNA was extracted from a culture of escherichia coli K12 MG1655 using the Blood & Cell Culture DNA Kit (Qiagen). Quality control performed by visualisation on the TapeStation (Agilent). Run metadata is shown in Table 1.\n\nEach file was compressed and tarred using each of five methods: no compression, gzip (applied after tarring, as per convention), picopore lossless, picopore deep-lossless and picopore raw. Each of these methods was run on a single core. Figure 1 shows that lossless achieves only slightly less compression than gzip, giving an average reduction in size of 25% compared to gzip’s 32%, while deep-lossless and raw perform significantly better, giving average reductions in size of 44% and 88%, respectively. A dependent sample t-test (R v3.3.0) was run on individual compressed file sizes. Table 2 shows that each successive method of compression (excluding gzip, which does not compress individual files) gives a significant reduction in size from the previous. Figure 2 shows that while all of Picopore’s compression methods are much slower than gzip, raw is the fastest of these, followed by lossless and deep-lossless. Note that the tarring time makes up a maximum of 0.04 s/read in each case and is largely negligible.\n\nTo demonstrate the effectiveness of Picopore’s multithreading, we ran deep-lossless, the most computationally expensive of the Picopore compression methods, on each dataset using 1, 2, 5, and 10 cores. Figure 3 shows an almost linear improvement in speed, showing that even on a small dataset, the multithreading overhead is relatively small.\n\n\nDiscussion\n\nIt is clear that, due to the enormous reduction in disk space and low total time requirements, Picopore’s raw compression is the optimal compression mode for users who have no need for the intermediate event detection and basecalling data. The superior running speed of lossless compression over deep lossless compression may mean that this is the preferred method for users who wish to retain all data and need to compress the data in real-time; however, for users with these data retention requirements who wish to store data long-term on their file server, for whom speed of compression is not an issue, deep lossless compression provides the best option. All Picopore compression methods provide significant improvements over uncompressed or traditionally compressed files, and lossless and raw methods carry the added benefit that files can be processed using analysis tools in compressed form.\n\nAlthough the compression has a high CPU cost, the capability of Picopore to run on multiple threads signifies that, if computing resources are available, the files can be compressed in a reasonably short period of time. Finally, extrapolating the running time per file to a real-time run of 500,000 reads over 48 hours (0.34 s/read), Picopore has the capability to run lossless (0.33 s/read) and raw (0.25 s/read) modes on a single core in real time. While deep-lossless requires multiple cores to keep pace with the MinION data generation, this adds little to the overall computational cost, and can reach real-time speed with just five cores (0.24 s/read). As data generation continues to increase in scale, further gains could be made by incorporating the compression methods used in Picopore into the basecalling software itself.\n\n\nConclusions\n\nONT’ MinION and PromethION sequencing devices promise to produce increasingly large datasets as the technology progresses toward commercial release. The disk space required to run and store one or multiple datasets from these poses a problem for service providers and users alike; Picopore provides three different solutions that cater to the different needs of users.\n\nAlthough the trade-off between data retention, computing time and disk space is a compromise that cannot be perfectly resolved, Picopore provides user options to reduce their ONT datasets to the minimum viable size based on their intended use. This may involve real-time compression for laptop disk space concerns, reduction of bandwidth usage for transfer of datasets between laboratories, or reduction of the storage footprint on shared data servers.\n\n\nSoftware and data availability\n\nSoftware for Linux or Mac OS available from: https://pypi.python.org/pypi/picopore\n\nSoftware for Linux, Mac OS and Windows available from: https://anaconda.org/bioconda/picopore\n\nSource code available from: https://github.com/scottgigante/picopore\n\nArchived source code from: https://doi.org/10.5281/zenodo.3219579\n\nLicense: GPLv3\n\nDataset 1. Output data generated using Picopore on four toy datasets. Each of the four toy datasets was compressed using each of Picopore’s three compression modes and GZIP. Datasets compressed using Picopore were tarred after compression, while the GZIP dataset was tarred before compression. doi, 10.5256/f1000research.11022.d15337010\n\nThe toy dataset used for the analyses in this paper is available on Zenodo: Toy datasets for compression by Picopore, doi: 10.5281/zenodo.32195911.\n\n\nAuthor endorsement\n\nChris Woodruff 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. Chris Woodruff declares he has no competing interests. Affiliation: Visiting Scientist at Bioinformatics Division of Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.",
"appendix": "Author contributions\n\n\n\nSG designed and developed the source code, published the software and wrote the article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\nAuthor Endorsement: Chris Woodruff 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. Chris Woodruff declares he has no competing interests. Affiliation: Visiting Scientist at Bioinformatics Division of Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.\n\n\nGrant information\n\nThe work discussed in this article was funded by the Speed Lab in the Bioinformatics Division of the Walter & Eliza Institute of Medical Research. The Speed Lab is supported by the Australian NHMRC Program (grant number 1054618).\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 author is grateful to Terry Speed and Chris Woodruff from the Walter & Eliza Hall Institute of Medical Research for their supervision of this and related work, to Alexis Lucattini and Lavinia Gordon from the Australian Genome Research Facility and Matthew Ritchie, Andrew Kinery and Marnie Blewitt from the Walter & Eliza Hall Institute of Medical Research for their assistance in providing MinION data and discussing ongoing analyses, to Biomedical Research Victoria and CSL Limited for the provision of a stipend to begin his work at the Walter & Eliza Hall Institute, and to the organisers and participants of PorecampAU for inspiring this work.\n\n\nReferences\n\nEisenstein M: Oxford Nanopore announcement sets sequencing sector abuzz. Nat Biotechnol. 2012; 30(4): 295–296. PubMed Abstract | Publisher Full Text\n\nQuick J, Loman NJ, Duraffour S, et al.: Real-time, portable genome sequencing for Ebola surveillance. Nature. 2016; 530(7589): 228–232. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIp CL, Loose M, Tyson JR, et al.: MinION Analysis and Reference Consortium: Phase 1 data release and analysis [version 1; referees: 2 approved]. F1000Res. 2015; 4: 1075. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTyson JR, O’Neil NJ, Jain M, et al.: Whole genome sequencing and assembly of a Caenorhabditis elegans genome with complex genomic rearrangements using the MinION sequencing device. bioRxiv. 2017. Publisher Full Text\n\nJain M, Olsen HE, Paten B, et al.: The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 2016; 17(1): 239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan der Walt S, Colbert SC, Varoquaux G: The numpy array: a structure for efficient numerical computation. Comput Sci Eng. 2011; 13(2): 22–30. Publisher Full Text\n\nLoman NJ, Quick J, Simpson JT: A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat Methods. 2015; 12(8): 733–735. PubMed Abstract | Publisher Full Text\n\nStoiber MH, Quick J, Egan R, et al.: De novo identification of DNA modifications enabled by genome-guided nanopore signal processing. bioRxiv. 2016. Publisher Full Text\n\nGigante S, Sjödin A: scottgigante/picopore: 1.0.0 Stable [Data set]. Zenodo. 2017. Data Source\n\nGigante S: Dataset 1 in: Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality. F1000Research. 2017. Data Source\n\nLucattini A, Kinery A: Toy datasets for compression by Picopore [Data set]. Zenodo. 2017. Data Source"
}
|
[
{
"id": "20761",
"date": "20 Mar 2017",
"name": "Matthew Loose",
"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\nPicopore is a well written package that installs quickly and easily, has clear guidance on its use and addresses a relevant issue in Nanopore sequencing at this time.\nThe tools function as described (certainly on OSX).\nI have some reservations about reporting speed in terms of reads/s. I would like to see metrics which take in to account the number of bases being processed per unit time as I suspect the alternative compression options will perform differently by this metric.\n\nI also have some reservations about the use of some modes of picopore compression. Users will need to think carefully about the application of modes which are not immediately compatible with existing tool chains. Given recent announcements from Nanopore with respect to provision of off line base calling I suspect a better long term storage of data will be as simple raw files with an associated fastq.gz.",
"responses": [
{
"c_id": "2625",
"date": "10 Apr 2017",
"name": "Scott Gigante",
"role": "Author Response",
"response": "Dear Dr. Loose, Thank you for your review. I would like to note that since the compression modes were tested all on the same dataset of a total of 160 files across four runs, the number of bases is fixed, with a total of 1.7 Mb in each instance. Thus, the comparison of speeds and sizes between compression modes will be equivalent by either metric. However, I agree that bases/s is a more useful metric than reads/s in comparing results between different protocols, and have included this in my analyses. In regards to your recommended mode of long term storage, this is indeed my expectation and is Picopore’s recommended mode for end-users. Picopore’s raw mode stores by default only the raw signal and the FASTQ, which is compressed using HDF5’s built-in GZIP compression. Picopore’s lossless and deep-lossless modes are aimed at developers who wish to retain the event data for active use or long-term storage respectively; I acknowledge that these use cases are only suitable for a particular subgroup of users. Thank you once again for your comments. Kind regards, Scott Gigante"
}
]
},
{
"id": "20757",
"date": "21 Mar 2017",
"name": "David A. Eccles",
"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's quite difficult to knock out a useful software tool for Oxford Nanopore devices before they change the protocol, or release their own tool that does similar things. Scott Gigante has done an admirable job in this regard by developing and publishing a much-needed tool within the space of time between two updates of the MinKNOW software (v1.4 -> v1.5). While the newest version of MinKNOW no longer produces event data by default in the FAST5 files (substantially reducing file sizes), Scott's tool will still be useful for existing file sets and event-called files in the future.\nManuscript\nThe manuscript is sufficiently verbose for a short software release publication: explaining modes of operation, demonstrating differences in file compression in different modes, and showing processing speed on different datasets.\nTitle / abstract\nThe title and abstract sufficiently summarise the manuscript\nIntroduction\n\"ONT'\" -> \"ONT's\" \"The drastic increase... the limiting factor in uptake of the technology\" -- cite, alter, or remove. My experience is that storage space is only an issue for *existing* users of the technology, not people deciding whether or not to use the MinION.\nOperation\nAt least one running example in the 'Execution' section would be useful, similar to the Usage section of the pypi repository. Something like this:\n'picopore --mode lossless --prefix shrunk '\nDiscussion\nThe introduction suggests ONT's internal runs are approaching 2M reads, yet the discussion suggests 0.5M reads per run. This is the difference between real-time processing and almost a week of waiting for processing to finish. The discussion mentions future potential capabilities for basecalling software, but not Picopore itself. Are there any planned updates on the horizon?\nSoftware testing\nThe current version of Picopore (installed by pip on 2017-Mar-17) had a few minor issues on my system that would prevent most users from being able to run the software. Once these issues were dealt with, Picopore was able to substantially reduce the file size of two FAST5 files with wildly different internal structures, while retaining important base call and raw signal information.\nInstallation\nThe program appeared to install fine on my Debian Linux desktop by running 'pip install picopore', as per the manuscript instructions. Unfortunately there was a problem with module import when showing the help dialog:\n\n$ picopore -h\n\nTraceback (most recent call last):\n\nFile \"/usr/local/bin/picopore\", line 7, in\n\nfrom picopore.__main__ import main\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/__main__.py\", line 22, in\n\nfrom picopore.parse_args import parseArgs, checkSure\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/parse_args.py\", line 20, in\n\nfrom builtins import input\n\nImportError: No module named builtins\nCommenting out the offending line in 'parse_args.py' fixed this error.\nUse of other data\nInstead of using the provided data, I did a stress test of sorts on Picopore by running it on two files which were put in subdirectories of a parent directory:\nA 2kb R7.3 tomato read produced by me in March 2016 (channel 342, read 13) [David Eccles' read] A 771kb R9.4 E. coli read produced by Nick Loman and Josh Quick in March 2017 [Nick Loman & Josh Quick's read]\nEquivalence testing\nIt is appreciated that Picopore includes a test for equivalence to make sure information is retained. Picopore was able to recursively descend through the directories, but the deep-lossless equivalence test reported failure for both of these sequences. In the case of the second file, it appears that the only failure was a missing //Picopore directory (which should probably be excluded from the failure modes):\n- Equivalence test 1\n\n$ picopore --prefix pico_ -t 10 --test --mode deep-lossless tested_picopore\n\nPerforming deep lossless compression on 2 files...\n\nNo conversion path for dtype: dtype('\n\nComplete.\n\nOriginal size:\n\n67048849\n\nCompressed size: 66616200\n\nChecking equivalence of /home/gringer/bioinf/reviews/tested_picopore/1/lambda_TEDxWellington_DavidEccles_3637_1_ch342_read13_strand.fast5 (file 1) and /home/gringer/bioinf/reviews/tested_picopore/1/picopore.test.lambda_TEDxWellington_DavidEccles_3637_1_ch342_read13_strand.fast5 (file 2)...\n\nFailure: //Analyses missing from file 2\n\nFailure: //Raw missing from file 2\n\nFailure: //Sequences missing from file 2\n\nFailure: //UniqueGlobalKey missing from file 2\n\nFailure: //Picopore missing from file 1\n\nTraceback (most recent call last):\n\nFile \"/usr/local/bin/picopore\", line 11, in\n\nsys.exit(main())\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/__main__.py\", line 80, in main\n\nrunTest(args)\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/__main__.py\", line 63, in runTest\n\ncheckEquivalent(f, compressedFile)\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/test.py\", line 72, in checkEquivalent\n\nrecursiveCheckEquivalent(file1, file2, group.name)\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/test.py\", line 56, in recursiveCheckEquivalent\n\nrecursiveCheckEquivalent(file1, file2, \"/\".join([name, key]))\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/test.py\", line 50, in recursiveCheckEquivalent\n\nif not attr2[key] == value:\n\nValueError: The truth value of an array with more than one element is ambiguous. Use a.any() or a.all()\n- Equivalence test 2\n\n$ picopore --prefix pico_ -t 10 --test --mode deep-lossless tested_picopore/2\n\nPerforming deep lossless compression on 1 files...\n\nNo conversion path for dtype: dtype('\n\nComplete.\n\nOriginal size:\n\n65686831\n\nCompressed size: 65688039\n\nChecking equivalence of /home/gringer/bioinf/reviews/tested_picopore/2/loman_771kb_ch181_read4882_strand.fast5 (file 1) and /home/gringer/bioinf/reviews/tested_picopore/2/picopore.test.loman_771kb_ch181_read4882_strand.fast5 (file 2)...\n\nFailure: //Picopore missing from file 1\n\nComplete.\nConfirmation dialog\nThe confirmation of writing files is also a great idea, but produces an error when both responses are given. I wonder if this is due to python version incompatibilities (and my attempted prior bugfix):\n- Confirmation test 1\n\n$ picopore --prefix pico_ -t 10 --mode raw tested_picopore\n\nPerforming raw compression with FASTQ and no summary on 2 files...\n\nAre you sure? (yes|no): no\n\nTraceback (most recent call last):\n\nFile \"/usr/local/bin/picopore\", line 11, in\n\nsys.exit(main())\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/__main__.py\", line 84, in main\n\nrun(args.revert, args.mode, args.input, args.y, args.threads, args.group, args.prefix, args.fastq, args.summary)\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/__main__.py\", line 34, in run\n\nif y or checkSure():\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/parse_args.py\", line 109, in checkSure\n\nresponse = input(\"Are you sure? (yes|no): \")\n\nFile \"\", line 1, in\n\nNameError: name 'no' is not defined\n- Confirmation test 2\n\n$ picopore --prefix pico_ -t 10 --mode raw tested_picopore\n\nPerforming raw compression with FASTQ and no summary on 2 files...\n\nAre you sure? (yes|no): yes\n\nTraceback (most recent call last):\n\nFile \"/usr/local/bin/picopore\", line 11, in\n\nsys.exit(main())\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/__main__.py\", line 84, in main\n\nrun(args.revert, args.mode, args.input, args.y, args.threads, args.group, args.prefix, args.fastq, args.summary)\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/__main__.py\", line 34, in run\n\nif y or checkSure():\n\nFile \"/usr/local/lib/python2.7/dist-packages/picopore/parse_args.py\", line 109, in checkSure\n\nresponse = input(\"Are you sure? (yes|no): \")\n\nFile \"\", line 1, in\n\nNameError: name 'yes' is not defined\nThe 'input' function in my version of python does an evaluation after reading input; replacing 'input(...)' with 'raw_input(...)' fixed this error.\nAfter these errors were fixed enough to allow the code to proceed, Picopore was able to successfully strip out event data (in 'raw' mode) from both the R7.3 and R9.4 FAST5 files, while retaining called FASTQ sequences and raw signal (i.e. it did what it said on the box). File sizes were reduced from 1.3MB down to 577kB for the R7.3 file, and 63Mb down to 9.8MB for the R9.4 file.\nPicopore also retained the 'Model' section from the R7.3 FAST5 files, indicating that it probably does a blacklist removal of known analysis components and retains unknown things in the file hierarchy; this should ensure that Picopore will be reasonably forward-compatible for future file format changes even without updates.\nThreaded mode\nI tried to run Picopore on the toy dataset in single-threaded mode, broke out of it because it was taking too long, then restarted in threaded mode and realised I wanted to stop that as well [Picopore leaves temporary files of a predictable name in the working directories that are not deleted on failure, and I had not deleted them between runs]. Unfortunately, when running Picopore in threaded mode, I was not able to break out of the running program (and needed to kill it using another console).\nEquivalence check on included dataset\nRunning the equivalence check in lossless mode on the provided toy dataset produced no errors. No additional testing was done on the toy dataset.",
"responses": [
{
"c_id": "2626",
"date": "10 Apr 2017",
"name": "Scott Gigante",
"role": "Author Response",
"response": "Dear Dr. Eccles, Thank you for your review. I have amended the manuscript according to your suggested revisions. You note that an equivalent to Picopore's raw compression is now the default behaviour for MinKNOW v1.5. I will continue to examine the output from the latest version of MinKNOW to find new mechanisms for size reduction; however, in the short term, I anticipate that Picopore's major use cases will be to reduce the size of older datasets, and to reduce the size of datasets produced by power users who continue to use MinKNOW with event data enabled. Thank you also for your extensive testing of Picopore. The errors you pointed out have been resolved in the latest version of Picopore, available on Pypi, Bioconda and Github. Thank you once again for your comments. Kind regards, Scott Gigante"
}
]
}
] | 1
|
https://f1000research.com/articles/6-227
|
https://f1000research.com/articles/6-1768/v1
|
27 Sep 17
|
{
"type": "Case Report",
"title": "Case Report: Spontaneous cholecystocutaneous fistula, a rare cholethiasis complication",
"authors": [
"Nunzio Maria Angelo Rinzivillo",
"Riccardo Danna",
"Vito Leanza",
"Melissa Lodato",
"Salvatore Marchese",
"Francesco Basile",
"Guido Nicola Zanghì",
"Nunzio Maria Angelo Rinzivillo",
"Riccardo Danna",
"Vito Leanza",
"Salvatore Marchese",
"Francesco Basile"
],
"abstract": "One of the most unusual complications in cholethiasis is spontaneous cholecystocutaneous fistula, which has only been reported a few times in the literature. We report the case of a 76 year old man who presented with a right hypochondrium subcutaneous abscess, with pain evoked through palpation. No comorbidity in the patient’s medical history were noted.\n\nConfirmation of cholecystocutaneous fistula was made using the proper diagnostic process, which is computed tomography with contrast media, followed by hepatobiliary MRI. This confirmed the presence of a fistulous pathway between the gallbladder and the skin. The patient underwent cholecystectomy surgery and open laparotomy with en block aponeurotic muscle, skin and fistula orifice excision.",
"keywords": [
"Cholecystocutaneous",
"Fistula",
"cholethiasis",
"abdominal fistula",
"gallbladder stones",
"gallbladder cancer",
"Bile Ducts",
"Laparotomy"
],
"content": "Introduction\n\nSpontaneous cholecystocutaneous abscess or fistula is an extremely uncommon complication of gallbladder disease. Less than 100 cases have been described in the literature. The first descriptions of cholecystocutaneous fistula was made by Thelisus in 1670. Later Courvoisier reported 169 cases of biliary fistula in the 19th century1. The natural history of the disease has changed from suppurative cholecystitis with spontaneous rupture to operative external drainage of an abscess2. Early and effective medical and surgical management of biliary tract disease can prevent this rare condition. Stone obstruction of the biliary tree plays a crucial role in the pathophysiology of the development of this condition; intra-gallbladder pressure can increase dramatically due to the obstruction of the cystic duct. The unresolved obstruction of the bile outflow compromise gallbladder wall blood circulation, as well as lymphatic drainage, resulting in necrosis of the gallbladder wall with the fistula formation. Once pierced, the gallbladder may drain into the peritoneal cavity, causing peritoneal localized abscess or the abscess can lead into an external fistula due to its adherence to the abdominal wall1–3.\n\n\nCase report\n\nA 76 year old man was admitted to our University Hospital “Ospedale Vittorio Emenuele” and seen in our surgical department. He presented with a 3 cm tumefaction of the right hypochondrium, surrounded by an erythematous skin area, with small secretion of a yellowing-green material, attributable to a bile leaking (Figure 1). The patient’s medical history was clear from previous medical disease and surgery; he only referred to previous upper right quadrant pain and nonspecific dyspeptic disorder.\n\nAn abdominal ultrasound examination revealed the presence of a lesion in the aponeurotic muscle wall, but the possible underlying pathology was unknown. No other signs of pathology were observed. Routine blood work was normal.\n\nAbdominal computed tomography scan with contrast media showed the gallbladder walls had diffuse thickened and blurred edges, and the right and transverse abdominal muscles were almost covered and embedded with minute hypo-dense ailments compatible with relapsing phlogistic processes (Figure 2). Hepatobiliary MRI detected that the gallbladder had anteriorly shifted and adhered to the right abdominal muscles. The side wall showed a break through with consensual purulent collection, which extruded through the thick abdominal wall (Figure 3). Eventually, several different-sized stones were revealed inside the cholecyst. Consequently, a diagnosis of spontaneous cholecystocutaneous fistula was made.\n\nLeft image, * indicates the gallbladder; right image, the arrows indicates the fistula.\n\nThe white arrows show the fistula. From left to right: coronal and sagittal fistula view.\n\nThe patient underwent cholecystectomy surgery 10 days after diagnosis, with an open laparotomy with en block aponeurotic muscle, skin and fistula orifice excision. In order to have a good abdominal wall reconstruction, a properly shaped Prolene Prosthesis was placed using fibrin glue4–7. The patient received broad-spectrum antibiotics after surgery (Piperacillin/Tazobactam 4.5 g, 3 times a day for 5 days via IV).\n\nConsidering the patient’s good general condition and good post-operative course, discharge was on the seventh day post-surgery. Surgical wound re-dressing was made one week after discharge in our facility, where the surgical stitches were removed. Scar appearance was good and without concern. No other dressing was needed and the scar was covered with a bandage. The first follow-up was scheduled 15 days after discharge, second one 60 days from discharge. For both follow-ups, routine blood work and surgical scar checking were performed, the results of which were normal and the scar was healing normally.\n\nA histological examination confirmed the diagnosis of chronic cholecystitis with gallstones and cholecystocutaneous fistula.\n\n\nDiscussion\n\nThanks to the progress made with medical imaging and surgical techniques, biliary fistula is today a very rare pathology8–11. Fistulas often represent the result of post-surgical12 or post-traumatic15 complications that generally involve the duodenum (77%) and colon (15%)16.\n\nSpontaneous cholecystocutaneous fistula represents a truly exceptional event, as confirmed by the analysis of the literature, which revealed only 28 cases published over the last 10 years (Table 1). This disease mainly affects female subjects over the age of 60. Etiology is generally due to an acute inflammatory process as a consequence of a cholecystitis or chronic gallstones disease17–20, although there are described cases of spontaneous cholecystocutaneous fistula in the absence of gallstones21. Rarely does cholecystocutaneous fistula evolve into a neoplastic process. Instead, fistula can be a sign of gallbladder cancer19,20. According to Sibakoti, polyarteritis nodosa with gallbladder vasculitis and prolonged use of high dose steroids can be considered predisposing factors21. Fistula primum movens is by cystic duct obstruction, which increases the pressure within the gallbladder, with wall distension and impaired vascularization, resulting in the formation of focal necrosis of the wall with perforation evolution and abscess formation to the surrounding area that will rupture in to the continuous structures. In the present case, the abscess drained through the abdominal wall and the fistulose pathway originated from the bottom of the gallbladder. This area is the most distant from the cystic artery and physiologically the least vascularized and therefore more susceptible to ischemia17.\n\nThe external fistular orifice is usually on the right upper quadrant, but other locations have been described, including the left hypocondrium, umbilical scar, right lumbar, and right iliac fossa, and rarely the right gluteus and breast region19,24,47.\n\nThe diagnostic process always begins with upper abdomen ultrasound and ends with hepatobiliary MRI to visualize the biliary tree. Considering that 11% of cholecystitis have concomitant presence of gallstones in the main bile duct, it is advisable to perform endoscopic retrograde cholangiopancreatography (ERCP)2–29. In our case CT with CM and hepatobiliary MRI confirmed the fistula presence and it was not necessary to execute the ERCP before surgery. Although an intraoperative cholangiogram was performed to check that the bile ducts were clear from gallstones. Cholecystocutaneous fistula has always been treated by two different strategies. The first includes a two-step approach: percutaneous drainage and antibiotic therapy, and subsequently cholecystectomy. The second directly involves laparotomy cholecystectomy execution with en block aponeurotic muscles, as well as skin and fistula orifice excision.\n\nThe second strategy is the most commonly used since the two-step approach treatment is reserved for patients with sepsis and poor general condition12,15,29.\n\nIn 1998, Kumar described the first case of gynecological fistula treated with laparoscopic technique, proposing to the scientific community the feasibility of this innovative approach27,30.\n\n\nConclusion\n\nRarity of this pathology confirms the great quality of progress made by early diagnostic techniques and medical treatment to prevent complication of cholethiasis. Although cholecystocutaneous spontaneous fistula is not common, it can lead to a serious condition. If not quickly treated, it can rapidly evolve into a generalized septic state with severe impairment prognosis. In our case, the patient was in good health arguably because the fistula was draining the most of the abscess outside the body and not in the peritoneum space. Surgical treatment was, however, essential to restore the physiologic bile flow and adequate broad-spectrum antibiotic prophylaxis lowed the risk of post-operative infections. Although laparoscopic approaches have been described since 1998, this pathology is, in most cases, continuing to be treated with open technique, most likely because it is easier and with fewer risks of post-surgical complications31–34.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of the patient’s clinical details and related images.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nFlora HS, Bhattacharya S: Spontaneous cholecystocutaneous fistula. HPB (Oxford). 2001; 3(4): 279–280. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGupta V, Benerjee S, Garg H, et al.: Spontaneous cholecysto-antral-cutaneous fistula: a consequence of neglected calculus cholecystitis. Singapore Med J. 2012; 53(10): e201–3. PubMed Abstract\n\nMathew G, Bhimji SS: Fistula, Cholecystocutaneous. University of Pelita Harapan, 2017. PubMed Abstract\n\nZanghì G, Rinzivillo NM, Caponnetto AM, et al.: Sentinel lymph node biopsy in breast cancer New indications and our experience. Ann Ital Chir. 2015; 86: 508–12. PubMed Abstract\n\nBiondi A, Tropea A, Monaco G, et al.: [Complications in the laparoscopic treatment of primary and secondary hernias of the abdominal wall]. 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Euromediterranean Biomedical Journal. 2016; 11(13): 101–106. Publisher Full Text\n\nVasanth A, Siddiqui A, O’Donnell K: Spontaneous cholecystocutaneous fistula. South Med J. 2004; 97(2): 183–5. PubMed Abstract | Publisher Full Text\n\nKumar SS: Laparoscopic management of a cholecystocutaneous abscess. Am Surg. 1998; 64(12): 1192–4. PubMed Abstract\n\nLeanza V, Zanghì G, Leanza G, et al.: A specific application of two psychological measures on pelvic organ prolapse patients. Giornale Italiano di Ostetricia e Ginecologia. 2015; 37(2): 82–86. Reference Source\n\nZanghì G, Leanza V, Vecchio R, et al.: Single-Incision Laparoscopic Cholecystectomy: our experience and review of literature. G Chir. 2015; 36(6): 243–246. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVecchio R, Marchese S, Famoso S, et al.: Colorectal cancer in aged patients. Toward the routine treatment through laparoscopic surgical approach. G Chir. 2015; 36(1): 9–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZanghì G, Di Stefano G, Caponnetto A, et al.: Breast cancer and sentinel lymph node micrometastases: indications for lymphadenectomy and literature review. G Chir. 2014; 35(11–12): 260–65. PubMed Abstract | Free Full Text\n\nMaynard W, McGlone ER, Deguara J: Unusual aetiology of abdominal wall abscess: cholecystocutaneous fistula presenting 20 years after open subtotal cholecystectomy. BMJ Case Rep. 2016; 2016: pii: bcr2015213326. PubMed Abstract | Publisher Full Text\n\nJayasinghe G, Adam J, Abdul-Aal Y: Unusual presentation of gallbladder perforation. Int J Surg Case Rep. 2016; 18: 42–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuardado-Bermúdez F, Aguilar-Jaimes A, Ardisson-Zamora FJ, et al.: [Spontaneous cholecystocutaneous fistula]. Cir Cir. (English Edition). 2015; 83(1): 61–64. PubMed Abstract | Publisher Full Text\n\nÁlvarez Florencia, Meraldi A, Emery NC, et al.: [Spontaneous cholecystocutaneous fistula in an elderly woman]. Rev Med Chil. 2014; 142(8): 1076–7. PubMed Abstract | Publisher Full Text\n\nDixon S, Sharma M, Holtham S: Cholecystocutaneous fistula: an unusual complication of a para-umbilical hernia repair. BMJ Case Rep. 2014; 2014: pii: bcr2013202417. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPripotnev S, Petrakos A: Cholecystocutaneous fistula after percutaneous gallbladder drainage. Case Rep Gastroenterol. 2014; 8(1): 119–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim M, Beenen E, Fergusson J: Spontaneous cholecystocutaneous fistula by gallstone erosion into abdominal wall. ANZ J Surg. 2014; 84(11): 888–89. PubMed Abstract | Publisher Full Text\n\nJayant M, Kaushik R, Attri AK: Spontaneous cholecystocutaneous abscess. Indian J Gastroenterol. 2014; 33(5): 498. PubMed Abstract | Publisher Full Text\n\nSodhi K, Athar M, Kumar V, et al.: Spontaneous cholecysto-cutaneous fistula complicating carcinoma of the gall bladder: a case report. Indian J Surg. 2012; 74(2): 191–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKapoor Y, Singh G, Khokhar M: Spontaneous cholecystocutaneous fistula-not an old time story. Indian J Surg. 2013; 75(Suppl 1): 188–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOzdemir Y, Yucel E, Sucullu I, et al.: Spontaneous cholecystocutaneous fistula as a rare complication of gallstones. Bratisl Lek Listy. 2012; 113(7): 445–47. PubMed Abstract | Publisher Full Text\n\nUgalde Serrano P, Solar García L, Miyar de León A, et al.: [Cholecystocutaneous fistula as a first sign of presentation of a gallbladder adenocarcinoma]. Cir Esp. 2013; 91(6): 396–97. PubMed Abstract | Publisher Full Text\n\nAndersen P, Friis-Andersen H: [Spontaneous cholecystocutaneous fistula presenting in the right breast]. Ugeskr Laeger. 2012; 174(18): 1235–36. PubMed Abstract\n\nIoannidis O, Paraskevas G, Kotronis A, et al.: Spontaneous cholecystocutaneous fistula draining from an abdominal scar from previous surgical drainage. Ann Ital Chir. 2012; 83(1): 67–69. PubMed Abstract\n\nCheng HT, Wu CI, Hsu YC: Spontaneous cholecystocutaneous fistula managed with percutaneous transhepatic gallbladder drainage. Am Surg. 2011; 77(12): E285–286. PubMed Abstract\n\nGordon PE, Miller DL, Rattner DW, et al.: Image of the Month—Quiz Case. Arch Surg. 2011; 146(4): 487–8. PubMed Abstract | Publisher Full Text\n\nSayed L, Sangal S, Finch G: Spontaneous cholecystocutaneous fistula: a rare presentation of gallstones. J Surg Case Rep. 2010; 2010(5): 5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPezzilli R, Barakat B, Corinaldesi R, et al.: Spontaneous Cholecystocutaneous Fistula. Case Rep Gastroenterol. 2010; 4(3): 356–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMetsemakers WJ, Quanten I, Vanhoenacker F, et al.: Spontaneous Cholecystocutaneous Abscess. JBR-BTR. 2010; 93(4): 198–200. PubMed Abstract | Publisher Full Text\n\nTallón Aguilar L, López Porras M, Molina García D, et al.: Fístula colecistocutánea. Una rara complicación de la colelitiasis. Gastroenterol Hepatol. 2010; 33(7): 553–54. Publisher Full Text\n\nKhan AA, Azhar MZ, Khan AA, et al.: Spontaneous Cholecystocutaneous Fistula. J Coll Physicians Surg Pak. 2005; 15(11): 726–27. PubMed Abstract\n\nHawari M, Wemyss-Holden S, Parry GW: Recurrent Chest Wall Abscesses Overlying a Pneumonectomy Scar: An Unusual Presentation of a Cholecystocutaneous Fistula. Interact Cardiovasc Thorac Surg. 2010; 10(5): 828–29. PubMed Abstract | Publisher Full Text\n\nMurphy JA, Vimalachandran CD, Howes N, et al.: Anterior abdominal wall abscess secondary to subcutaneous gallstones. Case Rep Gastroenterol. 2008; 2(2): 219–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIjaz S, Lidder S, Mohamid W, et al.: Cholecystocutaneous fistula secondary to chronic calculous cholecystitis. Case Rep Gastroenterol. 2008; 2(1): 71–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChatterjee S, Choudhuri T, Ghosh G, et al.: Spontaneous Cholecystocutaneous Fistula in a Case of Chronic Colculous Cholecystitis--a Case Report. J Indian Med Assoc. 2007; 105(11): 644, 646, 656. PubMed Abstract\n\nMalik AH, Nadeem M, Ockrim J: Complete laparoscopic management of cholecystocutaneous fistula. Ulster Med J. 2007; 76(3): 166–7. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "27856",
"date": "20 Nov 2017",
"name": "Matteo Novello",
"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 should clarify the reasons for the 10 days delay in the operative management of their patient.\nMore insight should be provided on the choice of aggressive operative treatment as compared to a percutaneous drainage. Perhaps the drainage would have been more readily available and also less expensive as compared to elective surgery. Keeping in mind the age of the patient, it might have been a reasonable option proceeding with conservative management and percutaneous drainage trial.\nDid the authors investigate the use of somatostatin (provided in the form of Octreotide) in such case? It is currently a first line approach for uncomplicated pancreatic-cutaneous fistulas and might as well work with the Gallbladder considering the physiological functions of the hormone.\nAll in all the case is interesting and, considering the rarity of the event, it deserves publication.\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": "28162",
"date": "21 Nov 2017",
"name": "Antonino Agrusa",
"expertise": [
"Reviewer Expertise General and emergency surgery"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMy compliments to the authors for the interesting and rare case report. I approve it for pubblication in F1000 Research.\nPlease give more explanation for the 10 days delay between patient admission and operative surgical management. On the basis of rare case report the authors should clarify the operative technique with more details.\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": []
},
{
"id": "28008",
"date": "24 Nov 2017",
"name": "Giuseppe Navarra",
"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 Case Report: Spontaneous cholecystocutaneous fistula, a rare cholethiasis complication covers a subject that nowadays is quite uncommon. It goes through the background of the case’s history, it provides details of diagnostic tests, treatment given and outcomes reached. It could be useful for other practitioners. It can be accepted for indexing with minor English revision.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1768
|
https://f1000research.com/articles/6-1752/v1
|
26 Sep 17
|
{
"type": "Method Article",
"title": "A method to estimate the number of neurons supporting visual orientation discrimination in primates",
"authors": [
"Ruben Coen-Cagli",
"Ingmar Kanitscheider",
"Alexandre Pouget",
"Ingmar Kanitscheider",
"Alexandre Pouget"
],
"abstract": "In this method article, we show how to estimate of the number of retinal ganglion cells (RGC), and the number of lateral genicular nucleus (LGN) and primary visual cortex (V1) neurons involved in visual orientation discrimination tasks. We reported the results of this calculation in Kanitscheider et al. (2015), where we were interested in comparing the number of neurons in the visual periphery versus visual cortex for a specific experiment. This calculation allows estimation of the information content at different stages of the visual pathway, which can be used to assess the efficiency of the computations performed. As these numbers are generally not readily available but may be useful to other researchers, we explain here in detail how we obtained them. The calculation is straightforward, and simply requires combining anatomical and physiological information about the macaque visual pathway. Similar information could be used to repeat the calculation for other species or modalities.",
"keywords": [
"visual cortex",
"vision",
"population coding"
],
"content": "Introduction\n\nWe would like to estimate:\n\n1) the number of RGC and LGN neurons activated by a given visual stimulus; and\n\n2) the number of V1 neurons that are activated by the same stimulus and are relevant to compute orientation.\n\nWe define as relevant those neurons that project to higher cortex, and separately consider the additional requirement that the neurons are tuned for orientation.\n\n\nMethods\n\nFirst, the number of neurons activated by a visual stimulus depends on the size and position of the image in the visual field. We use as a reference the setup of Dosher & Lu (1998) in experiments with human subjects, but the calculation reported below can be readily applied to visual stimuli of different size and position. Stimuli are presented parafoveally, at eccentricities between 2 and 5 degrees, and cover an area of 1×1 to 2×2 deg2 in the visual field. To illustrate the calculation, we assume a 1×1 deg2 stimulus at an eccentricity of 3 degrees.\n\nThe second factor is the volume of the cortex (surface x depth) that is activated by such stimuli. To estimate the surface we use cortical magnification factors (number of neurons per deg2, as a function of eccentricity). We use here the results of Van Essen et al. (1984) who found that the following equation captured the relation between eccentricity (E, in degrees) and cortical surface activated by a 1×1 deg2 stimulus (M, in mm2/deg2):\n\nM = 103(0.82 + E)–2.28 (1.1)\n\nHence, presenting a 1×1 deg2 stimulus at an eccentricity of 3 degrees should activate a cortical surface of approximately 5 mm2.\n\nTo determine cortical depth, we assume that the only V1 neurons used to solve a visual task are those that project to higher cortex, i.e. pyramidal neurons in layers 2/3/4B (Kandel et al., 2000, ch. 27). These layers constitute approximately 0.75 mm of cortical depth (Peters & Rockland, 1994, ch. 1), and approximately 80% of neurons in V1 are excitatory (Peters & Rockland, 1994, ch. 1), hence we consider an effective depth of 0.8×0.75=0.6 mm. Combining this with the estimated surface of 5 mm2, we obtain an effective volume of 3 mm3.\n\nWe then multiply this volume by cortical density, which in V1 is approximately 120,000 neurons/mm3 (O’Kusky & Colonnier, 1982). This leads to an estimate of 360,000 neurons/deg2. This number represents an upper bound, assuming that all neurons contribute to decoding orientation regardless of their individual tuning for orientation. This is a reasonable assumption as long as the variability of untuned neurons is correlated with that of tuned neurons (Zylberberg, 2017) or the tuning is not perfectly flat.\n\nTo obtain also a lower bound, we consider the alternative that only neurons selective for orientation are relevant for orientation discrimination. Ringach et al. (2002) characterized the distribution of orientation selectivity in V1 across layers. Using bandwidth (half of the tuning curve width at 1/2 height) as a measure of selectivity, if we include only neurons with bandwidth smaller than 30 degrees we find that, across layers 2/3/4B, approximately 75% of the neurons satisfy the criterion. The threshold of 30 degrees is arbitrary; we chose a rather small threshold to obtain a lower bound. Combining the above estimates, the lower bound on the number of V1 neurons that can be used by downstream areas for orientation discrimination in the experimental setting considered here is 270,000. This is the estimate reported in Kanitscheider et al. (2015).\n\nAn additional consideration is that typical extracellular recordings with single electrodes, as in Ringach et al. (2002), tend to be biased towards neurons that are visually responsive (i.e. activity evoked by their preferred stimulus is substantially larger than spontaneous activity) and have high firing rates (Olshausen & Field, 2005), raising the possibility that we overestimated the proportion of tuned neurons. Data recorded with chronically implanted multielectrode arrays (Kelly et al., 2007), which do not suffer from those biases, indicate that the proportion of tuned neurons in L2/3 is consistent with Ringach et al. (2002). However, if some neurons were entirely silent throughout a recording sessions, i.e. they did not fire even a spontaneous action potential, they would go undetected, thus positively biasing the proportion of tuned neurons. We are not aware of direct estimates of the number of such neurons in macaque, but we can use as a guidance recent studies of rodent V1. Using calcium imaging, Ko et al. (2014) found that 55% of all neurons in a small volume are responsive to at least one visual stimulus, indicating that silent neurons represent at most 45% of the population. If we further assume that silent neurons do not contribute to orientation discrimination, we are left with a proportion of 0.75*0.55, or approximately 150,000 neurons. This represents a loose lower bound.\n\nThe LGN volume and number of neurons activated by the same visual stimulus can be computed similarly from cell magnification factors derived by Malpeli et al. (1996) for parvocellular and magnocellular layers, respectively:\n\nNP = 1,011,688(2.9144 + E)–2.6798\n\nNM = 2,620.2(5.5638 + (E – 1.8322)2)–0.8012 (1.2)\n\nThe above leads to an estimate of approximately 9,000 LGN neurons for a 1×1 deg2 stimulus at an eccentricity of 3 degrees.\n\nLastly, to estimate the number of RGCs we used the cell magnification factors provided by Malpeli et al. (1996) figure 11 (based on Wässle et al., 1990), who assumed a constant fraction of RGCs projecting to LGN (90%) and binocular viewing conditions. We then interpolated linearly between the reported RGC densities at eccentricities of 5 degrees (3,000 cells/deg2) and 2 degrees (8,500 cells/deg2), and obtained an estimate of approximately 6,500 RGCs for a 1×1 deg2 stimulus at an eccentricity of 3 degrees.\n\n\nDiscussion\n\nWe have illustrated a method to estimate the number of neurons involved in a visual orientation discrimination task. The method involves considerations about experimental stimuli, including their size and position in the visual field, and considerations about the anatomy of the visual system, including magnification factors and neuron density. By considering different possible requirements for the subset of neurons that may be involved in the task, we have derived lower bounds on the estimates. Our results suggest an LGN-V1 expansion ratio between 17:1 and 40:1, similar to values reported previously for visual cortex (DiCarlo et al., 2012; Olshausen & Field, 2004), and other sensory pathways (Brecht & Sakmann, 2002; DeWeese et al., 2003; Mombaerts et al., 1996). Similarly, the RGC-V1 expansion ratio is between 23:1 and 55:1. The method could be readily applied to different stimuli and other visual areas.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Swiss National Science Foundation (31003A_143707) and the Simons Foundation.\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 Adam Kohn for helpful discussion.\n\n\nReferences\n\nBrecht M, Sakmann B: Dynamic representation of whisker deflection by synaptic potentials in spiny stellate and pyramidal cells in the barrels and septa of layer 4 rat somatosensory cortex. J Physiol. 2002; 543(Pt 1): 49–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeWeese MR, Wehr M, Zador AM: Binary spiking in auditory cortex. J Neurosci. 2003; 23(21): 7940–7949. PubMed Abstract\n\nDiCarlo JJ, Zoccolan D, Rust NC: How does the brain solve visual object recognition? Neuron. 2012; 73(3): 415–434. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDosher BA, Lu ZL: Perceptual learning reflects external noise filtering and internal noise reduction through channel reweighting. Proc Natl Acad Sci U S A. 1998; 95(23): 13988–13993. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKandel ER, Schwartz JH, Jessell TM: Principles of Neural Science. Fourth Edition, New York, McGraw-Hill. 2000. Reference Source\n\nKanitscheider I, Coen-Cagli R, Pouget A: Origin of information-limiting noise correlations. Proc Natl Acad Sci U S A. 2015; 112(50): E6973–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKelly RC, Smith MA, Samonds JM, et al.: Comparison of recordings from microelectrode arrays and single electrodes in the visual cortex. J Neurosci. 2007; 27(2): 261–264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKo H, Mrsic-Flogel TD, Hofer SB: Emergence of feature-specific connectivity in cortical microcircuits in the absence of visual experience. J Neurosci. 2014; 34(29): 9812–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMalpeli JG, Lee D, Baker FH: Laminar and retinotopic organization of the macaque lateral geniculate nucleus: Magnocellular and parvocellular magnification functions. J Comp Neurol. 1996; 375(3): 363–377. PubMed Abstract | Publisher Full Text\n\nMombaerts P, Wang F, Dulac C, et al.: Visualizing an olfactory sensory map. Cell. 1996; 87(4): 675–686. PubMed Abstract | Publisher Full Text\n\nO’Kusky J, Colonnier M: A laminar analysis of the number of neurons, glia, and synapses in the adult cortex (area 17) of adult macaque monkeys. J Comp Neurol. 1982; 210(3): 278–290. PubMed Abstract | Publisher Full Text\n\nOlshausen BA, Field DJ: How close are we to understanding v1? Neural Comput. 2005; 17(8): 1665–1699. PubMed Abstract | Publisher Full Text\n\nOlshausen BA, Field DJ: Sparse coding of sensory inputs. Curr Opin Neurobiol. 2004; 14(4): 481–487. PubMed Abstract | Publisher Full Text\n\nPeters A, Rockland KS eds: Cerebral Cortex: Volume 10 Primary Visual Cortex in Primates. Springer. 1994. Publisher Full Text\n\nRingach DL, Shapley RM, Hawken MJ: Orientation Selectivity in Macaque V1: Diversity and Laminar Dependence. J Neurosci. 2002; 22(13): 5639–5651. PubMed Abstract\n\nVan Essen DC, Newsome WT, Maunsell JH: The visual field representation in striate cortex of the macaque monkey: Asymmetries, anisotropies, and individual variability. Vision Res. 1984; 24(5): 429–448. PubMed Abstract | Publisher Full Text\n\nWässle H, Grünert U, Röhrenbeck J, et al.: Retinal ganglion cell density and cortical magnification factor in the primate. Vision Res. 1990; 30(11): 1897–911. PubMed Abstract | Publisher Full Text\n\nZylberberg J: Untuned But Not Irrelevant: A Role For Untuned Neurons In Sensory Information Coding. bioRxiv. 2017; 134379. Publisher Full Text"
}
|
[
{
"id": "28134",
"date": "28 Nov 2017",
"name": "Gregory W. Schwartz",
"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 a concise summary of the figures that go into estimating the number of neurons in retina, LGN, and V1 that are involved in a visual discrimination task. There are clearly a number of assumptions that go into such a calculation, but the authors do a good job of stating these assumptions explicitly and citing the relevant literature. One exception is the figure that 90% of RGCs project to LGN (specifically dorsal LGN in this case). This figure will also depend on eccentricity because the proportion of parasol and midget RGCs (which do project to dLGN) depends on eccentricity. In peripheral retina, the fraction of dLGN-projecting RGCs is far below 90% in primates. With this relatively simple fix, I think this short paper will provide a nice reference for calculations of cell counts in the early visual pathways.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "26423",
"date": "02 Jan 2018",
"name": "Denis G. Pelli",
"expertise": [
"Reviewer Expertise Perception",
"Object recognition"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Coen-Cagli et al. paper is useful, showing a reasonable way to use known physiology and anatomy to estimate the number of neurons responding to a stimulus. This may be useful towards the long term goal of figuring out how the brain puts together the activity of multiple neurons to make perceptual decisions.\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/6-1752
|
https://f1000research.com/articles/6-1319/v1
|
03 Aug 17
|
{
"type": "Research Note",
"title": "Giving eyespots a shiner: Pharmacologic manipulation of the Io moth wing pattern",
"authors": [
"Andrei Sourakov"
],
"abstract": "Our knowledge of wing pattern formation in Lepidoptera has advanced significantly in recent years due to the careful examination of several groups of butterflies. The eyespot is a prominent feature of Lepidoptera wing pattern, especially in the family Saturniidae. The present study examined how sulfated polysaccharides, which are known to simulate cold shock effect in nymphalid butterflies, affected the wing pattern formation of the Io moth, Automeris io (Saturniidae). Prepupae and pupae of this species were subjected to injections of heparin and cold shock. While the cold shock had little to no effect on wing pattern, the aberrations resulting from heparin injections consisted of moderate to profound increases in melanism around the eyespots. The resulting aberrations are dubbed ‘Black Eye’ and ‘Comet Eye.’ Most other known aberrations of Automeris io eyespots are summarized, illustrated and named.",
"keywords": [
"Eyespots",
"wing pattern",
"Lepidoptera",
"heparin",
"phenotypic plasticity",
"melanism",
"butterflies",
"moths"
],
"content": "Introduction\n\nWhile our understanding of the mechanisms involved in butterfly wing pattern development has been increasing exponentially in the recent two decades, the work has been largely limited to butterflies such as Junonia, Heliconius, Papilio and Bicyclus. Thanks to these ‘model’ genera, we now understand homologies among wing pattern elements and the adaptive radiation that led to the kaleidoscope of intriguing ‘designs’ found among ca. 160,000 Lepidoptera species (Martin & Reed, 2010).\n\nNatural and artificially generated aberrations serve as windows into the developmental mechanisms and evolutionary history of animals. In addition to many naturally occurring melanic aberrations and some melanic recessive phenotypes that can be obtained and/or maintained through inbreeding, the dark markings of Lepidoptera wings can sometimes be amplified by the timely application of a colder regime to the immature stages (e.g., Sourakov, 2015 and references within). Serfas & Caroll (2005) first demonstrated that injections of heparin into the early pupal stage can simulate cold shock and alter wing patterns in similar ways. Martin & Reed (2014) utilized heparin injections to understand genetic controls and homologies among separate wing pattern elements.\n\nEyespots are characteristic of many Lepidoptera, and considerable advances have been made towards understanding their evolution (Monteiro et al., 2006). In Automeris io, a species whose name, if anglicized (‘Eye’‘Oh’!), invokes associations with its pair of magnificent dorsal hindwing eyespots that are exposed when the moth (otherwise cryptic) is threatened. Several recessive mutations causing deformations of the black ring surrounding the dark blue eyespot with a white center have been obtained through inbreeding, conducted first by Thomas Manley (1978, 1990) and, more recently, myself (Table 1 below). However, the most dramatic aberration, which involves the melanization of almost the entire hindwing, was found in an A. io male collected in 1966. It was noticed only recently while the MGCL Saturniidae collection was being re-curated (Covell, 2012).\n\nIn the present study, an attempt has been made to replicate these aberrations with heparin injections to the prepupal and pupal stages, as well as by cold shock. The results of the former manipulations, while not replicas of previously known aberrations, are quite dramatic and are illustrated here along with some other aberrations, both known and those previously unrecorded.\n\n\nMethods\n\nRepresentatives of five broods of Automeris io from local stock (over 300 caterpillars) were reared on sugarberry under ambient condition in Gainesville, Florida, in the fall of 2016 resulting in 130 pupae. While the caterpillars were pupating in November, ten randomly selected pupae were injected, using a 10µl syringe, under a wing with ≈5mg (10µl (1 drop) of heparin dissolved in distilled water). Additionally, one prepupa was injected a day before pupation with half of that amount, and a dozen were subjected to cold shock in the refrigerator (4–5 C°) overnight. Ten other pupae were injected with 10µl of mannitol (saturated solution), and the rest were left untreated. All pupae were kept under ambient conditions during diapause, until they began emerging in May 2017.\n\n\nResults and discussion\n\nWhile most of the pupae that were injected did not emerge, one female with a strongly modified wing pattern emerged from a pupa injected with 5mg of heparin (Figure 1A.i). Injection must have damaged the right hindwing, so it did not spread properly (Figure 1A.ii), but the left side was structurally intact. The control sibling female is illustrated in Figures 1B.i and B.ii for comparison.\n\nA less aberrant male, whose prepupa was injected with 5mg of heparin a day before pupation, also emerged (Figures 2A.i, 2A.ii), different in its dorsal hindwing eyespots from all control siblings (e. g., Figure 2B). Most of the cold-shocked and the mannitol-injected individuals showed no obvious deviation from expected wing patterns. The slight wing pattern changes (Figures 2C and 2D) exhibited by two females, cold-shocked as prepupae, suggest that cold shock may have some melanization-inducing effect and perhaps, if administered differently, may potentially have a greater effect on the phenotype.\n\nA. “Black eye” aberration. A female of the Io Moth, Automeris io, with wing pattern altered by injection 5mg of heparin (sulfated polysaccharide) into the early pupal stage. Voucher FLMNH-MGCL#289216. B. A normal A. io female from the same brood. (i) dorsal, (ii) ventral surface. Photos by Andrei Sourakov.\n\nA. “Comet eye” aberration. A male of the Io Moth, Automeris io, from prepupa injected with 2.5mg of heparin a day prior to pupation. FLMNH-MGCL#289217. B. A normal A. io male from the same brood. Voucher FLMNH-MGCL#289218. C, D. Slight aberrations (“Comet eye” and “Barley eye”) of the black ring around eyespots in two A. io females cold-shocked as prepupae. Photos by Andrei Sourakov.\n\nOn the dorsal hindwing of aberrant female (Figure 1A.i), the blue eyespot center is smaller than that of the control siblings (e.g., Figure 1B.i), due to the infraction of melanin from the surrounding black ring. Heparin injection must have enhanced or prolonged the process of expansion of black pigment once it formed in the black ring around the blue scales. Judging by the wild aberrant male, such melanization process can go as far as eliminating the eyespot entirely (Figure 3A). In the heparin-injected aberrant female, the process of expansion mostly occurred outwards, so that the black ring around the eyespot merged with the normally thin black EIII line of the hindwing margin, which too had widened (Figure 1A.i).\n\nA. “Caecus” aberration. A wild-collected Automeris io male exhibiting a unique melanic aberration in which most wing pattern elements are obscured by melanin. (i) Dorsal side, (ii) Ventral side, (iii) Close-up of the eyespot area. Collected in Wascott Township, Douglas Co., Wisconsin, on 24 June 1966 by J.L. Boughner; Voucher FLMNH-MGCL#1000907. B, C. “Broken eye” aberrations resulting from recessive mutation reared by Thomas R. Manley (1978, 1990). (B) Male 67#3 resulting from cross of wild “broken eye” female crossed with sibling non-aberrant male; Collection of Peabody Museum; YPM No. 843761; (C) Female 67#1 sibling with the above male; YPM No. 843765. D. “Teardrop” aberration resulting from a recessive mutation reared by Thomas R. Manley. Cross 13-86 (1986) of normal “teardrop” brood male 10-85 with sib “teardrop” female 27-85. Collection of Peabody Museum, YPM No. 843755. E. “Winking eye” aberration resulting from a recessive mutation (expressed through 2nd consecutive full-sib crossing) reared by A. Sourakov in 2014. F. “Barley eye” aberration resulting from a recessive mutation (expressed through full-sib crossing) reared by A. Sourakov in 2014. Voucher FLMNH-MGCL #166627. Photos by Andrei Sourakov.\n\nA name “Black Eye” is proposed for the aberration shown in Figure 1A, following the tradition started by Manley (1978, 1990), who gave genetic aberrations of Automeris io names, such as “Broken eye,” (Figures 3B and 3C) and “Teardrop” (Figure 3D). Names of all aberrations, old and newly proposed, are summarized in Table 1.\n\nAs can be observed by comparing the “Black Eye” to its sibling (Figure 1B.i), the DI element of the forewing also underwent modification, as if it were smudged from its center along the veins toward the distal part of the wing. The ventral surface of the wing in “Black Eye” shows considerable expansion and diffusion of the small and compact black ring of control specimens around the small white ventral eyespot (Figures 1A.ii and 1B.ii).\n\nScholars engaged in wing pattern research have recently identified 27 genes associated with wing melanin production (Zhang et al., 2017). It is hoped that the present publication, while documenting unique aberrations in a single species, will be useful in the future work directed at understanding wing pattern evolution and development in general.",
"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\nI thank Larry Gall, the Entomology Collection Manager of the Peabody Museum at Yale University, for granting access to the collection and for permission to photograph and publish Automeris io aberrations reared by late T. R. Manley. I am also thankful to my family for their tolerance during the Thanksgiving break, when I was occupied with this experiment, and especially to my daughter Allie for proofreading this manuscript.\n\n\nReferences\n\nCovell VC Jr: A striking aberrant Automeris io. Association for Tropical Lepidoptera Notes. 2012; 4. Reference Source\n\nManley TR: Genetics of conspicuous markings of the Io moth. J Hered. 1978; 69(1): 11–18. Publisher Full Text\n\nManley TR: Heritable color variants in Automeris io (Saturniidae). Journal of Research on the Lepidoptera. 1990 (1991); 29(1–2): 37–53. Reference Source\n\nMartin A, Reed RD: Wingless and aristaless2 define a developmental ground plan for moth and butterfly wing pattern evolution. Mol Biol Evol. 2010; 27(12): 2864–2878. PubMed Abstract | Publisher Full Text\n\nMartin A, Reed RD: Wnt signaling underlies evolution and development of the butterfly wing pattern symmetry systems. Dev Biol. 2014; 395(2): 367–378. PubMed Abstract | Publisher Full Text\n\nMonteiro A, Glaser G, Stockslager S, et al.: Comparative insights into questions of lepidopteran wing pattern homology. BMC Dev Biol. 2006; 6(1): 52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSerfas MS, Carroll SB: Pharmacologic approaches to butterfly wing patterning: sulfated polysaccharides mimic or antagonize cold shock and alter the interpretation of gradients of positional information. Dev Biol. 2005; 287(2): 416–424. PubMed Abstract | Publisher Full Text\n\nSourakov A: Temperature-dependent phenotypic plasticity in wing pattern of Utetheisa ornatrix bella (Erebidae, Arctiinae). Tropical Lepidoptera Research. 2015; 25(1): 33–45. Reference Source\n\nZhang L, Martin A, Perry MW, et al.: Genetic Basis of Melanin Pigmentation in Butterfly Wings. Genetics. 2017; 205(4): 1537–1550. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "24773",
"date": "04 Aug 2017",
"name": "Arnaud Martin",
"expertise": [
"Reviewer Expertise Developmental Genetics",
"Lepidoptera"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript presents some exciting pattern aberrations observed in the eyespots of the Io Moth, including specimens obtained from drug injections, a wild-caught individual, and previously undescribed collection specimens.\nI enjoyed reading the manuscript and thank the author for publishing this. The reported phenotypes provide valuable information on the formation of eyespot rings in this particularly spectacular species.\n\nI noticed the following minor issues.\nWhat was the rationale for injecting mannitol?\n\nSerfas and Caroll (2005) -> Carroll with two r (typo)\n\nMethods: \"Ten randomly selected pupae were injected\" Please provide more details if possible on the staging. Were they tender and relatively fresh? Is there a way to determine the maximum age they were at, or a time estimate based on their cocoon spinning? 5mg of heparin is a large dose, so it may have killed the younger pupae, while the aberrant female came out by chance by being at a more resilient stage (or was accidentally injected with much less compound)\n\nMethods, please confirm the injected concentration of the injected heparin (apparently 0.5mg/uL), and provide the exact origin of the compounds (sodium salt? molecular weight? manufacturer?)\n\nFigure 1 Legend: the abbreviation for DI BR and EIII are missing.\n\nThe author is wrong calling the outer black line \"EIII\". Schwanwitsch 1956 and Henke 1936 (in 3 other Saturniids) suggest this is MI (central symmetry system outer band). I am personally inclined to say that while it may not be M1 in A. io ... it is certainly not EIII\n\n\"A less aberrant male, whose prepupa was injected with 5mg of heparin a day before pupation, also emerged\" I believe the author meant \"A less aberrant male, whose prepupa was injected with 2.5mg of heparin ONE day before pupation, also emerged\"\n\n\"Heparin injection must have enhanced or prolonged the process of expansion of black pigment once it formed in the black ring around the blue scales.\"\n\nThere is a way to deepen the discussion here, and I will try to explain briefly (feel free to use this suggestion).\n\nThe A. io Discal Spots (DI = Discalis I element of the Schwanwitsch pattern homology system) are stereotypical patterns that are always overlapping with the discal crossvein. Martin and Reed (2010) suggests that these spots always express the wingless morphogen in Lepidoptera, and Martin and Reed (2014) also shows that DI elements are responsive to heparin treatment in nymphalids. Interestingly, heparin is known to enhance wingless signaling in Drosophila: Baeg et al. (2001), Binari et al (1997) and Greco et al (2001).\nThus, the eyespot ring expansions observed upon heparin injection here suggest the exciting possibility that wingless, or perhaps other heparin-sensitive morphogens, are deployed during pre-pupal and pupal development to pattern different aspects of the discal ocelli structure.\n\nTo be honest, the current insight about melanization is a little too phenomenological, because melanin pigment synthesis happens much later in development and there is no known interaction between heparin and melanin biosynthesis...\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": []
},
{
"id": "24774",
"date": "17 Aug 2017",
"name": "Jeffrey M. Marcus",
"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 very promising approach to expanding the experimental study of color pattern development beyond the select group of model butterfly species that have received the bulk of the attention thus far. The Saturniid moth Automeris io was used for these experimental studies. Also described are A. io specimens derived from breeding experiments and wild-caught specimens with aberrant phenotypes.\n\nWhile I enjoyed reading the manuscript, there are a number of changes that I would like to suggest to the author:\n\nClarification of methods: There are a number of methodological details that should be clarified.\n“sugarberry”. Please give scientific name. Is this Celtis laevigata?\n\nStaging of pupae. As much detail as possible should be given here about when and how the injections were done relative to the pupal molt. How many hours at what temperature? Was the cuticle sclerotized yet? Where on the experimental specimens’ bodies did the injections take place? Under what conditions were the animals allowed to recover from their injections? What was the manufacturer and purity of the heparin used? For coldshock, how many hours is “overnight”?\n\nControl injections: what was the rationale for using manitol? Many experiments pair heparin with chondroitin sulfate B (keratan sulfate) as a negative control because it is structurally similar to heparin, but lacks the biological activity associated with heparin.\n\nOverwintering conditions: the author reports that the experimental animals were kept at ambient conditions until emergence the following spring. Were they kept indoors or outdoors? If indoors what was the typical temperature and lighting conditions during this period? If outdoors, try to provide descriptors of climatic conditions during the appropriate period using National Weather Service or other data.\n\nClarification of results: Additional clarification of the results is also required.\nThe author reports that most of the injected individuals did not emerge. How many failed to emerge? Were there any differences in eclosion rate between the heparin injected individuals and the chondroitin sulfate B injected individuals?\n\nWhat was the emergence rate of unmanipulated individuals? Was it different from the emergence rate of cold-shocked individuals?\n\nReinterpretation of Results and Elaboration of Discussion:\nThe author should keep in mind that Lepidopteran color patterns can be altered by manipulation of the developmental processes responsible for determining color patterns as well as by manipulation of the developmental processes responsible for color pattern differentiation. Determination processes might involve cell-cell signaling by signal transduction processes, while differentiation of color patterns involves the expression and regulation of biosynthetic pathways responsible for pigment synthesis. Manipulations such as cold-shock might have effects on both kinds of developmental processes, if they are taking place at the time of the manipulation. Additional references to prior work that examines the effects of coldshock (Nijhout 1984; Serfas and Carroll 2005; Mahdi et al. 2010; Dhungel and Otaki 2013) might be warranted.\n\nEverything that we know about the action of heparin suggests that it interacts with signal transduction pathways such as wingless/wnt (Binari et al. 1997), and there is no evidence that it interacts with melanin pigment biosynthesis. Discussing the experimental results of heparin injection from this study in reference to what is known about wingless/wnt signaling and its effects on color pattern determination in insects (Carroll et al. 1994; Monteiro et al. 2006; Martin and Reed 2010; Werner et al. 2010) would be highly desirable. I also urge the author to extend the discussion further and to connect his work with prior work on Saturniid moth color pattern development including both classic cautery studies (Henke 1933; Henke 1944) and studies of gene expression (Monteiro et al. 2006). Interestingly, the discal eyespots of Automeris io are positioned on top of crossveins, structures that also employ wingless/wnt signaling during their development (Conley et al. 1997; Marcus 2001). Relating eyespot development to underlying crossvein development in Saturniid moths, perhaps through modulation of the wingless/wnt pathway would be a very interesting research trajectory to pursue. Understanding how discal eyespots in some Saturniid moths are both similar to and different from border eyespots in butterflies would be a very interesting avenue for further studies of the evolution and development of Lepidopteran color patterns.\n\nI would like to encourage the author to consider these points in his revisions and also to continue his experimental explorations in future work.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1319
|
https://f1000research.com/articles/6-1749/v1
|
25 Sep 17
|
{
"type": "Case Report",
"title": "Case Report: Mucus plug in bronchus mimicking a bronchial solid foreign body obstruction",
"authors": [
"Kiran Devkota",
"Miao He",
"You Wei Zhang",
"Miao He",
"You Wei Zhang"
],
"abstract": "Bronchial foreign body obstruction is common in all clinical settings. Obstruction of the airway due to foreign bodies and foreign body aspiration are major causes of childhood mortality and morbidity, which are a big challenge to manage. Occasionally, bronchial obstruction may be due to mucus plugs or other endogenous factors. Here we describe a case of bronchial obstruction caused by mucus plug formation that was managed conservatively in a one-year old boy. The patient was suffering from a cough and noisy breathing for 2 days prior to coming to our hospital, when he experienced sudden onset of difficulty in breathing and a severe cough. At the time of presentation his vital sign readings were:- HR 186 bpm, RR 46/min, BP 78/40 MmHg, temp 36.9°C and SPO2 68%. He was given oxygen immediately and nebulization was started. Chest CT scan was performed that suggested the presence of a right bronchial foreign body with right sided obstructive emphysema. The patient was stable with oxygenation and nebulization with ipratropium bromide, albuterol, normal saline and budesonide before the CT scan. Therefore, we conclude that symptoms resembling foreign body obstruction are not always aspirated or inhaled, and sometimes secreted sputum forms a plug, which mimics the symptoms of foreign body obstruction.",
"keywords": [
"foreign body",
"bronchial obstruction",
"plastic bronchitis"
],
"content": "Introduction\n\nObstruction of the airway due to foreign bodies, and foreign body aspiration are major causes of childhood mortality and morbidity, which are a big challenge to manage. In the US, more than 17,000 patients visited the emergency department with foreign body aspiration in 2008, and 220 children aged <14 years died due to foreign body aspiration in 20091. In children younger than one year, airway obstruction by foreign bodies is the third most common cause of death due to unintentional injury1. In the US, 2900 deaths occur annually due to foreign body aspiration, as estimated by The National Safety Council2. Tracheal and bronchial foreign body obstruction is mainly seen among preschool children:- the most common is in infants and young children. More than 75% of cases due to the foreign body aspiration occur in children aged less than 3 years with 7% mortality rate3. In addition, foreign body aspiration is one of the major causes of infant and childhood mortality in developing countries3. The greatest significance of our case is that it provides another possibility in diagnosis of endogenous foreign bodies and its treatment.\n\n\nCase presentation\n\nA one year boy with a history of cough and noisy breathing for 2 days was brought to Renmin Hospital with a sudden onset of difficulty in breathing and severe cough for 5 hours. He had no history of fever, no cold and no history of vomiting or choking. Bowel and bladder habits were normal. He had no significant past medical history. The patient had normal birth history, normal growth, and immunization was up to date. At the time of presentation he was conscious but restless and had difficulty in breathing: more so for inspiration. He had a bluish discoloration of lips, and was pale, but not icteric. He had no signs of dehydration and had no any palpable lymph node. At the time of presentation his vital sign readings were: HR 186 bpm (90–140bpm), RR 46/min (22–37/min), BP 78/40 MmHg (86–106/42–63 MmHg), temp 36.9°C (34.7–37.3°C axillary) and SPO2 68% (95–98%). The throat was hyperemic, grade II enlargement of tonsils with congestion, but no pus point was noted. Chest examination showed bilateral symmetrical chest movement with intercostal retractions. On auscultation there was decreased air entry on the right lung, with wheezing. Heart sounds were not distinct. The abdomen was soft, non-tender. There was no sign of meningitis. Consequently, the patient was initially diagnosed with acute laryngitis with bronchial pneumonia.\n\nAs there was history of sudden onset of difficulty in breathing, severe cough, inspiratory dyspnea and decrease air entry on the right lung, we planned for a CT scan of the chest. By this time the patient had been given oxygen, IV fluids, continuous ECG and SPO2 monitoring and nebulization with ipratropium bromide (0.02% /2.5ml), albuterol (0.05mg/kg), normal saline (2ml) and budesonide (0.5mg/2ml). Intravenous methyl prednisolone (2mg/kg/day) was given and antibiotic Meropenem (50mg/kg/day) was started. Routine blood investigations, along with ABG and throat swab cultures were performed. All other investigations were within normal limits, except for ABG: pH 7.25 (7.35–7.45), Pao2 70mmHg (75–100 mmHg), PaCO2 55mmHg (35–45mmHg), HCO3 14.9mmol/L (23–31mmol/L). There was little increase in WBC count 12.93×109/L (4–10×109/L), Neutrophils 69.4% (50–75%), Lymphocytes 26% (37–52%), Monocytes 2.6%(3–8%) , Eosinophils 1.9%(0.5–5%), Basophils 0.1%(0–1%), and Hb 115gm/L (110–150gm/L).\n\nAfter one and half hours, the patient showed little improvement, and his vital signs were: HR 168 bpm, RR 40/min, BP 76/38 MmHg and SPO2 90% with oxygen via mask. He was continuously on oxygen, IV fluids at maintenance dose, and frequent nebulization with Albuterol (0.05mg/kg TID), ipratropium bromide (0.02% /2.5ml TID), budesonide (0.5mg/2ml BID), and normal saline. Dopamine (5mcg/kg/min) and Dobutamine (2mcg/kg/min) was started on low dose to improve blood circulation. A CT scan of chest was performed that showed increased right lung volume and increased transparency of lung field. A soft tissue density shadow was observed in the right main bronchus blocking the lumen (Figure 1). The trachea was shifted slightly to the left, but no obvious abnormity was seen. The chest CT was suggestive of the presence of a right bronchial foreign body, with right sided obstructive emphysema.\n\nThere is soft tissue density shadow seen in the right main bronchus blocking the lumen. The trachea is shifted slightly to the left, but no obvious abnormity is seen. The chest CT was suggestive of right bronchial foreign body, with right sided obstructive emphysema.\n\nAfter the CT scan, a consultation with the Consultant of Otolaryngology and Pulmonologist occurred. The consultant changed the diagnosis to right main bronchus foreign body obstruction probably due to mucus plug, and advised the possible need of a laryngeal mask airway or tracheal intubation, mechanical ventilation and bronchoscopic examination under anesthesia. However, the child was improving gradually, and was much calmer than before, with the exception of noisy breathing with an occasional cough. The patient started feeding after nine hours. The patient’s heart rate decreased to 130bpm RR 36/min and SPO2 92% with oxygen. On auscultation there was b/l conducted sound with wheezing on the right lung. Repeat ABG was pH 7.34, Pao2 85mmHg, PaCO2 46mmHg, HCO3 23.9mmol/L. Methyl prednisolone (2mg/kg/day OD) was continued and the antibiotic was switched to Cefotaxime (100mg/kg/day q12hr). Nebulization was continued and oxygen was given as needed. An additional CT scan of the chest was performed, which showed the infection of the right upper lobe and a narrow right upper lobe bronchus (Figure 2).\n\nThe patient improved gradually and was on same treatment for seven days. Then he was discharged on tapering dose of oral prednisolone. He has not any complained of cough or shortness of breath and chest is clear on auscultation on his follow up after 2 weeks.\n\n\nDiscussion\n\nAspiration of a foreign body in the airway mostly occurs in children younger than 15 years; children aged 1–3 years are the most susceptible. Foreign bodies can be exogenous, such as substances inhaled through the mouth (vegetables are the most common airway foreign body); the most common food item that are aspirated are peanuts. In young children, there is lack of molars for proper grinding of food and lack of coordination for swallowing and glottic closure, which is why they are most common age group for foreign body aspiration. Flexible fibro-optic bronchoscopy remains the gold standard for diagnosis and rigid bronchoscopy is the modality of choice in extracting airway foreign bodies4. Endogenous foreign bodies are thick mucus or sputum, bronchial casts, dry scabs, blood clots, pus etc. Endogenous sources may also obstruct airways in same the way as plastic bronchitis5. Plastic bronchitis is a rare disorder, in which there is the presence of gelatinous or rubbery bronchial casts that may be coughed up or found at bronchoscopy or in surgical specimens. Although the pathophysiology is not clearly understood, it is commonly seen in children with congenital heart diseases, cardiac and pericardial diseases in adults and in patients with chronic asthma6. Plastic bronchitis is more common in the lower lobe, but it may also occur in any segments of the bronchial tree7. As plastic bronchitis is common in the lower lobe, it is different to mucoid impaction, which tends to occur in the large segmental bronchi of the upper lobe; they are generally tightly adherent to the wall, and they are retained rather than being expectorated7. Mucoid impaction has a strong correlation with asthma8.\n\nChildren cannot cough out the sputum completely. When there is an infection, the glands on the respiratory passage secrete large amounts of mucous and debris, which lead to blockage of respiratory bronchioles and bronchus. This can later develop the symptoms similar to the foreign body obstruction. In the present case, the child presented with symptoms mimicking foreign body obstruction, but the clinical history was not suggestive. Imaging suggested a bronchial foreign body obstruction. By that time the patient’s symptoms were gradually improving by repeated nebulization with normal saline mix with Albuterol and Ipratropium bromide and steroids. This showed us that the sputum may sometimes block the respiratory passage and mimic foreign body bronchial obstruction. In this case we avoided fiberoptic bronchoscopy in order to reduce pain and risk during procedure, as well as reduce medical costs. A limitation in this case was that it is not fully clear whether it was foreign body or blockage due to sputum plug. Post treatment observation is necessary in such a condition. If the block is above the tracheobronchial bifurcation, the child obviously lacks O2 and it should be removed immediately by fiberoptic bronchoscope. If the block is below the tracheobronchial bifurcation, the patient can be treated conservatively and observed. Mucolytic agents, like N- Acetyl Cysteine, could have also been used. Salamone et al. (2017) published a case report where they mechanically removed bronchial tree-shaped mucous plug in a cystic fibrosis patient9. Park et al. mentioned that mucus impaction and plastic bronchitis are usually self-limited or responsive to medical therapy, with a good prognosis, although their patient did not respond to oral corticosteroid or intrabronchial instillation of acetylcysteine and continued to deteriorate despite medical therapy8.\n\n\nConclusion\n\nSymptoms resembling solid foreign body obstruction are not always aspirated or inhaled. Sometimes secreted sputum forming a plug also mimics the symptoms of foreign body obstruction. If there is no clear history of foreign body ingestion or aspiration of gastric content, immediate nebulization may help greatly.\n\n\nConsent\n\nWe have taken written informed consent from the child’s legal guardian (his Mother) to use her child’s medical case history for the publication of this article.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe wish to thank Li Yan, Nursing Chief and entire doctor and nursing staffs of Department of Pediatrics, Renmin Hospital, and to the child’s parents for the consent.\n\n\nReferences\n\nNational Safety Council: Injury, Death and Fatality Statistics. 2013.\n\nNational Safety Council: Accident Facts. 1992; 32.\n\nYetim TD, Bayarogullari H, Arica V, et al.: Foreign Body Aspiration in Children; Analysis of 42 Cases. J Pulmon Resp Med. 2012; 2: 121. Publisher Full Text\n\nSahil AM, Alfaki M, Alam-Elhuda DM: Airway foreign bodies: A critical review for a common pediatric emergency. World J Emerg Med. 2016; 7(1): 5–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlfageme I, Reyes N, Merino M: Aspirated foreign body. Int J Pulmon Med. 2007; 7: 5–6.\n\nWebb WA: Management of foreign bodies of the upper gastrointestinal tract: update. Gastrointest Endosc. 1995; 41(1): 39–51. PubMed Abstract | Publisher Full Text\n\nSomani SS, Naik CS: Bronchial cast: a case report. Indian J Otolaryngol Head Neck Surg. 2008; 60(3): 242–244. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPark JY, Elshami AA, Kang DS, et al.: Plastic bronchitis. Eur Respir J. 1996; 9(3): 612–614. PubMed Abstract | Publisher Full Text\n\nSalamone I, Mondello B, Lucanto MC, et al.: Bronchial tree-shaped mucous plug in cystic fibrosis: imaging-guided management. Respirol Case Rep. 2017; 5(2): e00214. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "32198",
"date": "23 Mar 2018",
"name": "Francesco Menzella",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case is well described and interesting. I have only a few minor observations:\nOn page 3, line 1 and 2, consultant and consultation is repeated several times: I would also use synonyms to avoid repeating the same words At least the use of some acronyms must be explicitly expanded, eg ABG, BP, HR Line 11 page 3: \"b / l\" must be written in full\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": "3545",
"date": "23 Mar 2018",
"name": "Kiran Devkota",
"role": "Author Response",
"response": "Respected Francesco MenzellaI am very grateful to you. Thank you so much for reveiw and valuable suggestion. My best regardsKiran Devkota"
}
]
},
{
"id": "31882",
"date": "23 Mar 2018",
"name": "Peng Hu",
"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\nAuthor has made good case report- Mucus plug in bronchus mimicking a bronchial solid foreign body obstruction. We often encounter a child with sudden shortness of breath on emergency department which after nebulization get stable. It would be better if they have done bronchoscopic examination. This case report helps to think other clinician that mucus plug can cause obstruction.\n\nAuthor describes the case history, clinical presentation and management in detail. I feel there is space to discuss more on obstruction by other agents.\n\nI hope this case report is good and help to other clinician as well.\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": [
{
"c_id": "3546",
"date": "23 Mar 2018",
"name": "Kiran Devkota",
"role": "Author Response",
"response": "Respected Hu PengThank you so much for your review and valuable suggestion. My best regardsKiran Devkota"
}
]
}
] | 1
|
https://f1000research.com/articles/6-1749
|
https://f1000research.com/articles/6-1631/v1
|
04 Sep 17
|
{
"type": "Research Article",
"title": "The evaluation of a virtual education system based on the DeLone and McLean model: A path analysis",
"authors": [
"Zohreh Mahmoodi",
"Sara Esmaelzadeh- Saeieh",
"Razieh Lotfi",
"Monir Baradaran Eftekhari",
"Mahnaz Akbari Kamrani",
"Zahra Mehdizadeh Tourzani",
"Katayoun Salehi",
"Sara Esmaelzadeh- Saeieh",
"Razieh Lotfi",
"Monir Baradaran Eftekhari",
"Mahnaz Akbari Kamrani",
"Zahra Mehdizadeh Tourzani",
"Katayoun Salehi"
],
"abstract": "Background: The Internet has dramatically influenced the introduction of virtual education. Virtual education is a term that involves online education and e-learning. This study was conducted to evaluate a virtual education system based on the DeLone and McLean model. Methods: This descriptive analytical study was conducted using the census method on all the students of the Nursing and Midwifery Department of Alborz University of Medical Sciences who had taken at least one online course in 2016-2017. Data were collected using a researcher-made questionnaire based on the DeLone and McLean model in six domains and then analyzed in SPSS-16 and LISREL-8.8 using the path analysis. Results: The goodness of fit indices (GFI) of the model represent the desirability and good fit of the model, and the rational nature of the adjusted relationships between the variables based on a conceptual model (GFI = 0.98; RMSEA = 0.014).The results showed that system quality has the greatest impact on the net benefits of the system through both direct and indirect paths (β=0.52), service quality through the indirect path (β=0.03) and user satisfaction through the direct path (β=0.73). Conclusions: According to the results, system quality has the greatest overall impact on the net benefits of the system, both directly and indirectly by affecting user satisfaction and the intention to use. System quality should therefore be further emphasized, to use these systems more efficiently.",
"keywords": [
"virtual education",
"evaluation",
"path analysis",
"model",
"DeLone and Mclean",
"education",
"system",
"e-learning"
],
"content": "Introduction\n\nInformation and communication technology (ICT) has attracted the attention of scientific circles and organizations over recent years, with a focus on knowledge and rationality, and in order to efficiently employ the power of thinking, transfer repetitive tasks to machines and remove communication constraints. This attention means that virtual education or e-learning is now considered a major achievement of ICT, and has caused a number of scientific, research and cultural advances1.\n\nClarke and Mayer define e-learning as presenting content through digital devices such as computers and cellphones, in order to improve learning2. Using this method, learning takes place in a virtual environment in which learners interact with their peers, instructors and educational equipment in a non-traditional environment. In the virtual learning environment, the educational content is offered to the students through software, multimedia resources, the Internet and video conferencing, and the students communicate with their instructors, peers and other people or resources with the help of a computer, in order to carry out individual and group learning activities3,4. One of the benefits of this approach is the full-time and pervasive access to educational resources, reduced transportation time and costs for the students, the expansion of education for all at a much lower cost, the ease of access to multiple and diverse educational resources, the possibility of learning at any time and place and the possibility to learn from the instructor at any time and place5. In spite of these benefits, implementing this form of education without analyzing whether the courses held have been adequately effective may lead to the failure of these courses as a means of education6. One of the best and most-cited models used to evaluate the success of information systems is the DeLone and McLean model. This model measures the success of information systems by measuring six variables, including information quality, system quality, intention to use, user satisfaction, service quality and net benefits (Figure 1). The success of a virtual medical education system is multidimensional and can be associated with system quality, information quality and service quality, which are the prerequisites of three other key structures, namely user satisfaction, intention to use and net benefits, which measure the effects of using the system after it is implemented7. The evaluation of the courses held is one of the most important tasks of any organization and this need is twice as important in universities as they are considered a hub for research. The present study was conducted to evaluate the virtual education system implemented at the Nursing and Midwifery Department of Alborz University of Medical Sciences, based on the DeLone and McLean model using a path analysis.\n\n\nMethods\n\nThis descriptive analytical study was conducted at the Nursing and Midwifery Department of Alborz University of Medical Sciences in Alborz, Iran, during the 2016–2017 academic years.\n\nStudy participants. All the undergraduate and graduate nursing and midwifery students who had taken at least one online course (n=127) were enrolled in the study through census sampling.\n\nData were collected using a researcher-made questionnaire based on the DeLone and McLean model (Supplementary File 1). The questionnaire was designed after a review of the literature and the relevant tools available, and was split into two sections:\n\n(1) The socio-demographic details of the students (gender, age, field of study and marital status),\n\n(2) System evaluation questions in six domains, including system quality (5 items), service quality (5 items), information quality (3 items), intention to use (3 items), user satisfaction (3 items) and net benefits (4 items), which were then scored based on a 5-point Likert scale (‘Totally disagree’ = 1 to ‘Totally agree’ = 5).\n\nOnce designed, the questionnaire was quantitatively and qualitatively validated using the face and content validity methods. For determining the face validity, the questionnaire was distributed among 15 students; for determining the content validity, it was distributed among ten faculty members in fields of nursing, reproductive health and management and they were asked to provide their feedback. All participants gave written consent for publication of potentially identifiable data. The reliability of the questionnaire was assessed using a test-retest with a two-week interval, and by measuring its Cronbach’s alpha coefficient.\n\nThe study began after obtaining the necessary permissions and a code of ethics from the university Ethics Committee (Abzums.Rec.1395.121). First, the eligible undergraduate and graduate nursing and midwifery students who had taken at least one online course in the 2016–2017 academic year were listed with the help of the Student Affairs Office. Arrangements were then made with this office in order to attend the online classes. At the final ten minutes of each class, the researcher went online and explained the objectives of the study, and obtained written consent from the students and provided them with the questionnaire to fill out.\n\nThe fit of a path analysis conceptual model (Figure 1) was assessed in this study in order to determine the correlation between the variables used to evaluate the virtual education system. The path analysis method is a generalization of simple regression that shows not only the direct effects, but also the indirect effects and the effects of each of the variables on the dependent variables, and the results of such a study can be used to provide a rational interpretation of the observed relationships and correlations. The data obtained in this study were analyzed in LISREL version 8 software.\n\n\nResults\n\nA total of 127 undergraduate and graduate students of the Nursing and Midwifery Department of Alborz University of Medical Sciences were recruited for this study. The mean age of the participants was 27.29 ± 1.5 years and the most frequent age group was 20–25 (49.6%). The majority of the participants were female (68.5%) and married (50.4%) (Table 1).\n\nBased on the findings of the quantitative part of the face validity assessment, the impact score of all the items was between 2.4 and 3.3. Since the values were all more than 1.5, all the items remained in the questionnaire in this stage. In the content validity assessment stage, the content validity ratio (CVR) of all the items was between 0.63 and 0.8 and exceeded 0.62; the content validity index (CVI) of the items was also between 0.76 and 1 and exceeded 0.7. As a result, no items were removed from the questionnaire in this stage either. The Content Validity Index average (S-CVI/Ave) of the questionnaire was calculated as 96.37% (±0.65). The Cronbach’s alpha coefficient of the researcher-made questionnaire was 0.762, and the test-retest correlation coefficient with a two-week interval was 0.712.The questionnaire was thus approved for extensive research.\n\nTo perform the path analysis, the correlation between the variables was investigated using the bivariate analysis. The net benefits of the system was the variable that had the highest correlation with user satisfaction (r = 0.81) and system quality (R = 0.72); (Table 2).\n\nIn the path analysis, the effects of variables including system quality, information quality, service quality, intention to use and user satisfaction were investigated for their usefulness (Figure 2).\n\nQS: quality of system, QI: Quality of information, QKH: Quality of service, SA: satisfactory, MO: Intention to use, US: usefulness.\n\nAccording to the results, the goodness of fit indices (GFI) of the model showed the desirability and good fit of the model and the rational nature of the adjusted relationships between the variables based on the conceptual model. As a result, the fitted model was not significantly different from the conceptual model (Table 3).\n\nAccording to the path diagram, user satisfaction (β = 0.73) was the variable that had the greatest impact on usefulness in the direct path and service quality (β = 0.03) the greatest impact in the indirect path. The only variable that had an impact through both the direct and indirect paths was system quality (β = 0.52).\n\nAccording to the results, net benefits will be greater when the system is in a favorable condition in terms of these variables. Table 4 shows the direct, indirect and overall effects of the noted variables on usefulness.\n\n\nDiscussion\n\nToday, e-learning has become a growing trend and an important strategy for promoting education in all major countries of the world.\n\nAccording to the findings of the study, system quality was the only variable that affected the benefits and usefulness of the virtual education system through both the direct and indirect paths. According to the DeLone and McLean model, system quality refers to aspects of the information system itself, such as data reception speed, easy access, system authenticity, responsiveness, flexibility and user-friendliness8–11. These findings are consistent with the results obtained by Pérez-Mira (2010) and Boroufar et al. (2014), who found that system quality has a positive impact on the benefits and usefulness of the system. According to their findings, system quality can be both encouraging and discouraging for the use of the system. They further argued that creating a quality e-learning system by increasing learner satisfaction has a positive effect on usefulness and benefits12,13. Chiu et al. (2005) also found that quality and motivation are two key factors that affect user satisfaction and continued use of virtual education. Machado et al. (2014) found that increased use has a direct relationship with increased system quality, and argued that people are encouraged to use virtual education systems that are user-friendly11.\n\nOf the variables exerting their effects only through one path, service quality had the greatest impact on benefits and usefulness by indirectly affecting the intention to use. According to the DeLone and McLean model, service quality is essential for implementing information systems and depends on the performance of the service providers, which require training in sales and customer support. In the present study, service quality was found to affect the net benefits and usefulness of the system by indirectly affecting learner’s intention. This finding is consistent with the results of a study by Boroufar et al. (2014), who found that service quality indirectly affects the net benefits and usefulness of a system by affecting user satisfaction. Kazemi and Nematollahi (2014) also found that if web-based learning meets the needs of the learners, they will be motivated and satisfied, and this motivation and satisfaction will then make the learners continue using the system14.\n\nOf the variables that directly affect the benefits of using the system, user satisfaction was the most effective. According to the DeLone and McLean model, user satisfaction refers to the response of the individual who uses the information system, such as overall satisfaction, enjoyable experiences and overall success. Chang et al. (2011) also found that the learners’ level of satisfaction has the greatest direct effect on net benefits (7 The term ‘net benefits’ refers to the main benefits achieved by user’s increased use and satisfaction8–11. The satisfaction of the users of a system plays a large role in the success and continued use of a system8. In the present study, student satisfaction was affected by system quality and service quality. These results are consistent with the results obtained by Bauk et al., who also found that user satisfaction is an effective measure of the contradictions between the expectations before and the perceived performance after receiving the education. User satisfaction is affected by system quality while it affects the net benefits of the system. Pérez-Mira B (2010), however, found different results and stated that despite there being relationship between user satisfaction and net benefits, there are no significant relationships between these two variables when entered into a path analysis model, which could be due to the lack of sufficient variation in measuring the satisfaction variable13.\n\nA major limitation of this study lies in the lack of variability with regard to the courses offered through the virtual education system, which were limited to the Nursing and Midwifery Department of Alborz University of Medical Sciences.\n\n\nConclusions\n\nAccording to the results, system quality has the greatest overall impact on net benefits through both the direct and indirect paths by affecting user satisfaction and intention to use. The continued use of virtual education systems and the more efficient use of these systems therefore requires special attention to system quality, which involves speed of data transfer, ease of access, system authenticity, responsiveness, flexibility and user-friendliness.\n\n\nData availability\n\nDataset 1: Raw data obtained from the questionnaire based on the DeLone and McLean model. Gender, 1-female, 2-male; job, 1-student; marriage, 1-single, 2-married, 3-divorced, 4-widowed; education, 1-dipolma, 2-BS, 3-MS; subject, 1-medicine, 2-pharmacy, 3-dentistry, 4-nursing, 5-midwifery, 6-MS in consultation; QS1-5, question on quality of the system; QI1-5, question on quality of information; QH1-3, question on quality of service; M1-3, question on intention to use; S1-3, question on satisfaction; u1-4, question on usefulness. doi, 10.5256/f1000research.12278.d17501116",
"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\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 express our gratitude to the Research Deputy for funding, Clinical Research Center of Kamali Hospital of Alborz University of Medical Sciences for counseling this study and all the persons who participated in the project.\n\n\nSupplementary material\n\nSupplementary File 1: Questionnaire based on the Delone and McLean model.\n\nClick here to access the data.\n\n\nReferences\n\nFaregzadeh N, Kashi A: An Evaluation Of Virtual Education Methods And Tools To Improve Teaching Quality From the point of view of the Faculty Members of Islamic Azad University of Khodabandeh. Quarterly Journal Of New Approach In Educational Administration. 2014; 5(17): 121–52. Reference Source\n\nClark RC, Mayer RE: E-learning and the science of instruction: Proven guidelines for consumers and designers of multimedia learning. John Wiley & Sons; 2016. Publisher Full Text\n\nCooper R: E-learning in the World. London: Falmer; 2004.\n\nPaola Torres Maldonado U, Feroz Khan G, Moon J, et al.: E-learning motivation and educational portal acceptance in developing countries. Online Inform Rev. 2011; 35(1): 66–85. Publisher Full Text\n\nActon T, Halonen R, Conboy K, et al.: DeLone & McLean success model as a descriptive tool in evaluating the use of a virtual learning environment. 2009. Reference Source\n\nFatehi Vajargah K, Pardakhtchi MH, et al.: EFFECTIVENESS evaluation of virtual learning courses in high education system of IRAN (Case of Ferdowsi University). Inform Commun Technol Educ Sci. 2011; 1(4): 5–21. Reference Source\n\nChang HC, Liu CF, Hwang HG: Exploring nursing e-learning systems success based on information system success model. Comput Inform Nurs. 2011; 29(12): 741–7. PubMed Abstract | Publisher Full Text\n\nBauk S, Šćepanović S, Kopp M: Estimating Students’ Satisfaction with Web Based Learning System in Blended Learning Environment. Educ Res Int. 2014; 2014. Publisher Full Text\n\nHalonen R, Acton T, Golden W, et al.: DeLone & McLean success model as a descriptive tool in evaluating a virtual learning environment. 2006.\n\nLagazian M, Nazemi S, Dadmand F: Evaluation of the success of Financial Information System of Ferdowsi University of Mashhad using Delon and McLean's Modified Model. Iran J Inform Proc Mag. 2012; 27(3): 577–96.\n\nMachado-Da-Silva FN, Meirelles FdS, Filenga D: Student Satisfaction Process in Virtual Learning System: Considerations Based in Information and Service Quality from Brazil's Experience. Turkish Online Journal of Distance Education. 2014; 15(3): 122–42. Publisher Full Text\n\nBoroufar A, Sadeghy S, Shokohyar S: Assess the success of e-learning system in the insurance system: Using a modified version Delon and McLean. Journal of E-learning University. 2014; 1(6): 71–84(persian).\n\nPérez-Mira B: Validity of Delone and Mclean’s model of information systems success at the web site level of analysis. Northwestern State University; 2010. Reference Source\n\nKazemi A, Nematollahi M: A Proposed Model for Sustainable Education in E-learning: the Effective Factors. Interdisciplinary Journal of Virtual Learning in Medical Sciences. 2014; 5(2): 79–86(persian). Reference Source\n\nZaied ANH: An integrated success model for evaluating information system in public sectors. Journal of Emerging Trends in Computing and Information Sciences. 2012; 3(6): 814–25. Reference Source\n\nMahmoodi Z, Esmaelzadeh-Saeieh S, Lotfi R, et al.: Dataset 1 in: The evaluation of a virtual education system based on the DeLone and McLean model: A path analysis. F1000Research. 2017. Data Source"
}
|
[
{
"id": "25652",
"date": "07 Sep 2017",
"name": "Mahboubeh Ahmadi Doulabi",
"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\nAccurate design and complete execution, key points are the main strength of this study.\nThe research topic is new and very interesting. However, it requires to answer some question.\nWhy was this project only in the Nursing and Midwifery faculty?\n\nWhy did they use this model for evaluation?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3003",
"date": "07 Sep 2017",
"name": "Zohreh Mahmoodi",
"role": "Author Response",
"response": "Dear Editor Thanks for your decision and your reviewer comments. Comments for the Author: 1- Why was this project only in the Nursing and Midwifery faculty?Answer: We did it in Nursing and Midwifery Department only because, the courses offered through the virtual education system, were limited to this Department and this subject was not presented in other department of Alborz University of Medical Sciences. I wrote it in limitation part.2- Why did they use this model for evaluation?Answer: One of the best and most-cited models used to evaluate the success of information systems is the DeLone and McLean model. This model measures the success of information systems by measuring six variables, including information quality, system quality, and intention to use, user satisfaction, service quality and net benefits"
}
]
},
{
"id": "25972",
"date": "13 Sep 2017",
"name": "Parisa Shojaee",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n\"...Student Affairs Office. Arrangements were then made with this office in order to attend the online classes. At the final ten minutes of each class, the researcher went online and explained the objectives of the study, and obtained written consent from the students and provided them with the questionnaire to fill out.\"\nIn my opinion, the completion time of the questionnaire was not suitable due to the stress of the exam. More exact details should be explained about the path analysis for readers of this paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3026",
"date": "14 Sep 2017",
"name": "Zohreh Mahmoodi",
"role": "Author Response",
"response": "Dear Editor Thanks for your decision and your reviewer comments. 1-In my opinion, the completion time of the questionnaire was not suitable due to the stress of the exam. More exact details should be explained about the path analysis for readers of this paper.Answer:The questionnaire was complete at the end of each class so the sentence 'At the final ten minutes of each class' means at the end of class not the exam time.Regards."
}
]
},
{
"id": "25649",
"date": "20 Sep 2017",
"name": "Laila Amini",
"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 total percentage of age in table is not 100% please correct Please change subgroups of sex in table 1 to male and female or women and men And marital statues change to married, divorced,... Introduction and discussion parts are not supported with enough documents and it is better researchers review more references in their 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?\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": "3043",
"date": "25 Sep 2017",
"name": "Zohreh Mahmoodi",
"role": "Author Response",
"response": "Dear Editor thank you for your decision. The total percentage of age in table is not 100% please correct Answer: It was corrected Please change subgroups of sex in table 1 to male and female or women and men Answer: It was corrected Introduction and discussion parts are not supported with enough documents and it is better researchers review more references in their article Answer: I reviewed it again"
}
]
}
] | 1
|
https://f1000research.com/articles/6-1631
|
https://f1000research.com/articles/6-677/v2
|
25 Sep 17
|
{
"type": "Case Report",
"title": "Case Report: Inflammatory myofibroblastic tumor causes formation of an ileal conglomerate in a patient previously treated for Wilms’ tumor",
"authors": [
"Julie Leganger",
"Rikke Raagaard Soerensen",
"Jacob Rosenberg",
"Jakob Burcharth",
"Rikke Raagaard Soerensen",
"Jacob Rosenberg",
"Jakob Burcharth"
],
"abstract": "Introduction: Inflammatory myofibroblastic tumors (IMTs) are uncommon mesenchymal lesions classified by WHO as neoplasms of uncertain behavior. Morphologically, IMTs are composed of proliferating spindled myofibroblastic cells accompanied by a marked - usually chronic - inflammatory infiltrate. The etiology is unknown, but several theories have been suggested, including an association with Wilms’ tumor. IMTs are rarely diagnosed in adults and have been reported in various organs. IMTs are considered benign but with a potential to recur at their primary site. Case report: A 44-year-old female experienced intermittent severe abdominal pain, loose stools and a visible abdominal bulge. In early childhood the patient had been treated for a Wilms’ tumor. At admission Meckel’s diverticulitis was suspected, but during surgery a tumor in the terminal ileum, creating a conglomerate of small intestinal loops, was observed and completely resected. The pathology report characterized the tumor as a histologically benign inflammatory myofibroblastic tumor. Postoperatively, the patient experienced several complications including an anastomotic leakage and subsequent formation of an abscess and transcutaneous fistula. Discussion: IMTs rarely arise in the small intestine, and to our knowledge the manifestation of a small intestine conglomerate has not been described previously. Making the diagnosis is difficult, and numerous differential diagnoses were possible in this case. Approximately 8-25% of IMTs in the gastrointestinal tract recur locally. Complete surgical resection is the treatment of choice, and re-excision is the preferred therapy for local recurrence. To our knowledge, no guidelines concerning follow-ups are available. Conclusion: IMTs in the terminal ileum can mimic Meckel’s diverticulitis and present with symptoms of obstructive ileus due to the formation of a conglomerate of small intestinal loops. Furthermore, IMTs should be considered as a diagnostic possibility in patients with a past medical history of Wilms’ tumor.",
"keywords": [
"Wilms’ tumor",
"inflammatory myofibroblastic tumors",
"IMTs",
"neoplasms of uncertain behavior",
"ileal conglomerate",
"ileus"
],
"content": "Introduction\n\nInflammatory myofibroblastic tumors (IMTs) are rare mesenchymal neoplasms composed of proliferating myofibroblastic spindle cells, and an accompanying inflammatory - usually chronic – infiltrate1. The etiology of IMTs is unknown, but an association with Wilms’ tumor, the most common primary renal malignancy in children, has been suggested2,3. Furthermore, theories suggesting a link to infectious agents, tumor associated factors, and cytokines have been proposed4. IMTs are most commonly seen in children and young adults1. IMTs were initially considered a pulmonary tumor2, but the lesions have subsequently been reported in various extrapulmonary organs including the mesentery and gastrointestinal tract4.\n\nThis report presents a case of an IMT in the terminal ileum in a female adult, treated for Wilms’ tumor in childhood. The tumor caused a conglomerate of small bowel and mimicked Meckel’s diverticulitis, which to our knowledge has not previously been described. We reported this case according to the CARE guidelines5.\n\n\nCase report\n\nA 44-year-old woman was admitted to the hospital due to intermittent severe abdominal pain, during which a visible bulge appeared in the right lower abdominal quadrant. Furthermore, the patient had experienced loose stools over a two year period. The initial blood tests were inconspicuous, with the exception of a slight neutrophilia. The past medical history included treatment for a Wilms’ tumor at the age of one with a right-sided nephrectomy and subsequent radiochemotherapy.\n\nIleus was initially suspected, and an abdominal CT scan was performed, showing a 3 × 5 × 4 cm mass in the small intestine. The imaging report described, that the lesion was composed of a solid and a necrotic portion plus an air-filled space (Figure 1), which lead to the tentative diagnosis of an underlying Meckel’s diverticulitis. A diagnostic laparoscopy was initiated, but converted to an open ileocecal resection with a primary anastomosis, as a tumor in the terminal ileum was discovered. There was no sign of intestinal perforation.\n\nCoronal (A) and horizontal (B) planes of the abdominal CT scan performed pre-operatively revealed a mass in relation to the small bowel (indicated by arrows).\n\nGrossly, the specimen consisted of a cecal pole that included the appendix and the distal 40 cm of the terminal ileum bound together in a conglomerate (Figure 2). 10 cm from the proximal resection margin a diverticulum-like pouch arising from the ileal wall was identified. The lesion contained a solid tumor of firm consistency, that measured 3 (depth) × 5 (width) cm. The cut surface of the tumor elicited heterogeneity with white/grayish areas and focal haemorrhage. The tumor was totally embedded.\n\nThe gross appearance of the tumor before (A) and after (B) formalin fixation. The tumor (indicated by arrows) infiltrated the wall of the small intestine and the adjacent mesoileum (B), creating a conglomerate of dilated small bowel (A).\n\nMicroscopy revealed a mesenchymal tumor infiltrating the ileal wall, the submucosa and adjacent mesentery. No true diverticulum was found. The tumor was composed of a dense proliferation of spindle-shaped cells primarily arranged in a fascicular growth pattern, focally also exhibiting a storiform architecture, admixed with a marked chronic inflammatory infiltrate and discrete areas of neutrophilic granulocytes (Figure 3). Immunohistochemically, the tumor cells were positive for actin, vimentin, desmin, and h-caldesmon, focally positive for factor 13A and CD68, and had a negative reaction for ALK1, CD34, CD117, DOG1, S100, AE1/AE3, CK18, CK7, CK19, beta-catenin and MDM2. Ki-67 staining showed a low proliferative rate. Although h-caldesmon reactivity was considered atypical for the tumor, the pathology report characterized the tumor as an IMT based on morphology and additional staining properties.\n\nThe tumor mass was built from bundles of spindled myofibroblastic cells with a low proliferative rate arranged in a crossing manner, accompanied by a pronounced lymphocytic infiltrate. Focally, infiltration with polynuclear granulocytes was observed (A, shown in the insert). Immunohistochemically, the tumor cells were positive for vimentin (B), actin (C), desmin (D) and h-caldesmon (E).\n\nPostoperatively, the patient suffered several complications including an intraabdominal abscess caused by a small anastomotic leakage. Subsequent percutaneous drainage of the abscess led to a phlegmone involving the abdominal wall, complicated by the development of a transcutaneous fistula with connection to the peritoneum. Six months after surgery the patient was still having symptoms from the transcutaneous fistula but was otherwise well. She was advised to have a control abdominal magnetic resonance imaging (MRI) after one year.\n\n\nDiscussion\n\nWe reported a case of an adult female patient presenting with an IMT in the terminal ileum causing a conglomerate of small intestinal loops leading to obstructive ileus. A previous study has reported that only 1.2% of IMTs arise from the small intestine3. It has been described that IMTs in the small bowel can cause intestinal obstruction due to intussusception1. However, in our case a conglomerate of small bowel was the cause of ileus, which to our knowledge has not been presented previously in the literature. In general, patients suffering from IMTs may clinically present with an abdominal mass or non-specific symptoms including abdominal pain6. In approximately 15–30% of cases the patients present with a constitutional syndrome of fever, weight loss, malaise and a variety of laboratory abnormalities such as anemia, thrombocytosis, leukocytosis, polyclonal hyperglobulinemia, or elevated erythrocyte sedimentation rate3. In this case the subject only presented with a few of these features.\n\nMacroscopically, IMTs in the gastrointestinal tract are most often characterized as solid, sessile, and solitary lesions, although cases with multiple lesions have been described4. In this case the tumor mimicked a diverticulum and based on the clinical presentation, numerous differential diagnoses were possible including Meckel’s diverticulitis. The histological differential diagnoses of IMTs depend on the dominant basic histological patterns, which involve the extent of proliferation and sclerosing, and the extent to which the IMT is fibromyxoid/vascular3. It is important to distinguish IMTs from other lesions in the family of inflammatory pseudotumors, as well as from non-neoplastic fibrosclerosing processes and malignant neoplasms with a prominent inflammatory infiltrate, e.g. fibromatosis, sarcoma, gastrointestinal stromal tumor, and mesenteric pannicilitis6. Immunohistochemically, the spindle cells in consideration in the diagnostic process of IMT are presented in Table 1. The tumor is reactive to antibodies directed against vimentin, smooth muscle actin, and muscle specific actin in the majority of cases. Anaplastic lymphoma kinase expression is detected in approximately 50% of cases2,4,6. The frequency of this finding decreases with age.\n\nA possible association with Wilms’ tumor has previously been suggested and different theories have been proposed. One theory is a shared genetic predisposition; another theory is that the treatment of Wilms’ tumor involving radiation and chemotherapy may damage the tissue and predispose the patient to development of IMTs. Notable that the latency time in this case is remarkable longer from the other reported cases2. The IMTs were at first considered a postinflammatory condition but is now acknowledge as a distinct neoplasm based on clonal rearrangements involving chromosome 2p6. The IMTs have been classified by WHO as a neoplasm of uncertain behavior2. The tumors are widely acknowledged as benign but with a potential to recur at the primary site. Approximately 8–25% recurs locally1,4. Rare examples are described with tumors undergoing malignant transformation2 and/or with metastasis4. Several studies have attempted to find predictors of aggressive behavior in IMTs without success6. However, it has been suggested that IMTs with a proliferating pattern, a multinodular presentation, or a manifest myofibroblastic or fibroblastic phenotype are more likely to recur4. Furthermore, IMTs arising in the gastrointestinal tract are more likely to recur compared with similar tumors arising elsewhere7. Anaplastic lymphoma kinase expression is associated with a lower risk of metastasis6,7. Complete surgical resection is the treatment of choice, and re-excision is the preferred therapy for local recurrence7. To our knowledge no guideline on IMT follow-up is available.\n\nAdapted from 6, to consider in the diagnostic process of IMT.\n\n\nConclusion\n\nAn IMT in the terminal ileum can mimic Meckel’s diverticulitis and the clinical manifestations can include intestinal obstruction due to the formation of a conglomerate of small intestinal loops. Furthermore, IMTs should be considered as a diagnostic possibility in patients with a past medical history of Wilms’ tumor2.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nJR contributed to the conception of the article. JL, RSS, JR, and JB contributed to the design of the work. JL contributed to the acquisition of data and prepared the first draft of the manuscript. RSS provided expertise in pathology and acquisition of clinical photographs. JL, RSS, JR, and JB were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAmbiru S, Nakamura S, Itabashi T, et al.: Inflammatory myofibroblastic tumor causing ileoileal intussusception: an unusual cause of an unusual neoplasm in an adult, with a clinicopathological review of the literature. Clin J Gastroenterol. 2009; 2(3): 194–8. PubMed Abstract | Publisher Full Text\n\nOrtiz MV, Rossi CT, Hill DA, et al.: Inflammatory myofibroblastic tumor as a second neoplasm after Wilms tumor. Pediatr Blood Cancer. 2015; 62(6): 1075–7. PubMed Abstract | Publisher Full Text\n\nCoffin CM, Humphrey PA, Dehner LP: Extrapulmonary inflammatory myofibroblastic tumor: a clinical and pathological survey. Semin Diagn Pathol. 1998; 15(2): 85–101. PubMed Abstract\n\nMakhlouf HR, Sobin LH: Inflammatory myofibroblastic tumors (inflammatory pseudotumors) of the gastrointestinal tract: how closely are they related to inflammatory fibroid polyps? Hum Pathol. 2002; 33(3): 307–15. PubMed Abstract | Publisher Full Text\n\nGagnier JJ, Kienle G, Altman DG, et al.: The CARE guidelines: consensus-based clinical case reporting guideline development. J Med Case Rep. 2013; 7: 223. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGleason BC, Hornick JL: Inflammatory myofibroblastic tumours: where are we now? J Clin Pathol. 2008; 61(4): 428–37. PubMed Abstract | Publisher Full Text\n\nChaudhary P: Mesenteric inflammatory myofibroblastic tumors. Ann Gastroenterol. 2015; 28(1): 49–54. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "26317",
"date": "26 Sep 2017",
"name": "Jiaqi Shi",
"expertise": [
"Reviewer Expertise Gastrointestinal",
"hepatobiliary",
"pancreatic pathology"
],
"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 case report described a presumable rare inflammatory myofibroblastic tumor (IMT) in the terminal ileum leading to an ileal conglomerate in a patient previously treated for Wilms’ tumor. Overall the case was well-presented with adequate discussion. However, the diagnosis of the tumor and the conclusion are not supported by the histology or immunohistochemical staining pattern, and therefore further studies are necessary in order to reach a definitive diagnosis. Detailed comments are listed below:\nThe findings are not diagnostic of IMT in this case based on the morphology of the tumor, lack of ALK1 expression, and diffuse h-caldesmon staining which is more typical for a smooth muscle tumor such as a leiomyoma or leiomyosarcoma. The rich lymphocytic infiltrate also raised the possibility of an EBV-associated smooth muscle tumor if the patient is immunocompromised. An EBER in-situ hybridization would be helpful to further evaluate for this possibility.\n\nAgain, the diffuse and strong h-caldesmon staining of this tumor is very unusual for IMT, and is not supportive of this diagnosis. All of the reported h-caldesmon positive IMT cases have only focal positivity. In my opinion, a FISH analysis for ALK1 translocation is necessary to reach the diagnosis of IMT in this case.\n\nIn Figure 1A, there are 3 arrows and it is not clear what one of the arrows is pointing to. Please clarify.\n\nIn Figure 2A, please add an arrow to indicate the mass. For both A and B, please modify the background so that it is a solid color without blood or other materials.\n\nPlease provide a 3 dimensional measurement of the tumor in gross description.\n\nMore discussion on the differential diagnosis of IMT based on histology and immunohistochemical profile is recommended.\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": "29440",
"date": "08 Jan 2018",
"name": "Jasmine Kamboj",
"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\nDoubt the diagnosis of IMT in absence of Alk positivity and positivity for h-caldesmon. Otherwise the case report is well written. Authors should consider an alternate diagnosis of a smooth muscle neoplasm also. The discussion section lacks detailed description of the differential diagnosis. Other spindle cell tumors should also be excluded by describing their histologic and immunohistochemical features.\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": []
}
] | 2
|
https://f1000research.com/articles/6-677
|
https://f1000research.com/articles/6-1262/v1
|
28 Jul 17
|
{
"type": "Data Note",
"title": "Preprocessed Consortium for Neuropsychiatric Phenomics dataset",
"authors": [
"Krzysztof J. Gorgolewski",
"Joke Durnez",
"Russell A. Poldrack",
"Russell A. Poldrack"
],
"abstract": "Here we present preprocessed MRI data of 265 participants from the Consortium for Neuropsychiatric Phenomics (CNP) dataset. The preprocessed dataset includes minimally preprocessed data in the native, MNI and surface spaces accompanied with potential confound regressors, tissue probability masks, brain masks and transformations. In addition the preprocessed dataset includes unthresholded group level and single subject statistical maps from all tasks included in the original dataset. We hope that availability of this dataset will greatly accelerate research.",
"keywords": [
"fMRI",
"human",
"cognition",
"preprocessed"
],
"content": "Introduction\n\nThe recently published Consortium for Neuropsychiatric Phenomics (CNP) dataset1 is large (272 participants), diverse (healthy controls as well as individuals diagnosed with schizophrenia, bipolar disorder, and attention deficit/hyperactivity disorder), and rich in phenotypic information (each participant filled 42 questionnaires) dataset. It is undoubtedly a rich resource for the academic community. However, before any brain behaviour relationships could be answered, computationally expensive and processing steps need to be performed2. In addition to requiring a lot of resources, a certain level of expertise in MRI data processing and fMRI task modelling is required before the data could be used to test scientific hypotheses.\n\nTo facilitate answering scientific questions using the CNP dataset, we have performed standard preprocessing as well as statistical modeling on the data, and are making the results of these analyses openly available. The preprocessing was designed to facilitate a wide range of analyses, and includes outputs in native (aligned with participants T1 weighted scan), MNI (volumetric) and fsaverage5 (surface) spaces. The data has not been denoised, but potential confound regressors have been calculated for each run, giving researchers the freedom to fit many different models that incorporate different denoising schemes. In addition, we also include group and single subject statistical maps for all tasks available in the original dataset. This preprocessed dataset joins the ranks of similar initiatives for other openly shared datasets3–5, and we hope it will be equally useful to the scientific community.\n\n\nMethods\n\nFor scanning parameters and details of the task fMRI paradigms, see 1. The input dataset was acquired from OpenfMRI.org6 - accession number ds000030, revision 1.0.3.\n\nResults included in this manuscript come from preprocessing performed using FMRIPREP version 0.4.4 (http://fmriprep.readthedocs.io), a Nipype7 based tool. FMRIPREP was run with the following command line arguments:\n\n--participant_label {sid} -w $LOCAL_SCRATCH --output-space T1w fsaverage5 template --nthreads 8 --mem_mb 20000\n\nWhere {sid} was the participant label and $LOCAL_SCRATCH was temporary folder for storing intermediate results.\n\nWithin the pipeline each T1 weighted volume was corrected for bias field using N4BiasFieldCorrection v2.1.08, skullstripped using antsBrainExtraction.sh v2.1.0 (using OASIS template), and coregistered to skullstripped ICBM 152 Nonlinear Asymmetrical template version 2009c9 using nonlinear transformation implemented in ANTs v2.1.010. Cortical surface was estimated using FreeSurfer v6.0.011.\n\nFunctional data for each run was motion corrected using MCFLIRT v5.0.912. Functional data was skullstripped using combination of BET and 3dAutoMask tools and was coregistered to the corresponding T1 weighted volume using boundary based registration with 9 degrees of freedom - implemented in FreeSurfer v6.0.013. Motion correcting transformations, transformation to T1 weighted space and MNI template warp were applied in a single step using antsApplyTransformations v2.1.0 with Lanczos interpolation.\n\nThree tissue classes were extracted from T1w images using FSL FAST v5.0.914. Voxels from cerebrospinal fluid and white matter were used to create a mask in turn used to extract physiological noise regressors using aCompCor15. The mask was eroded and limited to subcortical regions to limit overlap with grey matter, six principal components were estimated. Framewise displacement and DVARS16 was calculated for each functional run using Nipype implementation. In addition to those regressors global signal and mean white matter signal was also calculated.\n\nThe whole dataset was preprocessed in total three times. After each iteration the decision to modify the preprocessing was purely based on the visual evaluation of the preprocessed data and not based on results of model fitting. First iteration (using FMRIPREP 0.4.2) uncovered inconsistent output image field of view and issues with EPI skullstripping, second iteration (using FMRIPREP 0.4.3) uncovered two cases of failed normalization due to poor initialization. In the final iteration all those issues were resolved. In total the preprocessing consumed ~22,556 single CPU hours.\n\nFor more details of the pipeline see http://fmriprep.readthedocs.io/en/0.4.4/workflows.html.\n\nFor a full description of the paradigms for each task, please refer to1. We analysed the task data using FSL17 and AFNI18, implemented using Nipype7. Spatial smoothing was applied using AFNI’s 3dBlurInMask with a Gaussian kernel with FWHM=5mm. Activity was estimated using a general linear model (GLM) with FEAT17. Predictors were convolved with a double-gamma canonical haemodynamic response function19. Temporal derivatives were added to all task regressors to compensate variability in the haemodynamic response function. Furthermore, the following regressors were added to avoid confounding due to motion: standardised dvars, absolute dvars, the voxelwise standard deviation of dvars, framewise displacement, and the six motion parameters (translation in 3 directions, rotation in 3 directions).\n\nFor the Balloon Analog Risk Task (BART), we included 9 task regressors: for each condition (accept, explode, reject), we added a regressor with equal amplitude and durations of 1 second on each trial. Furthermore, we included the same regressors with the amplitude modulated by the number of trials before explosions (perceived as the probability of explosions). The modulator was mean centered to avoid estimation problems due to collinearity. For the conditions that require a response (accept, reject), a regressor was added with equal amplitude, and the duration equal to the reaction time. These regressors were orthogonalised with their fixed-duration counterpart to separate the fixed effect of the trial and the effect covarying with the reaction time. A regressor is added for the control condition.\n\nIn the retrieval phase of the Paired-Associate Memory Task (PAMRET), we modelled 4 conditions: true positives, false positives, true negatives, false negatives. For each condition, a regressor is modelled first with fixed durations (3s) and second with reaction time durations, with the latter orthogonalised with the former. With an extra regressor with control trials, there are 9 task regressors in total.\n\nIn the Spatial Capacity Task (SCAP), 25 task regressors were included. For each cognitive load (1 - 3 - 5 - 7) and each delay (1.5 - 3 - 4.5) with a correct response, two regressors were added: a regressor with fixed durations of 5 seconds and one with the duration equal to the reaction time, with the second orthogonalised with respect to the first. For both regressors, the onset is after the delay. The last regressor summarises all incorrect trials.\n\nFor the Stop-Signal Task (STOPSIGNAL), for each condition (go, stop - succesful, stop - unsuccesful), one task regressor was included with a fixed duration of 1.5s. For the conditions requiring a response (go and stop-unsuccesful), an extra regressor was added with equal amplitude, but the duration equal to the reaction time. Again, these regressors were orthogonalised with respect to the fixed duration regressor of the same condition. A sixth regressor was added with erroneous trials.\n\nIn the Task Switching Task (TASKSWITCH), all manipulations were crossed (switch/no switch, congruent/incongruent, CSI delay short/long), resulting in 8 task conditions. As in the SCAP task, we added for each condition two regressors: a regressor with fixed durations of 1 second, and one with the duration equal to the reaction time, with the second orthogonalised with respect to the first. There is a total of 16 regressors.\n\nFor subjects who are missing at least one regressor used in the contrasts, the task data are discarded. This is the case for example when no correct answers are registered for a certain condition in the SCAP task. For the SCAP task, we discarded 16 subjects; 14 subjects were removed for TASKSWITCH, 2 subjects for STOPSIGNAL, 2 subjects for BART, and 12 for PAMRET.\n\nAll modelled contrasts are listed in the Supplementary material. As is shown, all contrasts are estimated and tested for both a positive and a negative effect.\n\nThe total number of subjects modelled in the BART task is 259, while 244 subjects were modelled for the SCAP task. 254 subjects were included the TASKSWITCH task analysis, 197 subjects in the PAMRET task and 255 subjects in the STOPSIGNAL task.\n\nSubsequent to the single subject analyses, all subjects were entered in a one-sample group level analysis for each task. Three second level analysis strategies were followed: (A) ordinary least squares (OLS) mixed modelling using FLAME17, (B) generalized least squares (GLS) with a local estimate of random effects variance, using FSL17, and (C) non-parametric modelling (NP) using RANDOMISE20, with the whole brain first level parameter estimates for each subject as input, and 10,000 permutations. The first two analyses use a group brain mask with voxels that were present in 100% of all subjects. For the permutation tests, a group mask was created where voxels were discarded for further analysis if less than 80% of the subjects have data in those voxels.\n\nIn addition to group level statistical maps, activation count maps (ACMs) were generated to show the proportion of participants that show activation, rather than average activation over subjects21. These maps indicate whether the effects discovered in the group analyses are consistent over subjects. As in 21, the statistical map for each subject is binarized at z=+/-1.65. For each contrast, the average of these maps is computed over subjects. The average negative map (percentage of subjects showing a negative effect with z < -1.65) is subtracted from the average positive map to indicate the direction of effects.\n\n\nDataset validation\n\nTo validate the quality of volumetric spatial normalization we have looked at the overlap of the EPI derived brain masks in the MNI space (across all participants and runs - total of 1,969 masks - see Figure 1). The within subject coregistration and normalization worked well for the vast majority of participants, creating a very good overlap. All of the issues observed while processing the dataset are listed in Table 1.\n\nA selection of the tested contrasts in the task analyses is shown in Figures 2 to 6. Figures were generated using nilearn22.\n\nList of problems with the raw data we were aware of at the time of writing that impacted preprocessing.\n\nIn the left plot, the statistical map of the one-sample group test, computed with randomise. The right plot shows the difference between the positive and the negative activation count maps.\n\nIn the left plot, the statistical map of the one-sample group test, computed with randomise. The right plot shows the difference between the positive and the negative activation count maps.\n\nIn the left plot, the statistical map of the one-sample group test, computed with randomise. The right plot shows the difference between the positive and the negative activation count maps.\n\nIn the left plot, the statistical map of the one-sample group test, computed with randomise. The right plot shows the difference between the positive and the negative activation count maps.\n\nIn the left plot, the statistical map of the one-sample group test, computed with randomise. The right plot shows the difference between the positive and the negative activation count maps.\n\n\nData and software availability\n\nThe preprocessed images were deposited along the original dataset in the OpenfMRI repository – accession number: ds0000306, under the revision 1.0.4. The preprocessed data is organized according the draft BIDS derivatives specification. All FMRIPREP derivatives are organised under fmriprep/sub-<participant_label>/\n\nDerivatives related to T1 weighted files are in the anat subfolder:\n\n*T1w_preproc.nii.gz - bias field corrected T1 weighted file, using ANTS’ N4BiasFieldCorrection\n\n*T1w_brainmask.nii.gz - brain mask derived using ANTS\n\n*T1w_dtissue.nii.gz -tissue class map derived using FAST.\n\n*T1w_class-CSF_probtissue.nii.gz, *T1w_class-GM_probtissue.nii.gz, *T1w_class-WM_probtissue.nii.gz - probability tissue maps.\n\nAll of the above are available in native and MNI space.\n\n*T1w_smoothwm.[LR].surf.gii - smoothed GrayWhite surfaces.\n\n*T1w_pial.[LR].surf.gii - pial surface.\n\n*T1w_midthickness.[LR].surf.gii - MidThickness surfaces.\n\n*T1w_inflated.[LR].surf.gii - FreeSurfer inflated surfaces for visualization.\n\n*T1w_space-MNI152NLin2009cAsym_class-CSF_probtissue.nii.gz, *T1w_space-MNI152NLin2009cAsym_class-GM_probtissue.nii.gz, *T1w_space-MNI152NLin2009cAsym_class-WM_probtissue.nii.gz - probability tissue maps, transformed into MNI space.\n\n*T1w_target-MNI152NLin2009cAsym_warp.h5 Composite (warp and affine) transform to transform participant's T1 weighted image into the MNI space\n\nDerivatives related to EPI files are in the func subfolder:\n\n*bold_space-<space>_brainmask.nii.gz Brain mask for EPI files.\n\n*bold_space-<space>_preproc.nii.gz Motion-corrected (using MCFLIRT for estimation and ANTs for interpolation) EPI file\n\nAll of the above are available in the native T1 weighted space as well as the MNI space.\n\n*bold_space-fsaverage5.[LR].func.gii Motion-corrected EPI file sampled to surface.\n\n*bold_confounds.tsv A tab-separated value file with one column per calculated confound (see Methods) and one row per timepoint/volume\n\nThe results of the single subject task modeling are available in task/sub-<participant_label>/ and the group level results can be found in task_group/. Each subject-specific folder holds 5 folders - bart.feat, scap.feat, pamret.feat, stopsignal.feat, taskswitch.feat - with the results from the respective task modeling, organised as standard FEAT output. The group-level folder contains a folder for every task, in turn containing a folder for each contrast (see Supplementary material for naming conventions) and below those folders are the results of the three modeling strategies.\n\nIn addition, the dataset includes visual quality HTML reports (one per participant).\n\nThe results for each contrast in the one-sample group task analyses are deposited and can be interactively viewed in NeuroVault23: http://neurovault.org/collections/2606/.\n\nLatest source code used to produce the task analyses: https://github.com/poldracklab/CNP_task_analysis\n\nArchived source code as at the time of publication: http://doi.org/10.5281/zenodo.83231924.\n\nLicense: MIT license.\n\nAll code has been run through a singularity container25, created from a docker container poldracklab/cnp_task_analysis:1.0 available on docker hub (https://hub.docker.com/r/poldracklab/cnp_task_analysis/).",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work has been funded by the Laura and John Arnold Foundation. JD has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 706561. The acquisition of the original dataset was supported by the Consortium for Neuropsychiatric Phenomics (NIH Roadmap for Medical Research grants UL1-DE019580, RL1MH083268, RL1MH083269, RL1DA024853, RL1MH083270, RL1LM009833, PL1MH083271, and PL1NS062410).\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 of the developers and beta testers of the FMRIPREP package - especially Oscar Esteban, Chris Markiewicz, and Ross Blair.\n\n\nSupplementary material\n\nSupplementary File 1: Task fMRI Contrasts.\n\nClick here to access the data.\n\n\nReferences\n\nPoldrack RA, Congdon E, Triplett W, et al.: A phenome-wide examination of neural and cognitive function. Sci Data. 2016; 3: 160110. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPoldrack RA, Gorgolewski KJ: Making big data open: data sharing in neuroimaging. Nat Neurosci. 2014; 17(11): 1510–7, [cited 2014 Oct 28]. PubMed Abstract | Publisher Full Text\n\nPuccio B, Pooley JP, Pellman JS, et al.: The preprocessed connectomes project repository of manually corrected skull-stripped T1-weighted anatomical MRI data. Gigascience. 2016; 5(1): 45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBellec P, Chu C, Chouinard-Decorte F, et al.: The Neuro Bureau ADHD-200 Preprocessed repository. Neuroimage. 2017; 144(Pt B): 275–86. PubMed Abstract | Publisher Full Text\n\nGlasser MF, Sotiropoulos SN, Wilson JA, et al.: The minimal preprocessing pipelines for the Human Connectome Project. Neuroimage. 2013; 80: 105–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPoldrack RA, Barch DM, Mitchell JP, et al.: Toward open sharing of task-based fMRI data: the OpenfMRI project. Front Neuroinform. 2013; 7: 12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGorgolewski K, Burns CD, Madison C, et al.: Nipype: a flexible, lightweight and extensible neuroimaging data processing framework in python. Front Neuroinform. 2011; 5: 13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTustison NJ, Avants BB, Cook PA, et al.: N4ITK: improved N3 bias correction. IEEE Trans Med Imaging. 2010; 29(6): 1310–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFonov VS, Evans AC, McKinstry RC, et al.: Unbiased nonlinear average age-appropriate brain templates from birth to adulthood. Neuroimage. 2009; 47: S102. Publisher Full Text\n\nAvants BB, Epstein CL, Grossman M, et al.: Symmetric diffeomorphic image registration with cross-correlation: evaluating automated labeling of elderly and neurodegenerative brain. Med Image Anal. 2008; 12(1): 26–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDale AM, Fischl B, Sereno MI: Cortical surface-based analysis. I. Segmentation and surface reconstruction. Neuroimage. 1999; 9(2): 179–94. PubMed Abstract | Publisher Full Text\n\nJenkinson M, Bannister P, Brady M, et al.: Improved optimization for the robust and accurate linear registration and motion correction of brain images. Neuroimage. 2002; 17(2): 825–41. PubMed Abstract | Publisher Full Text\n\nGreve DN, Fischl B: Accurate and robust brain image alignment using boundary-based registration. Neuroimage. 2009; 48(1): 63–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang Y, Brady M, Smith S: Segmentation of brain MR images through a hidden Markov random field model and the expectation-maximization algorithm. IEEE Trans Med Imaging. 2001; 20(1): 45–57. PubMed Abstract | Publisher Full Text\n\nBehzadi Y, Restom K, Liau J, et al.: A component based noise correction method (CompCor) for BOLD and perfusion based fMRI. Neuroimage. 2007; 37(1): 90–101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPower JD, Mitra A, Laumann TO, et al.: Methods to detect, characterize, and remove motion artifact in resting state fMRI. Neuroimage. 2013; 84: 320–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJenkinson M, Beckmann CF, Behrens TE, et al.: FSL. Neuroimage. 2012; 62(2): 782–90. PubMed Abstract | Publisher Full Text\n\nCox RW: AFNI: software for analysis and visualization of functional magnetic resonance neuroimages. Comput Biomed Res. 1996; 29(3): 162–73. PubMed Abstract | Publisher Full Text\n\nGlover GH: Deconvolution of impulse response in event-related BOLD fMRI. Neuroimage. 1999; 9(4): 416–29. PubMed Abstract | Publisher Full Text\n\nWinkler AM, Ridgway GR, Webster MA, et al.: Permutation inference for the general linear model. Neuroimage. 2014; 92: 381–97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarch DM, Burgess GC, Harms MP, et al.: Function in the human connectome: task-fMRI and individual differences in behavior. Neuroimage. 2013; 80: 169–89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbraham A, Pedregosa F, Eickenberg M, et al.: Machine Learning for Neuroimaging with Scikit-Learn. arXiv [cs.LG]. 2014. Reference Source\n\nGorgolewski KJ, Varoquaux G, Rivera G, et al.: NeuroVault.org: a web-based repository for collecting and sharing unthresholded statistical maps of the human brain. Front Neuroinform. 2015; 9: 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDurnez J, Gorgolewski CJ, Poldrack RA: poldracklab/CNP_task_analysis: v0.1. Zenodo. 2017. Data Source\n\nKurtzer GM, Sochat V, Bauer MW: Singularity: Scientific containers for mobility of compute. PLoS One. 2017; 12(5): e0177459. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "24599",
"date": "08 Aug 2017",
"name": "Angela R. Laird",
"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 Data Note reports on the availability of an fMRI dataset from the Consortium for Neuropsychiatric Phenomics (CNP), which includes both original and processed data. The publication of shared fMRI datasets is strongly encouraged to amplify our community's efforts in promoting open science. Although dataset publications are on the rise, unfortunately, only a handful currently exist. I am delighted to see this work being published, and expect that it may serve as a representative fMRI dataset publication in the future. With that in mind, I think it would be helpful to revise the manuscript to include a more detailed description of the data, acquisition methods, and individual tasks.\n\nIntroduction: The first paragraph of the Introduction is extremely brief and should be expanded to include a description of the purpose of the study, participants, and the experimental protocol (imaging and behavioral). Only a very short mention of these three important aspects of the study are provided and collapsed into a single (somewhat awkward) first sentence. Such brevity might limit a reader's understanding of the overall context of the data that are being shared. The second sentence of the Introduction alludes to \"relationships\" being \"answered\" - this should be restated and expanded to more fully describe what questions may be asked from these data - again, this relates to the overall purpose of the CNP project. While such additional descriptions will result in a longer paper, the information will be helpful in allowing readers to understand how their specific research questions may be addressed by a deeper exploration of these data. Lastly, the Introduction should also state where the data may be downloaded and a summary of the different unprocessed and processed files that have been shared.\nMethods: Demographic information on the participants should be provided, as well as a statement of IRB approval. A description of the MRI scanner should be included, along with the data acquisition parameters. Each of the fMRI tasks should be fully described - this will help clarify subsequent reference to different conditions (e.g., “accept, explode, reject”). Much of the Methods is written for those who are already very familiar with the software packages that are utilized. It would be helpful to improve accessibility by including a brief description of some of the newer, less ubiquitious software tools. In particular, given how the Methods is framed around use of FMRIPREP, a short intro should be included. The total numbers of participants for each of the tasks (shown on page 3) doesn’t agree with the numbers of task datasets discarded for being incomplete - please note why the additional participants were omitted from the final dataset. Overall, the flow of the Methods section could be improved by adding a workflow or pipeline figure that summarizes the different analysis steps and the versions of the data (e.g., volume vs. surface approaches).\n\nI’m not convinced that “Dataset validation” is an appropriate heading. Figure 1 is a good sanity check, but it’s not clear how Figures 2 - 6 are a validation.\nData and software availability: Some readers may not be familiar with the BIDS format - please add a description of this. In addition, some readers may be looking for information about the DICOM and NIfTI images, so an explicit mention may be helpful.\nMinor comments: - page 2: first report of DVARS is capitalized, but later mentions are not - page 2: \"Temporal derivatives were added to all task regressors to compensate for variability in the haemodynamic response function\" - page 3: typo - “unsuccesful” - ensure past tense used consistently throughout Methods (e.g., “tasks data were discarded”, “no correct answers were registered” on page 3).\n\nIs the rationale for creating the dataset(s) clearly described? Partly\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": "3037",
"date": "22 Sep 2017",
"name": "Krzysztof J. Gorgolewski",
"role": "Author Response",
"response": "Dr. Laird,Thank you for your review and comments. They were very helpful in preparation of a new revision of the paper. We have extended the introduction to give a clearer view on the purpose and possibilities this dataset gives. In the methods sections, we have given demographic information on the participants, as well as IRB approval and a description of the MRI scanner, scanning parameters and tasks. We have changed the heading ‘Dataset validation’ to ‘Selected results’. We have expanded the description of preprocessing and introduction to the FMRIPREP package. We have clarified that not all subjects performed all of the tasks. We added a data processing overview figure. We have spelled out the BIDS acronym and added a reference to a paper with more information. We have added information about NIFTI and GIFTI file formats (DICOM files are not part of this dataset). Furthermore, in accordance with the review, we have fixed the reported typos and changed the tense of the paper."
}
]
},
{
"id": "24602",
"date": "22 Aug 2017",
"name": "Anderson M. Winkler",
"expertise": [
"Reviewer Expertise Medicine",
"statistics",
"medical imaging"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst of all, I would like to congratulate the authors for making the dataset available, which should allow interested scientists to explore the data and enrich the research they conduct with more information that can eventually lead to helpful new discoveries. It is remarkable that more than one processing stream was used (FSL and AFNI for functional, and volumetric and surface-based for structural), and further, three different inference approaches were considered, all of which are a great bonus in terms of comparisons among methods.\nI have very few concerns about the current version of the manuscript (v1, dated 28/July/2017):\nPage 2, 1st column, 2nd paragraph of the Introduction: \"giving researchers the freedom to fit many different models that incorporate different denoising schemes\": as stated, it may suggest that it would be adequate to simply run multiple models, without attention to excess error due to multiple testing. Perhaps a different wording such as \"giving researchers the freedom to choose their own denoising schemes\" could still accommodate what the authors may have wished to state.\n\nI cannot find information about the subjects. Who were them? Where was the data collected? With what scanner and sequences? Who approved the protocol? Presumably this information is on reference #1 but it cannot hurt to have that information here.\n\nIt would be good if a few more details on what exactly FMRIPREP does could be given, without having to rely completely on external links that may no longer be available in the future. Even more important considering that results did change with after minor version changes.\n\nPage 2, 1st column, 4th paragraph of the Methods: As written, a reader may think that the input images to FreeSurfer were those non-linearly aligned to the MNI space, which surely was not the case. But if that was, then the FS analysis would have to re-done as the warps affect thickness and area measurements.\n\nPage 3, 2nd column: Regarding the masks, one would have thought that using the mask from FEAT/FLAME could have been a good shortcut instead of creating a new one for randomise. Why wasn't that done?\n\nPage 3, Validation section: The strategy using the mask contours to investigate between-subjects registration is surely not a good one for not showing how the overlap between structures. A hyperslab across subjects would have been more informative. Moreover, the contours shown in Figure 1 are a bit concerning for suggesting somewhat suboptimal registration.\n\nStill in the Validation section: what exactly is being validated here? It doesn't seem to show that the dataset would be valid or not valid in any particular aspect. Consider investigating some specific validation parameters over different aspects (e.g., registration, bias correction, surface reconstruction, the tasks eliciting expected response, etc), or remove this section altogether, as it can be misleading for suggesting that the dataset is \"valid\" somehow.\n\nPage 6: The description of the files is extremely helpful. I note that one of the files listed has extension .h5. Is this HDF5? If yes, please state so. I believe this format was used for the lack of another option, but in fact, this is a great format that probably should in the future be an option for most imaging data we use (both surface-based and volume-based).\n\nOf the FreeSurfer surfaces, the white is the most important one, not pial or midthickness. The pial is computed after the white already exists, and its exactness depends on the white. The midthickness does not match any particular tissue border, and if one measures surface area from it, that area will depend on thickness, which would make it a poor phenotype. It would be great if the white surface files could be provided.\n\nPage 7: I find it concerning that information and resources about this dataset are scattered over the internet: There is the current paper (PDF) and its Supplementary Material on F1000, then there are results stored in NeuroVault, source code on Github and Zenodo, and finally, a Docker container on DockerHub. Could not a copy of all these pieces be on a single place that can be simply downloaded and maintained on the long term, e.g., in DataDryad? How can the readers be sure that all these links will be alive in 10 or 20 years?\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": "3038",
"date": "22 Sep 2017",
"name": "Krzysztof J. Gorgolewski",
"role": "Author Response",
"response": "Dr. Winkler,Thank you for the detailed review. Your comments helped us to improve the manuscript in the following way: We have applied the suggested changes to the second paragraph of the introduction We have added information about the subjects We have clarified that the FS analysis was done in parallel with the alignment. We have added a few more details about preprocessing and an overview figure. Furthermore we have mode sure that the online documentation of the version of FMRIPREP used to generate this data has been deposited in the Internet Archive for long term preservation. We have clarified why different mask strategies were applied. We have added the hyperslab figure to the manuscript. The 95% and 85% overlap brain mask overlap contours show good agreement across the normalized masks with signal dropout in areas usually affected by susceptibility distortion artifacts. The 100% overlap is much worse since it requires voxels to be present in all of the 1,969 evaluated masks. We have changed the header ‘Validation’ to ‘Selected Results’ to not give the wrong impression that we validated the analyses. We added clarification on the file format for the .h5 files (indeed it’s HDF5!) The white surface is provided (both in GIfTI and native FreeSurfer formats) - we made this information more prominent. We have clarified that there is long-term storage of the data and code."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1262
|
https://f1000research.com/articles/6-1718/v1
|
21 Sep 17
|
{
"type": "Software Tool Article",
"title": "lakemorpho: Calculating lake morphometry metrics in R",
"authors": [
"Jeffrey Hollister",
"Jemma Stachelek",
"Jemma Stachelek"
],
"abstract": "Metrics describing the shape and size of lakes, known as lake morphometry metrics, are important for any limnological study. In cases where a lake has long been the subject of study these data are often already collected and are openly available. Many other lakes have these data collected, but access is challenging as it is often stored on individual computers (or worse, in filing cabinets) and is available only to the primary investigators. The vast majority of lakes fall into a third category in which the data are not available. This makes broad scale modelling of lake ecology a challenge as some of the key information about in-lake processes are unavailable. While this valuable in situ information may be difficult to obtain, several national datasets exist that may be used to model and estimate lake morphometry. In particular, digital elevation models and hydrography have been shown to be predictive of several lake morphometry metrics. The R package lakemorpho has been developed to utilize these data and estimate the following morphometry metrics: surface area, shoreline length, major axis length, minor axis length, major and minor axis length ratio, shoreline development, maximum depth, mean depth, volume, maximum lake length, mean lake width, maximum lake width, and fetch. In this software tool article we describe the motivation behind developing lakemorpho, discuss the implementation in R, and describe the use of lakemorpho with an example of a typical use case.",
"keywords": [
"limnology",
"R",
"lake morphometry",
"lake depth",
"lake volume"
],
"content": "Introduction\n\nThe study and quantification of lake shape (i.e. lake morphology and morphometry) is one of the foundations of limnology, and for students of limnology, some of the first lessons are centered around a typical suite of metrics and how to calculate them1. It is also widely accepted that the morphometry of lakes and ponds can impact available nutrients and thus overall productivity. For instance, the widely used Vollenweider input-output models that are used to estimate nutrient concentrations rely on hydraulic residence time and sometimes mean depth, both of which are derived from total lake volume2,3. Also, clear water versus turbid water states in lakes have been linked in part to lake morphometry, in particular mean depth4,5. In short, limnologists have long recognized the importance of lake morphology as one factor controlling a variety of ecological processes in lakes.\n\nTraditional methods for calculating lake morphometry metrics have relied upon the use of paper bathymetry maps, planimeters, or simple heuristics6–9. In addition, detailed bathymetry is a requirement for the calculation of most lake morphometry metrics, but is generally only available for a relatively small number of lakes. Although this is not a problem when the focus of a study is a single lake, a small number of lakes, or a group of well-studied lakes, reliance on complete bathymetry becomes a limitation when attempting to conduct regional or national-scale lake studies. For instance, Soranno et al. found that for some water quality datasets lake depth, in spite of its importance, was not always available10. In cases such as these, alternative approaches for estimating lake morphometry are required.\n\nRecent work has demonstrated the ability to estimate many of these metrics from ubiquitous spatial data9,11–13. For instance, maximum depth and lake volume may be predicted using the lake polygon and surrounding topography9,11 provided by the National Hydrography Dataset Plus and the National Elevation Dataset, respectively14,15. These methods were initially developed with proprietary tools, thus limiting their accessibility. In an effort to reach a broader audience, the tools were converted to R, expanded to include a more complete suite of lake morphometry metrics and compiled into an R Package.\n\n\nMethods\n\nUsing R as a Geographic Information System is now possible, as several packages provide spatial data handling, geospatial analysis, and visualization. It is because of these packages that lakemorpho was implemented as an R package16. In particular, lakemorpho relies on the following packages: maptools, rgdal, raster, rgeos, sp, and geosphere17–23. In addition to these packages, two external libraries, the Geospatial Data Abstraction Library (GDAL) and Geometry Engine, Open Source (GEOS), are needed. Their availability to R and installation varies by operating system24,25.\n\nThe lakemorpho package includes one function to create a lakeMorpho object, several functions to calculate morphometry metrics, a default plotting function, two example datasets, and an example lakeMorpho object.\n\nA typical workflow for using lakemorpho to calculate lake metrics would include pulling spatial data into R (e.g. as shapefiles, tiff, etc.), creating a lakeMorpho object and calculating the desired lake morphometry metrics. The following sections provide details on the type of input data required and demonstrate use of the available functions with the provided example data.\n\nThe lakeMorpho Class and lakeSurroundTopo function. Many of the lake morphometry metrics rely on the same information about the lake. For instance, the functions to estimate maximum depth, mean depth, and volume rely on statistical summaries of the surrounding topography as well as the maximum in-lake distance to shoreline9,11. To avoid recalculating these values, a lakeMorpho object was created to link information on surrounding topography to the original datasets and facilitate default plotting of the outputs. All lake morphometry functions in the lakemorpho package require an object of class lakeMorpho as input. Some functions also return an updated lakeMorpho object that includes calculated spatial objects as output. At a minimum, a lakeMorpho object contains (see Figure 1):\n\n\"lake\" - A SpatialPolygons or SpatialPolygonsDataFrame object of the original input lake data.\n\n\"elev\" - A RasterLayer representing the elevation in a suitably large area around the lake.\n\n\"surround\" - A SpatialPolygons or SpatialPolygonsDataFrame object representing the land area defined as the surrounding topography.\n\n\"lakeDistance\" - A RasterLayer object of the euclidean distance from the shoreline to center of each pixel. Maximum value is equal to the maximum in-lake distance.\n\n\"lakeOnEdge\" - A logical value indicating if the \"surround\" polygon falls on the edge of the \"elev\" raster (i.e. would contain missing (i.e. NA) elevation data).\n\nThe lakeSurroundTopo function is the primary mechanism for creating a lakeMorpho object. There are two required inputs and one optional input for lakeSurroundTopo. The first required input is a SpatialPolygons or SpatialPolygonsDataFrame of the lake21. Only a single lake is accepted as input, although this lake may be composed of multiple polygons (i.e. a lake with islands). If metrics for multiple lakes are required they will need to be passed to the suite of lakemorpho functions separately. The second required input is a RasterLayer of the elevation surrounding the lake22. The default raster size is taken from the resolution of the input elevation data but may be specified separately. The third input specifies the area representing the surrounding topography. By default, this is a buffer of the lake shoreline, with the buffer width equal to the maximum in-lake distance. An optional SpatialPolygons object of any polygon intersecting the lake (e.g. catchments) can be used to define the surrounding topography instead of the default buffer. An object of class lakeMorpho is returned from lakeSurroundTopo.\n\nIn addition to providing the required inputs, users should pay attention to both the extent of the input elevation dataset as well as the coordinate reference systems being used. First, the elevation data must be of a large enough extent so that the surrounding topography does not include land area outside that extent (i.e. would return NA values). As noted above, the lakeOnEdge item indicates if the surrounding topography is on the edge of the user supplied elevation and thus would return some missing data. Second, all of the functions of lakemorpho assume that projections have been handled prior to creating the lakemorpho class or calculating the metrics. If the input data are not of the same projection, lakeSurroundTopo will return an error. The data must be re-projected into the same coordinate reference system (CRS). The units of all metrics are determined by the CRS and care must be taken to make sure that the vertical units of the elevation are the same as horizontal units of the projection. For instance, elevation data may be available in meters yet the CRS is specified in feet. In cases such as these, a conversion of the vertical data should be done. Lastly, care must be taken in choosing an appropriate CRS for the area under consideration. This is because all CRS will distort area, distance, shape, or direction. Thus a projection that minimizies distortions of distance and area are preferrable. A useful reference for further exploring coordinate reference system is Iliffe and Lott's 2008 book on the topic26.\n\nUsage of lakeSurroundTopo and generation of a lakeMorpho object from the example data included with lakemorpho is done as follows:\n\n\n\nThe resulting object contains the minimum set of components that make up a lakeMorpho object. We can verify that the components are of the expected class with the following command:\n\n\n\nLake Morphometry Functions. Each of the remaining functions expects a lakeMorpho object as input and returns a numeric value. Some of the functions also have a side effect of adding a spatial object to the input lakeMorpho object.\n\ncalcLakeMetrics\n\nThe calcLakeMetrics function is a convenience function that will calculate all of the lakemorpho metrics for a single lakeMorpho object. It requires an input lakeMorpho object, a bearing for calculating lakeFetch, and pointDens for maximum lake length and width (defined below).\n\n\n\nlakeFetch\n\nFetch is the maximum open water distance in a given direction and can be used an indicator of mixing as greater fetch implies greater potential for waves due to wind effects27. The lakeFetch function calculates fetch along an input bearing. The input bearing may be any value from 0 to 360 where 0 and 360 both represent north, and the fetch for opposite directions (e.g. east and west) are identical.\n\nTo calculate the fetch of an input lake use:\n\n\n\nlakeMajorAxisLength\n\nThe major axis of a lake is defined as the longest line intersecting the convex hull formed around its polygon while passing through its center. In contrast to lakeMaxLength, its value represents the distance across a lake without regard to land-water configuration.\n\nTo calculate the major axis length of an input lake use:\n\n\n\nlakeMaxDepth\n\nMaximum lake depth provides information that may be used, along with flow rates, to estimate the residence time of a lake. While there is no substitute for field verified measurements, maximum lake depth may be estimated from the surrounding topography. The lakeMaxDepth function uses the methods outlined in Hollister et al.11 to provide an estimate of the maximum lake depth. It requires only a lakeMorpho object as input. Optionally, a correction factor based off of verified depth data may be specified if one is known.\n\nTo calculate maximum depth use:\n\n\n\nIt is important to note that the accuracies of these maximum depth predictions do vary across regions and often a correction factor using field measured data is required. For example, Hollister et al.11 demonstrate that for the New England and Mid-Atlantic regions of the United States East coast, the initial predictions were larger than the true values and needed to be reduced.\n\nlakeMaxLength\n\nMaximum lake length is the longest open water distance within a lake and, similar to fetch, is a metric that can be used to estimate mixing potential1,28. The current implementation of this calculation in lakemorpho places points at equal distances along the shoreline of the lake and then finds the longest point-to-point distance that also does not intersect land (e.g. peninsulas or islands). The optional parameter addLine has a default value of TRUE and allows the SpatialLines object to be stored on the input lakeMorpho object (Figure 2).\n\nTo calculate maximum lake length use:\n\n\n\nThe pointDens parameter can have an impact on both the processing time and the resulting value and both of these can vary as a function of the complexity of the shape of the lake with less complex lakes providing more consistent lake length across a number of points. Given this caveat, care must be taken in choosing an appropriate number of points (and thus lines) to use to calculate maximum lake length. Several densities should be tested and the smallest number of points that produce a stable estimate should be used.\n\nlakeMaxWidth\n\nMaximum lake width is the maximum shore to shore distance that is perpendicular to the line representing maximum lake length and is another metric related to mixing1,28. The lakeMaxWidth function requires a lakeMorpho object and pointDens value which is used to determine the number of points along the maximum lake length line. The issue with pointDens, discussed above, also exists for the use of pointDens with lakeMaxWidth and care should be taken to determine an appropriate number of lines to test.\n\nUsage of lakeMaxWidth is:\n\n\n\nlakeMeanDepth\n\nMean depth of a lake is calculated as the volume of the lake divided by the area1,28. This function requires only a lakeMorpho object and returns a numeric value of the mean depth. Usage of the function is:\n\n\n\nThere is an optional zmax argument that allows a user to specify a maximum lake depth if one is available. If not supplied, the maximum depth will be estimated using lakeMaxDepth. For instance, in the above example, the maximum depth without using a correction factor is estimated at 99 meters which results in a mean depth estimate of 28.95. The measured maximum depth, 32 meters, is much less than the estimate depth. To use this information you would simply add the measured valued in for the zmax argument.\n\n\n\nlakeMeanWidth\n\nThe mean width of a lake is defined as lake area divided by maximum lake length1,28. Input for this function is a lakeMorpho object that has the maximum lake length line added via 'lakeMaxLength`. This requirement is checked and returns an error if the maximum length line is missing.\n\n\n\nlakeMinorAxisLength\n\nThe minor axis of a lake is defined as the shortest line intersecting the convex hull formed around the lake polygon while passing through its center. In contrast to lakeMaxWidth, its value represents the distance across a lake with regard to the the convex hull and without consideration of the land-water configuration.\n\n\n\nlakeMinorMajorRatio\n\nThe ratio of the lake major axis length to the minor axis length is also known as the aspect ratio. Circular lakes have aspect ratios approaching 1 while thin-elongated lakes have aspect ratios approaching 0. If major and minor axis length have not already been added to the lakeMorpho object, these are calculated. The addLine argument adds the lines for the lake's minor and major axes to the lakeMorpho object.\n\n\n\nlakeShorelineDevelopment\n\nThe shoreline development metric provides a measure of the complexity of the shoreline. It is a ratio of the perimeter of the lake to the perimeter of a circle of the same area. Values will be 1 or greater with value of 1 indicating a circular lake. This metric is used as an indicator of potential habitat1,28. It only requires a lakeMorpho object as input.\n\n\n\nlakeShorelineLength and lakeSurfaceArea\n\nShoreline length is simply the total perimeter of the lake polygon and, as with all other functions, requires a lakeMorpho object as input. To calculate the shoreline length:\n\n\n\nSimilarly, surface area for a lake is the total area of the lake polygon. It is calculated via:\n\n\n\nlakeVolume\n\nThe lakeVolume function uses maximum lake depth (see lakeMaxDepth) and methods outlined by Hollister et al.9 to estimate lake volume. The method assumes that the maximum in-lake distance (Dmax) from the shoreline is also the deepest part of the lake (Zmax). The lakeVolume function creates a raster of the in-lake distance to shoreline and converts those distances, using Zmax:Dmax, to depths and then summing the volume of each pixel to estimate total lake volume.\n\n\n\nSimilar to lakeMeanDepth, there is a zmax argument to be used for a known maximum lake depth.\n\n\nUse case\n\nA common application of lakemorpho is to calculate the full suite of lake metrics for multiple lakes. This use case demonstrates how to do that with a commonly encountered GIS data file, the shapefile. To do this we iterate through the lakes, calculate metrics for each lake and include the metrics on an output shapefile. The data for this use case is from Rhode Island (Figure 3). The data for the lakes were downloaded from the Rhode Island Geographic Information Systems (RIGIS)29 and the elevation data are from Amazon Web Services Terrain Tiles via the elevatr package30.\n\nThis use case relies on the sp and rgdal packages for the spatial data handling. These are dependencies for lakemorpho, thus no additional installs are required. To read in the data we utilized rgdal::readOGR and read in the ri_lakes.shp from the current directory. This file is available for download from https://github.com/USEPA/lakemorpho_manuscript/blob/master/ri_lakes.zip.\n\n\n\n\n\nIn R, there are many ways to iterate. For simplicity and clarity we use a for loop to iterate through all lakes and calculate the full suite of lake metrics with calcLakeMetrics. We will utilize the elevatr package which provides access to elevation data from various sources30. In this example we will use the Amazon Web Services terrain tiles. The vertical elevation data are in meters and the Rhode Island lake data are projected in Rhode Island State Plane Feet, thus we will convert the elevation data into feet.\n\n\n\nWe can now merge the morphometry metrics back to the lake polygons.\n\n\n\n\nConclusions\n\nThe lakemorpho package provides functions to calculate common lake morphometry metrics in R. For those conducting lake analyses in R this allows for streamlined analysis workflows. Also, lakemorpho provides a foundation for additional metrics. For instance, it might be possible to combine hydrological methods for estimating stream flow into and out of lakes with lake volume and add a function for calculating residence time.\n\nBeyond adding additional metrics, more fundamental rewriting of the package may also be useful. For instance, lakemorpho currently is built on top of the current spatial data standard for R, sp. This allows a clean interface with many existing tools; however, it is likely that sp will be replaced in the next several years by the sf package21,31. Future versions of lakemorpho might benefit from using the sf tool chain and the \"tidy data\" framework32.\n\nIn summary, lakemorpho provides limnologists and aquatic ecologists with a consistent framework in R for calculating a suite of the most common lake morphometry metrics. This paper outlines the currently available functions and provides an example through a typical use case of calculating many metrics for several lakes.\n\n\nSoftware availability\n\nThe lakemorpho version 1.1.0 package is currently available directly from the Comprehensive R Archive Network (CRAN) and may simply be installed and loaded in R via:\n\n\n\nTo access the help pages (including a version of this manuscript) use:\n\n\n\nThere are tentative plans to continue developing new functions for lakemorpho and these new features will be available first through the development version on GitHub at http://github.com/usepa/lakemorpho.\n\nTo install and load the development version requires use of the devtools package. This may be done with:\n\n\n\nArchived source code as at the time of publication:\n\nhttp://doi.org/10.5281/zenodo.86305133",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to thank Stephen Shivers (ORISE), Betty Kreakie (US EPA, Atlantic Ecology Division), Bryan Milstead (US EPA, Atlantic Ecology Division), Joe LiVolsi (US EPA, Atlantic Ecology Division), Tim Gleason (US EPA, Atlantic Ecology Division), and Wayne Munns (US EPA, Atlantic Ecology Division) for constructive reviews of this paper. Special thanks to TBD for reviews of the submitted manuscript. The views expressed in this article are those of the authors and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency. Any mention of trade names, products, or services does not imply an endorsement by the U.S. Government or the U.S. Environmental Protection Agency. The EPA does not endorse any commercial products, services, or enterprises. This contribution is identified by the tracking number ORD-022603 of the Atlantic Ecology Division, Office of Research and Development, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency.\n\n\nReferences\n\nWetzel R: Limnology, 3 e. lake and river ecosystems. Academic Press, California. 2001; 850 p. Reference Source\n\nVollenweider RA: Input-output models. Schweizerische Zeitschrift für Hydrologie. 1975; 37(1): 53–84. Publisher Full Text\n\nMilstead WB, Hollister JW, Moore RB, et al.: Estimating summer nutrient concentrations in Northeastern lakes from SPARROW load predictions and modeled lake depth and volume. PLoS One. 2013; 8(11): e81457. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGenkai-Kato M, Carpenter SR: Eutrophication due to phosphorus recycling in relation to lake morphometry, temperature, and macrophytes. Ecology. 2005; 86(1): 210–219. Publisher Full Text\n\nScheffer M, van Nes EH: Shallow lakes theory revisited: Various alternative regimes driven by climate, nutrients, depth and lake size. Hydrobiologia. 2007; 584(1): 455–466. Publisher Full Text\n\nKalff J: Limnology: Inland water ecosystems. Prentice Hall New Jersey. 2002; 592 p. Reference Source\n\nWelch P: Limnology. McGraw-Hill, New York. 1935. Reference Source\n\nWetzel RG, Likens G: Limnological analyses. 3rd editon. Springer Verlag, New York. 2000. Publisher Full Text\n\nHollister J, Milstead WB: Using GIS to estimate lake volume from limited data. Lake and Reservoir Management. 2010; 26(3): 194–199. Publisher Full Text\n\nSoranno PA, Bissell EG, Cheruvelil KS, et al.: Building a multi-scaled geospatial temporal ecology database from disparate data sources: fostering open science and data reuse. Gigascience. 2015; 4: 28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHollister JW, Milstead WB, Urrutia MA: Predicting maximum lake depth from surrounding topography. PLoS One. 2011; 6(9): e25764, Accessed 28 Jun 2013. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMessager ML, Lehner B, Grill G, et al.: Estimating the volume and age of water stored in global lakes using a geo-statistical approach. Nat Commun. 2016; 7: 13603. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOliver SK, Soranno PA, Fergus CE, et al.: Prediction of lake depth across a 17-state region in the united states. Inland Waters. 2016; 6: 314–324. Reference Source\n\nUSEPA U: National hydrography dataset plus–NHDPlus. 2005. Reference Source\n\nGesch D, Evans G, Mauck J, et al.: The national map-elevation: US geological survey fact sheet 2009–3053. 2009; 4 p. Reference Source\n\nHollister JW, Stachelek J: Lakemorpho: Lake morphometry in R. 2016. Reference Source\n\nBivand R, Lewin-Koh N: Maptools: Tools for reading and handling spatial objects. 2014. Reference Source\n\nBivand R, Keitt T, Rowlingson B: Rgdal: Bindings for the geospatial data abstraction library. 2014. Reference Source\n\nBivand R, Rundel C: Rgeos: Interface to geometry engine - open source (geos). 2014. Reference Source\n\nBivand RS, Pebesma EJ, Gómez-Rubio V: Applied spatial data analysis with R. Springer. 2008. Publisher Full Text\n\nPebesma EJ, Bivand RS: Classes and methods for spatial data in R. R news. 2005; 5: 9–13. Reference Source\n\nHijmans RJ: Raster: Raster: Geographic data analysis and modeling. 2014. Reference Source\n\nHijmans RJ: Geosphere: Spherical trigonometry. 2014. Reference Source\n\nTeam GD: GDAL - geospatial data abstraction library, version 1.9.2. Open Source Geospatial Foundation. 2012. Reference Source\n\nFoundation OSG: GEOS - geometry engine - open source. Open Source Geospatial Foundation. 2013. Reference Source\n\nIliffe J, Lott R: Datums and map projections for remote sensing, gis and surveying. 2nd ed. Whittles Publishing. 2008. Reference Source\n\nScheffer M: Ecology of shallow lakes. Springer Science & Business Media. 2004. Publisher Full Text\n\nLAKEWATCH F: Department of fisheries and aquatic sciences, a beginner’s guide to water management-lake morphometry. 2001. Reference Source\n\n(RIGIS) RIGIS: Lakes and ponds (1: 5000); lakes5k10. 2010. Reference Source\n\nHollister J, Shah T: Elevatr: Access elevation data from various apis. 2017. Reference Source\n\nPebesma E: Sf: Simple features for R. 2017. Reference Source\n\nWickham H: Tidy data. J Stat Softw. 2014; 59: 1–23. Reference Source\n\nHollister JW, Stachelek J, Ortiz J: jhollist/lakemorpho_manuscript: Submission version for F1000Research. Zenodo. 2017. Data Source"
}
|
[
{
"id": "26223",
"date": "23 Oct 2017",
"name": "Samantha K. Oliver",
"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 lakemorpho package fills a need in the limnological community (and beyond) for easily deriving lake morphometry metrics in a repeatable and documented way. The package is relatively simple to use and understand, and implements common, published methods for calculate lake morphometry metrics. Additionally, the authors implement a tool for predicting lake depth, which is a difficult lake parameter to obtain. I only a have minor suggestions for changes in the language and documentation for the package. Most of these changes are related to the order in which ideas are presented in the paper. While reading the paper, I had several questions that were later answered, but I think the authors can avoid the questions altogether by simply rearranging some of the text (see specific comments below). Additionally, I think it’s important for the authors to include an error estimate of the lake depth predictions – prediction error is notoriously high for lake depth, and I think it’s important for users to understand this. I recommend this paper for publication after the following changes are considered – and I look forward to using the package!\nMinor revisions: - Recommend removing “…and for students of limnology, some of the first lessons are centered around a typical suite of metrics and how to calculate them.” from the first sentence and instead lead with morphometry affecting water quality. - What is a “suitably large area around the lake”? 50m, 100m? - I think you can remove bulleted list from the first paragraph of the lakeMorpho and lakeSurroundTopo section. It only roughly introduces pieces of the lakeMorpho object, and left me with more questions than answers (e.g., previous question RE: suitably large area around the lake which you answer later). You answered all of the questions immediately in the next paragraphs, which left me feeling like you could just leave out the list, or use it as a summary list at the end of the section. - “If metrics for multiple lakes are required they will need to be passed to the suite of lakemorpho functions separately.” This seems to be in direct contradiction with the purpose of the package – which is to derive morphometry metrics for a large number of lakes. It becomes clear later in the text that you address this with a use-case. I think you should state this up front – “Although each lake must be passed through lakemorpho functions separately, we provide a use case to show how a user can easily calculate these metrics for several lakes”. - This sentence is really confusing: “First, the elevation data must be of a large enough extent so that the surrounding topography does not include land area outside that extent (i.e. would return NA values)”. My interpretation is: “The extent of the elevation data must be equal to or greater than the extent of the area surrounding the lake that you are summarizing”. I think this is confusing because you are not using “surrounding topography” in a general sense but as a defined area that is part of the user input. Perhaps use a different naming convention? You’re describing the surrounding topography (through summarizing the elevation data) within a specified area. Perhaps elevation = your fabric and buffer area = your stencil? This is adopting language from the USGS geoknife package. It may not make sense to use fabric since you only have one type of raster input, but I think stencil is a nice word to use for your variable buffer/watershed area. I see you call this “surround” in the lakemorpho object, so that may be a good option as well. - Maybe give some error measurement from the max depth predictions in the Hollister paper? I think it’s important for users to understand there is substantial error in these models. - I think you should briefly describe the Hollister methods for calculating zmax in this paper. Also, the mean depth calculation is unclear from the text. How is volume calculated? By estimating depth at each pixel of the lake? Do you know anything about the error of these estimates? Ok – now I see that you give a good description of volume. Maybe move volume up earlier (before mean depth)? - In the lake shoreline development section: “This metric is used as an indicator of potential habitat” It is also used an indicator of potential terrestrial-aquatic linkages1.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "27253",
"date": "20 Nov 2017",
"name": "Luke A. Winslow",
"expertise": [
"Reviewer Expertise Global Limnology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Hollister and Stachelek outline the functionality and use of the lakemorpho R package. The package includes a number of tools that allow users to calculate morphological, including a some generally complex metrics that are not easy to recreate using existing functionality in available spatial packages. Packages such as these go a long way to preventing the constant re-inventing of the wheel by graduate students and other researchers in generating code to calculate these metrics. This package is a significant contribution to the field and this paper does a reasonable job of describing the package.\nI have a few minor suggestions that I think will improve the manuscript and applicability of the package.\nSome of these metrics are not immediately intuitive to most readers. It would be super useful to have a figure, maybe with a series of panels, that shows the reader what each of these metrics are on some example lakes where it matters. This would be great to have accompanying the section “Lake Morphology Functions”.\nThere needs to be a description of what the option ‘pointDens’ means. Does it have units? Is it relative to the projection or independent of it? Further, while this may be difficult, would it be possible to show the sensitivity of output metrics to pointDens for complex lakes? It would be great to know if there was a reliable metric that could be used, perhaps a default informed by the total lake area or SDF? Such a default or heuristic would be really useful to the purported use here (calculations on a large number of less-studied lakes). Basically, if I use this on 1 million lakes at the continental scale, I really need an informed way to pick that pointDens value.\nRight now, this package has depth-related calculations for lakes which do not have observed bathymetry. It would be really useful if the package could be generalized to use observed bathymetry rasters in the calculation of max and mean depth. Such data are increasingly available (e.g. http://bathybase.org).\nA lot of 1-D hydrodynamic models need an estimate of the hypsometric curve. It would be great if an additional function could be included that would use either an estimated bathymetry or a provided bathy raster (see earlier comment) and output the lake’s hypsometric curve. Very useful in hydrodynamic modeling.\n\nHere are a few additional publications you might consider referencing. At the moment, references to existing lake morphological literature is a little thin. Especially check out other work of Håkanson as he’s a morphology master.\nHåkanson, L. (2005), The Importance of Lake Morphometry for the Structure and Function of Lakes1\nDunalska, J. a. (2011), Impact of morphometric and catchment variables on summer organic carbon richness in deep temperate lakes2 Read, E. K. et al. (2015), The importance of lake-specific characteristics for water quality across the continental United States3\nI look forward to using this useful tool!\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1718
|
https://f1000research.com/articles/6-1717/v1
|
21 Sep 17
|
{
"type": "Opinion Article",
"title": "Make researchers revisit past publications to improve reproducibility",
"authors": [
"Clare Fiala",
"Eleftherios P. Diamandis",
"Clare Fiala"
],
"abstract": "Scientific irreproducibility is a major issue that has recently increased attention from publishers, authors, funders and other players in the scientific arena. Published literature suggests that 50-80% of all science performed is irreproducible. While various solutions to this problem have been proposed, none of them are quick and/or cheap. Here, we propose one way of reducing scientific irreproducibility by asking authors to revisit their previous publications and provide a commentary after five years. We believe that this measure will alert authors not to over sell their results and will help with better planning and execution of their experiments. We invite scientific journals to adapt this proposal immediately as a prerequisite for publishing.",
"keywords": [
"Scientific irreproducibility",
"revisit past publications",
"reflection on past publications",
"improve scientific reproducibility",
"inflated research",
"bias",
"accountability"
],
"content": "Introduction\n\nHardly a day goes by without a screed against perverse incentives in research. It goes like this: Scientists get better rewards for announcing breakthroughs than for producing solid work. The achievements needed to win grants, jobs, and publications - combined with researchers’ (often noble) ambitions - encourage them to build castles in the air.\n\nAfter that comes a plea for large-scale change. One recent proposal would require scientists to complete rigid, time-consuming confirmation studies before publishing a single paper1.\n\nWe propose something that is quicker, cheaper, and simpler: Require researchers to write post-publication reflections five years after their papers appear.\n\nIn these self-reviews, researchers would assess how their claims held up. They should describe whether an invention or discovery was translated or commercialized, and how (or whether!) others could build on their work. The practice would provide a straightforward, non-stigmatized way to identify errors, misinterpretations, and other roadblocks.\n\nFor many, these-self-reviews would be a welcome opportunity for clarification, celebration, and even self-promotion. But the main advantage is that self-reviews would encourage scientists to think in advance how they might be wrong.\n\n\nCauses of irreproducibility\n\nHow might this work? Let’s consider the sources of irreproducibility. We put this down to a half-dozen causes: Often several occur together in the same paper! Fraud captures the most attention, but is rare. Self-deception, or bias, occurs aplenty. It is easier to attribute an observation to a hoped-for reason than to imagine trivial causes. Who wants to believe that a test result depends on the brand of test tube or day of the week rather than the earliest detectable sign of disease?\n\nThen there are unrecognized technical deficiencies; researchers who know how to operate a machine, but lack enough experience to recognize artifacts and infelicities. They enter the wrong parameters or use the wrong pipette tips without realizing that they have rendered their data meaningless. Similarly, big data and data crunchers readily produce false interpretations. In 2007, one crystallographer had to retract five prominent papers after discovering a small computer glitch2.\n\nAll of these problems are exacerbated by fragmented science. Projects are now executed in pieces in various laboratories and results knitted together without anyone knowing exactly what happened at each site, so no one is able to bring sufficient scrutiny to bear.\n\nIn each of these cases, the problems are clear with hindsight. If post-publication self-review was commonplace, some of these problems would become clear as experiments were being planned and conducted.\n\nIn our own lab, we have made a habit of reflecting on our papers (though not necessarily with a strict five-year timeframe). Though several papers led to work taken up by biotech companies and other scientists, others proved much less valuable than we had hoped. Bias and technical deficiencies are the most prominent reasons behind our papers that did not ‘succeed.’ That realization has made one of us a better mentor and supervisor over time. It has also led to several publications pointing out flaws in common reagents and lab practices.\n\nWork by the psychologists Philip Tetlock and Jessica Lerner suggests that simple steps meant to hold people accountable for their judgment calls actually improves their judgment3. They become more accurate in their thinking and more objective when they evaluate evidence.\n\nAccountability in science is ad hoc. Researchers get credit for a publication well before enough time has passed for the scientific community to really know whether the paper has made a valuable contribution. No wonder that researchers bent on submitting a paper are obsessed with making the best possible case for its acceptance rather than illustrating its limitations. If researchers are forced to consider how well their paper will stand up five years hence, they will be more careful when doing the work and more critical in their analysis.\n\nAbout ten years ago, one of us came up with the idea of a new journal, tentatively titled Reflections in Medicine, in which authors of prominent papers could publish their post-publication thoughts, and contacted about 20 prospective authors, who all ignored or refused the request. We believe some did not want to revisit problematic results.\n\nWith the advent of electronic publishing, it is now possible for journals (or funders or other platforms, such as PubMed) to create a space for these five-year reflections and to connect them with the original paper.\n\nSelf-evaluation, based on strict criteria and instructions, can be revealing even if the authors try to inflate the impact of old work. For example, the boldest claims in a scientific paper should be annotated and addressed directly in authors’ reflections. Researchers could also be asked a series of straightforward yes/no questions about whether the results of a paper have changed clinical or scientific practice.\n\nJournals, funders, or research institutions could oblige scientists to write self-reflections. Failing to do so would be a red flag. One can imagine a system in which publications in reference lists or literature databases could be annotated as lacking self-review, and so taken less seriously.\n\nWith luck, care, and enthusiasm, this simple, inexpensive step would counter perverse incentives. Instead of being stigmatized for correcting a paper, researchers would be stigmatized for failing to do so. Junior scientists would learn by example how to read papers critically and design more-rigorous experiments. The public would learn that a paper is not a definitive statement, but a single contributor to a gradually emerging picture of how nature works.\n\nIn short, self-reflections could demote scientific papers to their rightful place and turn a vicious cycle into a virtuous one.",
"appendix": "Competing interests\n\n\n\nEleftherios P Diamandis declares he is a consultant/advisory role with Abbott Diagnostics. Clare Fiala has nothing to declare.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMogil JS, Macleod MR: No publication without confirmation. Nature. 2017; 524(7642): 409–11. PubMed Abstract | Publisher Full Text\n\nMiller G: Scientific publishing. A scientist's nightmare: software problem leads to five retractions. Science. 2006; 314(5807): 1856–7. PubMed Abstract | Publisher Full Text\n\nLerner JS, Tetlock PE: Accounting for the effects of accountability. Psychol Bull. 1999; 125(2): 255–75. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "26217",
"date": "25 Sep 2017",
"name": "Georgios Pampalakis",
"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\nScientific irreproducibility is indeed a serious problem nowadays. In the current opinion article, the authors state the reason that govern irreproducibility and for the first time they provide a potential method to treat such results. Importantly, their suggested method is based on a self-evaluation by the authors of a published article after a 5-year period. Indeed, this “self-review” process is simple, quick and with no additional cost. The article is very well-written for a broad audience.\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": "27535",
"date": "06 Nov 2017",
"name": "Edward W. Randell",
"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 long term relevance of our published works is a point for both secret pride and disappointment for many continuing in careers with active engagement in research. This opinion article suggests that compelling researchers to reflectively evaluate their past publications and to produce a published commentary on these works (after 5 years) will improve the reliability of published work. In my opinion, this is an idea that may have reached its time and in some ways is long overdue. While it remains to be seen if this strategy improves the quality of published work (if implemented), the user friendly and low cost proposal made by these authors at least represents a feasible approach toward improving the quality of publications by causing researchers to think twice about what they submit for publication. And this by knowing that publication involves a long term commitment and accountability to the ideas presented and to be revisited by reflective review 5 years later. Certainly, this is a well written and interesting concept that in my opinion merits consideration by journal editors and publishers, and the support of researchers committed to research excellence.\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": "26215",
"date": "07 Nov 2017",
"name": "Morley D. Hollenberg",
"expertise": [
"Reviewer Expertise Molecular Pharmacology",
"Endocrinology",
"Signal Transduction",
"G-protein-coupled receptor signalling",
"G-protein-coupled Proteinase-activated Receptors"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSYNOPSIS:\n\nThis overview deals with an enlarging key issue related to the reproducibility of published data in journals of all stripes, ranging from ‘high’ to ‘low’ impact quality. A ‘checkpoint’ process is proposed requiring authors to write a 5-year-post-publication ‘reflection’ on the previously published data. Failure to do so would place a ‘red flag’ beside references to that author’s work in subsequent databases and presumably also in subsequent manuscript reference sections quoting that work. The authors suggest a ‘simple’ yes/no checklist to determine whether the results of a paper have changed clinical or scientific practice.\n\nCRITIQUE\n\nThis contribution is a well-written, thoughtful and concise overview of a deepening thorn in the side of published peer-reviewed data in the literature. The strength of the commentary is that the issue of long-term ‘accountability’ for those who publish the data is raised; and a ‘checkpoint’ solution is well described. The weakness in the overview is that the challenge of implementing the ‘simple questionnaire’ and the alternatives for failure of compliance are not dealt with in the text.\n\nFor instance, one of the authors would have needed to respond to reflect on over 170 publications over the past 5 years. To determine an accurate answer about the subsequent reproducibility of the 170 findings or about the impact of the findings on clinical or basic science practice would represent a considerable challenge; and the request would be an invitation to side-step accuracy in favour of expediency to get the annoying fly off the desk. This issue is not dealt with in the text. Further, although author accountability is of prime importance, the suggested ‘solution’ would not surface the frustration of those e.g. other laboratories or even new trainees in the authors’ laboratories, who have difficulties repeating the published data. Those ‘negative’ findings, which are of immense importance, almost never see the light of day. The article does not deal with that key issue, which merits attention.\n\nSUMMARY AND RECOMMENDATION:\n\nThis brief, well-written overview surfaces a key issue plaguing the scientific literature and offers a valuable process to enhance author accountability via a 5-year post-publication ‘reflection’ mechanism. That said, the above comments raise two issues that the authors may wish to deal with as they finalize their text. Thus, the possible outcomes of their approach (i.e. expected lack of compliance; or even worse, continued mis-representation of the data) could be dealt with and alternatives to poor outcomes for their process could be suggested. Further, a process that would enable the ‘reporting’ of lack of reproducibility from others in the field, with an opportunity for the authors to respond with ‘further reflections’ would enhance the process of accountability. As an example, this reviewer has had the opportunity to follow an enzyme isolation procedure exactly as described in a JBC manuscript; and the enzyme activity of the product was essentially as expected. That said, a biochemical sequencing of the enzyme product (NOT done in the previous JBC manuscript) showed that the enzyme was not at all the one claimed to be yielded by the published procedure. How would the ‘reflection’ process deal with that issue? One solution would be for the journal to be alerted as to the lack of reproducibility; and for the journal to insist on accountability from EACH AND EVERY ONE of those who have their names on the originally published article. The authors are encouraged to suggest an expanded ‘accountability’ process that would enhance the ‘reflection’ mechanism suggested and surface the lack of reproducibility by others of findings of published 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": []
},
{
"id": "27534",
"date": "13 Nov 2017",
"name": "Jake Cosme",
"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 review article brings forward a recommendation of mitigating the current issues of reproducibility in academic research by encouraging authors to perform post-publication self-review on their studies in 5 years.\n\nThe authors of the review article suggest that the implementation of self-review will allow for academic research to be undertaken with better judgment, as accountability in their work is emphasized. The value of self-review post-publication provides in principle, an excellent platform for authors to reflect on the impact and influence of their research while also providing insight on what led to possible reproducible concerns. The review article appropriately includes limitations of their proposal, such as the adoption rate by the scientific community and implementation into research programs. I believe this is an important point that should be emphasized as the value of self-review is highly dependent on the acceptance of the community. The authors insightfully recommend that self-review be included as terms for publication by \"Journals, funders, and research institutions\" to promote this process.\n\nIn summary, this is a well-written review article introducing self-reflection as a means to identify sources of irreproducibility by the authors of academic research. By encouraging self-review, peers will be able to properly place academic findings into the appropriate place in their knowledge base.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1717
|
https://f1000research.com/articles/4-67/v1
|
13 Mar 15
|
{
"type": "Opinion Article",
"title": "From where to what: a neuroanatomically based evolutionary model of the emergence of speech in humans",
"authors": [
"Oren Poliva"
],
"abstract": "In the brain of primates, the auditory cortex connects with the frontal lobe via the temporal pole (auditory ventral stream; AVS) and via the inferior parietal lobule (auditory dorsal stream; ADS). The AVS is responsible for sound recognition, and the ADS for sound-localization, voice detection and audio-visual integration. I propose that the primary role of the ADS in monkeys/apes is the perception and response to contact calls. These calls are exchanged between tribe members (e.g., mother-offspring) and are used for monitoring location. Perception of contact calls occurs by the ADS detecting a voice, localizing it, and verifying that the corresponding face is out of sight. The auditory cortex then projects to parieto-frontal visuospatial regions (visual dorsal stream) for searching the caller, and via a series of frontal lobe-brainstem connections, a contact call is produced in return.Because the human ADS processes also speech production and repetition, I further describe a course for the development of speech in humans. I propose that, due to duplication of a parietal region and its frontal projections, and strengthening of direct frontal-brainstem connections, the ADS converted auditory input directly to vocal regions in the frontal lobe, which endowed early Hominans with partial vocal control. This enabled offspring to modify their contact calls with intonations for signaling different distress levels to their mother. Vocal control could then enable question-answer conversations, by offspring emitting a low-level distress call for inquiring about the safety of objects, and mothers responding with high- or low-level distress calls. Gradually, the ADS and the direct frontal-brainstem connections became more robust and vocal control became more volitional. Eventually, individuals were capable of inventing new words and offspring were capable of inquiring about objects in their environment and learning their names via mimicry.",
"keywords": [
"Speech",
"Evolution",
"Auditory dorsal stream",
"Contact calls",
"Auditory cortex",
"Vocal production"
],
"content": "1. Introduction\n\nIn the past five decades, gorillas, orangutans, chimpanzees and bonobos were shown capable of learning sign language (Blake, 2004; Gibson, 2011). An important cognitive distinction between the language used by humans and the language used by other apes is with the ability to ask questions. This was first noted by (Premack & Premack, 1984) who reported that, although their chimpanzee, Sarah, showed no difficulty answering questions or repeating questions before answering them, she never used the question signs for inquiring about her own environment. Jordania (2006), in his review of the literature, noted that other signing apes did not utilize questions and that their initiation of conversations was limited to commands (e.g., “me more eat”) and observational statements (e.g., “bird there”). This absence of a questioning mind is in direct contrast to human toddlers and children, who are renown for their incessant use of questions. My interpretation of this human-ape distinction is that during human evolution, we transitioned from the display of curiosity toward items that are present in our environment (i.e., observational statements) to curiosity toward items that are absent in our environment (i.e., WH questions). Developing curiosity about out of sight events and objects could thus explain the rapid migration of humans across the globe. Furthermore, this curiosity toward the unknown is the driving force behind scientific exploration and technological development. One could hence argue that it is the ability to ask that separates us from other animals and makes the human species unique.\n\nAlthough no non-human primate has been reported to ask questions, they were reported to exchange calls for monitoring location (i.e., contact calls). For example, when a mother and her infant are physically separated, each emits in turn a call to signal the other their location. This emission of contact calls could therefore be interpreted as akin in meaning to the question “where are you?” If human communication and contact calls are related, it suggests that the preliminary urge to learn about the unknown is derived from infants and mothers seeking to reunite. In the present paper, based on findings collected from brain research, genetics and paleoarcheology, I demonstrate that human speech and contact calls use the same brain structures, and consequently argue that human speech emerged from contact call exchange. I then argue that by modifying their contact calls with intonations, infants were capable of signaling their mothers whether they were under high- or low-level of distress. Given the turn taking nature of these calls, and as both mothers and infants were capable of modifying their calls with intonations, the ability to choose the call type eventuated with the first yes-no conversation structure. In this scenario infants were capable of inquiring about the safety of objects in their environment (i.e., with a low-level distress call) and mothers were capable of responding to that question with a high-level distress call to signal danger or a low-level distress call to signal safety. As the use of intonations became more prevalent, vocal control became more volitional. Eventually, individuals became capable of enunciating novel calls, and the question-answer conversation pattern further evolved for infants inquiring their mothers for the names of objects in their surrounding and then mimicking the mother’s vocal response.\n\nThroughout the 20th century, our knowledge of language processing in the brain was dominated by the Wernicke-Lichtheim-Geschwind model (Geschwind, 1965; Lichtheim, 1885; Wernicke & Tesak, 1974). This model is primarily based on research conducted on brain-damaged individuals who were reported to possess a variety of language related disorders. In accordance with this model, words are perceived via a specialized word reception center (Wernicke’s area) that is located in the left temporoparietal junction. This region then projects to a word production center (Broca’s area) that is located in the left ventrolateral prefrontal cortex. Because almost all language input was thought to funnel via Wernicke’s area and all language output to funnel via Broca’s area, it became extremely difficult to identify the basic properties of each region. This lack of clear definition for the contribution of Wernicke’s and Broca’s regions to human language rendered it extremely difficult to identify their homologues in other primates. (For one attempt, see Aboitiz & García, 1997). With the advent of the MRI and its application for lesion mappings, however, it was shown that this model is based on incorrect correlations between symptoms and lesions and is therefore flawed (Anderson et al., 1999; Dronkers et al., 1999; Dronkers, 2000; Dronkers et al., 2004; Poeppel et al., 2012; Rauschecker & Scott, 2009 - Supplemental Material; Vignolo et al., 1986). The refutation of such an influential and dominant model opened the door to new models of language processing in the brain, and as will be presented below, to formulating a novel account of the evolutionary origins of human language from a neuroscientific perspective.\n\nIn the last two decades, significant advances occurred in our understanding of the neural processing of human auditory processing. In parallel to the refutation of the classical model, comparative studies reported on homologies between the auditory cortices of humans and other primates. Based on histological staining, functional imaging and recordings from the auditory cortex of several primate species, 3 auditory fields were identified in the primary auditory cortex, and 9 associative auditory fields were shown to surround them (Figure 1 top left; Bendor & Wang, 2006; Kaas & Hackett, 2000 - review; Petkov et al., 2006; Rauschecker et al., 1995). Anatomical tracing and lesion studies further indicated of a separation between the anterior and posterior auditory fields, with the anterior primary auditory fields (areas R-RT) projecting to the anterior associative auditory fields (areas AL-RTL), and the posterior primary auditory field (area A1) projecting to the posterior associative auditory fields (areas CL-CM; la Mothe et al., 2006; Morel et al., 1993; Rauschecker et al., 1997). Recently, evidence accumulated that indicates homology between the human and monkey auditory fields. In humans, histological staining studies revealed two separate auditory fields in the primary auditory region of Heschl’s gyrus (Sweet et al., 2005; Wallace et al., 2002), and by mapping the tonotopic organization of the human primary auditory fields with high resolution fMRI and comparing it to the tonotopic organization of the monkey primary auditory fields, homology was established between the human anterior primary auditory field and monkey area R (denoted in humans as area hR) and the human posterior primary auditory field and the monkey area A1 (denoted in humans as area hA1; Da Costa et al., 2011; Humphries et al., 2010; Langers & van Dijk, 2012; Striem-Amit et al., 2011; Woods et al., 2010). Intra-cortical recordings from the human auditory cortex further demonstrated similar patterns of connectivity to the auditory cortex of the monkey. Recording from the surface of the auditory cortex (supra-temporal plane) reported that the anterior Heschl’s gyrus (area hR) projects primarily to the middle-anterior superior temporal gyrus (mSTG-aSTG) and the posterior Heschl’s gyrus (area hA1) projects primarily to the posterior superior temporal gyrus (pSTG) and the planum temporale (area PT; Figure 1 top right; Gourévitch et al., 2008; Guéguin et al., 2007). This connectivity pattern is also corroborated by a study that recorded activation from the lateral surface of the auditory cortex and reported of simultaneous non-overlapping activation clusters in the pSTG and mSTG-aSTG while listening to sounds (Chang et al., 2011).\n\nTop: The auditory cortex of the monkey (left) and human (right) is schematically depicted on the supratemporal plane and observed from above (with the parieto-frontal operculi removed). Bottom: The brain of the monkey (left) and human (right) is schematically depicted and displayed from the side. Orange frames mark the region of the auditory cortex, which is displayed in the top sub-figures. Top and Bottom: Blue colors mark regions affiliated with the ADS, and red colors mark regions affiliated with the AVS (dark red and blue regions mark the primary auditory fields). Abbreviations: AMYG-amygdala, HG-Heschl’s gyrus, FEF-frontal eye field, INS-insula, IPS-intra parietal sulcus, MTG-middle temporal gyrus, PC-pitch center, PP-planum polare, PT-planum temporale, TP-temporal pole, Spt-sylvian parietal-temporal, DLPFC/VLPFC- dorsolateral/ventrolateral prefrontal cortex, pSTG/mSTG/aSTG-posterior/middle/anterior superior temporal gyrus, CL/ML/AL/RTL-caudo-/middle-/antero-/rostrotemporal-lateral belt area, CPB/RPB-caudal/rostral parabelt fields.\n\nDownstream to the auditory cortex, anatomical tracing studies in monkeys delineated projections from the anterior associative auditory fields (areas AL-RTL) to the ventrolateral prefrontal cortex (VLPFC; Munoz et al., 2009; Romanski et al., 1999) and amygdala (Kosmal et al., 1997). Cortical recording and functional imaging studies in macaque monkeys further elaborated on this processing stream by showing that acoustic information flows from the anterior auditory cortex to the temporal pole (TP) and then to the VLPFC (Perrodin et al., 2011; Petkov et al., 2008; Poremba et al., 2004; Romanski et al., 2004; Russ et al., 2007; Tsunada et al., 2011). This pathway is commonly referred to as the auditory ventral stream (AVS; Figure 1, bottom left-red arrows). In contrast to the anterior auditory fields, tracing studies reported that the posterior auditory fields (areas CL-CM) project primarily to the dorsolateral prefrontal cortex (although some projections do terminate in the VLPFC; Cusick et al., 1995; Romanski et al., 1999). Cortical recordings and anatomical tracing studies in monkeys further provided evidence that this processing stream flows from the posterior auditory fields to the prefrontal cortex via a relay station in the intra-parietal sulcus (IPS; Cohen, 2004; Deacon, 1992; Lewis & Van Essen, 2000; Roberts et al., 2007; Schmahmann et al., 2007; Seltzer & Pandya, 1984). This pathway is commonly referred to as the auditory dorsal stream (ADS; Figure 1, bottom left-blue arrows). Comparing the white matter pathways involved in communication in humans and monkeys with diffusion tensor imaging techniques indicates of similar connections of the AVS and ADS in the two species (Monkey: Schmahmann et al., 2007; Human: Catani et al., 2004; Frey et al., 2008; Makris et al., 2009; Menjot de Champfleur et al., 2013; Saur et al., 2008; Turken & Dronkers, 2011). In humans, the pSTG was shown to project to the parietal lobe (sylvian parietal-temporal junction-inferior parietal lobule; Spt-IPL), and from there to the dorsolateral prefrontal cortex (Figure 1, bottom right-blue arrows), and the aSTG was shown to project to the anterior temporal lobe (middle temporal gyrus-temporal pole; MTG-TP) and from there to the VLPFC (Figure 1 bottom right-red arrows).\n\nOn the basis of converging evidence collected from monkeys and humans, it has been established that the AVS is responsible for the extraction of meaning from sounds (see appendix A for a review of the literature). Specifically, the anterior auditory cortex is ascribed with the perception of auditory objects, and downstream, the MTG and TP are thought to match the auditory objects with their corresponding audio-visual semantic representations (i.e., the semantic lexicon). This recognition of sounds in the AVS, although critical for intact communication, appears to contribute less to the uniqueness of human language than the ADS. This is demonstrated by the universality of sound recognition, as many mammalian species use it for localizing prey, predators or potential mates. As an example, dogs were reported capable of recognizing spoken words and extract their meaning (Kaminski et al., 2004; Pilley & Reid, 2011), and with fMRI this sound recognition ability was localized to the TP of the AVS (Andics et al., 2014). Studies also provided evidence that the sound recognition of non-human apes is equivalent in complexity to ours. Apes trained in human facilities were reported capable of learning human speech and comprehending its meaning (e.g., the bonobos, Kanzi and Panbanisha, were reported to recognize more than 3000 spoken English words; Blake, 2004; Gibson, 2011). Moreover, a study that compared humans and a chimpanzee in their recognition of acoustically distorted spoken words, reported no differences between chimpanzee and human performance (Heimbauer et al., 2011). Finally, a diffusion tensor imaging study that compared the white matter of humans and chimpanzees demonstrated significant strengthening of ADS connectivity, but not AVS connectivity (Rilling et al., 2011). This study thus indicates that it is the ADS, and not the AVS, that separates us from our apian relatives.\n\nIn contrast to the AVS, the ADS has a diverse range of seemingly unrelated functions that process language. These functions, which will be detailed throughout this paper, include auditory localization, audio-visual integration, and voice detection in monkeys. In humans, the ADS has been further ascribed with speech articulation, speech repetition and perception, and production of linguistic prosody. In the present paper, I interpret the functional differences between the ADS of monkeys and humans as evidence of intermediate stages in the development of human speech.\n\nThe most established role of the ADS is with audio-spatial processing. This is evidenced via studies that recorded neural activity from the auditory cortex of monkeys, and correlated the strongest selectivity to changes in sound location with the posterior auditory fields (areas CM-CL), intermediate selectivity with primary area A1, and very weak selectivity with the anterior auditory fields (Benson et al., 1981; Miller & Recanzone, 2009; Rauschecker et al., 1995; Woods et al., 2006). In humans, behavioral studies of brain damaged patients (Clarke et al., 2000; Griffiths et al., 1996) and EEG recordings from healthy participants (Anourova et al., 2001) demonstrated that sound localization is processed independently of sound recognition, and thus is likely independent of processing in the AVS. Consistently, a working memory study (Clarke et al., 1998) reported two independent working memory storage spaces, one for acoustic properties and one for locations. Functional imaging studies that contrasted sound discrimination and sound localization reported a correlation between sound discrimination and activation in the mSTG-aSTG, and correlation between sound localization and activation in the pSTG and PT (Ahveninen et al., 2006; Alain et al., 2001; Barrett & Hall, 2006; De Santis et al., 2006; Viceic et al., 2006; Warren & Griffiths, 2003), with some studies further reporting of activation in the Spt-IPL region and frontal lobe (Hart et al., 2004; Maeder et al., 2001; Warren et al., 2002). Some fMRI studies also reported that the activation in the pSTG and Spt-IPL regions increased when individuals perceived sounds in motion (Baumgart et al., 1999; Krumbholz et al., 2005; Pavani et al., 2002). EEG studies using source-localization also identified the pSTG-Spt region of the ADS as the sound localization processing center (Tata et al., 2005a; Tata et al., 2005b). A combined fMRI and MEG study corroborated the role of the ADS with audio-spatial processing by demonstrating that changes in sound location results in activation spreading from Heschl’s gyrus posteriorly along the pSTG and terminates in the IPL (Brunetti et al., 2005). In another MEG study, the IPL and frontal lobe were shown active during maintenance of sound locations in working memory (Lutzenberger et al., 2002).\n\nIn addition to localizing sounds, the ADS appears also to encode the sound location in memory, and to use this information for guiding eye movements. Evidence for the role of the ADS in encoding sounds into working memory is provided via studies that trained monkeys in a delayed matching to sample task, and reported of activation in areas CM-CL (Gottlieb et al., 1989) and IPS (Linden et al., 1999; Mazzoni et al., 1996) during the delay phase. Influence of this spatial information on eye movements occurs via projections of the ADS into the frontal eye field (FEF; a premotor area that is responsible for guiding eye movements) located in the frontal lobe. This is demonstrated with anatomical tracing studies that reported of connections between areas CM-CL-IPS and the FEF (Cusick et al., 1995; Stricanne et al., 1996), and electro-physiological recordings that reported neural activity in both the IPS (Linden et al., 1999; Mazzoni et al., 1996; Mullette-Gillman, 2005; Stricanne et al., 1996) and the FEF (Russo & Bruce, 1994; Vaadia et al., 1986) prior to conducting saccadic eye-movements toward auditory targets.\n\nIn the visual system, it is well established that the inferior temporal lobe processes the identity of visual objects (visual ventral stream; purple arrow in Figure 2), and that the IPS and FEF process visuospatial properties of objects and convert them into appropriate motor behaviors (visual dorsal stream; pink arrow in Figure 2; Goodale & Milner, 1992; Tanné-Gariépy et al., 2002; Ungerleider & Haxby, 1994). Given the dual role of the IPS-FEF pathway in audiospatial and visuospatial processing, it is tempting to assume that spatial processing in both modalities occur in parallel. Cumulating evidence, however, suggests that audiospatial input is first converted into a visuospatial code and then processed via a visuospatial network. In monkeys, electrophysiological studies that recorded activity in the IPS reported that almost all the neurons in this area that are selective for auditory locations, are also selective to visual locations (Linden et al., 1999; Mazzoni et al., 1996). It was also shown that neurons in the IPS responded first to visual stimuli, and only after training do they become responsive to auditory stimuli (Grunewald et al., 1999). Retrograde tracing from the IPS revealed that there were much less connections from the auditory cortex (primarily from areas CM-CL) than from the visual cortex (Lewis & Van Essen, 2000). The encoding of auditory information in visual working memory in the ADS is further demonstrated via a monkey fMRI study that correlated integration of auditory and visual stimuli with activation in the posterior, but not anterior, auditory cortex (Kayser et al., 2009), and a behavioral working memory study of monkeys that demonstrated audio-visual integration to be susceptible to visual, but not auditory, interferences (Colombo & Graziano, 1994). Human studies also indicate that the ADS encodes sound locations in visual working memory. For example, an fMRI study that compared cortical activation during a visual motion discrimination task and an auditory motion discrimination task reported of overlapping activation in both modalities in the IPS (Lewis et al., 2000). A subsequent cross-modal integration task then revealed heightened activation in the IPS that is selective to the combined auditory and visual stimuli. On this account, the researchers endowed the IPS with the role of audio-visual integration of spatial information. An fMRI study that compared the brain areas active during rehearsal of sound locations with rehearsal of visual locations in working memory reported that the IPS is the only region that is always active in both tasks (Martinkauppi et al., 2000). An fMRI study that contrasted spatial orienting to sounds with spatial orienting to visual objects also reported of overlapping parietal and frontal activation in both tasks (Smith et al., 2010b). Supporting the maintenance of sound locations in visual working memory in humans is also a study that reported of spatial bias in sound localization while wearing visuospatially distorting goggles (prism goggles; Zwiers et al., 2003). Finally, a working memory study demonstrated that rehearsal of sound locations in working memory is more susceptible to visual interference than auditory interference (Clarke et al., 1998). In contrast, rehearsal of simple tone sounds in working memory, which in the context of the present model is associated with processing in the AVS, is more susceptible to auditory interference than visual interference.\n\nThe model depicts three stages in the early development of language, each characterized with its own neuroanatomical modifications (these changes are depicted here on schematized chimpanzee brains). Left: In extant monkeys/apes, sound recognition is processed via the auditory ventral stream (AVS; red arrows). Contact calls, however, are perceived in the pSTG. The pSTG then projects (blue arrow) to the IPS (pink asterisk), which is part of the visual dorsal stream (VDS, pink arrow). The IPS then projects to the VLPFC and FEF (pink arrow). The FEF guides saccadic eye-movements to the source of the call; the VLPFC initiates the vocal response by activating a cascading series of descending connections (green arrows). First, the VLPFC activates a network of limbic structures (e.g., amygdala; AMYG). The limbic network then projects to the midbrain periaqueductal grey (PAG), which in turn projects to the brainstem motor nuclei (BMN). Some elements of the vocal response are mediated via a direct VLPFC-BMN pathway (brown arrow). Middle: Approximately 2.5 million years ago, the Homo genus emerged as a result of duplication of the IPS (pink and blue asterisks) and subsequent duplication of its frontal projections (pink and blue arrows). Since the auditory cortex targeted the more proximal of these duplicated parietal regions (blue asterisk), a new pathway dedicated for auditory processing emerged (i.e., auditory dorsal stream; ADS-blue arrows). By strengthening pre-existing direct VLPFC-BMN connections (brown arrow), the ADS also acquired partial vocal control. This development of an audio-vocal pathway enabled individuals to modify contact calls with intonations for signaling high- and low-level distress calls. Right: Given that the ability to choose whether the emitted intonation signaled distress or safety was partially volitional, the modification of contact calls with intonations enabled question-answer conversations about other topics besides location. This use of intonations for more complex communication resulted with strengthening of the ADS and the descending VLPFC-BMN pathway (blue and brown arrows). Eventually, the ADS-VLPFC-BMN pathway acquired complete control over the vocal apparatus and speech became volitional.\n\nIn addition to processing the locations of sounds, evidence suggests that the ADS further integrates sound locations with auditory objects. Demonstrating this integration are electrophysiological recordings from the posterior auditory cortex (Recanzone, 2008; Tian et al., 2001) and IPS (Gifford & Cohen, 2005), as well a PET study (Gil-da-Costa et al., 2006), that reported neurons that are selective to monkey vocalizations. One of these studies (Tian et al., 2001) further reported neurons in this region (CM-CL) that are characterized with dual selectivity for both a vocalization and a sound location. Consistent with the role of the pSTG-PT in the localization of specific auditory objects are also studies that demonstrate a role for this region in the isolation of specific sounds. For example, two functional imaging studies correlated circumscribed pSTG-PT activation with the spreading of sounds into an increasing number of locations (Smith et al., 2010a-fMRI; Zatorre et al., 2002-PET). Accordingly, an fMRI study correlated the perception of acoustic cues that are necessary for separating musical sounds (pitch chroma) with pSTG-PT activation (Warren et al., 2003).\n\nWhen elucidating the role of the primate ADS in the integration of a sound’s location with calls, it remains to be determined what kind of information the ADS extracts from the calls. This information could be then used to make inferences about the function of the ADS. Studies from both monkeys and humans suggest that the posterior auditory cortex has a role in the detection of a new speaker. A monkey study that recorded electrophysiological activity from neurons in the posterior insula (near the pSTG) reported neurons that discriminate monkey calls based on the identity of the speaker (Remedios et al., 2009a). Accordingly, human fMRI studies that instructed participants to discriminate voices reported an activation cluster in the pSTG (Andics et al., 2010; Formisano et al., 2008; Warren et al., 2006). A study that recorded activity from the auditory cortex of an epileptic patient further reported that the pSTG, but not aSTG, was selective for the presence of a new speaker (Lachaux et al., 2007-patient 1). The role of this posterior voice area, and the manner in which it differs from voice recognition in the AVS (Andics et al., 2010; Belin & Zatorre, 2003; Nakamura et al., 2001; Perrodin et al., 2011; Petkov et al., 2008), was further shown via electro-stimulation of another epileptic patient (Lachaux et al., 2007-patient 2). This study reported that stimulation of the aSTG resulted in changes in the perceived pitch of voices (including the patient’s own voice), whereas stimulation of the pSTG resulted in reports that her voice was “drifting away.” This report indicates a role for the pSTG in the integration of sound location with an individual voice. Consistent with this role of the ADS is a study that reported patients with AVS damage but spared ADS (surgical removal of the anterior STG/MTG) were no longer capable of isolating environmental sounds in the contralesional space, whereas their ability of isolating and discriminating human voices remained intact (Efron et al., 1983). Preliminary evidence from the field of fetal cognition suggests that the ADS is capable of identifying voices in addition to discriminating them. By scanning fetuses of third trimester pregnant mothers with fMRI, the researchers reported of activation in area Spt when the hearing of voices was contrasted to pure tones (Jardri et al., 2012). The researchers also reported that a sub-region of area Spt was more selective to maternal voice than unfamiliar female voices. Based on these findings, I suggest that the ADS has acquired a special role in primates for the localization of conspecifics.\n\nTo summarize, I have argued that the monkey’s ADS is equipped with the algorithms required for detecting a voice, isolating the voice from the background cacophony, determining its location, integrating the location of this voice into a visuospatial map of the area, and guiding eye movements for the origin of the call. An example of a behavior that utilizes all these functions is the exchange of contact calls, which are used by extant primates to monitor the location or proximity of conspecific tribe members (Biben et al., 1986; Sugiura, 1998). The utilization of these ADS functions during the exchange of contact calls was demonstrated in studies of squirrel monkeys and vervet monkeys (Biben, 1992; Biben et al., 1989; Cheney & Seyfarth, 1980; Symmes & Biben, 1985). In both species, mothers showed no difficulty in isolating their own infant’s call, localizing it, and maintaining this location in their memory while approaching the source of the sound. A similar use of contact calls has been documented in our closest relatives, chimpanzees. The exchange of pant-hoot calls was documented between chimpanzees that were separated by great distances (Goodall, 1986; Marler & Hobbett, 1975) and was used for re-grouping (Mitani & Nishida, 1993). Because infants respond to their mother’s pant-hoot call with their own unique vocalization (staccato call; Matsuzawa, 2006), the contact call exchange appears also to play an important role in the ability of mothers to monitor the location of their infants. It is also worth noting that when a chimpanzee produced a pant-hoot call and heard no call in response, the chimpanzee was reported to carefully scan the forest before emitting a second call (Goodall, 1986). This behavior demonstrates the relationship between the perception of contact calls, the embedding of auditory locations in a map of the environment, and the guidance of the eyes for searching the origin of the call. Further corroborating the involvement of the ADS in the perception of contact calls are intra-cortical recordings from the posterior insula (near area CM-A1) of the macaque, which revealed stronger selectivity for a contact call (coo call) than a social call (threat call; Remedios et al., 2009a). Contrasting this finding is a study that recorded neural activity from the anterior auditory cortex, and reported that the proportion of neurons dedicated to a contact call was similar to the proportions of neurons dedicated to other calls (Perrodin et al., 2011).\n\nPerceiving a contact call can be viewed as a three-step process. The individual is required to detect a voice, integrate it with its location and verify that no face is visible in that location (Figure 3). In the previous paragraphs, I provided evidence for the involvement of the ADS in the first two stages (voice detection and localization). Evidence for the role of the ADS in the integration of faces with their appropriate calls is provided by a study that recorded activity from the monkey auditory cortex (areas A1 and ML; Ghazanfar, 2005). The monkeys were presented with pictures of a monkey producing a call in parallel to hearing the appropriate call, or only saw the face or heard the call in isolation. Consistent with the prediction from the present model that visual perception of faces inhibits processing of contact calls, the face-call integration was much more enhanced for the social call (grunt call) than for the contact call (coo call). Associating this integration of faces with calls with processing in the ADS is consistent with the evidence presented earlier that ascribe the ADS with audio-visual integration (e.g., a monkey fMRI study correlated audio-visual integration with activation in the posterior, but not in the anterior, auditory fields; Kayser et al., 2009).\n\nIn accordance with the model, the original function of the ADS is for the localization of and the response to contact calls that are exchanged between mothers and their infants. When an infant emits a contact call (A), the mother perceives it (B) by integrating in the pSTG-IPS the identity of the speaker (B1) with the location of the call (B2) and maintains this information in visual working memory. Then, if the corresponding face is absent in that location (B3), the mother emits a contact call in return (C).\n\nHitherto, I have argued that the ADS is responsible for the perception of contact calls. However, as the perception of a contact call leads to producing a contact call in return, it is also desirable to suggest a pathway through which the ADS mediates vocal production. Monkey studies have demonstrated that the ADS doesn’t directly process vocal production. This was shown through studies that damaged the temporoparietal and/or the VLPFC regions and reported that such lesions had no effect on spontaneous vocal production (Aitken, 1981; Sutton et al., 1974). This conclusion is also consistent with comprehensive electro-stimulation mappings of the monkey’s brain, which reported no spontaneous vocal production during stimulation of the temporal, occipital, parietal, or frontal lobes (Jürgens & Ploog, 1970; Robinson, 1967). These studies, however, reported emission of vocalizations after stimulating limbic and brainstem regions (amygdala, anterior cingulate cortex, basal forebrain, hypothalamus, mid-brain periaqueductal gray (PAG)). Moreover, based on a study that correlated chemical activation in the mid-brain PAG with vocal production, it was inferred that all the limbic regions project to central pattern generators in the PAG, which orchestrates the vocal production (Zhang et al., 1994). In a series of tracing studies and electrophysiological recordings, it was also shown that the PAG projects to pre-motor brainstem areas (Hage & Jürgens, 2006; Hannig & Jürgens, 2005), which in turn project to brainstem motor nuclei (BMN; green arrows in Figure 2; Holstege, 1989; Holstege et al., 1997; Lüthe et al., 2000; Vanderhorst et al., 2001; Vanderhorst et al., 2000). The BMN then activates the individual muscles of the vocal apparatus. Because documented calls of non-human primates (including chimpanzees) were shown with very little plasticity (Arcadi, 2000) and were observed only in highly emotional situations (Goodall, 1986), these limbic-brainstem generated calls are likely more akin to human laughter, sobbing, and screaming than to human speech. In relation to contact calls, a likely candidate for mediating the ADS with the limbic-brainstem vocal network is the VLPFC. This is because electrophysiological recordings (Cohen, 2004) and anatomical tracing (Deacon, 1992; Roberts et al., 2007) studies in monkeys demonstrated this region to receive parietal afferents and to project to several limbic structures (Roberts et al., 2007). Corroborating the role of the VLPFC in mediating vocal production of contact calls are studies that recorded neural activity from the VLPFC of macaques and reported neural discharge prior to cued or spontaneous contact call production (coo calls), but not prior to production of vocalizations-like facial movements (i.e., silent vocalizations; Coudé et al., 2011; see also Gemba et al., 1999 for similar results). Consistently, a study that sacrificed marmoset monkeys immediately after responding to contact calls (phee calls) measured highest neural activity (genomic expression of cFos protein) in the posterior auditory fields (CM-CL), and VLPFC (Miller et al., 2010). Monkeys sacrificed after only hearing contact calls or only emitting them showed neural activity in the same regions but to a much smaller degree (See also Simões et al., 2010 for similar results in a study using the protein Egr-1). Further supporting this ability of the VLPFC in regulating limbic-brainstem generated calls is the result of a tracing study that reported direct connections between the cortical motor area of the mouth and a brainstem motor nucleus that executes tongue movements (hypoglossal nucleus; Jürgens & Alipour, 2002). Hence, this study suggests that in addition to the VLPFC-AMYG-PAG-BMN pathway (green arrows in Figure 2), a second direct VLPFC-BMN pathway (brown arrow in Figure 2) has evolved in monkeys. The role of this direct VLPFC-BMN pathway is not yet known, but its anatomical connectivity implies that it is capable of bypassing the limbic-brainstem vocal network, and therefore dominates vocal production.\n\nAccording to Falk’s evolutionary hypothesis (2004), due to bipedal locomotion and the loss of hair in early Hominins, mothers were not capable of carrying their infants while foraging. As a result, the mothers maintained contact with their infant through a vocal exchange of calls that resemble contemporary “motherese” (the unique set of intonations that caregivers use when addressing infants). Following this model, Masataka (2009) provided evidence that macaque mothers are capable, to a limited extent, of modifying their contact calls to acoustically match those of their infants and further suggested that the human mother-infant prosodic vocal exchange evolved from the exchange of contact calls between our apian ancestors. Support for the use of prosody in contact calls are studies of squirrel monkeys and macaque monkeys that reported of small changes in the frequencies of contact calls, which resulted with the caller and responder emitting slightly different calls (Biben et al., 1986; Sugiura, 1998). Evidence supporting the transition from contact call expression to volitional speech is provided by a study where macaque monkeys spontaneously learned to modify the vocal properties of their contact call for requesting different objects from the experimenter (Hihara et al., 2003). Anecdotal reports of more generalized volitional vocal control, albeit rudimentary, in apes (Hayes & Hayes, 1952; Hopkins et al., 2007; Kalan et al., 2015; Koda et al., 2007; Koda et al., 2012; Lameira et al., 2015; Taglialatela et al., 2003; Wich et al., 2008) further indicates that the ability to modify calls with intonations was enhanced prior to our divergence from our apian relatives.\n\nSupporting evidence for a role of the ADS in the transition from mediating contact calls into mediating human speech includes genetic studies that focused on mutation to the protein SRPX2 and its regulator protein FOXP2 (Roll et al., 2010). In mice, blockage of SRPX2 or FOXP2 genes resulted in pups not emitting distress calls when separated from their mothers (Shu et al., 2005; Sia et al., 2013). In humans, however, individuals afflicted with a mutated SRPX2 or FOXP2 were reported with speech dyspraxia (Roll et al., 2006; Watkins et al., 2002). A PET imaging study of an individual with a mutated SRPX2 gene correlated this patient’s disorder with abnormal activation (hyper-metabolism) along the ADS (pSTG-Spt-IPL; Roll et al., 2006). Similarly, an MRI study that scanned individuals with mutated FOXP2 reported increased grey matter density in the pSTG-Spt and reduced density in the VLPFC, further demonstrating abnormality in ADS‘ structures (Belton et al., 2003). A role for the ADS in mediating speech production in humans has also been demonstrated in studies that correlated a more severe variant of this disorder, apraxia of speech, with IPL and VLPFC lesions (Deutsch, 1984; Edmonds & Marquardt, 2004; Hillis et al., 2004; Josephs, 2006; Kimura & Watson, 1989; Square et al., 1997). The role of the ADS in speech production is also demonstrated via a series of studies that directly stimulated sub-cortical fibers during surgical operations (Duffau et al., 2008-review), and reported that interference in the pSTG and IPL resulted in an increase in speech production errors, and interference in the VLPFC resulted in speech arrest (see also Acheson et al., 2011; Stewart et al., 2001 for similar results using magnetic interference in healthy individuals).\n\nFurther support for the transition from contact call exchange to human language are provided by studies of hemispheric lateralization (Petersen et al., 1978). In one study, Japanese macaques and other old world monkeys were trained to discriminate contact calls of Japanese macaques, which were presented to the right or left ear. Although all the monkeys were capable of completing the task, only the Japanese macaques were noted with right ear advantage, thus indicating left hemispheric processing of contact calls. In a study replicating the same paradigm, Japanese macaques had an impaired ability to discriminate contact calls after suffering unilateral damage to the auditory cortex of the left, but not right, hemisphere (Heffner & Heffner, 1984). This leftward lateralization of contact call perception is similar to the long established role of the human left hemisphere in the processing human language (Geschwind, 1965).\n\nConsidering Falk’s and Masataka’s hypotheses, evidence also indicates that the ADS was involved in the transition of contact calls into human speech through a transitory prosodic phase. This view is consistent with an fMRI study that instructed participants to rehearse speech, and reported that perception of prosodic speech, when contrasted with flattened speech, results in a stronger activation of the PT-pSTG of both hemispheres (Meyer et al., 2004). In congruence, an fMRI study that compared the perception of hummed speech to natural speech didn’t identify any brain area that is specific to humming, and thus concluded that humming is processed in the speech network (Ischebeck et al., 2007). fMRI studies that instructed participants to analyze the rhythm of speech also reported of ADS activation (Spt, IPL, VLPFC; Geiser et al., 2008; Gelfand & Bookheimer, 2003). An fMRI study that compared speech perception and production to the perception and production of humming noises, reported in both conditions that the overlapping activation area for perception and production (i.e., the area responsible for sensory-motor conversion) was located in area Spt of the ADS (Hickok et al., 2003). Supporting evidence for the role of the ADS in the production of prosody are also studies reporting that patients diagnosed with apraxia of speech are additionally diagnosed with expressive dysprosody (Odell et al., 1991, Odell et al., 2001; Shriberg et al., 2006 - FOXP2 affected individuals). Finally, the evolutionary account proposed here from vocal exchange of calls to a prosodic-based language is similar to the recent development of whistling languages, since these languages were documented to evolve from exchanging simple calls used to report speakers’ locations into a complex semantic system based on intonations (Meyer, 2008).\n\nIn section 3, I presented evidence that in monkeys audio-spatial input is integrated with visual stimuli in the IPS prior to guiding eye movements. However, human studies that explore the neuroanatomical correlates of inner and outer speeches report that these are processed in a purely auditory network. This was first shown by Conard (1962), who instructed participants to rehearse a sequence of letters and showed that, at recall, they tend to confuse letters that sound similar, but not letters that look similar. Following this discovery, in a series of studies Baddeley & Hitch (1974) demonstrated that simultaneous performance of visual and verbal working memory tasks was nearly as efficient as performance of the same visual or verbal tasks in isolation. In contrast, these researchers showed that simultaneous performance of two separate verbal working memory tasks, or two visual working memory tasks, is less efficient than when performing each task in isolation (e.g., recitation of the alphabet, but not discrimination of faces, interferes with rehearsal of a sequence of digits in working memory). These findings led the researchers to propose the existence of two working memory systems, one for visuospatial material (i.e., the visuospatial sketchpad) and another for verbal material (i.e., the phonological loop). The neuroanatomical correlate of the phonological loop was identified in fMRI studies that compared the activation pattern of individuals when they listened to speech, and the pattern when they produced speech externally or internally (Buchsbaum et al., 2001; Hickok et al., 2003; Wise et al., 2001). These studies reported that in both speech perception and production (covert or overt) area Spt became active, and thus associated this region with the conversion of auditory stimuli into appropriate articulations. The role of the ADS in verbal rehearsal is in accordance with other functional imaging studies that localized activation to the same region during speech repetition tasks (Giraud & Price, 2001; Graves et al., 2008; Karbe et al., 1998). An intra-cortical recording study that recorded activity throughout most of the temporal, parietal and frontal lobes also reported of activation during speech repetition in regions along the ADS (areas Spt, IPL and VLPFC; Towle et al., 2008). The association of the ADS with rehearsal is also consistent with neuropsychological studies that correlated the lesion of individuals with speech repetition deficit, but intact auditory comprehension (i.e., conduction aphasia), to the temporoparietal junction (Axer et al., 2001; Baldo et al., 2008; Bartha & Benke, 2003; Buchsbaum et al., 2011; Fridriksson et al., 2010; Kimura & Watson, 1989; Leff et al., 2009; Selnes et al., 1985), and with studies that applied direct intra-cortical electro-stimulation to this same region and reported of a transient speech repetition deficit (Anderson et al., 1999; Boatman et al., 2000; Ojemann, 1983; Quigg & Fountain, 1999; Quigg et al., 2006).\n\nIn a review discussing the role of the ADS in humans, Warren, et al., (2005) noted that there is similarity in function between the conversion of visual input into eye movements in the IPS, and the conversion of auditory input into articulations in area Spt. Given this dual role of the parietal lobe in sensory-motor transformation of both audio-spatial and verbal information, I propose that during Hominin evolution there was a cortical field duplication, with the IPS (pink asterisk in Figure 2) duplicating to form area Spt (blue asterisk in Figure 2). Such duplication is a common phenomenon in mammalian evolution and was reported in several cortical regions (Butler & Hodos, 2005). Consequently, because area Spt was closer to the auditory cortex than the IPS, area Spt received the majority of the auditory afferents. Moreover, because of the preexisting connections of the IPS with the VLPFC (Figure 2 pink arrow), I suggest that the duplication of the IPS resulted in further duplication of its projections to the VLPFC (Figure 2 blue arrow). The development of connections from the auditory cortex to area Spt, and from there to the VLPFC, thus resulted with a pathway dedicated for audio-vocal conversion. The cortical field duplication hypothesis is consistent with an fMRI study that reported visual and auditory working memory to activate neighboring regions in the VLPFC (Rämä & Courtney, 2005). Further support for the cortical field duplication comes from research of autism. This is because individuals with autism report that they think in pictures instead of words (Grandin, 2008; Sahyoun et al., 2009), which implies ADS impairment. This conclusion is also consistent with an fMRI study that reported weaker activation in the IPL and VLPFC of autistic patients than healthy participants (Just et al., 2007).\n\nEvidence for cortical duplication in the IPL also derives from the fossil record. A study that reconstructed the endocranium of early Hominins noted that Homo habilis, but not any of its Australopith ancestors, is characterized by a dramatic heightening (but not widening) of the IPL and less dramatic enlargement of the VLPFC, whereas the rest of the endocranium remains extremely similar to the endocranium of modern apes (Tobias, 1987). It is also worth reporting that the recently discovered Australopithecus sediba (Carlson et al., 2011), which is the closest known relative to the Australopith predecessor of Homo habilis, also has a very ape-like parietal and frontal lobes (although some modifications of the orbitofrontal surface were noted). Based on these findings, I propose that the cortical field duplication in the IPL occurred 2.3–2.5 million years ago and resulted with the brain enlargement that characterizes the Homo genus (Kimbel et al., 1996; Schrenk et al., 1993; Wood & Baker, 2011). This development equipped early Hominans (i.e., members of the genus Homo; Wood & Richmond, 2000) with partial control of lip and jaw movements, and thus endowed them with sufficient vocal control for modifying innate calls with intonations.\n\nIn the opening paragraph of this paper, I described the inability of apes to ask questions, and proposed that the ability to ask questions emerged from contact calls. Because the ability to ask questions likely co-emerged with the ability to modify calls with prosodic intonations, I expand Falk’s and Masataka’s views regarding the prosodic origins of vocal language, and propose that the transition from contact calls to prosodic intonations could have emerged as a means of enabling infants to express different levels of distress (Figure 4). In such a scenario, the modification of a call with intonations designed to express a high level of distress is akin in meaning to the sentence “mommy, come here now!”. Hence, the modification of calls with intonations could have served as a precursor for the development of prosody in contemporary vocal commands. On the other hand, the use of intonations for expressing a low-level of distress is akin in meaning to the sentence “mommy, where are you?”. Therefore, this use of prosody for asking the first question could have served as the precursor for pragmatically converting calls into questions by using prosody as well. This transition could be related to the ability of present-day infants of using intonations for changing the pragmatic utilization of a word from a statement to a command/demand (“mommy!”) or a question (“mommy?”). Evidence supporting a relationship between the ability to ask questions and the ADS is derived from the finding that patients with phonological dementia, who are known to suffer from degeneration along the ADS and show signs of ADS impairment (Gorno-Tempini et al., 2008; Rohrer et al., 2010), were impaired in distinguishing whether a spoken word was a question or a statement (Rohrer et al., 2012).\n\nIn accordance with the model, vocal control began with the ADS modifying the rigid limbic-brainstem-generated vocalizations with intonations (prosody). This modification could have originated for the purpose of expressing different levels of distress. In this figure, we see a Homo habilis child using prosody to modify the contact call to express a high level of distress (A) or a low level of distress (C). The child’s mother then registers the call by integrating his prosodic intonation and voice, the location, and the absence of his face to recognize whether her child requires immediate (B) or non-immediate (D) attention.\n\nA possible route for the transition from emitting low-distress contact calls to asking questions is by individuals starting to utilize the former to signal interest about objects in their environment. Given that both contact call exchange and contemporary speech are characterized with turn taking, early Hominans could have responded to the low-level distress calls with either high or low level distress calls. For example, when an infant expressed a low-level distress call prior to eating berries, his/her mother could have responded with a high-level distress call that indicated the food is dangerous or a low-level distress call that indicated the food is safe (Figure 5). Eventually, the infant emitted the question call and waited for an appropriate answer from their mother before proceeding with their intended action. This conversation structure could be the precursor to present-day yes/no questions. As intonations became more prevalent and questions became more complex, the ADS and VLPFC-BMN pathways (blue and brown arrows in Figure 2) became more robust, and as a consequence individuals acquired more volitional control over the vocal apparatus. Consistent with the role of the ADS in speech repetition (see section 6), the increase in volitional vocal control could have then been used to invent new calls and teach them to offspring via vocal mimicry. This desire of offspring to learn about their environment by mimicking their mother’s calls and then encoding the new words to long-term memory could have been the guiding force that sparked the curiosity to explore the unknown. Discussing the transition from exchanging low-level distress contact calls into complex vocal language, however, is beyond the scope of the present paper and a model for such transition is discussed in length in a sibling paper titled ‘Vocal Mimicry as the Sculptor of the Human Mind. A Neuroanatmically based Evolutionary Model of The Emergence of Vocal Language’ (Poliva, in preparation).\n\nIn accordance with the model, the modification of contact calls with intonations for reporting distress levels eventually transitioned into question-answer conversations about items in their environment. In this figure, a child is using low-level distress call (A,C) to ask permission to eat an unfamiliar food (berries). The mother can then respond with a high-level distress call (D) that signals danger or a low-level distress (B) that signals safety.\n\nFollowing in the footsteps of Dean Falk and Nobuo Masataka, the present model argues that human speech emerged from the exchange of contact calls via a transitory prosodic phase. Since the principle of natural selection was first acknowledged by the scientific community however, several other accounts of language evolution were proposed. Here, I’ll present two schools of thought, and discuss their validity in the context of the present model.\n\nThe earliest model for language evolution was proposed by Charles Darwin. In his book, The Descent of Man (1871), Darwin equated speech exchange to bird song, and proposed that the perception and production of songs during mating rituals were the precursor to human language (singing ape hypothesis). Similar accounts suggesting music to participate in the evolutionary development of speech were also proposed by more recent researchers (Jordanaia, 2006; Masataka, 2009; Mithen, 2006). However, so far the idea of music as precursor to language has not taken hold in the scientific community due to lack of substantiating evidence. In appendix A, I cite evidence that the perception of melodies occurs in the aSTG of the AVS. Given the mounting evidence indicating that speech is processed primarily in the ADS, we would expect that precursors to speech would also be processed in the same pathway (although, see the review by Stewart, 2006 who suggests roles also for other auditory fields in music perception). Since I hypothesize that singing-like calls were utilized for communication prior to complex vocal language, the idea of music perception and production isn’t too different from the present model. However, arguing that music served as precursor to speech is different than arguing that music and speech emerged from a common proto-function. Investigating whether music served as a precursor to vocal language is problematic since such a model implies that music perception is a unique human trait. Therefore, in order to resolve the conundrum of music evolution and its level of contribution to the emergence of vocal language, future studies should first attempt to determine whether non-human primates can perceive music. (See Remedios et al., 2009b for preliminary findings).\n\nA more recent school of thought argues that language with complex semantics and grammar was first communicated via the exchange of gestures and only recently became vocal (Gestural language model; Arbib, 2008; Corballis, 2010; Donald, 2005; Gentilucci & Corballis, 2006; Hewes, 1973; Studdert-Kennedy, 2005). In accordance with this model, speech could have served for increasing communication distance and enabling communication under low visibility conditions (e.g., night, caves). This model is primarily based on the natural use of gestural communication between non-human primates, the ability of apes to learn sign language, and the natural development of sign languages in deaf communities. This model also received increased popularity since the discovery of mirror neurons, as these neurons are interpreted by proponents of the model as evidence of a mechanism dedicated to the imitation of gestures. From a neuroanatomical perspective it is plausible that vocal communication emerged from gestures. For instance, earlier I presented evidence that in monkeys the IPS encodes auditory stimuli into the visual dorsal stream. Given that the primary function of the visual dorsal stream is to convert visual stimuli into motor actions, it is possible that in addition to vocally responding to contact calls, this pathway served for converting visually observed gestures into producing gestures. This view is also consistent with an fMRI study that correlated hearing animal calls with bilateral activation in the mSTG-aSTG, whereas hearing tool sounds (e.g., hammer, saw) correlated with activation in the pSTG and IPL of the hemisphere contralateral to the dominant hand (Lewis et al., 2006). This recognition of tool sounds in the ADS instead of AVS is surprising because it could suggest that the teaching of tool use, which required gestures, was associated with speech production. Based on these findings I find the hypothesis that speech and gestures co-evolved compelling. However, given that my model delineates a course for the development of proto-conversations from calls that are used by extant primates, it is incongruent with the argument that a gestural language with complex grammar and semantics preceded vocal language.\n\nIn the present paper, I delineate a course for the early development of language by proposing five hypotheses: 1. In non-human primates, the ADS is responsible for perceiving and responding to contact calls; 2. The ADS of non-human primates integrates auditory locations into the visual dorsal stream; 3. Duplication of the ape’s IPS and its frontal projections (visual dorsal stream) resulted with a pathway (ADS) dedicated for converting auditory stimuli into articulations; 4. Mother-offspring vocal exchange was the predominant force that guided the emergence of speech in the ADS; 5. Speech emerged from modifying calls with intonations for signaling a low-level and high-level of distress, and these calls are the precursor to our use of intonations for converting words into questions and commands, respectively. Cumulative and converging evidence for the veracity of each of these hypotheses was provided throughout the paper. However, as the veracity of a model can only be measured by its ability to predict experimental results, I will present here outlines for 5 potential studies that can test each of these hypotheses.\n\nIn accordance with the first hypothesis, the ADS of non-human primates is responsible for the perception and vocal response to contact calls. A possible way of testing this hypothesis is by inducing bilateral lesions to the temporo-parietal junction of a monkey and then measuring whether the monkey no longer responds vocally to contact calls or responds less than before the lesion induction.\n\nIn accordance with the second hypothesis, audiospatial information is integrated in the IPS into visual regions and processed via the visual dorsal stream. This conclusion, although supported by many studies, is primarily derived from the study of Grunewald et al. (1999), who reported that, prior to training, neurons in the IPS responded only to visual stimuli and to auditory stimuli only after training. This study therefore needs to be replicated in more primate species to determine its veracity.\n\nIn accordance with the third hypothesis, the Homo genus emerged as a result of duplicating the IPS and its frontal projections. This duplication resulted with area Spt and its projections to the VLPFC. In contrast to the visual dorsal stream that processes audiovisual spatial properties, the human ADS processes inner and outer speech. I therefore predict that fMRI studies scanning participants while rehearsing auditory locations, visual locations and sentences, will find that the first two tasks activate a region more dorsal in the frontal lobe than the latter task.\n\nIn accordance with the fourth hypothesis, mother infant interaction was the guiding force that endowed the ADS with its role in speech. This hypothesis is primarily based on the finding that a sub-region of area Spt in human fetuses was shown selective to the voice of their mothers (Jardri et al., 2012). Future studies should further explore whether this region remains active in the brain of infants and toddlers and whether mothers also possess a region in the ADS that is selective to the voice of their children.\n\nIn accordance with the fifth hypothesis, the ADS originally served for discriminating calls that signal different levels of distress by analyzing their intonations. At present day, this development is reflected in our ability to modify intonations for converting spoken words into questions and commands. A way of testing this hypothesis is by using fMRI to compare the brain regions active when participants discriminate spoken words into questions and commands, with the brain regions active when they discriminate these words based on their emotional content (e.g., scared and happy). I predict that the former will activate the ADS whereas the latter the AVS.",
"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\nFirst, I would like to thank my advisor and mentor, Robert Rafal for his advice, comments and support when writing this paper. I would also like to thank Ben Crossey, Iva Ivanova, Cait Jenkins, Ruth Fishman and Catherine Le Pape for their help with reviewing this paper; and to the editors of American Journal Experts, Journal Prep and NPG language editing for their participation in the editing, proofreading and reviewing of this paper at its different stages.\n\n\nAppendix A: The auditory ventral stream and its role in sound recognition\n\nAccumulative converging evidence indicates that the AVS is involved in recognizing auditory objects. At the level of the primary auditory cortex, recordings from monkeys showed higher percentage of neurons selective for learned melodic sequences in area R than area A1 (Yin et al., 2008), and a study in humans demonstrated more selectivity for heard syllables in the anterior Heschl’s gyrus (area hR) than posterior Heshcl’s gyrus (area hA1; Steinschneider et al., 2004). In downstream associative auditory fields, studies from both monkeys and humans reported that the border between the anterior and posterior auditory fields (Figure 1-area PC in the monkey and mSTG in the human) processes pitch attributes that are necessary for the recognition of auditory objects (Bendor & Wang, 2006). The anterior auditory fields of monkeys were also demonstrated with selectivity for con-specific vocalizations with intra-cortical recordings (Perrodin et al., 2011; Rauschecker et al., 1995; Russ et al., 2007) and functional imaging (Joly et al., 2012; Petkov et al., 2008; Poremba et al., 2004). One fMRI monkey study further demonstrated a role of the aSTG in the recognition of individual voices (Petkov et al., 2008). The role of the human mSTG-aSTG in sound recognition was demonstrated via functional imaging studies that correlated activity in this region with isolation of auditory objects from background noise (Scheich et al., 1998; Zatorre et al., 2004) and with the recognition of spoken words (Binder et al., 2004; Davis & Johnsrude, 2003; Liebenthal, 2005; Narain, 2003; Obleser et al., 2006a; Obleser et al., 2007; Scott et al., 2000), voices (Belin & Zatorre, 2003), melodies (Benson et al., 2001; Leaver & Rauschecker, 2010), environmental sounds (Lewis et al., 2006; Maeder et al., 2001; Viceic et al., 2006), and non-speech communicative sounds (Shultz et al., 2012). A study that recorded neural activity directly from the left pSTG and aSTG reported that the aSTG, but not pSTG, was more active when the patient listened to speech in her native language than unfamiliar foreign language (Lachaux et al., 2007-patient 1). Consistently, electro stimulation to the aSTG, but not pSTG, resulted in impaired speech perception (Lachaux et al., 2007-patient 1; see also Matsumoto et al., 2011 for similar finding). Intra-cortical recordings from the right and left aSTG of another patient further demonstrated that speech is processed laterally to music (Lachaux et al., 2007-patient 2). Recordings from the anterior auditory cortex of monkeys while maintaining learned sounds in working memory (Tsunada et al., 2011), and the debilitating effect of induced lesions to this region on working memory recall (Fritz et al., 2005; Stepien et al., 1960; Strominger et al., 1980), further implicate the AVS in maintaining the perceived auditory objects in working memory. In humans, area mSTG-aSTG was also reported active during rehearsal of heard syllables with MEG (Kaiser et al., 2003) and fMRI (Buchsbaum et al., 2005). The latter study further demonstrated that working memory in the AVS is for the acoustic properties of spoken words and that it is independent to working memory in the ADS, which mediates inner speech.\n\nIn humans, downstream to the aSTG, the MTG and TP are thought to constitute the semantic lexicon, which is a long-term memory repository of audio-visual representations that are interconnected on the basis of semantic relationships. (See also the reviews by Hickok & Poeppel, 2007 and Gow, 2012, discussing this topic). The primary evidence for this role of the MTG-TP is that patients with damage to this region (e.g., patients with semantic dementia or herpes simplex virus encephalitis) are reported with an impaired ability to describe visual and auditory objects and a tendency to commit semantic errors when naming objects (i.e., semantic paraphasia; Noppeney et al., 2006; Patterson et al., 2007). Semantic paraphasias were also expressed by aphasic patients with left MTG-TP damage (Dronkers et al., 2004; Schwartz et al., 2009) and were shown to occur in non-aphasic patients after electro-stimulation to this region (Hamberger et al., 2007) or the underlying white matter pathway (Duffau et al., 2008). Two meta-analyses of the fMRI literature also reported that the anterior MTG and TP were consistently active during semantic analysis of speech and text (Binder et al., 2009; Vigneau et al., 2006); and an intra-cortical recording study correlated neural discharge in the MTG with the comprehension of intelligible sentences (Creutzfeldt et al., 1989).\n\nIn contradiction to the Wernicke-Lichtheim-Geschwind model that implicates sound recognition to occur solely in the left hemisphere, studies that examined the properties of the right or left hemisphere in isolation via unilateral hemispheric anesthesia (i.e., the WADA procedure; Hickok et al., 2008) or intra-cortical recordings from each hemisphere (Creutzfeldt et al., 1989) provided evidence that sound recognition is processed bilaterally. Moreover, a study that instructed patients with disconnected hemispheres (i.e., split-brain patients) to match spoken words to written words presented to the right or left hemifields, reported vocabulary in the right hemisphere that almost matches in size with the left hemisphere (Zaidel, 1976). (The right hemisphere vocabulary was equivalent to the vocabulary of a healthy 11-years old child). This bilateral recognition of sounds is also consistent with the finding that unilateral lesion to the auditory cortex rarely results in deficit to auditory comprehension (i.e., auditory agnosia), whereas a second lesion to the remaining hemisphere (which could occur years later) does (Poeppel, 2012; Ulrich, 1978).\n\n\nReferences\n\nAboitiz F, García VR: The evolutionary origin of the language areas in the human brain. A neuroanatomical perspective. Brain Res Brain Res Rev. 1997; 25(3): 381–396. 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}
|
[
{
"id": "8933",
"date": "17 Jul 2015",
"name": "Amy Poremba",
"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 contribution is a wide-ranging theory of how speech evolved in humans, which incorporates the dorsal and ventral auditory processing streams, but primarily focused on the auditory dorsal stream.\n\nThere are several large leaps in the proposed trajectory for language evolution such as, “eventually, individuals were capable of inventing new words and offspring were capable of inquiring about objects in their environment and learning their names via mimicry.” While the first part of the overall proposed theory is well supported, these latter stages are under-supported by current knowledge, particularly when moving to discussing individuals that became capable of enunciating novel calls (e.g., last paragraph of introduction); (some publications that may be helpful, comment by Meguerditchian et al., 2014; original article, Ackermann et al., 2014). The steps proposed for inventing new words and inquiring about objects are likely to require a large number of processes and the theory does not specify what those steps might be. Overall, Poliva’s theory as set forth does generate some interesting, testable, hypotheses as demonstrated in section 9, and the leaps in the logical flow do not negate these as the hypotheses are more closely related to the current knowledge base. As this is a theory of “From where to what,” missing for me was a better description of how the dorsal and ventral streams might interact in this theory. Calls still need to be “recognized” as auditory objects and imaging and recording studies have indicated the ventral stream does process this type of information. The ventral stream was given much less prominence and described in the appendix. It would be nice to include a paragraph or two on how the two systems may work together or how the ventral stream object identification comes to participate or interact with word formation and questions about objects. In the first paragraph of the introduction, curiosity toward the unknown may be related to non-human primates’ tendency to pick novel objects from known objects. This is also true in many lower animals. The development of curiosity of objects that are absent from our environment as Poliva suggests must also be related to memory development. One must be able to remember that objects exist and have detailed memories in order to determine if an object is indeed missing. There are aspects of work by Mishkin and colleagues suggesting that the lack of robust, or expansive, long-term auditory memory may relate to the absence of complex communication systems in non-human primates, such as rhesus macaques. Clearly, visual memory is much more extensive and robust than auditory memory and the sign language that other non-human primates have demonstrated may be related to the robust nature of visual memory. The issue of memory mechanisms necessary for identifying that auditory objects are indeed missing from the environment, and how these may differ and interact between auditory and visual systems, should at least be mentioned in passing.",
"responses": [
{
"c_id": "1750",
"date": "21 Jan 2016",
"name": "Oren Poliva",
"role": "Author Response",
"response": "This contribution is a wide-ranging theory of how speech evolved in humans, which incorporates the dorsal and ventral auditory processing streams, but primarily focused on the auditory dorsal stream. There are several large leaps in the proposed trajectory for language evolution such as, “eventually, individuals were capable of inventing new words and offspring were capable of inquiring about objects in their environment and learning their names via mimicry.” While the first part of the overall proposed theory is well supported, these latter stages are under-supported by current knowledge, particularly when moving to discussing individuals that became capable of enunciating novel calls (e.g., last paragraph of introduction); (some publications that may be helpful, comment by Meguerditchian et al., 2014; original article, Ackermann et al., 2014). The steps proposed for inventing new words and inquiring about objects are likely to require a large number of processes and the theory does not specify what those steps might be. Overall, Poliva’s theory as set forth does generate some interesting, testable, hypotheses as demonstrated in section 9, and the leaps in the logical flow do not negate these as the hypotheses are more closely related to the current knowledge base.Response: I agree with the reviewer that the final evolutionary stages show a leap and are not strongly substantiated with evidence. As mentioned in the paper, in depth discussion of these stages is presented in a sibling paper, which is currently in writing. Nonetheless, in the revised manuscript, I made more effort to describe possible transition to mimicry. Moreover, I removed discussing this issue from the abstract and introduction, as it is not the primary concern of the present paper. As this is a theory of “From where to what,” missing for me was a better description of how the dorsal and ventral streams might interact in this theory. Calls still need to be “recognized” as auditory objects and imaging and recording studies have indicated the ventral stream does process this type of information. The ventral stream was given much less prominence and described in the appendix. It would be nice to include a paragraph or two on how the two systems may work together or how the ventral stream object identification comes to participate or interact with word formation and questions about objects. Response: As mentioned above, in the revised manuscript I downplayed the role of the ADS in object naming and mimicry, and limited the discussion to speech. In depth discussion into the role of the AVS in these functions will be presented in the sibling paper. There was simply too many hypotheses and topics to cover, which made it impossible to include them all in a single paper. In the first paragraph of the introduction, curiosity toward the unknown may be related to non-human primates’ tendency to pick novel objects from known objects. This is also true in many lower animals. Response: The hypothesis that curiosity to novel objects prompted our curiosity to the unknown is an interesting alternative hypothesis. Humans, however, since the beginning of written history, were also documented with another curiosity: desire to explore unknown places. In the present paper, I present evidence that the primary drive for the emergence of speech was by lost infants and mothers seeking to reunite. This model seems to explain both the emergence of speech and our unique curiosity for the unknown and is thus parsimonious. Presenting an alternative explanation would entail evidence for a different evolutionary course, and is thus beyond the scope of the present paper. Saying that, I’ll be very interested to read about evidence for an evolutionary course that explains the curiosity to the unknown from this perspective. In the present model, I argue that the first question ever asked was “where are you”. It leaves me wondering that if the curiosity to the unknown was prompted by curiosity to novel objects, then what could have been the first question? The development of curiosity of objects that are absent from our environment as Poliva suggests must also be related to memory development. One must be able to remember that objects exist and have detailed memories in order to determine if an object is indeed missing. There are aspects of work by Mishkin and colleagues suggesting that the lack of robust, or expansive, long-term auditory memory may relate to the absence of complex communication systems in non-human primates, such as rhesus macaques. Clearly, visual memory is much more extensive and robust than auditory memory and the sign language that other non-human primates have demonstrated may be related to the robust nature of visual memory. The issue of memory mechanisms necessary for identifying that auditory objects are indeed missing from the environment, and how these may differ and interact between auditory and visual systems, should at least be mentioned in passing. Response: The hypothesis that expansion of auditory memory contributed to the development of language is very interesting and I do appreciate that the reviewer brought this research to my attention. However, in the present paper I only describe an evolutionary course up to the advent of the first conversation. Enhancement of auditory memory likely occurred in later stages of language development, and is thus beyond the scope of the present paper. As a final note, I want to thank the reviewer for her insightful comments and opinions, and hope that she enjoys the revised version of the paper."
}
]
},
{
"id": "7964",
"date": "24 Dec 2015",
"name": "Josef Rauschecker",
"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 contribution to the literature on language evolution. The first two sections ('Introduction' and 'Models of Language Processing in the Brain...') are a joy to read. Later sections are more controversial and contain serious flaws that have to be brought up to speed with the current literature. These concerns are summarized here: 1) The terminology is quite fuzzy. For instance, when the author refers to 'perception' he seems to mean 'detection' or 'processing'. In most people's minds, and in most extant models of perception and action, perception is specifically tied to the ventral stream. Therefore, it can, almost by definition, not also be a property of the dorsal stream. This is best exemplified in the Abstract: The author states: 'I propose that the primary role of the auditory dorsal stream (ADS) in monkeys/apes is the perception and response to contact calls.' This misstatement can be fixed by replacing 'perception' with 'detection'. Similarly, in a later sentence ('Perception of contact calls occurs by the ADS detecting a voice...'), 'Perception' can be substituted by 'Processing'. Thirdly, in the Abstract's second paragraph, the following sentence does not make any sense: 'Because the human ADS processes also speech production and repetition...'. Here, 'processes' needs to be replaced with 'performs'. 2) In the third section, the author first makes a strong case for a role of the ADS in auditory spatial processing, for encoding of sound location in memory and for use of this information in guiding eye movements. The published literature is well represented, though a key reference is missing here (Tian et al., Science, 2001). Then, in a surprising turnaround, the author suddenly concludes that 'audiospatial input is first converted into a visuospatial code and then processed via a visuospatial network'. The evidence cited stems from 15-year old studies of monkey area LIP, which is part of a visuospatial network; auditory signals, however, are relayed to a different part of IPS (area VIP; Lewis & VanEssen, 2000), for which corresponding studies have not been performed. Figure 2, which pertains to this section, reflects this misinterpretation: While the version on the left is neuroanatomically acceptable (with the only difference that parietal cortex is not just a visuospatial but a multisensory or amodal network, the versions in the center and on the right are incorrect on multiple grounds, most notably by postulating the 'duplication of the IPS [pivoting around an imaginary blue asterisk] and subsequent duplication of its frontal projections'. The assumptions about anatomical connections of the IPL with ventrolateral prefrontal cortex (VLPFC) are largely unsubstantiated and the characterization of VLPFC as a motor representation is plain wrong. I assume what the author may be referring to is ventral premotor cortex (PMv), which is indeed the terminal point of the auditory dorsal stream and is closely interfacing with Broca’s area. 3) According to a third hypothesis put forward by the author, \"the Homo genus emerged as a result of duplicating the IPS and its frontal projections. This duplication resulted with area Spt and its projections to the VLPFC. In contrast to the visual dorsal stream that processes audiovisual spatial properties, the human ADS processes inner and outer speech.\" This hypothesis is seriously flawed, because both ADS and VDS process spatial properties and both process sensorimotor signals. In fact, they may be one and the same structure. Thus, there is no fundamental difference between visual and auditory processing that would require duplication of IPS or its projections or special evolution of speech (see Bornkessel et al., 2015). 4) Additional citations that the author should add: DeWitt and Rauschecker, 2012 1DeWitt and Rauschecker, 2013 2 Bornkessel-Schlesewsky, et al., 2015 3 Mesulam, et al., 2015 4 Roux, et al., 2015 5",
"responses": [
{
"c_id": "1749",
"date": "21 Jan 2016",
"name": "Oren Poliva",
"role": "Author Response",
"response": "This is an interesting contribution to the literature on language evolution. The first two sections ('Introduction' and 'Models of Language Processing in the Brain...') are a joy to read. Later sections are more controversial and contain serious flaws that have to be brought up to speed with the current literature. These concerns are summarized here: 1) The terminology is quite fuzzy. For instance, when the author refers to 'perception' he seems to mean 'detection' or 'processing'. In most people's minds, and in most extant models of perception and action, perception is specifically tied to the ventral stream. Therefore, it can, almost by definition, not also be a property of the dorsal stream. This is best exemplified in the Abstract: The author states: 'I propose that the primary role of the auditory dorsal stream (ADS) in monkeys/apes is the perception and response to contact calls.' This misstatement can be fixed by replacing 'perception' with 'detection'. Similarly, in a later sentence ('Perception of contact calls occurs by the ADS detecting a voice...'), 'Perception' can be substituted by 'Processing'. Thirdly, in the Abstract's second paragraph, the following sentence does not make any sense: 'Because the human ADS processes also speech production and repetition...'. Here, 'processes' needs to be replaced with 'performs'. Response: As far as I understand it, perception refers to all elements of the external world that reach our awareness. In accordance with this definition, through the AVS we perceive the identity of sounds and through the ADS we perceive the location of sounds. As human speech production is also processed in the ADS, I would expect that we also perceive elements of speech preparation through the ADS. A good example is a study that reported of patients who were electrically stimulated in the left inferior parietal lobule and consequently believed they produced sounds, when in fact they didn’t (Desmurget et al., 2009). This study can be argued to demonstrates perception of speech preparation in the ADS. Nonetheless, considering that different researchers might have different definitions for perception, I replaced instances that describe perception with detection wherever it was applicable. Desmurget M, Reilly KT, Richard N, Szathmari A, Mottolese C, Sirigu A. Movement intention after parietal cortex stimulation in humans. Science. 2009 May 8;324(5928):811–3. 2) In the third section, the author first makes a strong case for a role of the ADS in auditory spatial processing, for encoding of sound location in memory and for use of this information in guiding eye movements. The published literature is well represented, though a key reference is missing here (Tian et al., Science, 2001). Then, in a surprising turnaround, the author suddenly concludes that 'audiospatial input is first converted into a visuospatial code and then processed via a visuospatial network'. The evidence cited stems from 15-year old studies of monkey area LIP, which is part of a visuospatial network; auditory signals, however, are relayed to a different part of IPS (area VIP; Lewis & VanEssen, 2000), for which corresponding studies have not been performed. Figure 2, which pertains to this section, reflects this misinterpretation: While the version on the left is neuroanatomically acceptable (with the only difference that parietal cortex is not just a visuospatial but a multisensory or amodal network, the versions in the center and on the right are incorrect on multiple grounds, most notably by postulating the 'duplication of the IPS [pivoting around an imaginary blue asterisk] and subsequent duplication of its frontal projections'. …. According to a third hypothesis put forward by the author, \"the Homo genus emerged as a result of duplicating the IPS and its frontal projections. This duplication resulted with area Spt and its projections to the VLPFC. In contrast to the visual dorsal stream that processes audiovisual spatial properties, the human ADS processes inner and outer speech.\" This hypothesis is seriously flawed, because both ADS and VDS process spatial properties and both process sensorimotor signals. In fact, they may be one and the same structure. Thus, there is no fundamental difference between visual and auditory processing that would require duplication of IPS or its projections or special evolution of speech (see Bornkessel et al., 2015). Response: Although I don’t entirely agree with the reviewer’s perspective in this regard, given that the paper is already rich in evidence and hypotheses, I removed the sections (last paragraph of section 3 and section 7) discussing these hypotheses in the revised version. Also, I removed figure 2 from the revised version, and accordingly modified the manuscript to accommodate this change. 3) …I assume what the author may be referring to is ventral premotor cortex (PMv), which is indeed the terminal point of the auditory dorsal stream and is closely interfacing with Broca’s area. Response: Thinking back, I agree with the reviewer that referring to this region as the ‘ventral premotor cortex’ is more accurate. The reason I referred to this region as the ventrolateral prefrontal cortex is to be consistent with previous papers (e.g., Romansky et al., 1999). As it possible that the area most often referred to as Broca’s area encompasses both parts of the ventrolateral prefrontal cortex and ventral premotor cortex, in the revised manuscript I replaced the term ‘ventrolateral prefrontal cortex’ with its anatomical equivalent, the ‘inferior frontal gyrus’. Romanski LM, Bates JF, Goldman-Rakic PS. Auditory belt and parabelt projections to the prefrontal cortex in the rhesus monkey. J Comp Neurol. 1999 Jan 11;403(2):141–57. As a final note, I want to thank the reviewer for his time and effort, and hope he finds the revised version even more enjoyable to read."
}
]
},
{
"id": "11960",
"date": "18 Jan 2016",
"name": "Michael A Arbib",
"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 see this paper as a first draft of what could become an important contribution to neurally based approaches to the study of the evolution of the human brain’s capacity for language. Its importance is three-fold:It treats the ventral and dorsal streams for both the auditory and visual modalities.It regards monkey calls not in terms of perception alone or production alone but rather in terms of their role in the interaction between two individuals in the context of their environment.It places the ability to ask and answer questions at the heart of language use.Below, I will offer several comments on how some shortcomings of the current version might be removed in future work by Poliva, but first a disclosure: I have emphasized the role of the two visual streams in relation to both the production and comprehension of language with an emphasis on the role of manual gesture and protosign in language evolution, and in terms of visual perception of what an utterance may be about (Arbib, 2013). By contrast, Bornkessel-Schlesewsky and Schlesewsky (2013) offer hypotheses on the roles of the auditory streams in the perception of sentences of a spoken language, linking them to neurolinguistic data from their lab and others. I have attempted a preliminary synthesis of these approaches (Arbib, 2015). More recently, they have co-authored a review of relevant data on the auditory streams in both monkey and human with the claim that no major evolutionary innovations were required in these streams to make language possible (Bornkessel-Schlesewsky, Schlesewsky, Small, & Rauschecker, 2015) – a claim with which I (and, I suspect, Poliva) would disagree. I hope to support the counter-claim in a forthcoming article in the Journal of Neurolinguistics. I believe Poliva’s assessment of these articles would enrich his work, but now let me turn to other issues.I endorse the key points of Amy Poremba’s review: (i) The dorsal auditory stream was over-emphasized at the expense of assessing the role of the ventral stream and how these streams are integrated. (ii) Poremba notes the relevance of work from Mishkin’s lab on auditory memory – see, e.g., Fritz, Mishkin, and Saunders (2005) which “raises the possibility that language is unique to humans not only because it depends on speech but also because it requires long-term auditory memory.” I would add that Aboitiz and his colleagues have emphasized the expansion of working memory capacity as a key element in evolving a language-ready brain (see Aboitiz, 2012, for a recent review of this approach). (iii) The leap from contact calls to “individuals … capable of inventing new words and offspring … capable of inquiring about objects in their environment and learning their names via mimicry” is essentially unbridged. Since there are many monkey calls, it seems unclear why, if one is to use these calls as the core for evolving a brain with language, one should focus on contact calls alone. Including other calls might add more “evolutionary opportunities.” In this regard, note the argument of Seyfarth and Cheney that one may see the structure of language prefigured in the “rules” monkeys develop for social cognition (Cheney & Seyfarth, 2005; Seyfarth & Cheney, 2014). I suspect that further work in language evolution will reveal a “mosaic” of innovations, some of which are apparent in different monkey or ape species. One may hope that studies of the brains of different species will reveal diverse cues that illuminate, perhaps, the convergent evolution of different tiles of the language-supporting neural mosaic of the human brain. Consider, for example, the capability for turn taking in geladas (Gustison, le Roux, & Bergman, 2012; Richman, 1987) and marmosets (Miller, Thomas, Nummela, & de la Mothe, 2015; Takahashi, Narayanan, & Ghazanfar, 2013) as just one of the diverse components of language-ready brain that are differentially evident in different species of nonhuman primate. Figure 1 shows dual stream connectivity between the auditory cortex and frontal lobe of monkeys and humans. What can be said about the intersection of the 2 streams in VLPFC? And what can be said about the interaction of DLPFC and VLPFC? Figure 2 depicts the “From Where to What model” via three stages of neuroanatomical modifications. It might be useful to first provide a diagram focusing on VVS and VDS (initial V for Visual) and discussing the relation in both anatomy and function of these paths with each other. It might also be helpful to present pieces of the model along with the exposition of the related data, postponing this integrative figure until the pieces are in place. A valuable feature of Poliva’s model is its suggestion of how the response to an auditory call might initiate visual search as the basis for action (he emphasizes the mother emitting a call if the child is not seen; a related scenario would be movement toward the child if it were seen). This issue of integration of communication and action, which may (but need not) integrate audition with vision, is an important feature which too few studies take into account. My question is whether he unduly emphasizes cortical pathways involving the frontal eye fields and shortchanges subcortical interactions involving the superior colliculus (noting of course that these are open to cortical influences modulated by the basal ganglia). In Figure 2, Poliva asserts: (i) “Approximately 2.5 million years ago, the Homo genus emerged as a result of [my italics] duplication of the IPS and subsequent duplication of its frontal projections” (a) Surely, many more changes led to the emergence of Homo. (b) At the end of Section 7, Poliva suggests the relevance of endocast data to this claim. Are there relevant data on apes that could help us assess this transition? (ii) “Since the auditory cortex targeted the more proximal of these duplicated parietal regions, a new pathway dedicated for auditory processing emerged (i.e., auditory dorsal stream; ADS.” But monkey data show an ADS, so what is the transition being suggested here? Picking up on the issue in (5), one needs to better understand the division of labor between ADS and subcortical mechanisms (as well as AVS, to reiterate Poremba’s point). Poliva claims to review “evidence for a role of the ADS in the transition from mediating contact calls into mediating human speech” but simply cites data correlating ADS impairment with disorders like speech apraxia. Nothing in the data privileges contact calls over other vocal productions – and, anyway, clear articulation is a far cry [sic] from mechanisms supporting the role of syntax and semantics in language production and perception. In relation to 6(i), Poliva notes the dual role of the parietal lobe in sensory-motor transformation of both audio-spatial and verbal information, and proposes that during Hominin evolution there was a cortical field duplication, of the IPS with further duplication of its projections to the VLPFC which resulted in a pathway dedicated for audio-vocal conversion. How would this serve people who employ a signed language? (Of course, those who advocate a gestural origin of language must face the complementary question of how visuo-manual pathways came to support audio-vocal signals – which they must do because other primates lack vocal learning, let alone the use of syntax and semantics in either domain.) Poliva stresses that the ability to ask and answer questions is an essential feature of language use. I agree. Future work on language evolution should pay more attention to the challenge of explaining how this evolved. However the focus on modifying contact calls with prosodic intonations seems to me too narrow (I may be wrong, but more argument would be needed) and (as Poremba observed) the account of the transition remains too sketchy. Poliva cites “the ability of present-day infants of using intonations for changing the pragmatic utilization of a word from a statement to a command/demand (“mommy!”) or a question (“mommy?”),” but one must be careful to distinguish these infant “communicative acts” from the ability to deploy grammar to formulate an open-ended repertoire of commands and questions using the structures of a language – let along being able to marshal answers to questions of even modest complexity. In any case, it seems mistaken to place exclusive emphasis on the role of ADS in the transition – one might thus assess the hypotheses of Bornkessel-Schlesewsky and Schlesewsky (2013) on the roles of both ADS and AVS (and frontal areas) in speech comprehension. However, a companion paper is promised: “Discussing the transition from exchanging low-level distress contact calls into complex vocal language, however, is beyond the scope of the present paper and a model for such transition is discussed [at] length in a sibling paper titled ‘Vocal Mimicry as the Sculptor of the Human Mind. A Neuroanatomically based Evolutionary Model of The Emergence of Vocal Language’ (Poliva, in preparation).” Perhaps it would be better if less were said about this topic in the present paper so that the implications of the evidence on ADS function and evolution could be better assessed for their merits irrespective of the contact call hypothesis.",
"responses": [
{
"c_id": "1777",
"date": "21 Jan 2016",
"name": "Oren Poliva",
"role": "Reader Comment",
"response": "I want to thank the reviewer for his positive review and for his insightful and constructive comments. Below are my responses:I endorse the key points of Amy Poremba’s review: (i) The dorsal auditory stream was over-emphasized at the expense of assessing the role of the ventral stream and how these streams are integrated.Response: I agree with the reviewer that the article focuses on the ADS, and pay little attention to the AVS. I also agree that the AVS partakes an important role in the perception and production of human language, and that it interacts with the ADS. However, as I also previously responded to Poremba, in the present paper I propose a model for the emergence of speech and not language, and speech appears to be primarily or solely a function of the ADS. A possible course for the transition from speech to language and the role the AVS in such functions is discussed in detail in the second paper (mentioned in the article).Poremba notes the relevance of work from Mishkin’s lab on auditory memory – see, e.g., Fritz, Mishkin, and Saunders (2005) which “raises the possibility that language is unique to humans not only because it depends on speech but also because it requires long-term auditory memory.” I would add that Aboitiz and his colleagues have emphasized the expansion of working memory capacity as a key element in evolving a language-ready brain (see Aboitiz, 2012, for a recent review of this approach).Response: I agree with the reviewer that expansion of auditory memory (or its ability to sustain interferences as shown by Scott, Mishkin & Yin, 2012) took an important part in the evolution of language. However, as I previously responded to Poremba, this change likely occurred after Hominins acquired volitional control over the vocal apparatus, and thus is beyond the scope of the present paper. This issue is also discussed in detail in the second paper.Scott BH, Mishkin M, Yin P. Monkeys have a limited form of short-term memory in audition. Proceedings of the National Academy of Sciences. 2012 Jul 24;109(30):12237–41. The leap from contact calls to “individuals … capable of inventing new words and offspring … capable of inquiring about objects in their environment and learning their names via mimicry” is essentially unbridged…..In any case, it seems mistaken to place exclusive emphasis on the role of ADS in the transition – one might thus assess the hypotheses of Bornkessel-Schlesewsky and Schlesewsky (2013) on the roles of both ADS and AVS (and frontal areas) in speech comprehension. However, a companion paper is promised: “Discussing the transition from exchanging low-level distress contact calls into complex vocal language, however, is beyond the scope of the present paper and a model for such transition is discussed [at] length in a sibling paper titled ‘Vocal Mimicry as the Sculptor of the Human Mind. A Neuroanatomically based Evolutionary Model of The Emergence of Vocal Language’ (Poliva, in preparation).” Perhaps it would be better if less were said about this topic in the present paper so that the implications of the evidence on ADS function and evolution could be better assessed for their merits irrespective of the contact call hypothesis.Response: I agree with the reviewer that the article doesn’t delve enough into the transition from speech to vocal mimicry. As I responded to Poremba, and mentioned in the paper, this topic is discussed in detail in the second paper. As the primary concern of the present paper is the emergence of speech, I removed from the abstract and introduction any mentioning of the transition from speech to vocal mimicry based language, and limited its discussion to a short paragraph near the end of the paper. Since there are many monkey calls, it seems unclear why, if one is to use these calls as the core for evolving a brain with language, one should focus on contact calls alone. Including other calls might add more “evolutionary opportunities.” In this regard, note the argument of Seyfarth and Cheney that one may see the structure of language prefigured in the “rules” monkeys develop for social cognition (Cheney & Seyfarth, 2005; Seyfarth & Cheney, 2014).Response: The reviewer presents an interesting question when he suggests that contact calls might not be special. The paper he cites suggests that rule based alarm calls could serve as a potential precursor to human language. In my opinion, contact calls are a more likely candidate precursor to present day vocal conversation than alarm calls. Like present day vocal conversations, contact call are characterized with turn taking and require interaction between (at least) two participants. The content of contact calls is also similar to present day question answer dialogue (as if similar to the question ‘where are you?’ and the answer ’I’m here, Where are you?’). Alarm calls in contrast, although context dependent and thus likely under cortical influence, do not require vocal response and thus don’t resemble conversation. Moreover, as I present in the paper, converging evidence suggests that both human speech and contact call exchange in non-human primates are processed in the ADS. As far as I’m aware of, no study provided evidence that alarm calls are processed in the ADS. (Given its dependence on observing emotive stimuli, I would assume that expressing alarm calls occurs through processing in the visual ventral stream and amygdala, and response to alarm calls occurs through the auditory ventral stream and amygdala.)I suspect that further work in language evolution will reveal a “mosaic” of innovations, some of which are apparent in different monkey or ape species. One may hope that studies of the brains of different species will reveal diverse cues that illuminate, perhaps, the convergent evolution of different tiles of the language-supporting neural mosaic of the human brain. Consider, for example, the capability for turn taking in geladas (Gustison, le Roux, & Bergman, 2012; Richman, 1987) and marmosets (Miller, Thomas, Nummela, & de la Mothe, 2015; Takahashi, Narayanan, & Ghazanfar, 2013) as just one of the diverse components of language-ready brain that are differentially evident in different species of nonhuman primate.Response: I admit I got confused from the reviewer’s comment. The reviewer argues that turn taking occurs in gelada monkeys. The studies he cite however don’t mention such behavior. The reviewer then proceed to cite turn taking vocal behavior in marmoset monkeys, as an alternative explanation to how humans developed turn taking in conversations. The reviewer, however, cite studies that explore turn taking in the exchange of contact calls, which further support the discussed model.Figure 1 shows dual stream connectivity between the auditory cortex and frontal lobe of monkeys and humans. What can be said about the intersection of the 2 streams in VLPFC? And what can be said about the interaction of DLPFC and VLPFC? Response: In the paper I describe two pathways connecting the auditory cortex with the prefrontal cortex. The prefrontal cortex is primarily ascribed with planning and problem solving. When detecting and responding to contact calls, the prefrontal cortex likely mediates high level processing, such as determining the best way to overcome an obstacle in order to reach the caller. In the present model, I attempt to demonstrate that the detection and production of contact calls occur in the same pathway as speech in humans, and on that account attribute a relationship between them. The role of the prefrontal cortex in such high level processing is not necessary for establishing this relationship and is thus beyond the scope of the present paper.Figure 2 depicts the “From Where to What model” via three stages of neuroanatomical modifications. It might be useful to first provide a diagram focusing on VVS and VDS (initial V for Visual) and discussing the relation in both anatomy and function of these paths with each other. It might also be helpful to present pieces of the model along with the exposition of the related data, postponing this integrative figure until the pieces are in place…..In Figure 2, Poliva asserts: (i) “Approximately 2.5 million years ago, the Homo genus emerged as a result of [my italics] duplication of the IPS and subsequent duplication of its frontal projections” (a) Surely, many more changes led to the emergence of Homo. (b) At the end of Section 7, Poliva suggests the relevance of endocast data to this claim. Are there relevant data on apes that could help us assess this transition? (ii) “Since the auditory cortex targeted the more proximal of these duplicated parietal regions, a new pathway dedicated for auditory processing emerged (i.e., auditory dorsal stream; ADS.” But monkey data show an ADS, so what is the transition being suggested here? Picking up on the issue in (5), one needs to better understand the division of labor between ADS and subcortical mechanisms (as well as AVS, to reiterate Poremba’s point)….In relation to 6(i), Poliva notes the dual role of the parietal lobe in sensory-motor transformation of both audio-spatial and verbal information, and proposes that during Hominin evolution there was a cortical field duplication, of the IPS with further duplication of its projections to the VLPFC which resulted in a pathway dedicated for audio-vocal conversion. How would this serve people who employ a signed language? (Of course, those who advocate a gestural origin of language must face the complementary question of how visuo-manual pathways came to support audio-vocal signals – which they must do because other primates lack vocal learning, let alone the use of syntax and semantics in either domain.)Response: I agree with the reviewer that in depth description of the visual streams and addition of evidence for the parietal duplication hypothesis could add more depth to the paper. However, reviewer 2 (Josef Rauschecker) argued that the section of the paper discussing the relationship between the auditory and visual streams is problematic, and overall disagreed with the parietal duplication hypothesis. Although I don’t entirely agree with his perspective, given that the paper is already rich in hypotheses and evidence, I chose to remove the sections discussing this topic from the paper. Possibly, the parietal duplication hypothesis will be presented in the future in its own paper.A valuable feature of Poliva’s model is its suggestion of how the response to an auditory call might initiate visual search as the basis for action (he emphasizes the mother emitting a call if the child is not seen; a related scenario would be movement toward the child if it were seen). This issue of integration of communication and action, which may (but need not) integrate audition with vision, is an important feature which too few studies take into account. My question is whether he unduly emphasizes cortical pathways involving the frontal eye fields and shortchanges subcortical interactions involving the superior colliculus (noting of course that these are open to cortical influences modulated by the basal ganglia)Response: I agree with the reviewer that area LIP in the intraparietal sulcus likely guides eye movements via projections to the frontal eye field and the superior colliculi. Such connections from the area LIP to the superior colliculi were described in tracing studies (Lynch et al., 1985). However, to the best of my knowledge no study so far demonstrated that this parieto-collicular pathway carries auditory information. It would also be very difficult to demonstrate that auditory influence on the superior colliculus occurs via connections from area LIP and not via ascending connections from the inferior colliculi. Given the lack of evidence of an auditory parieto-collicular pathway I chose at this point not to include it in the revised paper.Lynch, J. C., AMs Graybiel, and L. J. Lobeck. \"The differential projection of two cytoarchitectonic subregions of the inferior parietal lobule of macaque upon the deep layers of the superior colliculus.\" Journal of Comparative Neurology 235.2 (1985): 241-254.Poliva claims to review “evidence for a role of the ADS in the transition from mediating contact calls into mediating human speech” but simply cites data correlating ADS impairment with disorders like speech apraxia. Response: In addition to the paragraph discussing the role of the ADS in speech production, I present throughout the paper many other studies that indirectly show a role of the human ADS in speech production, such as fMRI studies that compare speech production to the production of melodies (Hickok et al., 2003) and many studies that ascribe the ADS with a role in speech repetition (Hickok et al., 2007). Nothing in the data privileges contact calls over other vocal productionsResponse: Many studies have shown that the ADS (associated in human with speech production) has a special role in the detection and production of contact calls. For example:“Further corroborating the involvement of the ADS in the perception of contact calls are intra-cortical recordings from the posterior insula (near area CM-A1) of the macaque, which revealed stronger selectivity for a contact call (coo call) than a social call (threat call; Remedios et al., 2009a). Contrasting this finding is a study that recorded neural activity from the anterior auditory cortex, and reported that the proportion of neurons dedicated to a contact call was similar to the proportions of neurons dedicated to other calls (Perrodin et al., 2011).”Also:“Consistently, a study that sacrificed marmoset monkeys immediately after responding to contact calls (phee calls) measured highest neural activity (genomic expression of cFos protein) in the posterior auditory fields (CM-CL), and VLPFC (Miller et al., 2010). Monkeys sacrificed after only hearing contact calls or only emitting them showed neural activity in the same regions but to a much smaller degree (See also Simões et al., 2010 for similar results in a study using the protein Egr-1).” – and, anyway, clear articulation is a far cry [sic] from mechanisms supporting the role of syntax and semantics in language production and perception.Response: I agree with the reviewer that arguing that the ADS processes speech does not necessitate that the ADS process more complex linguistic functions such as semantics and syntax. This is why in the paper I only present a model for the emergence of speech. More complex linguistic functions and possible evolutionary course will be discussed in the second paper. Poliva stresses that the ability to ask and answer questions is an essential feature of language use. I agree. Future work on language evolution should pay more attention to the challenge of explaining how this evolved. However the focus on modifying contact calls with prosodic intonations seems to me too narrow (I may be wrong, but more argument would be needed) and (as Poremba observed) the account of the transition remains too sketchy. Poliva cites “the ability of present-day infants of using intonations for changing the pragmatic utilization of a word from a statement to a command/demand (“mommy!”) or a question (“mommy?”),” but one must be careful to distinguish these infant “communicative acts” from the ability to deploy grammar to formulate an open-ended repertoire of commands and questions using the structures of a language – let along being able to marshal answers to questions of even modest complexity.Response: I agree with the reviewer that adults often use complex syntax to ask questions. However, given that children (and occasionally adults) can express a question with a single word using intonations, suggests, in my opinion, that such question asking method could have preceded syntax, and thus indicate of an intermediate stage in the evolution of language. A transition from a single word question to syntax likely occurred at later evolutionary stages, and is thus beyond the scope of the present paper."
}
]
}
] | 1
|
https://f1000research.com/articles/4-67
|
https://f1000research.com/articles/6-1710/v1
|
20 Sep 17
|
{
"type": "Correspondence",
"title": "On the interpretability and computational reliability of frequency-domain Granger causality",
"authors": [
"Luca Faes",
"Sebastiano Stramaglia",
"Daniele Marinazzo",
"Sebastiano Stramaglia",
"Daniele Marinazzo"
],
"abstract": "This Correspondence article is a comment which directly relates to the paper “A study of problems encountered in Granger causality analysis from a neuroscience perspective” (Stokes and Purdon, 2017). We agree that interpretation issues of Granger causality (GC) in neuroscience exist, partially due to the historically unfortunate use of the name “causality”, as described in previous literature. On the other hand, we think that Stokes and Purdon use a formulation of GC which is outdated (albeit still used) and do not fully account for the potential of the different frequency-domain versions of GC; in doing so, their paper dismisses GC measures based on a suboptimal use of them. Furthermore, since data from simulated systems are used, the pitfalls that are found with the used formulation are intended to be general, and not limited to neuroscience. It would be a pity if this paper, even if written in good faith, became a wildcard against all possible applications of GC, regardless of the large body of work recently published which aims to address faults in methodology and interpretation. In order to provide a balanced view, we replicate the simulations of Stokes and Purdon, using an updated GC implementation and exploiting the combination of spectral and causal information, showing that in this way the pitfalls are mitigated or directly solved.",
"keywords": [
"Granger-Geweke causality",
"frequency-domain connectivity",
"time series analysis",
"directed coherence",
"vector autoregressive models",
"spectral decomposition",
"brain connectivity",
"physiological oscillations"
],
"content": "Correspondence\n\nGranger causality (GC)1 is an extremely popular statistical tool used to analyze directed interactions from multivariate time series measured from coupled dynamical systems. A particularly appealing aspect of the notion of GC is that it can be formulated in the frequency domain, and is thus eligible for the analysis of signals that are rich of oscillatory content such as those commonly encountered in neuroscience and physiology2,3. The spectral formulation of GC is obtained by elaborating in the frequency domain the parameters of the linear vector autoregressive (VAR) model that fit the observed multivariate time series. A main approach to do this was developed by Geweke4,5, yielding bivariate and conditional frequency domain measures of the so-called Granger-Geweke causality (GGC). An alternative framework stems from the works of Kaminski et al6. and Baccalà et al7,8., who derived measures like the directed coherence (DC) and the partial DC (PDC), quantifying the total and direct directed influence of one time series over another in a fully multivariate setting (see references 9–11 for comprehensive treatments).\n\nIn their recent work12, Stokes and Purdon performed a critical evaluation of frequency-domain GC computed within the Geweke framework, evidencing in two simulation studies some computational and interpretational problems associated with the GGC measures. Specifically, they showed that – even when the systems generating the observed data belong to the finite-order VAR model class – spectral GGC cannot be reliably estimated and cannot recover the functional oscillatory structure underlying the data. These observations led the authors to conclude that the notion of causality quantified by GGC, and by other Granger causality measures in general, often yield counterintuitive and misleading results, thus being incompatible with the objectives of many neuroscience studies.\n\nWe definitely agree that GC and lag-based data-driven methods in general cannot provide measures of “causality” as intended in other applications (see references 9 and 13 for a thoughtful distinction between data-driven and model-based approaches). We also share the view that the assumptions of linearity and stationarity, as well as the presence of unobserved variables, noise or inappropriate sampling may pose theoretical and practical problems which can severely impair both the formulation and the computation of spectral GC measures – this has been stated by Stokes and Purdon12 and in previous studies2,3,14. On the other hand we think that, based on the way simulated data have been analyzed and interpreted by Stokes and Purdon12, frequency domain GC methods have been dismissed based on a suboptimal (even though frequently applied) formulation of GGC, and based on the lack of direct consideration of the DC/PDC framework.\n\nIn this contribution, we repeat the simulations of Stokes and Purdon12, and suggest that the negative conclusions based on the results of such simulations are overstated. We show that spectral GGC estimates can be obtained with a high computational reliability if proper estimation approaches are employed, and the interpretation of frequency domain causality measures can be meaningfully performed if spectral and causal information are properly combined. The codes for running our analyses are based on existing Matlab© toolboxes11,15,16 and are provided as supplementary data alongside this article.\n\nThe first simulation of Stokes and Purdon12 shows that, due to the modeling choices required to compute spectral GGC, this measure cannot be reliably estimated even for simple systems. By generating 100 realizations of this simulation with the same parameters and data length we confirm that, by applying the standard method of fitting separate full and reduced VAR models, spectral GGC estimates display a strong bias (Figure 1A) or a very large variability (Figure 1B), depending on the choice of the model order. As explained by Stokes and Purdon12, this tradeoff between bias and variance arises from the incorrect representation of the reduced model as a VAR process of finite order. Exactly for this reason however, the problem can be overcome employing the state-space (SS) approach16, which allows to compute GGC in closed form from the SS parameters of any observed VAR process. Here we show that this approach yields highly accurate spectral estimates of GGC, which closely follow the expected profiles over the coupled directions and have negligible magnitude over the uncoupled direction (Figure 1C); the higher reliability of the SS estimator compared with the standard VAR-based method is evident also looking at single process realizations (Figure 1D).\n\nGGC is computed along the two coupled directions (f1→2, f2→3) and along a direction with no coupling (f3→1). Columns report the distribution of GGC estimates (median and 5th–95th percentiles) computed using classical vector autoregressive (VAR) estimation of full and reduced models performed with the true model order p=3 (a) and with an increased order p=20 (b), and using state space (SS) estimation (c), as well as estimates obtained for a single simulation run (d); in each plot, the true causality values computed from the original model parameters are reported in red. Results evidence the lower bias and variability of spectral GGC computed using the SS method compared to the classical VAR approach.\n\nThe second simulation of Stokes and Purdon12 shows that, due to the independence of GGC measures from the intrinsic dynamics of the “receiver” process, the spectral GGC profiles linking this process to its putatively causal “transmitter” process are often misleading, because different systems can have identical causality functions but different receiver dynamics. In Figure 2 we confirm this result both in terms of GGC and using the DC, a spectral causality measure taken from the VAR framework7 that for bivariate processes like the one simulated here is analytically related to the spectral GGC15. However, this invariance property is in our view absolutely reasonable, because the DC has a clear-cut interpretation as the relative amount of spectral power that, at each frequency, arrives to the receiver starting from the transmitter11. Nevertheless, the DC is also useful to fully recover the functional oscillatory structure of the observed processes, because it shapes the receiver spectrum to reveal the portion of its spectral power that is “causally” due to the transmitter; this is depicted in the spectral decomposition of Figure 2.\n\nThe system is studied setting a resonance frequency of 50 Hz for the transmitter and of 10 Hz (top row panels), 30 Hz (mid row panels) and 50 Hz (bottom row panels) for the receiver. The fact that the power spectral density (PSD) of the transmitter (S11(f), a) is the same for the three cases induces, together with the unaltered coefficients determining the causal effects, the same profile for the directed coherence DC1→2(f) (c) and the spectral GGC measure GG1→2(f) (d). However, the different causal contribution of the transmitter on the receiver is revealed by the partial spectrum S2|1(f)=S22(f)·DC1→2(f) (orange line in (b)), which quantifies the portion of the overall PSD of the receiver (S22(f), purple line in (b)) that is causally explained by the transmitter; the non-explained part (S2|2(f), green line in (b)) reflects the autonomous dynamics of the receiver.\n\nIn conclusion, while thanking Stokes and Purdon12 for pointing out some weaknesses of GGC measures, we think that proper formulations can provide meaningful results of directed dynamical influence, whose interpretation still is bound to the knowledge and good faith of those who write and read related scientific literature.\n\n\nData availability\n\nDataset 1: Codes to compute frequency-domain Granger causality in linear stochastic processes. The package contains Matlab scripts and functions that allow to reproduce the simulations of Stokes and Purdon, and to compare for these simulations: (i) the standard vector autoregressive estimator and the updated state space estimator of the spectral Granger-Geweke causality measure; (ii) the spectral Granger-Geweke causality measure with the directed coherence measure.\n\nDOI, 10.5256/f1000research.12694.d17815917.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe work was funded by the Department of Industrial Engineering, University of Trento, Italy (40300314).\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\nGranger CWJ: Investigating Causal Relations by Econometric Models and Cross-spectral Methods. Econometrica. 1969; 37(3): 424–438. Publisher Full Text\n\nSeth AK, Barrett AB, Barnett L: Granger Causality Analysis in Neuroscience and Neuroimaging. J Neurosci. 2015; 35(8): 3293–3297. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPorta A, Faes L: Wiener–Granger causality in network physiology with applications to cardiovascular control and neuroscience. Proc IEEE. 2016; 104(2): 282–309. Publisher Full Text\n\nGeweke J: Measurement of Linear Dependence and Feedback between Multiple Time Series. J Am Stat Assoc. 1982; 77(378): 304–313. Publisher Full Text\n\nGeweke JF: Measures of Conditional Linear Dependence and Feedback between Time Series. J Am Stat Assoc. 1984; 79(388): 907–915. Publisher Full Text\n\nKamiński M, Ding M, Truccolo WA, et al.: Evaluating causal relations in neural systems: Granger causality, directed transfer function and statistical assessment of significance. Biol Cybern. 2001; 85(2): 145–157. PubMed Abstract | Publisher Full Text\n\nBaccalá LA, Sameshima K, Ballester G, et al.: Studying the Interaction Between Brain Structures via Directed Coherence and Granger Causality. Appl Signal Process. 1998; 5(1): 40–48. Reference Source\n\nBaccalá LA, Sameshima K: Partial directed coherence: a new concept in neural structure determination. Biol Cybern. 2001; 84(6): 463–474. PubMed Abstract | Publisher Full Text\n\nBressler SL, Seth AK: Wiener-Granger causality: a well established methodology. Neuroimage. 2011; 58(2): 323–329. PubMed Abstract | Publisher Full Text\n\nChicharro D: On the spectral formulation of Granger causality. Biol Cybern. 2011; 105(5–6): 331–347. PubMed Abstract | Publisher Full Text\n\nFaes L, Erla S, Nollo G: Measuring connectivity in linear multivariate processes: definitions, interpretation, and practical analysis. Comput Math Methods Med. 2012; 2012: 18, 140513. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStokes PA, Purdon PL: A study of problems encountered in Granger causality analysis from a neuroscience perspective. Proc Natl Acad Sci U S A. 2017; 114(34): E7063–E7072. PubMed Abstract | Publisher Full Text | Free Full Text\n\nValdes-Sosa PA, Roebroeck A, Daunizeau J, et al.: Effective connectivity: influence, causality and biophysical modeling. Neuroimage. 2011; 58(2): 339–361. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlorin E, Gross J, Pfeifer J, et al.: Reliability of multivariate causality measures for neural data. J Neurosci Methods. 2011; 198(2): 344–358. PubMed Abstract | Publisher Full Text\n\nFaes L, Nollo G: Measuring frequency domain granger causality for multiple blocks of interacting time series. Biol Cybern. 2013; 107(2): 217–232. PubMed Abstract | Publisher Full Text\n\nBarnett L, Seth AK: Granger causality for state-space models. Phys Rev E Stat Nonlin Soft Matter Phys. 2015; 91(4): 040101. PubMed Abstract | Publisher Full Text\n\nFaes L, Stramaglia S, Marinazzo D: Dataset 1 in: On the interpretability and computational reliability of frequency-domain Granger causality. F1000Research. 2017. Data Source"
}
|
[
{
"id": "26204",
"date": "02 Nov 2017",
"name": "Luiz Antonio Baccala",
"expertise": [
"Reviewer Expertise I work in statistical signal analysis and estimation with interest in applications to multivariate time series contexts as applied to neuroscience and acoustical imaging applications. I have been co-proposer of partial directed coherence (PDC) mentioned in the text."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI am glad that Faes et al. have not fallen prey to these considerations and have mounted a fair and elegant, albeit brief, critical appraisal of the PNAS Stokes and Purdon (S/P) paper. Fael et al. have restricted themselves to nailing just three obvious crucial facts that may go unnoticed by the casual reader for whom the authoritativeness of a vehicle like PNAS may stand as a certificate of validity.\nThe first point is that the S/P paper fails to mention how their VAR models were computed. My own AsympPDC package (The AsympPDC Package 3.0 is directly downloadable from http://www.lcs.poli.usp.br/~baccala/pdc/asymp_package_v3.zip. A preliminary version is also available through [7]. Visit http://www.lcs.poli.usp.br/~baccala/pdc/ for future version updates.) provides five different methods: the simplest naive and most popular approach, the least squares method, is the worst performer thanks to error propagation alone. It is important to stress that accurate VAR model estimation is crucial to whichever approach to GC is chosen. Faes et. al correctly supply an alternative method of estimation where no theoretically meaningless ‘negative’ GGC value is observed. The Faes et al. paper has the added obvious merit of including the actual value of GGC computed from the actual true model absentmindedly lacking in the original S/P paper.\nThe second point raised almost in passing by Faes et al. is that the S/P paper may possibly induce its readers to completely disregard frequency domain causality descriptions while S/P mostly glosses over the alternative DC/PDC framework. This is a huge oversight since currently only DC/PDC estimators have statistical theoretically rigorous computable confidence intervals and objective null hypothesis threshold tests 1-2 which may be applied using the freely downloadable AsympPDC package. This fact alone sets the DC/PDC methodologically apart. In today’s internet era of information no more than a google away, this omission is unforgivable.\nThe third point concerns the two time series case of Example 2. Indeed here is a point that Faes et al correctly argue that S/P fail to grasp. GC was conceived by Granger 3 to decompose pairwise relationships into exposing factors that aid predictability. This was later shown to be equivalent to detecting and characterizing the presence of feedback by Sims 4. In this case, GGC, DC and PDC address the so called connection ‘detection’ problem by focusing on the connection. If some given influence is present and if an influenced subsystem is affected, how it responds resonantly or otherwise is its own business.\nDo the valid Faes et al. criticisms imply the S/P paper is worthless? Despite its many additional shortcomings, the S/P paper has the important merit of stripping bare some of the field’s reigning confusion, enough to call for added discussion. This only stresses the relevance of the present criticisms and the urgent need for clarification.\nFinally I would like to put forward some thoughts that may explain the present state of conceptual disarray regarding causality. The first issue is ignorance about time series estimation — of the ‘a little knowledge is dangerous thing' kind. One cannot expect correct and reasonable results by just downloading some package, pressing some buttons without knowing the precise limitations of each available tool - time series analysis still has some elements of an art. The second problem has to do with the notions of Causality - it is tempting to ask the methodology to provide a glimpse on actual mechanisms. Sometimes, on physical grounds, this desire may be fulfilled, but true (mechanical) causality determination requires observer intervention 5-6; what GC does is to allow the exclusion of some tentative mechanisms. Thirdly, even when it reflects actual mechanisms, but more than two time series are simultaneously examined, different descriptions, as is the case for the complementarity between DC and PDC, they reflect different system properties so that one must break the GC concept into more general ideas: Granger Connectivity and Granger Influentiability 5-6. Last but not least, popular descriptions of large scale connectivity sometimes qualified as ‘effective’ versus ‘functional’ have further added to the present state of confusion due to their shear inconsistent application throughout the literature (se a discussion in 5-7.\nI think we should thank Faes et al. for pointing out some of these problems. Perhaps this is a good opportunity to start clearing up these issues once and for all.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes",
"responses": []
},
{
"id": "26209",
"date": "06 Nov 2017",
"name": "Karin Schiecke",
"expertise": [
"Reviewer Expertise computational neurosience",
"complex systems",
"nonlinear dynamics",
"connectivity analysis"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe correspondence is a comment directly related to “A study of problems encountered in Granger causality analysis from a neuroscience perspective1. I completely agree with the authors that there are interpretation issues of Granger causality (and by the way also a lot of wrong use/wrong adaptation in the field) and therefore the whole concept has to be applied with care. But – like the authors clearly stated in their comment – Stokes and Purdon uses 1) a version of frequency-domain Granger causality which is a least sub-optimal and 2) the results they were able to show by means of simulated data should be a general problem not only occurring in the case of neuroscience data. Therefore, I explicitly support the comment of Faes, Stramaglia and Marinazzo.\n\nThe comment is well written. Basic information concerning the concept of Granger causality and in particular the frequency-domain approach are given (including the coverage of related literature). The main idea behind the paper of Stokes and Purdon and the reason why the authors felt the need to comment on this paper are clearly stated. Then, the simulations of Stokes and Purdon are repeated using an updated Granger causality implementation and a combination of spectral and causal information. Technical details are given, results are correctly presented and well described, and conclusions are drawn.\n\nI do not have any major or minor concerns.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1710
|
https://f1000research.com/articles/6-1707/v1
|
19 Sep 17
|
{
"type": "Correspondence",
"title": "Unintended consequences of machine learning in medicine?",
"authors": [
"Laura McDonald",
"Sreeram V. Ramagopalan",
"Andrew P. Cox",
"Mustafa Oguz",
"Sreeram V. Ramagopalan",
"Andrew P. Cox",
"Mustafa Oguz"
],
"abstract": "Machine learning (ML) has the potential to significantly aid medical practice. However, a recent article highlighted some negative consequences that may arise from using ML decision support in medicine. We argue here that whilst the concerns raised by the authors may be appropriate, they are not specific to ML, and thus the article may lead to an adverse perception about this technique in particular. Whilst ML is not without its limitations like any methodology, a balanced view is needed in order to not hamper its use in potentially enabling better patient care.",
"keywords": [
"machine learning",
"healthcare",
"medicine",
"artificial intelligence"
],
"content": "\n\nThere is significant interest in the use of machine learning (ML) in medicine. ML techniques can ‘learn’ from the vast amount of healthcare data currently available, in order to assist clinical decision making. However, a recent article1 highlighted a number of consequences that may occur with increased ML use in healthcare, including physician deskilling, and that the approach is a ‘black box’ and unable to use contextual information during analysis.\n\nWhilst we agree that Cabitza et al’s concerns are justified1, we believe that a more balanced discussion could have been provided with regards to ML-based decision support systems (ML-DSS). As it stands, an impression is given that ML is flawed, rather than the issue being the way in which it is applied. The concerns raised are generally applicable to many analytical approaches, and reflect poor study design and/or a lack of analytical rigour than the particular technique being used.\n\nThe authors cite two examples to claim that ML-DSS could potentially reduce physician diagnostic accuracy. The mammogram example2 shows reduction in sensitivity for 6 of the most discriminating of 50 radiologists. However, the mammogram ML-DSS referred to is old2, and it is not clear how the underlying model was trained and evaluated. The model may perform well for some types of cancer, but not as well for others as a result of the training data. Indeed updates have been shown to increase detection sensitivity3. ML models can be refined by providing more data and results need to be critically appraised in this context. Additionally, no mention is made of the possible benefits of ML-DSS for less experienced staff. In the mammogram example, an improvement in sensitivity for 44 out of 50 radiologists was seen for easier to detect cancers. There was also an increased overall diagnostic accuracy when using ML-DSS in the electrocardiogram study4. Accuracy loss for experienced readers when using ML-DSS is valid, but more reflective of training needed and not an outcome specific to ML-DSS. A knowledgeable doctor may have no need for an ML-DSS, but the tool could greatly assist less experienced staff.\n\nCabitza et al. also argue that the confounding caused by asthma in the outcome of patients with pneumonia would have not been observed in a neural network model. There are, however, methods to obtain the feature importance and the direction of the relationship between predictor variables and outcome in neural networks5. Further, some ML approaches, such as random forest, are more transparent than others and ML can easily be coupled with clinical expertise to develop risk models that have their benefits over traditional statistical modelling6.\n\nThe issues highlighted by Cabitza et al. are more concerned with the studies themselves rather than an intrinsic flaw in ML methodology. To fully leverage ML or any other approach, users must have a good understanding of the caveats. In summary, we agree that ML-based approaches are not without their limitations, but the growing application of ML in healthcare has the potential to significantly aid physicians, especially in increasingly resource constrained environments. Informed, appropriate use of ML-DSS could, therefore, enable better patient care.",
"appendix": "Competing interests\n\n\n\nLM and SR are employees of Bristol-Myers Squibb Company. AC and MO are employees of Evidera Inc.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCabitza F, Rasoini R, Gensini GF: Unintended Consequences of Machine Learning in Medicine. JAMA. 2017; 318(6): 517–518. PubMed Abstract | Publisher Full Text\n\nPovyakalo AA, Alberdi E, Strigini L, et al.: How to discriminate between computer-aided and computer-hindered decisions: a case study in mammography. Med Decis Making. 2013; 33(1): 98–107. PubMed Abstract | Publisher Full Text\n\nKim SJ, Moon WK, Kim SY, et al.: Comparison of two software versions of a commercially available computer-aided detection (CAD) system for detecting breast cancer. Acta Radiol. 2010; 51(5): 482–490. PubMed Abstract | Publisher Full Text\n\nTsai TL, Fridsma DB, Gatti G: Computer decision support as a source of interpretation error: the case of electrocardiograms. J Am Med Inform Assoc. 2003; 10(5): 478–483. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOlden J, Jackson DA: Illuminating the \"black box\": a randomization approach for understanding variable contributions in artificial neural networks. Ecol Model. 2002; 154(1–2): 135–150. Publisher Full Text\n\nAyer T, Chhatwal J, Alagoz O, et al.: Informatics in radiology: comparison of logistic regression and artificial neural network models in breast cancer risk estimation. Radiographics. 2010; 30(1): 13–22. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "26485",
"date": "30 Oct 2017",
"name": "Arturo Gonzalez-Izquierdo",
"expertise": [
"Reviewer Expertise biomedical informatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 publication of this letter is both important and timely given the increased interest that statistical learning approaches applied to healthcare data are receiving.\n\nThe original article emphasized a negative perception of potential adverse consequences of machine learning (ML). It did not fully highlight the current benefits of using large amount of information for clinical decision making and the potential for methodological improvement with regards to statistical learning approaches. The very field of ML is rapidly evolving as illustrated by the rapid growth of deep learning over the past years.\nMinor comments\nContent could potentially be enhanced with a discussion on the notion that, precisely due to the outlined potential misuses and consequences, a systematic and strategic use of ML approaches must be developed and used to facilitate the robust application of such methods to healthcare data.\n\nThe authors of the letter rightly point out that ML has similar advantages and drawbacks as any other analytical approach applied in a clinical setting. However, they fail to explain how clinician deskilling, which can be a real consequence of automation, could be averted or indeed whether it is an acceptable outcome, given overall positive effects. This is separate issue to that of algorithm predictive accuracy.\n\n\"Accuracy loss for experienced readers when using ML-DSS is valid, but more reflective of training needed” - Is this training to improve interpretation of ML-DSS output in cases where it hinders correct reading? This could be relevant in the context of a study, however, it leaves open the possibility that in busy clinical settings, readers would be more likely to rely on computer aided detection.\n\nThe authors could also highlight the potential for upskilling clinicians in the understanding of ML methods, which can enhance their interpretation of DSS and other data-driven processes. Clinicians could be invaluable in spotting when algorithms go wrong, even (and perhaps especially) for cases where they’ve been shown to overall outperform humans.\n\nFinally, the authors would make their case stronger by citing examples that demonstrate \"proof of clinically important improvements in relevant outcomes compared with usual care, along with the satisfaction of patients and physicians”, as a concrete counter-balance to the negative or inconclusive examples of the original article.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Partly\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Partly\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes",
"responses": []
},
{
"id": "27518",
"date": "23 Nov 2017",
"name": "Hugo Schnack",
"expertise": [
"Reviewer Expertise (bio)medical data analysis"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMachine learning (ML) methods are currently being applied in a wide range of fields. Theoretically, the ability to extract meaningful relations from large datasets holds a great promise for health care and could potentially offer new, unexpected insights into disease and recovery.\nHowever, more critical voices have emerged warning of potential issues surrounding the use of ML and a number of points were addressed in the original paper by Cabitza et al. The authors of the current F1000 paper (McDonald et al) indicate they consider the issues raised as justified but call for a more balanced discussion regarding ML use in medicine. Indeed, Cabitza et al’s view is mainly negative. McDonald and colleagues correctly argue that it is often bad study design that leads to unreliable or unwanted outcomes and not the technique that is used. While a valid point, given how common bad study design or misuse of more common statistical techniques is, it is not unlikely that such issues will also arise from use of ML techniques in health care. Given the far reaching implications of unreliable AI-based decision support systems in health care careful scrutiny of the techniques is necessary.\nIn their discussion of the reduced sensitivity of radiologists evaluating mammograms, McDonald and colleagues bring to the defense of ML techniques that the model that led to unwanted outcomes was old and that it might work better or worse depending on which type of cancer is being studied. In addition they state that it was not clear how the model was trained and evaluated and that having appropriate feedback loops in place can improve the accuracy of a model considerably. These are obviously important points that should always be taken into account both when evaluating a specific model as well as for the evaluation of the use of ML in medicine in general. The quality of the algorithm but also the quality of available data will ultimately decide how useful a ML approach is for a given medical problem. This only goes to show that ML models should be applied with care and does not negate the original concerns put forward by Cabitza et al.\nMcDonald and colleagues also propose the use of more transparent ML techniques to prevent potential confounding variables that would not show in a black-box model. However, the predictive power of ML models often increases with their complexity, making transparency either very difficult or even impossible to obtain. As stated by the authors, users of ML models should have a good understanding of the limitations of the techniques. More often than not, ML models will be implemented in a collaboration between technical experts without medical knowledge and medical experts with limited technical expertise. Ensuring an optimal level of transparency of the models combined with the right amount of clinical expertise is therefore vital and while the authors mention this, there are no specific recommendations proposed to actually achieve this. With the increased application of ML models in health care there is a need for guidelines on how to optimally make use of the most technically advanced techniques while making sure bias in the data and confounding variables are accounted for. One recommendation the authors could have made is the need to bring the two worlds (medical and ML-technical) together, bridging the gap by educating people to become professionals with a (bio)medical and technical expertise, as well as training medical doctors in critically working with computerized predictions.\nAnother point that could have been addressed is how to deal with the differences in experience between medical doctors. Superior performance of the most-experienced doctors could be used to improve the computerized models, while less-experienced doctors may likely gain expertise by reviewing the computerized predictions –provided the ML tool allows for a clear interpretation how it did come to its diagnosis. This property of ML algorithms should be further explored and developed, because it can and, in fact, should lead to further insight in the underlying mechanisms of diseases.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Partly\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? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1707
|
https://f1000research.com/articles/6-1702/v1
|
18 Sep 17
|
{
"type": "Case Report",
"title": "Case Report: Hyperprolactinemia and growth hormone deficiency associated with Morning Glory Syndrome; with a review of the literature",
"authors": [
"Ravi Bhavsar",
"Maia Pavlovic",
"Afsoon Razavi",
"Muhammad Umair",
"Harsha Senapathi",
"Issac Sachmechi",
"Maia Pavlovic",
"Afsoon Razavi",
"Muhammad Umair",
"Harsha Senapathi",
"Issac Sachmechi"
],
"abstract": "Morning Glory Syndrome (MGS) is a rare congenital malformation of the optic nerve that is caused by a failure of the closure of the choroidal embryonic fissure in utero. The syndrome is usually seen in association with midline cranial defects, such as transsphenoidal and basal encephaloceles. Although MGS usually presents as an isolated ocular finding, it can be associated with endocrinological abnormalities. We report a case of a 32 year old female with MGS with hyperprolactinemia and growth hormone (GH) deficiency. She was diagnosed with MGS at the age of three and her past medical history was significant for left eye blindness, hyperprolactinemia and GH deficiency. She has received GH replacement and oral contraceptive pills in the past. Our investigations revealed elevated prolactin levels (63mg/l) and borderline low GH levels. Magnetic resonance imaging revealed an abnormality involving the optic chiasm, left optic nerve and compression of the pituitary gland by a basal encephalocele. Genetic studies were positive for a mutation in Paired box 6 gene (PAX6). She is being currently treated with cabergoline for her hyperprolactinemia. Our aims of this report are to highlight the hormonal manifestations of MGS and to review the etiopathogenesis of this rare disorder.",
"keywords": [
"morning glory syndrome",
"hyperprolactinemia",
"growth hormone deficiency",
"basal encephalocele"
],
"content": "Introduction\n\nMorning Glory Syndrome (MGS) was first reported in German literature in 1929, but has been more frequently reported since Kindler named it in 19701. The name was based on the condition’s resemblance to the funnel-shaped excavation of the posterior fundus incorporating the optic nerve to a tropical morning glory flower1. It is a rare congenital malformation of the optic nerve that is caused by a failure of the closure of the choroidal embryonic fissure in utero. We report a unique case of a 32 year old female with MGS with hyperprolactinemia and growth hormone (GH) deficiency. Our case highlights the various endocrinological presentations that can present concomitantly with this rare syndrome.\n\n\nCase report\n\nA 32 year old woman came to our clinic for the evaluation of amenorrhea and hyperprolactinemia. She was diagnosed with MGS at the age of 3 and at the age of 17 she was diagnosed with hyperprolactinemia and GH deficiency causing her to have a short stature (4ft 1 inch). She had no family history of similar or related issues. She was treated with GH replacement in the past, which led to an increase in her height (5ft) and she received oral contraceptives until the age of 28. Due to hyperprolactinemia and anovulatory cycles, she was treated with cabergoline. At the age of 31, she delivered a healthy baby via in vitro fertilization.\n\nHer most recent physical and vital parameters were under normal limits (blood pressure: 110/70 mmHg; pulse: 72 per minute; height: five feet; weight: 126 pounds). Visual acuity to finger counting was 20/20 in the right eye and 20/200 in the left eye. She had hypertelorism and strabismus of the left eye. Laboratory investigations revealed: fasting glucose levels: 87 mg/dl (65–99); Prolactin: 62 mg/l (2–14); GH: 0.2 ng/ml (0.0–10.0); GH arginine stimulation test: <2ng/ml; Insulin-like growth factor 1: 22ng/ml (71–241); luteinizing hormone: 1.3 miu/ml (0.0–4.0); follicle stimulating hormone: 5.0 miu/ml (0.0–5.0); Estradiol: 8.6 pg/ml (12.5–166.0); Dehydroepiandrosterone: 323 ng/dl (31–701); adrenocorticotropic hormone: 33.1 pg/ml (7.2–63.3); thyroid stimulating hormone: 1.48 mIU/L (0.45–4.5); free T4: 1.2 ng/dl (0.58–1.6).\n\nMagnetic resonance imaging (MRI) performed at this time revealed an abnormality involving the optic chiasm, left optic nerve and mild compression of the pituitary gland by a basal encephalocele (BE), with a normal sized pituitary gland. Genetic studies were positive for a mutation in Paired box 6 gene (PAX6). She continues to receive 0.5 mg of cabergoline once daily in view of her elevated prolactin levels. Our patient does not have any other symptoms and is being followed up regularly at our clinic.\n\n\nDiscussion\n\nMGS is a rare congenital malformation of the optic nerve that is caused by a failure of the closure of the choroidal embryonic fissure in utero. It is characterized by an enlarged, funnel shaped optic disk with a central mass of white glial tissue, surrounded by a raised pigmented chorioretinal ring. MGS usually presents as a unilateral malformation without gender predisposition with a median diagnosis of two years. The pathogenesis of MGS is relatively unknown and studies are currently being done to understand the syndrome clearly.\n\nMGS usually presents as an isolated ocular manifestation with decreased visual acuity, strabismus, myopia and astigmatism. The most common visual field defect is a central scotoma2 and MGS is also commonly associated with midline cranial defects, such as transsphenoidal and basal encephaloceles. Transsphenoidal encephalocele or BE have been largely associated with MGS, with 67% of people with BE also having MGS3.\n\nCranial defects may present with wide heads, flat noses, cleft lip/palate, hypertelorism, agenesis of the corpus callosum, hypopituitarism, posterior pituitary ectopia, basal and transsphenoidal encephaloceles. BE is a herniation of tissue through the sphenoid bone or cribriform plate of the ethmoid bone4. BE may present as a mass in the pharynx, nasal cavity and orbits4. Literature suggests that the association of MGS with craniofacial abnormalities may be linked to an embryogenic effect. Kissel et al. theorized that defects in neural crest cells are responsible for the craniofacial malformations5. This is the most probable mechanism by which BE occurs, due to the failure of closure of the anterior neuropore, which normally occurs by 4 weeks in utero5. The embryological findings support the neurologic and craniofacial manifestations seen with MGS.\n\nHormonal dysfunctions are seen with approximately 50–60% of BE patients. GH deficiency (66.7%), hypogonadotropic hypogonadism, hypothyroidism, hyperprolactinemia (13.3%) and diabetes insipidus are the most common hormonal disorders reported3. Eustis et al. postulated that the dysplastic optic discs in association with endocrine abnormalities are products of reduced trophic stimulation of the pituitary gland caused by abnormal hypothalamic control or an abnormal portal hypophysial system6. Table 1 lists several cases of MGS associated with endocrinopathies that have been reported in literature.\n\nGlory Syndrome associated with different endocrinopathies that have been reported in the literature. Abbreviations: M: Male, F: Female, ADH: Anti-Diuretic Hormone, GH: Growth Hormone, TSH: Thyroid Stimulating Hormone, PRL: Prolactin, LH: Luteinizing Hormone, NP: Not Performed.\n\nStudies by Asakura et al. suggest that MGS may be associated with a heterozygous Prokineticin receptor 2 (PROKR2) gene mutation7. The PROKR2 pathway plays a vital role in early pituitary development and the development of gonadotropin releasing hormone neurons. This could possibly explain the pituitary malformation and the hormonal imbalance seen in our case report Hormonal disorders are common in patients with BE induced MGS, possibly due to malformed cranial structures, which exert pressure on the pituitary gland causing gland compression, thereby restricting production of hormones, such as GH and prolactin at healthy rates.\n\nGenetic studies performed on our patient revealed a mutation in the PAX6 gene. PAX6 gene mutations are commonly implicated in congenital ocular malformations. PAX6 gene is responsible for activating genes involved in the formation of the eyes, brain, spinal cord, and pancreas during embryonic development8. As far as MGS is concerned, the PAX6 protein is an excellent resource to study in patients, as it is responsible for ocular embryogenesis and regulating the expression of other genes involved in the other structures of the eye.\n\nMGS is a complex disease, but can be diagnosed best through a fundus examination and radiological studies, such as CT scans and cranial MRI scans. The white glial tissue mass in the malformation causes the pupil to look a whitish-color (leukokoria), which is a classic and telling symptom. The diagnostic measures should be accompanied by a complete physical and ophthalmological examination and appropriate laboratory investigations to rule out hormonal dysfunction.\n\nThere are still under 100 cases of MGS reported worldwide. It is still a very rare medical anomaly that has not been greatly researched until more recent decades. Treatments are directed towards preventing and treating possible existing complications associated with the syndrome such as hormone replacement for hormonal imbalance and suitable correction lenses for myopia and astigmatism.\n\n\nConclusion\n\nOur case aims to highlight the endocrinological manifestations of MGS. Although the association of MGS with pituitary hormonal imbalance is relatively well known, the diagnosis was established much later in our case. Early recognition of these features through physical examination and lab investigations should prompt appropriate intervention.\n\n\nConsent statement\n\nWritten informed consent was obtained from the patient for the publication of the patient’s details.",
"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\nOur team presented this case as an abstract (#1349) at the American Association of Clinical Endocrinologists meeting in 2014. The considerable interest received regarding the case promoted the authors to write this article.\n\n\nReferences\n\nKindler P: Morning glory syndrome: unusual congenital optic disk anomaly. Am J Ophthalrnol. 1970; 69(3): 376–384. PubMed Abstract | Publisher Full Text\n\nBeyer WB, Quencer RM, Osher RH: Morning glory syndrome. A functional analysis including fluorescein angiography, ultrasonography, and computerized tomography. Ophthalmology. 1982; 89(12): 1362–7. PubMed Abstract | Publisher Full Text\n\nOyakawa Barcelli Y, García Durruti P, Enes Romero P, et al.: Morning glory syndrome associated with transsphenoidal encephalocele and panhypopituitarism. Endocrinol Nutr. 2014; 61(4): 222–224. PubMed Abstract | Publisher Full Text\n\nHope-Ross M, Johnston SS: The Morning Glory syndrome associated with sphenoethmoidal encephalocele. Ophthalmic Paediatr Genet. 1990; 11(2): 147–53. PubMed Abstract | Publisher Full Text\n\nKissel P, Andre JM, Jacquier A: The Neurocristopathies. New York, NY: Mas-son Publishing; 1981. Reference Source\n\nEustis HS, Sanders MR, Zimmerman T: Morning glory syndrome in children. Association with endocrine and central nervous system anomalies. Arch Ophthalmol. 1994; 112(2): 204–207. PubMed Abstract | Publisher Full Text\n\nAsakura Y, Muroya K, Hanakawa J, et al.: Combined pituitary hormone deficiency with unique pituitary dysplasia and morning glory syndrome related to a heterozygous PROKR2 mutation. Clin Pediatr Endocrinol. 2015; 24(1): 27–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuerra-Junior G, Spinola-Castro AM, Siviero-Miachon AA, et al.: Absence of mutations in Pax6 gene in three cases of morning glory syndrome associated with isolated growth hormone deficiency. Arq Bras Endocrinol Metabol. 2008; 52(8): 1221–7. PubMed Abstract | Publisher Full Text\n\nPollock JA, Newton TH, Hoty WF: Transsphenoidal and transethmoidal encephaloceles. A review of clinical and roentgen features in 8 cases. Radiology. 1968; 90(3): 442–53. PubMed Abstract | Publisher Full Text\n\nWiese GM, Kempe LG, Hammon WM: Transsphenoidal meningohydroencephalocele. Case report. J Neurosurg. 1972; 37(4): 475–8. PubMed Abstract | Publisher Full Text\n\nManelfe C, Starling-Jardim D, Touibi S, et al.: Transsphenoidal encephalocele associated with agenesis of corpus callosum: value of metrizamide computed cisternography. J Comput Assist Tomogr. 1978; 2(3): 356–61. PubMed Abstract | Publisher Full Text\n\nLarsen JL, Bassøe HH: Transsphenoidal meningocele with hypothalamic insufficiency. Neuroradiology. 1979; 18(4): 205–9. PubMed Abstract | Publisher Full Text\n\nEllyin F, Khatir AH, Singh SP: Hypothalamic-pituitary functions in patients with transsphenoidal encephalocele and midfacial anomalies. J Clin Endocrinol Metab. 1980; 51(4): 854–6. PubMed Abstract | Publisher Full Text\n\nNishi Y, Muraki K, Sakoda K, et al.: Hypopituitarism associated with transsphenoidal meningoencephalocele. Eur J Pediatr. 1982; 139(1): 81–4. PubMed Abstract | Publisher Full Text\n\nTakezawa N, Sato M, Yanagisawa T, et al.: [Pituitary dwarfism associated with morning glory syndrome and transsphenoidal encephalocele: a case report]. No To Hattatsu. 1987; 19(6): 492–6. PubMed Abstract | Publisher Full Text\n\nDurham LH, Mackenzie IJ, Miles JB: Transphenoidal Meningohydroencephalocoele. Br J Neurosurg. 1988; 2(3): 407–409. PubMed Abstract | Publisher Full Text\n\nRice JF, Eggers DM: Basal transsphenoidal encephalocele: MR findings. AJNR Am J Neuroradiol. 1989; 10(5 suppl): S79. PubMed Abstract\n\nKobayashi S, Miyazaki M, Miyagi O, et al.: [A case of transsphenoidal meningoencephalocele]. No Shinkei Geka. 1990; 18(11): 105–70. PubMed Abstract\n\nMorioka M, Marubayashi T, Masumitsu T, et al.: Basal encephaloceles with morning glory syndrome, and progressive hormonal and visual disturbances: case report and review of the literature. Brain Dev. 1995; 17(3): 196–201. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "26768",
"date": "10 Oct 2017",
"name": "Thiago Gonçalves dos Santos Martins",
"expertise": [
"Reviewer Expertise Ophthalmology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe background of the case’s history and progression is described in sufficient detail. The case is presented with sufficient detail\nThe authors should consider a differential diagnosis. The coloboma of optic disk, which is a differential diagnosis, is characterized as excavation, normally in inferior part, without glial tissue typically present in Morning Glory syndrome. Coloboma of the optic nerve is a congenital anomaly of the optic disc in which there is a defect of the inferior aspect of the optic nerve. The issue stems from incomplete closure of the embryonic fissure while in utero. A varying amount of glial tissue typically fills the defect, manifests as a white mass. Although both optic nerve colobomas and morning glory disc anomaly (MGDA) involve mutations of the PAX6 gene, these two separate diseases represent two distinct causes. An optic nerve coloboma is easily differentiated from morning glory anomaly. Colobomas affect only the inferior aspect of the nerve as it represents an incomplete closure of the embryonic fissure, whereas MGDA encompasses all aspects of the nerve and represents more generally a dysgenesis of the mesoderm.\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": "27942",
"date": "03 Jan 2018",
"name": "Pamela Garcia-Filion",
"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\nIs the background of the case’s history and progression described in sufficient detail? Partly\nComment: The background fails to describe the ophthalmic diagnostic characteristics of Morning Glory Syndrome.\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\nComment: Case describes only a patient with decreased visual acuity, and nothing specific to Morning Glory. Authors don’t provide information about the clinical eye exam to establish the 3 defining clinical features: i.e., enlarged disc, chorioretinal pigmentary changes around the optic disc, a glial tuft overlying the disc. Decreased visual acuity is insufficient to establish diagnosis. There is no opthalmoscopic findings, no funduscopic findings. The MR findings don’t match those of Morning Glory. Additionally, the MR details don’t mention whether the scan involved orbital cuts.\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\nComment: The discussion is the best part of the case report. The relevance is questionable since the case is unlikely to be Morning Glory Syndrome.\n\nIs the case presented with sufficient detail to be useful for other practitioners? No\nCommentary: Detail is insufficient and this case absolutely shouldn’t be available to clinicians.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1702
|
https://f1000research.com/articles/6-1489/v1
|
16 Aug 17
|
{
"type": "Research Note",
"title": "Professional medical writing support and the reporting quality of randomized controlled trial abstracts among high-impact general medical journals",
"authors": [
"Ira Mills",
"Catherine Sheard",
"Meredith Hays",
"Kevin Douglas",
"Christopher C. Winchester",
"William T. Gattrell",
"Catherine Sheard",
"Meredith Hays",
"Kevin Douglas",
"Christopher C. Winchester",
"William T. Gattrell"
],
"abstract": "Background: In articles reporting randomized controlled trials, professional medical writing support is associated with increased adherence to Consolidated Standards of Reporting Trials (CONSORT). We set out to determine whether professional medical writing support was also associated with improved adherence to CONSORT for Abstracts. Methods: Using data from a previously published cross-sectional study of 463 articles reporting randomized controlled trials published between 2011 and 2014 in five top medical journals, we determined the association between professional medical writing support and CONSORT for Abstracts items using a Wilcoxon rank-sum test. Results: The mean proportion of adherence to CONSORT for Abstracts items reported was similar with and without professional medical writing support (64.3% vs 66.5%, respectively; p=0.30). Professional medical writing support was associated with lower adherence to reporting study setting (relative risk [RR]; 0.40; 95% confidence interval [CI], 0.23–0.70), and higher adherence to disclosing harms/side effects (RR 2.04; 95% CI, 1.37–3.03) and funding source (RR 1.75; 95% CI, 1.18–2.60). Conclusions: Although professional medical writing support was not associated with increased overall adherence to CONSORT for Abstracts, important aspects were improved with professional medical writing support, including reporting of adverse events and funding source. This study identifies areas to consider for improvement.",
"keywords": [
"randomized controlled trials",
"medical writing",
"CONSORT guidelines",
"abstracts",
"adherence",
"adverse events",
"funding source"
],
"content": "Introduction\n\nPrior studies demonstrate low levels of adherence to Consolidated Standards of Reporting Trials (CONSORT) guidelines1,2, as well as CONSORT for Abstracts3,4 in reporting randomized controlled trials (RCTs). Professional medical writing support, correctly acknowledged, is endorsed by Good Publication Practice (GPP3)5, and its prevalence increased between 2001/2002 and 2009/2010, with a reported doubling to nearly 35% of industry-sponsored studies6. Professional medical writing support is associated with increased adherence to CONSORT in articles reporting RCTs; in a sample of open-access journals, the number of articles that completely reported ≥50% of the studied CONSORT items was significantly higher with professional medical writing support (39%) than without professional medical writing support (21%; p<0.05)7,8.\n\nThe purpose of this study was to determine whether professional medical writing support was also associated with improved adherence to CONSORT for Abstracts by analyzing a published dataset from five high-impact general medical journals with overall variable and incomplete adherence9.\n\n\nMethods\n\nWe examined data from a published cross-sectional study of 463 articles reporting RCTs9. The RCTs were published between 2011 and 2014 in five top medical journals: The New England Journal of Medicine, Annals of Internal Medicine, The Lancet, The BMJ, and JAMA. We determined the association between professional medical writing support and the reporting of CONSORT for Abstracts items10 (Table 1) using a Wilcoxon rank-sum test. One author (CS), who was blinded to the CONSORT for Abstracts scores using de-identified original dataset outputs, identified articles as being prepared with professional medical writing support using automated searching followed by manual review if they acknowledged the involvement of one of the following: medical writer, medical writing, writing services, writing assistance, editorial assistance, or editorial support. The context of these terms was also examined.\n\nMean proportions of CONSORT for Abstract adherence with and without professional medical writing support was compared using a Wilcoxon rank-sum test, and then tested with additional effect of variable journal adherence using an analysis of variance (ANOVA). The relative risk (RR) and 95% confidence interval (CI) for each item’s adherence with and without professional medical writing support were calculated using the command “oddsratio” in the R package fmsb 0.5.211. All statistical analyses were performed in R 3.2.212.\n\n\nResults\n\nFrom the original published dataset of 463 abstracts from RCTs reported in five journals, acknowledged professional medical writing support was observed in 66 articles (14.3%). Two articles identified in the automated search were excluded on manual review, one of which stated13, “there was no writing assistance from anyone who is not listed as an author,” and the other14, “the Writing committee drafted the report… without editorial assistance.” The mean proportion of CONSORT for Abstracts items reported in articles with (n=66) and without (n=397) professional medical writing support was 64.3% versus 66.5%, respectively; p=0.3044 (Wilcoxon rank-sum test). This difference remained nonsignificant when journal variation in CONSORT for Abstracts adherence was considered (ANOVA, p=0.1347). Overall, reporting of the individual CONSORT for Abstracts items was similar with and without professional medical writing support (RR 0.97; 95% CI, 0.88–1.07) (Figure 1). However, a lower rate of reporting the study setting (item 4) was observed in articles with professional medical writing support (RR 0.40; 95% CI, 0.23–0.70). Conversely, professional medical writing support was associated with higher adherence to reporting both harms and side effects (item 16) (RR 2.04; 95% CI, 1.37–3.03) and source of funding (item 19) (RR 1.75; 95% CI, 1.18–2.60).\n\nCONSORT, Consolidated Standards of Reporting Trials; PMWS, professional medical writing support. Significant associations are shaded.\n\n\nDiscussion\n\nAlthough professional medical writing support was not associated with increased overall adherence to CONSORT for Abstracts, important aspects were improved with professional medical writing support, including reporting of adverse events and funding source. These data confirm prior evidence showing that professional medical writing support is associated with improved safety reporting15, and serve to support the important role of professional medical writers in promoting adherence to Medical Publishing Insights & Practices recommendations to improve adverse event reporting in clinical trial publications16. Reporting of funding source was also improved with professional medical writing support, most likely reflecting the emphasis placed on transparency about funding by GPP3 guidelines5,17. In articles with professional medical writing support, we observed 100% adherence for other important CONSORT for Abstract items, including reporting of the clinical trials registration number (item 18), vital for transparency and study tracking, which has recently been automated using technology such as the TrialsTracker18.\n\nHowever, we were disappointed to see that professional medical writing support was not associated with improvements in reporting of other CONSORT for Abstract items, including specification in the title of the design of the study (item 2) and that it was randomized (item 1), and reporting of the numbers randomized and analyzed (items 12 and 13). Indeed, professional medical writing support was actually associated with worse reporting of one item, study setting (item 4). These areas represent clear areas in which professional medical writers can help further improve the reporting of clinical trials in the abstracts of journal articles.\n\nAlthough this was a post-hoc analysis, it has the advantage that CONSORT for Abstracts adherence was assessed before our study question was posed. Additionally, the presence of professional medical writing support was assigned by an assessor who was blinded to the CONSORT for Abstracts score. However, in the original study, inter-rater agreement for scoring was 84%, which is suboptimal9. Furthermore, the original dataset was limited to a sample of high-impact journals, and so may not be generalizable to the biomedical literature as a whole. Indeed, in this dataset of high-impact journals, adherence to CONSORT for Abstracts is likely to have been influenced by the journal’s in-house scientific editing; consequently, the impact of professional medical writing support on adherence to CONSORT for Abstracts may be greater in journals without professional in-house editing. These data were potentially confounded by funding source; because professional medical writing support is typically restricted to industry-funded studies, it is possible that the review processes followed by industry19, rather than professional medical writing support per se, caused the improvements in reporting that we observed. However, in industry-funded articles, professional medical writing support was associated with a greater than two-fold increase in ≥50% adherence to CONSORT items studied compared with industry-funded articles prepared without this support (38% vs 18%, p<0.05)7,8. In addition, industry funding alone had no impact on the quality of CONSORT reporting in the absence of professional medical writing support7,8. Nevertheless, it can be difficult to correctly ascribe the role of the funder from the details provided in manuscripts as, for example, investigator-led studies typically undergo a different review process to those conducted with full industry support20.\n\nIn summary, although professional medical writing support was not associated with increased overall adherence to CONSORT for Abstracts, important aspects were improved with professional medical writing support, including reporting of adverse events and funding source. Ensuring adherence to reporting guidelines is a complex task, so we believe that there is a role for reporting professionals such as professional medical writers to work with authors and journals, to provide training, writing and reviewing, and thereby improve the quality of reporting of clinical trials.\n\n\nData availability\n\nDataset 1: Dataset for reporting of CONSORT for Abstracts items in articles with and without acknowledged professional medical writing support. Dataset used per Hays et al.9 with the addition of column C for this subanalysis by professional medical writing support (yes: 1; no: 0). doi, 10.5256/f1000research.12268.d17243721",
"appendix": "Competing interests\n\n\n\nMills I is an employee of PAREXEL International. Sheard C, Hays M, and Douglas K have no disclosures to report. Gattrell WT is an employee of Ipsen Biopharma. Winchester CC is an employee and Director of Oxford PharmaGenesis Ltd, and a Director and shareholder of Oxford PharmaGenesis Holdings Ltd.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe authors wish to acknowledge Ronald Gruber, M.S, M.L.S, of QWKINSIGHTS, New York, NY, for early support in the design and conception of this study, and Lindy Dunlop and Megan Misukonis of PAREXEL for editorial assistance.\n\nThis study was presented at the 13th Annual Meeting of ISMPP, May 1–3, 2017, National Harbor, MD, USA.\n\n\nReferences\n\nHopewell S, Dutton S, Yu LM, et al.: The quality of reports of randomised trials in 2000 and 2006: comparative study of articles indexed in PubMed. BMJ. 2010; 340: c723. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPlint AC, Moher D, Morrison A, et al.: Does the CONSORT checklist improve the quality of reports of randomised controlled trials? A systematic review. Med J Aust. 2006; 185(5): 263–267. PubMed Abstract\n\nGhimire S, Kyung E, Kang W, et al.: Assessment of adherence to the CONSORT statement for quality of reports on randomized controlled trial abstracts from four high-impact general medical journals. Trials. 2012; 13: 77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHopewell S, Ravaud P, Baron G, et al.: Effect of editors' implementation of CONSORT guidelines on the reporting of abstracts in high impact medical journals: interrupted time series analysis. BMJ. 2012; 344: e4178. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBattisti WP, Wager E, Baltzer L, et al.: Good Publication Practice for Communicating Company-Sponsored Medical Research: GPP3. Ann Intern Med. 2015; 163(6): 461–464. PubMed Abstract | Publisher Full Text\n\nKim MR, Nilsen J, Smith G, et al.: Trends in medical writing acknowledgment in medical journals over the last decade. Curr Med Res Opin. 2011; 27(S1): S13. Reference Source\n\nGattrell W, Hopewell S, Young K, et al.: Professional medical writing support improves the quality of reporting of randomized controlled trials. Curr Med Res Opin. 2015; 31(S1): S20. Reference Source\n\nGattrell WT, Hopewell S, Young K, et al.: Professional medical writing support and the quality of randomised controlled trial reporting: a cross-sectional study. BMJ Open. 2016; 6(2): e010329. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHays M, Andrews M, Wilson R, et al.: Reporting quality of randomised controlled trial abstracts among high-impact general medical journals: a review and analysis. BMJ Open. 2016; 6(7): e011082. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHopewell S, Clarke M, Moher D, et al.: CONSORT for reporting randomized controlled trials in journal and conference abstracts: explanation and elaboration. PLoS Med. 2008; 5(1): e20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNakazawa M: fmsb:Functions for medical statistics book with some demographic data. R package version 0.5.2. Accessed January 6, 2017. Reference Source\n\nR Core Team:R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Accessed January 6, 2017. Reference Source\n\nJüttler E, Unterberg A, Woitzik J, et al.: Hemicraniectomy in older patients with extensive middle-cerebral-artery stroke. N Engl J Med. 2014; 370(12): 1091–1100. PubMed Abstract | Publisher Full Text\n\nPROVE Network Investigators for the Clinical Trial Network of the European Society of Anaesthesiology, Hemmes SN, Gama de Abreu M, et al.: High versus low positive end-expiratory pressure during general anaesthesia for open abdominal surgery (PROVHILO trial): a multicentre randomised controlled trial. Lancet. 2014; 384(9942): 495–503. PubMed Abstract | Publisher Full Text\n\nJacobs A: Adherence to the CONSORT guideline in papers written by professional medical writers. The Write Stuff. 2010; 19: 196–200. Reference Source\n\nLineberry N, Berlin JA, Mansi B, et al.: Recommendations to improve adverse event reporting in clinical trial publications: a joint pharmaceutical industry/journal editor perspective. BMJ. 2016; 355: i5078. PubMed Abstract | Publisher Full Text\n\nGraf C, Battisti WP, Bridges D, et al.: Research Methods & Reporting. Good publication practice for communicating company sponsored medical research: the GPP2 guidelines. BMJ. 2009; 339: b4330. PubMed Abstract | Publisher Full Text\n\nPowell-Smith A, Goldacre B: The TrialsTracker: Automated ongoing monitoring of failure to share clinical trial results by all major companies and research institutions [version 1; referees: 2 approved]. F1000Res. 2016; 5: 2629. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWager E, Woolley K, Adshead V, et al.: Awareness and enforcement of guidelines for publishing industry-sponsored medical research among publication professionals: the Global Publication Survey. BMJ Open. 2014; 4(4): e004780. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLundh A, Sismondo S, Lexchin J, et al.: Industry sponsorship and research outcome. Cochrane Database Syst Rev. 2012; 12: MR000033. PubMed Abstract | Publisher Full Text\n\nMills I, Sheard C, Hays M, et al.: Dataset 1 in: Professional medical writing support and the reporting quality of randomized controlled trial abstracts among high-impact general medical journals. F1000Research. 2017. Data Source"
}
|
[
{
"id": "25066",
"date": "17 Aug 2017",
"name": "Ana Marusic",
"expertise": [
"Reviewer Expertise Dissemination bias",
"publication integrity"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study presents secondary analysis of a study of adherence to CONSORT reporting guidelines for Abstracts. While the original study (Hays et al, 2016) looked at this adherence in a sample of abstracts from several high-profile medical journals, this study performed a subgroup analysis, looking at whether the mention of professional writing assistance was associated with the score for different items on the reporting checklist. The study is thus quite methodologically biased, and it is not clear whether the observed differences are real, or random. For example, the finding that the acknowledgment of professional writing assistance decreases the frequency of adequately reporting the setting of the study is difficult to explain. The potential randomness of the significant findings should be addressed in the Discussion section.\nMaking a publication out of the original publication by looking at a single characteristic does not really warrant the publication of the submitted manuscript. It would be preferable if other characteristics were explored in relation to reporting completeness in abstracts, such as the type of funding, phase of the trial, institutions where trials were performed.\nThe manuscript is well written and structured, and there are no major comments or suggestions for improvement except the conceptual one described above. In the Methods section, the extraction of data for this study is not fully explained - how many experts extracted the data, how were different terms in the statements on writing assistance interpreted and categorized, etc.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? 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": "3018",
"date": "14 Sep 2017",
"name": "Ira Mills",
"role": "Author Response",
"response": "We thank Ana Marušić for her insight and providing suggestions to improve our paper. We feel that the data described herein fulfill the criteria for publication as a “Research Note” in F1000 Research, designed to report “single-finding papers that can be reported with one or two illustrations.” Our intention was to conduct a post hoc analysis to specifically examine the role of professional medical writing support on CONSORT adherence as previously described by two of the authors. In the discussion, we acknowledge that there was substantial risk of false positives given that separate confidence intervals were computed for 19 different CONSORT items. However, the p value for setting (item 4) was 0.0010, for harms or side effects (item 16) was 0.0004, and for source of funding (item 19) was 0.0055; the first two survive a Bonferroni correction, which would conservatively demand that p<0.0026, and all three pass the less stringent Benjamini-Hochberg procedure. We now include a note in the discussion that the limited number of articles with acknowledged professional medical writing support did not allow further exploration of the results (e.g. type of funding, trial phase, institutions where performed) and also mention that it would be of interest to examine whether similar trends are observed in separate studies of CONSORT for Abstracts. As described in the original submission, one author performed the data extraction conducting a blinded automated search per the defined terms followed by reexamining for context. We note two articles that were excluded from the analysis by manual review and provide rationale. In one case there was no writing assistance from anyone not listed as an author and in the second case the “Writing committee drafted the report…without editorial assistance.” We have made it clear that this was performed using the full text articles."
}
]
},
{
"id": "25067",
"date": "23 Aug 2017",
"name": "Jackie M. Marchington",
"expertise": [
"Reviewer Expertise Professional medical writing",
"publication ethics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 subanalysis of a previous study detailing the completeness of clinical study reporting in abstracts according to the CONSORT for abstracts checklist. The original publication was restricted to a sample of clinical trial publications in 5 high-ranking general medical journals. This subanalysis investigated differences in adherence to CONSORT for abstracts between papers with or without acknowledged professional medical writing support.\nWith respect to the co-authors' previous study, citation of the full paper (ref 8) is sufficient. Citing the poster of the same study is not necessary (ref 7).\nRegarding the methods, it is not clear how automated searching would have identified the presence of professional medical writing support. In this reviewer's experience, that has always required retrieval of the full text article. If the automated search was performed on each full text article, I think it would be clearer to state this.\nIn the discussion, the authors point out funding source as a potential confounder of their results. It would perhaps be of interest to examine industry-funded studies written up without acknowledged professional medical writing support as an additional comparator group to determine whether industry funding was driving differences in CONSORT adherence.\nI also have a niggling concern that although GPP3 encourages transparency around the involvement of non-author contributors such as professional medical writers in industry-funded publications, there may be papers in this cohort that used unacknowledged medical writers, papers where a professional writer was included as an author, so not acknowledged as such, or non-industry funded papers where the authors used institutional editorial/writing resource that is not acknowledged. Perhaps the authors could comment on this potential limitation in the discussion.\nAlthough this is a fairly straightforward subanalysis of a previous study, I think it makes an important contribution to the evidence base about the role of professional medical writers, even though the findings are neutral in terms of adherence to CONSORT for abstracts.\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": "3017",
"date": "14 Sep 2017",
"name": "Ira Mills",
"role": "Author Response",
"response": "We thank Jackie Marchington for her insight and providing suggestions to improve our paper. Although Good Publication Practice guidelines (GPP3) encourage transparency of professional medical writing support, it remains possible that professional medical writing support was not consistently acknowledged in the studied dataset, and cited evidence in the literature of its prevalence. Any unacknowledged professional medical writing support would tend towards the null hypothesis enhancing the confidence in the observed statistical differences. As noted by the referee, we have pointed out funding source as a potential confounder of the results and provided discussion as to some of these considerations. The relatively low number of articles with acknowledged professional medical writing support did not allow us to conduct an analysis of professional medical writing support with and without industry support with any statistical validity. In our paper to support cited information, we have listed both reference 7 (full publication) and reference 8 (corresponding abstract for a prior congress presentation) as the abstract reported p values not found in the full publication. We have swapped the order of the references to have the full publication cited first."
}
]
}
] | 1
|
https://f1000research.com/articles/6-1489
|
https://f1000research.com/articles/6-1316/v1
|
03 Aug 17
|
{
"type": "Method Article",
"title": "A simple method to measure CLOCK-BMAL1 DNA binding activity in tissue and cell extracts",
"authors": [
"Maud Gillessen",
"Pieter Bas Kwak",
"Alfred Tamayo",
"Maud Gillessen",
"Pieter Bas Kwak"
],
"abstract": "The proteins CLOCK and BMAL1 form a heterodimeric transcription factor essential to circadian rhythms in mammals. Daily rhythms of CLOCK-BMAL1 DNA binding activity are known to oscillate with target gene expression in vivo. Here we present a highly sensitive assay that recapitulates native CLOCK-BMAL1 DNA binding rhythms from crude tissue extracts, which we call the Clock Protein-DNA Binding Assay (CPDBA). This method can detect less than 2-fold differences in DNA binding activity, and can deliver results in two hours or less using 10 microliters or less of crude extract, while requiring neither specialized equipment nor expensive probes. To demonstrate the sensitivity and versatility of this assay, we show that enzymatic removal of phosphate groups from proteins in tissue extracts or pharmacological inhibition of casein kinase I in cell culture increased CLOCK-BMAL1 DNA binding activity by ~1.5 to ~2 fold, as measured by the CPDBA. In addition, we show that the CPDBA can measure CLOCK-BMAL1 binding to reconstituted chromatin. The CPDBA is a sensitive, fast, efficient and versatile probe of clock function.",
"keywords": [
"circadian clock",
"DNA binding assay",
"CLOCK",
"BMAL1",
"phosphorylation",
"chromatin"
],
"content": "Introduction\n\nThe maintenance or disruption of circadian rhythms contribute significantly to several areas of health and disease (Asher & Schibler, 2011; Chen & Yang, 2015; Puram et al., 2016; Roenneberg & Merrow, 2016; Sahar & Sassone-Corsi, 2009; Takahashi et al., 2008). Circadian rhythms are daily biological rhythms synchronized by light and dark cycles of the day/night continuum. Underlying circadian rhythms are oscillations of gene expression occurring in nearly all tissues and cells observed to date (Koike et al., 2012; Lamia et al., 2008; Lande-Diner et al., 2015; Zhang et al., 2014). Daily cycles of gene transcription and translation are driven by circadian clocks (Gustafson & Partch, 2015; Mendoza-Viveros et al., 2017; Takahashi, 2017).\n\nAn essential component of circadian clocks is the CLOCK-BMAL1 heterodimeric transcription factor (Bunger et al., 2000; Gekakis et al.,1998; Hogenesch et al., 1998; Huang et al., 2012; Lande-Diner et al., 2013; Tamayo et al., 2015). CLOCK-BMAL1 drives the expression of many proteins, including its own repressors, forming the basis of a negative feedback loop (Kume et al., 1999; Sato et al., 2006). The oscillating abundance of transcriptional repressors leads to daily cycles of CLOCK-BMAL1 target gene expression.\n\nRhythmic CLOCK-BMAL1 binding to target genes is likely critical to the generation of circadian rhythms (Koike et al., 2012; Rey et al., 2011; Ripperger & Schibler, 2006). We have previously demonstrated that immobilized DNA oligonucleotides containing E-box DNA binding motifs (CACGTG) can be used to capture native CLOCK-BMAL1, as measured by mass spectrometry (Tamayo et al., 2015). Here we present a sensitive and versatile method to measure native CLOCK-BMAL1 DNA and chromatin binding from virtually any tissue or cell source in a fast and efficient manner.\n\n\nMaterials and methods\n\nThe mouse strain C57/BL6J (The Jackson Laboratory) was used as wildtype (WT), unless the experiment called for a genetically modified animal, in which case an appropriate control animal was utilized. Bmal1−/− animals were bred from heterozygotes in our facility, therefore WT animals were homozygous Bmal1+/+ littermates (C57/BL6J background). WT controls for Per2-FH animals were mixed C57BL/6J × 129 genetic background (The Jackson Laboratory). Bmal1−/− and Per2-FH (generated by the Weitz Laboratory of Harvard Medical School) have been previously described (Bunger et al., 2000; Duong et al., 2011). Mice were entrained to a 12:12 hr light-dark cycle for at least 2 weeks and then were kept in constant darkness for 24 hrs before sacrificing at the indicated circadian time (CT). CT0 corresponds to the time the lights would turn on, CT4 to 4 hours after that point, and so forth. Mice were euthanized under infrared light, and tissues were dissected under room light. Studies were performed in accordance with the protocol approved by the Harvard Medical School Standing Committee on Animals (protocol #03376).\n\nTissue and lysate were kept on ice or at 4°C through all steps. As a first step to each tissue extraction, liver was finely minced using a razor then washed with PBS (phosphate buffered saline) and centrifuged (400 × g, 5 min) until wash solution was clear (8-10×).\n\nNuclear extracts were prepared as previously described (Kim et al., 2014), with some amendments. Briefly, tissue was Dounce homogenized with pestle A, centrifuged, then Dounce homogenized with pestle B, followed by ultracentrifugation through a 2M sucrose cushion (84,000 × g for 1 hr at 4°C) to isolate nuclei (pellet). Nuclei were lysed in nuclear lysis buffer (10 mM Tris-HCl, 300 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.2% TX-100, pH 7.4) containing protease inhibitor cocktail without EDTA (Roche) and phosphatase inhibitor cocktails 2/3 (Sigma-Aldrich). Upon a final centrifugation (20,000 × g, 30 min), the remaining supernatant was liver nuclear extract. For whole tissue extract, the tissue pellet was weighed and resuspended in whole cell extract buffer (10mM Tris-HCl, 300 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.5% TX-100 [Sigma-Aldrich], pH 7.4, protease and phosphatase inhibitors) in a volume 4× the weight of tissue (e.g. 4 ml/1 g) then Dounce homogenized with pestle A (8 strokes). The homogenate was incubated on ice for 30 min, then centrifuged (20,000 × g, 30 min). The remaining supernatant was whole cell extract. Cytoplasmic extracts were prepared as previously described (Song et al., 2006), with some amendments. Tissue pellet was weighed and resuspended in Cyto Lysis Buffer (250 mM Sucrose, 10 mM HEPES, pH 7.6, protease and phosphatase inhibitors) in a volume 4 times the weight of tissue (e.g. 4 ml/1 g), then Dounce homogenized with pestle A (8 strokes). The resulting homogenate was then subjected to a series of centrifugation steps whereupon only the supernatant was retained. Step 1, 1,000 × g, 10min. Step 2, 2,000 × g, 15min. Step 3, 8,000 × g, 5min. Step 4, 20,000 × g, 30min. The supernatant remaining after the final centrifugation was the cytoplasmic extract.\n\nSDS-PAGE/immunoblotting was performed using standard methods. NuPAGE 4–12% polyacrylamide gels (Life Technologies) and NuPAGE LDS sample buffer (with β-mercaptoethanol) were used for SDS-PAGE, and proteins were wet-transferred using a Bio-Rad system (20 mM Tris-HCl, 150 mM glycine, 20% methanol, 0.02% SDS) to PVDF membranes (Millipore) for immunoblotting. 5% skim milk (Millipore) in TBS-T (Tris-HCl buffered saline, 0.01% tween-20) was used as a blocking agent, and TBS-T was used in all washing steps. ECL Prime (GE) was used as an HRP chemiluminescent detection substrate, followed by exposure to film (Denville). CN (clear native) PAGE for the detection of mononucleosomes was performed similarly to previously described BN (blue native) PAGE, except Coomasie blue G dye was omitted from all buffers (Kim et al., 2014). The sample and 1Kb Plus DNA Ladder (NEB) were mixed with loading dye to a final concentration of 0.17 mg/ml Orange G dye (TCI) and 5% glycerol in 10 mM Bis-Tris (pH 7.0). Samples and DNA ladder were separated on Native PAGE 4–16% Bis-Tris gel (Life Technologies) in 1× anode buffer (50 mM Bis-Tris/HCl [pH 7.0]) and 1× cathode buffer (50 mM Tricine [pH 7.0], 15 mM Bis-Tris) at 4°C.\n\nThe following antibodies were used:\n\n– anti-CLOCK (Abcam, cat# ab3517),\n\n– anti-BMAL1 (generated by the Weitz Laboratory of Harvard Medical School against TDKDDPHGRLEYAEHQGRC and previously described in Tamayo et al., 2015),\n\n– anti-PER2 (ADI, cat# PER21-A),\n\n– anti-CRY1 (Abcam, cat# ab54649),\n\n– anti-HISTONE3 (Abcam, cat# ab1791),\n\n– ECL anti-Rabbit IgG-HRP linked Ab (GE, cat# NA9340V).\n\nAll antibodies were polyclonal and raised in rabbits.\n\nTwo oligonucleotide designs were used:\n\n1) Binding/quantitation oligonucleotides,\n\n2) Mononucleosome assembly oligonucleotides.\n\nEach design had two forms: E-box DNA, and control DNA. E-box DNA-binding/quantitation oligonucleotides contained three known CLOCK-BMAL1 binding sites from the Per1 locus, consisting of a canonical E-box sequence (CACGTG) and 10 bp of flanking sequence (Gekakis et al., 1998). E-box DNA-mononucleosome assembly oligonucleotides contained two copies of each E-box binding site (total of 6 E-box sequences) for a total length of 166bp. Mononucleosome formation requires a minimum of 145bp (Luger et al., 1997). Control DNA forms were identical to E-box DNA, except that the E-box sequences were scrambled (GCCTGA). All oligonucleotides contained three restriction enzyme sites (SmaI, XhoI, and HpaI) near the 5’ end for native protein elution. The sense strand of each oligonucleotide pair was labeled with a single biotin moiety at the 5’ end.\n\nDNA binding of native clock proteins was performed as previously described (Tamayo et al., 2015) with some amendments. Briefly, sense and anti-sense strands of ssDNA binding/quantitation oligonucleotides were combined (1 µM final) and heated to 94°C for 10 min in high salt annealing buffer (10 mM Tris-HCl, 300 mM NaCl, 2.5 mM MgCl2, 0.05% tween-20), then allowed to cool for 1 hr at 25°C to form dsDNA. 150 µl of dsDNA was incubated with 50 µl Dynabeads M-270 Streptavidin (Life Technologies) for 30 min at room temperature (RT). Unbound DNA was washed away with nuclear lysis buffer or cyto lysis buffer. 50 µl of immobilized DNA was incubated with 150 µl of tissue extract (nuclear or cytoplasmic), and incubated for 1 hr at 4°C. Beads were then washed 3× with nuclear lysis buffer prior to elution by 50 µl LDS sample buffer at 98°C for 5 min.\n\nIn PCR tubes (Axygen), 10 µl of Dynabeads M-270 Streptavidin (Life Technologies) were incubated with 100 µl of a concentration range between 1 nM and 100 nM dsDNA binding/quantitation oligonucleotide for 15 min at RT, then washed 3× with high salt annealing buffer. Beads were incubated with 6–10 µl of extract (unless a range of extract concentrations is specified) in a final volume of 100 µl nuclear lysis buffer for 30 min at 4°C, then washed 3× with nuclear lysis buffer. Bead-DNA-clock protein conjugates were then incubated with primary antibody (anti-CLOCK or anti-BMAL1) at 1:1000 dilution in TBS-T for 10 min at RT, washed 3× with TBS-T, then incubated with anti-Rabbit IgG (HRP linked) at 1:1000 for 10 min at RT, then washed 3× with TBS-T. The equivalent of 2.5 µl of beads (unless a range is specified) was diluted into 50 µl (final volume) TBS-T in a black/clear bottom 96-well plate (Greiner). 50 µl of ECL Prime chemiluminescent substrate (GE) was added to the well. Data was collected using a Victor3V multi-label reader (Perkin-Elmer) with a 425/60 nm filter. Data analysis and figure preparation were performed using Excel and PowerPoint 2013 (Microsoft).\n\nImmunoprecipitation of PER2-FH and associated proteins was performed as previously described (Kim et al., 2014). Briefly, nuclear extracts from livers of Per2-FH mice were incubated with FLAG-M2 agarose beads (Sigma-Aldrich) for 2 hr at 4°C. Beads were washed four times with Buffer C (50 mM Tris-HCl, 250 mM NaCl, 1.5 mM MgCl2, and 0.2% TX-100, pH 7.5). PER2-FH complexes were eluted with 100 μg/ml FLAG peptide (Sigma-Aldrich) in Buffer C for 30 min at 4°C.\n\n1.7 µl of 800,000 units/mg λ-phosphatase (NEB) were combined with every 6 µl of nuclear extract and incubated in 100 µl of NEBuffer for PMP (50 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.01% Brij 35, pH 7.5) supplemented with 1 mM MnCl2, for 45 min at 30°C. This reaction was then analyzed with the CPDBA.\n\nMouse hepatoma cells Hep-1c1c7 (ATCC, CRL-2026) were chosen because they are a mouse cell line derived from liver tissue and have been shown to possess functional circadian clocks (Tong et al., 2010; Yin et al., 2010). Cells were grown in DMEM (Gibco, 1 g/L glucose, L-glutamine, 110 mg/L sodium pyruvate) supplemented with 10% heat inactivated FBS (Atlas), penicillin/streptomycin (Corning) and MEM nonessential amino acids (Cellgro) at 37°C. Cells were passaged using Trypsin/EDTA (Corning).\n\nHep-1c1c7 cells were allowed to grow for an additional 48 hrs after they reached 90% confluency prior to treatment. Cell density was an important determinant for successful CPDBA. Cells were incubated with 10 μM of PF670462 or equal volume of DMSO (vehicle) for 48 hrs. To prepare whole cell extracts, PBS washed cells were resuspended in whole cell extract buffer (10mM Tris-HCl, 300mM NaCl, 1.5mM MgCl2, 1mM EDTA, 0.5% TX-100, pH 7.4, protease and phosphatase inhibitors) in a volume 4× the weight of tissue (e.g. 4 ml/1 g), incubated on ice for 30 min, then centrifuged at 20,000 × g for 30 min at 4°C. The remaining supernatant was whole cell extract. 10 µl of whole cell extract diluted in 100 µl final volume of whole cell extract buffer were used for the CPDBA.\n\nPAGE purified ssDNA mononucleosome assembly oligonucleotides (IDT) were annealed by incubation in high salt annealing buffer (10 mM Tris-HCl, 300 mM NaCl, 2.5 mM MgCl2, 0.05% Tween-20) for at least 1 hr at RT. The salt concentration of the annealed product was diluted to 150 mM NaCl using low salt annealing buffer (10 mM Tris-HCl, 50 mM NaCl, 1.5 mM MgCl2, 0.05% Tween-20) and run on a 2.5% agarose (TBE) gel. The gel between 100 bp and 200 bp was excised, and DNA was extracted using the QIAEX II Agarose gel extraction protocol (Qiagen). The concentration and quality of the resulting dsDNA mononucleosome assembly oligonucleotides were estimated by a spectrophotometer (NanoDrop). The Chromatin Assembly Kit (Active Motif) was used to form mononucleosomes with a few modifications to the protocol. Concentrations of chaperones (hNAP-1 and ACF complex) and HeLa core histones were doubled, and the final incubation was performed for 30 min at 37°C. Mononucleosome formation was observed by running samples on 4–16% native PAGE Bis-Tris gels (Life Technologies) using CN PAGE conditions, as described above, and probing for DNA by incubating the gel in SYBR Gold Nucleic Acid Stain (Thermo Fisher Scientific). To immobilize mononucleosomes, 20 μl of the mononucleosome assembly reaction were incubated with 25 μl of Dynabeads M270 (Life Technologies) for 45 min at 4°C. Beads bound to mononucleosomes were washed 1× with high salt buffer (10 mM Tris pH7.4, 300 mM NaCl, 1.5 mM MgCl2, 0.1% Igepal-CA 630 [Sigma-Aldrich]) and 2× with low salt annealing buffer. Varying concentrations of liver nuclear extracts were incubated with immobilized mononucleosomes for 30 min at RT. Beads were washed 1× with high salt buffer and 2× with wash buffer (10 mM Tris-HCl, 150 mM NaCl, 1.5 mM MgCl2, 0.1% TX-100). Beads were analyzed by the CPDBA as described above, or proteins were eluted with LDS sample buffer as described above for SDS-PAGE/immunoblotting.\n\nE-box DNA-binding/quantitation oligonucleotide sense:\n\n5’AGTAGTGTTAACCCCGGGCTCGAGCAGTATTTAGCCACGTGACAGTGTAAGCACACGTGGGCCCTCAAGTCCACGTGCAGGGA3’\n\nE-box DNA-binding/quantitation oligonucleotide anti-sense:\n\n5’TCCCTGCACGTGGACTTGAGGGCCCACGTGTGCTTACACTGTCACGTGGCTAAATACTGCTCGAGCCCGGGGTTAACACTACT3’\n\nControl DNA-binding/quantitation oligonucleotide sense:\n\n5’AGTAGTGTTAACCCCGGGCTCGAGCAGTATTTAGCCTGAGCACAGTGTAAGCACTGAGCGGCCCTCAAGTCCTGAGCCAGGGA3’\n\nControl DNA-binding/quantitation oligonucleotide anti-sense:\n\n5’TCCCTGGCTCAGGACTTGAGGGCCGCTCAGTGCTTACACTGTGCTCAGGCTAAATACTGCTCGAGCCCGGGGTTAACACTACT3’\n\nE-box DNA-mononucleosome assembly sense:\n\nBiotin5’AGTAGTGTTAACCCCGGGCTCGAGCAGTATTTAGCCACGTGACAGTGTAAGCACACGTGGGCCCTCAAGTCCACGTGCAGGGAAGTAGTGTTAACCCCGGGCTCGAGCAGTATTTAGCCACGTGACAGTGTAAGCACACGTGGGCCCTCAAGTCCACGTGCAGGGA3’\n\nE-box DNA-mononucleosome assembly anti-sense:\n\n5’TCCCTGCACGTGGACTTGAGGGCCCACGTGTGCTTACACTGTCACGTGGCTAAATACTGCTCGAGCCCGGGGTTAACACTACTTCCCTGCACGTGGACTTGAGGGCCCACGTGTGCTTACACTGTCACGTGGCTAAATACTGCTCGAGCCCGGGGTTAACACTACT3’\n\nControl DNA-mononucleosome assembly sense:\n\nBiotin5’AGTAGTGTTAACCCCGGGCTCGAGCAGTATTTAGCCTGAGCACAGTGTAAGCACTGAGCGGCCCTCAAGTCCTGAGCCAGGGAAGTAGTGTTAACCCCGGGCTCGAGCAGTATTTAGCCTGAGCACAGTGTAAGCACTGAGCGGCCCTCAAGTCCTGAGCCAGGGA3’\n\nControl DNA-mononucleosome assembly anti-sense:\n\n5’TCCCTGGCTCAGGACTTGAGGGCCGCTCAGTGCTTACACTGTGCTCAGGCTAAATACTGCTCGAGCCCGGGGTTAACACTACTTCCCTGGCTCAGGACTTGAGGGCCGCTCAGTGCTTACACTGTGCTCAGGCTAAATACTGCTCGAGCCCGGGGTTAACACTACT3’\n\n\nResults\n\nWe have developed a method to quantitate native CLOCK-BMAL1 DNA binding on immobilized E-box DNA, termed the clock protein-DNA binding assay, or CPDBA (Figure 1A). 10 µl of nuclear extract from wildtype mouse liver (WT extract) was incubated with immobilized E-box DNA or scrambled E-box DNA (Control DNA) at a single concentration. As an additional control for specificity, we performed parallel experiments using nuclear extracts harvested from Bmal1−/− knockout animals (BKO Extract). Upon extract incubation and anti-BMAL1/secondary antibody incubation with immobilized DNA, varying amounts of immobilized-DNA-protein-antibody (or CLOCK-BMAL1-DNA in the case of WT extract incubated with E-box DNA) were incubated with HRP chemiluminescent substrate, and luminescence data was collected. The presence of BMAL1, as detected by HRP chemiluminescence, was shown to be virtually linear within a given range of CLOCK-BMAL1-DNA, and significantly greater than all control signals (Figure 1B/CPDBA data). This experiment was repeated with a single concentration of CLOCK-BMAL1-DNA using anti-CLOCK (Figure 1C/CPDBA data) and anti-BMAL1 for detection (Figure 1D/CPDBA data), demonstrating a ~5 fold and ~10 fold signal increase over control conditions, respectively. The control conditions were nearly identical to each other, indicating that little or no CLOCK binds to E-box DNA in the absence of BMAL1 in vitro. In addition, BMAL1 quantitation showed a dose-dependent relationship with both tissue extract (Figure 1E/CPDBA data) and DNA concentration (Figure 1F/CPDBA data).\n\n(A) CPDBA design. E-box DNA (or E-box scrambled Control DNA) is immobilized onto a bead substrate. Immobilized DNA is incubated with cell or tissue extract, washed, incubated with primary antibody against CLOCK or BMAL1, washed, incubated with secondary antibody (HRP linked), then washed a final time. The immobilized antibody-protein-DNA complex is then incubated with chemiluminescent substrate (ECL), and analyzed by spectrophotometry (luminescence at 425/60 nm). Counts are arbitrary units. (B) CPDBA was applied to WT nuclear extracts or Bmal1−/− nuclear extracts (KO). The x-axis represents the amount of immobilized antibody-protein-DNA complex (in µl of magnetic beads), used in the final step of the CPDBA, and in the case of WT extract incubated with E-box DNA, corresponds to CLOCK-BMAL1-DNA probed withanti-BMAL1. This experiment was repeated (n=3) with a single volume of immobilized antibody-protein-DNA complex using anti-CLOCK (C) or anti-BMAL1 (D). BMAL1 binding to immobilized DNA was measured from a series of WT nuclear extract dilutions while keeping the DNA concentration constant (E), or a series of DNA concentrations were used while keeping the extract concentration constant (F).\n\nAs a quality control, we performed similar DNA binding experiments using SDS-PAGE/Immunoblotting to qualitatively assess clock protein binding to E-box DNA (see Supplementary Material). We observed that very little CLOCK and BMAL1 were bound to PER2 from cytoplasmic extracts (C) as compared to nuclear extracts (N), as shown by Anti-FLAG co-immunoprecipitation experiments from extracts containing PER2-FLAG-HA (Figure S1A/Uncropped Figure S1A-B). Since PER2 and CRY1 are known to bind E-box DNA through their interactions with CLOCK-BMAL1, this observation allowed us to use cytoplasmic extracts as an additional negative control for clock protein DNA binding in vitro. Nuclear or cytoplasmic extracts were incubated with immobilized E-box DNA or Control DNA (scrambled E-box), and bound proteins were analyzed by SDS-PAGE/immunoblotting for PER2, CRY1, CLOCK and BMAL1. Nuclear but not cytoplasmic PER2, CRY1, CLOCK and BMAL1 bound to E-box DNA (Figure S1B/Uncropped Figure S1C), further demonstrating specific clock protein interactions with E-box DNA in vitro. Nuclear and cytoplasmic markers were distributed as expected (Figure S2/Uncropped Figure S2). While these results do not preclude the existence of BMAL1 in the cytoplasm, as previously reported by Kwon et al., 2006; Lipton et al., 2015, they suggest that BMAL1 is a predominately nuclear protein.\n\nTaken together, these experiments demonstrate that native CLOCK-BMAL1 DNA binding can be relatively quantitated using tissue extracts as a source of protein and naked DNA as a binding substrate.\n\nSeveral studies have demonstrated the circadian rhythmicity of CLOCK-BMAL1 E-box DNA binding in vivo, as observed by chromatin immunoprecipitation (ChIP) (Duong et al., 2011; Koike et al., 2012; Ripperger & Schibler, 2006). We asked if CLOCK-BMAL1 DNA binding activity would also oscillate when measured by the CPDBA.\n\nNuclear extracts were prepared from livers harvested from wildtype mice every 4 hours across circadian time or CT (see methods), and analyzed by SDS-PAGE/immunoblotting for CLOCK, BMAL1, PER2 and CRY1 (Figure 2A/Uncropped Figure 2). CLOCK and BMAL1 levels were mostly stable across the day, while PER2 and CRY1 levels were highly rhythmic. We then applied the CPDBA to these extracts to monitor CLOCK (Figure 2B/CPDBA data) and BMAL1 (Figure 2C/CPDBA data) DNA binding. In both cases, DNA binding reached its peak at CT4 and its trough 12 hours later at CT16, revealing very similar binding patterns to those demonstrated by in vivo ChIP experiments. These results show that rhythmic CLOCK-BMAL1 DNA binding activity can be recapitulated using the CPDBA, validating its use as a probe of CLOCK-BMAL1 function.\n\n(A) Nuclear extracts were prepared from mouse livers harvested over circadian time (CT). Extracts were analyzed by SDS-PAGE/immunoblotting for the presence of CLOCK, BMAL1, PER2 and CRY1. (B, C) CPDBA was applied to these extracts to measure DNA binding by CLOCK (B) or BMAL1 (C) to E-box DNA. Data from technical replicates using extracts from a single mouse are displayed for CLOCK (n=3), and normalized data from multiple mice are displayed for BMAL1 (n=3) The y-axis represents the amount of CLOCK or BMAL1 binding to DNA as measured by the CPDBA (luminescence).\n\nPhosphorylation is the most extensively studied post-translational modification involved in circadian clocks, and has been implicated in the regulation of CLOCK-BMAL1 DNA binding (Dardente et al., 2007; Kondratov et al., 2006; Lee et al., 2014; Reischl & Kramer, 2011; Yoshitane et al., 2009; Wang et al., 2013). Nuclear extracts prepared from mouse liver were treated with λ-phosphatase or mock conditions, and analyzed by SDS-PAGE/immunoblotting for CLOCK and BMAL1 (Figure 3A/Uncropped Figure 3A), indicating similar levels in both conditions. CPDBA was then applied to these extracts to quantitate CLOCK (Figure 3B/CPDBA data) and BMAL1 (Figure 3C/CPDBA data) DNA binding to E-box DNA or scrambled E-box DNA (Control DNA). Data across experiments were normalized to the mock treated/E-box DNA bound sample. DNA binding activity increased between 1.5- to 2-fold upon treatment of tissue extract with phosphatase using either antibody.\n\n(A) Liver nuclear extracts were treated with lambda phosphatase (λPPase) or mock buffer (Mock). Extracts were analyzed by SDS-PAGE/immunoblotting for the presence of PER2, CLOCK and BMAL1. (B, C) CPDBA was applied to these extracts to measure binding of CLOCK (B) or BMAL1 (C) to E-box DNA. All data were normalized to the E-box DNA/mock treated condition (normalized to 1). (* P<0.0001, two-tailed, unequal variance). (D) Extracts were made from Hep-1c1c7 cells incubated with vehicle (DMSO) or 10 µM CKIϵ/δ inhibitor PF670462. Extracts were analyzed by SDS-PAGE/immunoblotting for PER2, CLOCK, BMAL1 and CKIδ. (E, F) CPDBA was applied to these extracts to measure binding of (E) CLOCK and (F) BMAL1 to E-box DNA. All data were normalized to the E-box DNA/vehicle treated condition (normalized to 1). (*P=<0.0001, two-tailed, unequal variance).\n\nPharmacological inhibition of CKIϵ/δ has previously been shown to severely disrupt circadian rhythms (Isojima et al., 2009; Meng et al., 2010). In this study, mouse hepatoma cells in culture were incubated with a specific kinase inhibitor of CKIϵ/δ (PF670462, IC50 for CKIϵ and CKIδ are 7.7 nM and 14 nM, respectively) or DMSO (vehicle) for 48 hours. This treatment did not discernably affect cell viability. Whole cell extracts were analyzed by SDS-PAGE/immunoblotting for CLOCK and BMAL1 (Figure 3D/Uncropped Figure 3B), indicating similar levels in each condition. CPDBA was applied to these extracts to quantitate CLOCK (Figure 3E/CPDBA data) or BMAL1 (Figure 3F/CPDBA data), and the data across experiments were normalized to the vehicle treated/E-box DNA incubated set. Pharmacological inhibition of CKIϵ/δ resulted in a ~1.5-fold increase of CLOCK-BMAL1 DNA binding activity. Taken together, these results demonstrate the sensitivity and versatility of the CPDBA by capturing less than 2-fold differences in CLOCK-BMAL1 DNA binding activity in a tissue extract and cell culture model of clock modulation.\n\nPrevious studies have indicated that chromatin modifications alter clock protein access to gene regulatory sites (Brown et al., 2005; Doi et al., 2006; Duong et al., 2011; Duong & Weitz, 2014; Etchegaray et al., 2003; Kim et al., 2014; Koike et al., 2012; Ripperger & Schibler 2006; Tamayo et al., 2015). Here we asked if native CLOCK-BMAL1 binding to reconstituted nucleosomes can be measured using the CPDBA.\n\nMononucleosomes were reconstituted using histone octamers, chromatin assembly chaperones and a biotin tagged 166 base pair oligonucleotide containing E-box sequences or scrambled E-box sequences (Control DNA), then analyzed by CN PAGE/fluorescence DNA labeling (Figure 4A). Reaction mixtures containing histone octamers shifted the oligonucleotide’s apparent molecular weight to ~600bp, indicating the formation of mononucleosomes (Figure 4A) (White et al., 2016). Mononucleosomes containing E-box sequences or scrambled E-box sequences (Control DNA) were immobilized using streptavidin coated magnetic beads, then incubated with nuclear extracts prepared from mouse liver tissue. Bound proteins were analyzed by SDS-PAGE/immunoblotting for CLOCK, BMAL1 and HISTONE3 (Figure 4B/Uncropped Figure 4), demonstrating that CLOCK-BMAL1 bound specifically to E-box sequences within mononucleosomes in vitro. We applied the CPDBA to varying concentrations of nuclear extract using anti-CLOCK (Figure 4C/CPDBA data), demonstrating a relationship between extract concentration and E-box specific CLOCK binding to mononucleosomes.\n\n(A) Free dsDNA containing E-box DNA or scrambled E-box DNA (Control DNA) was incubated with or without core histones, and reconstituted mononucleosomes were analyzed using CN-PAGE stained with SYBR gold. (B) Immobilized mononucleosomes were incubated with liver nuclear extracts. CLOCK-BMAL1 binding to mononucleosomes was assessed using SDS-PAGE/immunoblotting for CLOCK, BMAL1, and HISTONE3. (C) Immobilized mononucleosomes were used in place of naked DNA in CPDBA to measure CLOCK binding to E-box DNA within mononucleosomes using a series of nuclear extract concentrations (n=3).\n\n\nDiscussion\n\nPreviously, we coupled DNA binding selection to quantitative mass spectrometry and discovered novel CLOCK-BMAL1 interacting proteins and chromatin modifying activities (Tamayo et al., 2015). Here, we present a simple method to measure native CLOCK-BMAL1 DNA and chromatin binding activity from tissue or cell extracts that we term the clock protein-DNA binding assay (CPDBA). Using the CPDBA, we reproduced rhythmic CLOCK-BMAL1 binding from crude tissue extracts in a manner strikingly similar to previously reported chromatin immunoprecipitation (ChIP) patterns (Duong et al., 2011; Etchegaray et al., 2003; Kim et al., 2014; Koike et al., 2012; Rey et al., 2011; Ripperger & Schibler, 2006; Tamayo et al., 2015). In addition, we show that the CPDBA can be adapted to quantify CLOCK-BMAL1 binding to reconstituted chromatin, in the form of mononucleosomes. These results indicate that the CPDBA is a viable tool for measuring native CLOCK-BMAL1 DNA binding activity. As such, the CPDBA could complement a variety of research approaches that require monitoring of circadian clock function.\n\nTo demonstrate the versatility of the CPDBA, we used it to measure CLOCK-BMAL1 DNA binding activity in both tissue and cell culture extracts, while also using two different approaches to modulate CLOCK-BMAL1 activity. Phosphatase treatment of tissue extracts increased CLOCK-BMAL1 DNA binding as measured by the CPDBA, while treating cells with a specific inhibitor of Casein Kinase ϵ/δ (CKIϵ/δ) yielded similar results. In both cases, the differences between control and experimental conditions were 2-fold or less, demonstrating the sensitivity of the CPDBA. We have successfully performed the CPDBA with as little as 6 µl of nuclear extract (~6 µg protein) in 2 hours. While this is already fast and efficient, the CPDBA can likely be improved by further optimization.\n\nThe CPDBA is similar to previously reported assays developed for different DNA binding proteins (Brand et al., 2010; Fischer et al., 2016). While not modeled upon previously described assays, the CPDBA shares features that make it amenable to high throughput approaches, with the potential for automation (Brand et al., 2013). Furthermore, this method can theoretically be applied to tissue or cells of virtually any source; an important feature given that functional clocks have been observed in most tissues. In conclusion, we submit the CPDBA as a sensitive, fast, efficient and versatile probe of clock function.\n\n\nData availability\n\nDataset 1: Uncropped images of SDS-PAGE/Immunoblots used to construct Figures 2A, 3A, 3D, 4B, S1A, S1B and S2. DOI, 10.5256/f1000research.11685.d169055 (Gillessen et al., 2017a)\n\nDataset 2: CPDBA data. Raw data generated by the CPDBA used to construct Figures 1B–F, 2B, 2C, 3B, 3C, 3E, 3F and 4C. DOI, 10.5256/f1000research.11685.d169056 (Gillessen et al., 2017b)",
"appendix": "Author contributions\n\n\n\nA.G.T. conceived of the study and designed experiments. M.G., P.B.K. and A.G.T. carried out the research. M.G. contributed as part of a student internship (University of Namur, Belgium) at Harvard Medical School. A.G.T. prepared the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the G. Harold and Leila Y. Mathers Charitable Foundation (CJW) and the U.S. National Institutes of Health Training Grant in Sleep, Circadian, and Respiratory Neurobiology (T32HL07901 to AGT).\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 Charles J. Weitz in the Department of Neurobiology of Harvard Medical School for allowing to us to perform this work in his laboratory. We thank Michael Gebert (currently at Baxter International) for contributing to extract preparations. We thank Ming Liu (Harvard Medical School) for managing mouse colonies. We also thank Charles J. Weitz, Hao A. Duong and Rajindra P. Aryal for critical readings of this manuscript during preparation (Harvard Medical School).\n\n\nSupplementary material\n\nFigure S1. Native clock proteins bind specifically to immobilized E-box DNA sequences. Cytoplasmic (C) and nuclear (N) extracts were prepared from the livers of mice expressing PER2-FLAG-HA at CT16. (A) SDS-PAGE/immunoblotting was used to assess the presence of PER2, CLOCK, CRY1 and BMAL1 (equal loading by volume). Anti-FLAG (Sigma-Aldrich) or IgG negative control was used to co-immunoprecipitate clock proteins from C or N extracts. (B) Immobilized dsDNA containing E-box sequences (E-box DNA) or scrambled E-box sequences (Control DNA) were incubated with either C or N extracts. Bound proteins were eluted under denaturing conditions, then analyzed by SDS-PAGE/immuno-blotting for clock proteins.\n\nClick here to access the data.\n\nFigure S2. Segregation of nuclear and cytoplasmic markers from mouse liver extracts. Nuclear and cytoplasmic extracts were prepared from mouse livers harvested over circadian time. Extracts were analyzed by SDS-PAGE/immuno-blotting for the presence of RNA polymerase II (nuclear marker) and tubulin (cytoplasmic marker).\n\nClick here to access the data.\n\n\nReferences\n\nAsher G, Schibler U: Crosstalk between components of circadian and metabolic cycles in mammals. Cell Metab. 2011; 13(2): 125–37. PubMed Abstract | Publisher Full Text\n\nBrand LH, Henneges C, Schüssler A, et al.: Screening for protein-DNA interactions by automatable DNA-protein interaction ELISA. 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}
|
[
{
"id": "24786",
"date": "17 Aug 2017",
"name": "Yoshitaka Fukada",
"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 present work, the authors developed a novel method to detect DNA binding activity of CLOCK-BMAL1 complex with a fixed DNA probe containing an E-Box repeat. The method is based on a simple procedure and could be useful for studies including high-throughput investigation of the regulatory mechanism on CLOCK-BMAL1.\nIt is desirable to compare the new method with previously established ones, e.g., EMSA and ChIP-PCR in parallel experiments by using the same samples. Then, readers could easily understand advantage/disadvantage of the present assay, in terms of detection limit, the range, and sensitivity etc.\nThe data in Figure 3 demonstrate enhanced DNA-binding activities of CLOCK:BMAL1 by pre-treatment with lambda phosphatase and CKI-epsilon/delta inhibitor (PF670462). We are interested in a potential change of the CLOCK-BMAL1 activity under previously described conditions. For example, we reported several treatments that reduce the transactivation ability of CLOCK-BMAL1 complex: MAPK-dependent phosphorylation of BMAL1, phospho-mimic mutation in CLOCK, reduced CLOCK-BMAL1 dimerization under CaMKII inhibition [1,2,3]\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "3008",
"date": "08 Sep 2017",
"name": "Alfred Tamayo",
"role": "Author Response",
"response": "Dear Dr. Fukada and Dr. Kon. A new version of this manuscript has been submitted with the changes indicated below.1.) “It is desirable to compare the new method with previously established ones, e.g., EMSA and ChIP-PCR in parallel experiments by using the same samples. Then, readers could easily understand advantage/disadvantage of the present assay, in terms of detection limit, the range, and sensitivity etc.”Answer: While it is beyond the scope of this work to perform parallel experiments with other techniques, we comment on their comparison in our revised Discussion section: “In addition, we show that the CPDBA can be adapted to quantify CLOCK-BMAL1 binding to reconstituted chromatin, in the form of mononucleosomes. This variation of the CPDBA could be used to provide in vivo ChIP studies with mechanistic insights.… We also performed related experiments using an electrophoretic mobility shift assays (EMSA) to monitor the effect of phosphatase on purified nuclear PER complex (containing CLOCK-BMAL1), and we again detected an increase in DNA binding upon phosphatase treatment (Aryal and Kwak et al., 2017). EMSA may surpass the CPDBA in sensitivity and detection limit, since it can use radiolabeled DNA probes to quantify protein-DNA binding. However, the CPDBA is less technically cumbersome and more scalable than EMSAs.”2.) “The data in Figure 3 demonstrate enhanced DNA-binding activities of CLOCK:BMAL1 by pre-treatment with lambda phosphatase and CKI-epsilon/delta inhibitor (PF670462). We are interested in a potential change of the CLOCK-BMAL1 activity under previously described conditions. For example, we reported several treatments that reduce the transactivation ability of CLOCK-BMAL1 complex: MAPK-dependent phosphorylation of BMAL1, phospho-mimic mutation in CLOCK, reduced CLOCK-BMAL1 dimerization under CaMKII inhibition [1,2,3]”Answer: The complex relationship between phosphorylation and CLOCK-BMAL1 regulation is indeed interesting and requires a comprehensive understanding of the causal relationship between site-specific phosphorylation, biochemical activity and biological consequence. As you point out, it would be interesting to interrogate the roles of other kinases, such as MAPKs and CaMKII in CLOCK-BMAL1 DNA binding using the CPDBA. We should note that dose and exposure time would need to be optimized in any kinase inhibitor study, as was done for PF670462, though we do not mention it in the manuscript. We must also point out however that this assay cannot assess CLOCK-BMAL1 transactivation activity downstream of DNA binding.Also, we have changed how your work is cited in the Results section to better distinguish it from the other works cited.Best Regards"
}
]
},
{
"id": "24783",
"date": "21 Aug 2017",
"name": "Katja A. Lamia",
"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\nGillessen, Kwak, and Tamayo describe a method for measuring the interaction of CLOCK and BMAL1 purified from cellular or tissue extracts with either naked DNA or nucleosomes including an established CLOCK-BMAL1 binding sequence containing several E-boxes. The manuscript is well written, and the method will be broadly useful, especially as it could be adapted to other transcription factors by altering the target DNA sequences. Given that it is a method description, it is important that all details of the protocol are clear and I include here some suggestions for clarification.\n\nMaterials and methods:\nIn the description of the preparation of tissue extracts, the following is insufficiently clear: “…tissue was Dounce homogenized with pestle A, centrifuged, then Dounce homogenized with pestle B, followed by…” Was the supernatant discarded after centrifugation between homogenization with pestle A and pestle B? If so, was the pellet resuspended again in the same buffer?\n\nPlease provide detailed composition and volumes of buffers used for initial homogenization and sucrose cushion centrifugation.\n\nIn the list of antibodies for the last item, either delete “ECL” or include the full product name “Amersham ECL Rabbit IgG, HRP-linked F(ab’)2 fragment (from donkey)”. Also, I can’t find a product number with a “V” at the end – is that a typo?\n\nEither place the oligonucleotide sequences within the section describing “DNA-binding oligonucleotide design” or include a sentence stating that the sequences are listed below.\n\nNowhere in the manuscript does it state the concentration of extracts that is used in the assays and this is critical information. Perhaps this was meant to be included in the section describing the CPDBA where it states “Beads were incubated with 6-10 l of extract (unless a range of concentrations is specified)…” ?\n\nThe section titled “Pharmacological inhibition of CKIe/d”also includes details of preparation of cell extracts, which might fit more logically as a separate section following preparation of tissue extracts.\n\nResults :\nRelated to point #5 above, there is no mention of the concentration of extract used in experiments shown in Fig. 1. What does arbitrary units refer to on the x and y axes in Fig. 1B? In Fig. 1E and Fig. 4C, the x axis is labeled “fold dilution” or “l extract” but it would be much more informative to know what concentration of extract is included in the assay.\n\nIn Figs 3A and 3D, the legend refers to Western blot detection of PER2 and CKId that does not appear in the figure.\n\nThe text states that there are similar levels of CLOCK and BMAL1 protein in each condition in Fig 3D but the figure shows much less CLOCK and BMAL1 protein in the extract treated with PF671462. Both Fig. 3A and 3D should include a loading control blot as well. Since the change in protein amounts appears to be opposite to the change in binding measured, this does not invalidate the conclusions but the data should be described accurately in the text.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "3009",
"date": "12 Sep 2017",
"name": "Alfred Tamayo",
"role": "Author Response",
"response": "Dear Dr. Lamia. A new version of this manuscript has been submitted with the changes indicated below. 1.)”In the description of the preparation of tissue extracts, the following is insufficiently clear: “…tissue was Dounce homogenized with pestle A, centrifuged, then Dounce homogenized with pestle B, followed by…” Was the supernatant discarded after centrifugation between homogenization with pestle A and pestle B? If so, was the pellet resuspended again in the same buffer?” Answer: Please see changes made to Materials and Methods: “The pellet was resuspended withn 8 ml/1 gr Dounce Buffer (3 ml PBS + 5ml Homogenization Buffer: 2.2 M sucrose, 15 mM KCl, 2 mM EDTA, 10 mM HEPES, pH 7.6), then diluted with 22 ml Homogenization Buffer (assuming 1 gr tissue)” 2.) “Please provide detailed composition and volumes of buffers used for initial homogenization and sucrose cushion centrifugation.” Answer: Requested information added to Materials and Methods: “…Hypotonic Lysis Buffer (250 mM Sucrose, 10 mM HEPES, pH 7.6, protease and phosphatase inhibitors) in a volume 4 times the weight of tissue (e.g. 4 ml/1 g), thencentrifuged at 400 × g for 5 min. The pellet was resuspended with 8 ml/1 gr Dounce Buffer (3 ml PBS + 5ml Homogenization Buffer: 2.2 M sucrose, 15 mM KCl, 2 mM EDTA, 10 mM HEPES, pH 7.6), then diluted with 22 ml Homogenization Buffer (assuming 1 gr tissue) and Dounce homogenized with pestle B, followed by ultracentrifugation through a 10 ml sucrose cushion (2.05 M sucrose, 15 mM KCl, 2 mM EDTA, 10 mM HEPES, pH 7.6) at84,000 × g for 1 hr at 4°C to isolate nuclei (pellet)…” 3.)”In the list of antibodies for the last item, either delete “ECL” or include the full product name “Amersham ECL Rabbit IgG, HRP-linked F(ab’)2 fragment (from donkey)”. Also, I can’t find a product number with a “V” at the end – is that a typo?” Answer: Yes, the typo has been corrected and the requested information modified and added. 4.) “Either place the oligonucleotide sequences within the section describing “DNA-binding oligonucleotide design” or include a sentence stating that the sequences are listed below.” Answer: Requested sentence has been added. 5.) “Nowhere in the manuscript does it state the concentration of extracts that is used in the assays and this is critical information. Perhaps this was meant to be included in the section describing the CPDBA where it states “Beads were incubated with 6-10 l of extract (unless a range of concentrations is specified)…” ?” Answer: Changes were made to the Materials and Methods to better indicate the protein concentrations used: “Beads were incubated with ~8 µg protein or 6–10 µl (~1 µg/µl) at a final concentration of ~80 ng/µl of extract (unless a range of extract concentrations is specified)…” 6.) “The section titled “Pharmacological inhibition of CKIe/d”also includes details of preparation of cell extracts, which might fit more logically as a separate section following preparation of tissue extracts.” Answer: A separate section describing the preparation of cell extracts has been added to Materials and Methods. 7.) “Related to point #5 above, there is no mention of the concentration of extract used in experiments shown in Fig. 1. What does arbitrary units refer to on the x and y axes in Fig. 1B? In Fig. 1E and Fig. 4C, the x axis is labeled “fold dilution” or “l extract” but it would be much more informative to know what concentration of extract is included in the assay.” Answer: As noted above in the answer to point #5, a better description of the protein concentrations of extracts used in all assays, unless a concentration series is stated, has been added to Materials and Methods. In addition, the concentration equivalent to 1 in Figure 1E is now noted in the legend: “1 = 16 µg of extract or 160 ng/µl final extract concentration with magnetic beads.” In Figure 1B, AU in the x-axis is a typo and has been changed (x-axis in this case is ul of magnetic beads, as noted in the legend). Y-axis AUs are raw luminescence counts from a plate reader. 8.) “In Figs 3A and 3D, the legend refers to Western blot detection of PER2 and CKId that does not appear in the figure.” Answer: This was a typo and has been corrected. 9.)”The text states that there are similar levels of CLOCK and BMAL1 protein in each condition in Fig 3D but the figure shows much less CLOCK and BMAL1 protein in the extract treated with PF671462. Both Fig. 3A and 3D should include a loading control blot as well. Since the change in protein amounts appears to be opposite to the change in binding measured, this does not invalidate the conclusions but the data should be described accurately in the text.” Answer: Please see revised Results section addressing the description of the data: “Whole cell extracts were analyzed by SDS-PAGE/immunoblotting for CLOCK and BMAL1 indicating similar or lower levels (as shown) of CLOCK and BMAL1 in the PF670462 treated condition ( Figure 3D/Uncropped Figure 3B)…” Also, to Figures 3A and 3D we have added portions of SDS-PAGE/coomassie stained gels which we believe are equal or better than western blot controls to assess equal loading. Best Regards"
}
]
},
{
"id": "24900",
"date": "23 Aug 2017",
"name": "Joanna C. Chiu",
"expertise": [
"Reviewer Expertise Molecular chronobiology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGillessen et al. presents a simple assay to measure DNA binding activity of CLOCK-BMAL1 heterodimers, the major transcriptional activators of clock-regulated genes in animals. They show that the Clock Protein-DNA Binding Assay (CPDBA) can recapitulate CLOCK-BMAL1 DNA binding rhythms from tissue nuclear extracts, detect less than 2-fold differences, and can be used as an alternative to approaches including ChIP-qPCR and gel-shift (EMSA). As opposed to ChIP-qPCR and EMSA, CPDBA may be more amenable to automation and/or high throughput screening of mutations or chemicals to modulate CLOCK-BMAL1 DNA binding activity. I applaud them for going one step further by confirming the functionality of their assay in the context of reconstituted chromatin (mononucleosomes). Overall, this is an excellent and efficient tool, and should not be too difficult for chronobiology colleagues to adopt, especially compared to ChIP and EMSA. I, for one, am excited to try it out.\n\nSpecific comments that the authors should address are detailed below. Most of them are minor.\nThe authors emphasize on a number of occasions that crude tissue extracts can serve as input for the CPDBA (e.g. in the Abstract). In most of the experiments, nuclear extracts as opposed to whole cell extracts were used. Obviously CLOCK-BMAL1 DNA binding takes place in the nucleus, and tissue nuclear extracts will likely produce the cleanest results. Have the authors compare the results of CPDBA using whole cell extracts vs. nuclear extracts? Is it necessary to perform fractionation to collect nuclear extracts for CPDBA. If yes, perhaps the authors should include this recommendation.\n\nIn the Abstract, the authors mention the assay uses less than 10 microliters or less of crude extract. Perhaps they should specify the protein amount instead of volume. They do state in the Discussion that they assume 1 microliter equals roughly 1microgram (for their extract), but maybe they need to clarify that in the Abstract.\n\nThe CLOCK-centered Introduction is clear, concise, and well-written, highlighting the importance of studying CLOCK-BMAL1 interaction to DNA in the context of mammalian clock. The authors should consider adding a paragraph discussing factors/modifications that are known to affect DNA binding activity of CLOCK-BMAL1.\n\nCPDBA uses HRP conjugated secondary antibodies and chemiluminescent detection. Have the authors consider the use of fluorescent-labeled antibodies and multiplexing? Would fluorescent-labeled antibodies be more quantitative than chemiluminescent detection, and present less problems with signal saturation?\n\nThe mobility shift the authors intend to show in Figure 3A (and 3D) in cell extracts that are untreated and treated with lambda phosphatase is not obvious at all. Are the authors expecting a significant shift in mobility shift? The authors could try using phostag gel to accentuate the shift.\n\nIn the section for “Oligonucleotides”, the authors need to add “Biotin” to the first and third primers, just as they did for the mononucleosome assembly primers.\n\nCan the authors explain the discrepancies in Y-axis values (DNA binding activity) for Figure 2B vs 2C? The difference in CLOCK (Fig. 2B) and BMAL (Fig. 2C) binding can perhaps be explained by the use of different antibodies. But what about the difference between Figure 2C and Figure 1? The values for Figure 2C seems oddly low, given that both figures are using liver nuclear extracts.\n\nSince I am interested in the regulation of clock protein function by post-translational modifications such as phosphorylation, I would be interested to see the authors discuss the possibility of using phosphospecific antibodies (instead of the polyclonal antibodies they list) to detect if specific isoforms of CLOCK and BMAL1 bind preferentially to DNA.\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": "3010",
"date": "12 Sep 2017",
"name": "Alfred Tamayo",
"role": "Author Response",
"response": "Dear Dr. Chiu. A new version of this manuscript has been submitted with the changes indicated below. Also find below answers to your questions and comments. 1.) “The authors emphasize on a number of occasions that crude tissue extracts can serve as input for the CPDBA (e.g. in the Abstract). In most of the experiments, nuclear extracts as opposed to whole cell extracts were used. Obviously CLOCK-BMAL1 DNA binding takes place in the nucleus, and tissue nuclear extracts will likely produce the cleanest results. Have the authors compare the results of CPDBA using whole cell extracts vs. nuclear extracts? Is it necessary to perform fractionation to collect nuclear extracts for CPDBA. If yes, perhaps the authors should include this recommendation.”Answer: It is not necessary to use nuclear extracts in the CPDBA, whole cell extracts also work. However we have not performed head-to-head comparisons of nuclear extracts versus whole cell extracts, so we cannot recommend one over the other. 2.) “In the Abstract, the authors mention the assay uses less than 10 microliters or less of crude extract. Perhaps they should specify the protein amount instead of volume. They do state in the Discussion that they assume 1 microliter equals roughly 1microgram (for their extract), but maybe they need to clarify that in the Abstract.”Answer: A better description of the protein concentrations of extracts used in all assays, unless a concentration range is stated, has been added to Materials and Methods: “Beads were incubated with ~8 µg protein or 6–10 µl (~1 µg/µl) at a final concentration of ~80 ng/µl of extract (unless a range of extract concentrations is specified)…” In addition, the concentration equivalent to 1 in Figure 1E is now noted in the legend: “1 = 16 µg of extract or 160 ng/µl final extract concentration with magnetic beads.” 3.) “The CLOCK-centered Introduction is clear, concise, and well-written, highlighting the importance of studying CLOCK-BMAL1 interaction to DNA in the context of mammalian clock. The authors should consider adding a paragraph discussing factors/modifications that are known to affect DNA binding activity of CLOCK-BMAL1.”Answer: A more detailed discussion of factors affecting DNA binding may not be in keeping with the spirit of a methods article. However, such a discussion is certainly worth having in light of our findings, so we included it in this comment (below), which is also publically available.The molecular underpinnings governing CLOCK-BMAL1 DNA binding activity are not fully elucidated. Repressor proteins physically interact with CLOCK-BMAL1, suggesting steric or post-translational regulation of CLOCK-BMAL1 activity (Ye et al. 2011, Ye et al. 2014, Chiou et al. 2016, Michael et al. 2017). In addition, a body of work indicates epigenetic regulation of DNA accessibility as an underlying cause (Etchegary et al. 2003, Brown et al. 2005, Doi et al. 2006, Ripperger and Schibler 2006, Duong et al. 2011, Koike et al. 2012). Direct repression of CLOCK-BMAL1 activity and gating of chromatin accessibility are not mutually exclusive, therefore, how are these modalities integrated?Phosphorylation dependent restriction of CLOCK-BMAL1 DNA binding has been implicated by previous reports. Mutagenic replacement of serine residues with phospho-mimicking amino acids within the basic helix-loop-helix (bHLH) domains of either CLOCK (S38, S42) or BMAL1 (S78) disrupts DNA binding (Yoshitane et al. 2009, Wang et al. 2013). However, CLOCK phosphorylation at S440, S441, and S446 is correlated with CLOCK-BMAL1 activation (Robles et al. 2016). These findings point to an intricate coordination of clock protein phosphorylation tied to structure-function relationships, which we may begin to unravel using methods described in this study. 4.) “CPDBA uses HRP conjugated secondary antibodies and chemiluminescent detection. Have the authors consider the use of fluorescent-labeled antibodies and multiplexing? Would fluorescent-labeled antibodies be more quantitative than chemiluminescent detection, and present less problems with signal saturation?”Answer: Yes we have considered using fluorescent-labeled antibodies as well as other alterations to the original technique such as TR-FRET (time resolved fluorescence energy transfer) and ALPHAScreen technology, however, we haven’t tried these adaptations as yet. 5.) “The mobility shift the authors intend to show in Figure 3A (and 3D) in cell extracts that are untreated and treated with lambda phosphatase is not obvious at all. Are the authors expecting a significant shift in mobility shift? The authors could try using phostag gel to accentuate the shift.” Answer: In general, upon phosphatase treatment of extract, we did not see a mobility shift for CLOCK, we did see a subtle shift for BMAL1 and a significant shift for PER2 (not shown) using standard SDS-PAGE/immunoblotting. As you point out, a lack of shift does not exclude effective dephosphorylation. Using phostag gels is an excellent suggestion, though we have not attempted. MS analysis of immunoprecipitated proteins from phosphatase treated extracts would also be informative, if less practical. However, a more thorough analysis of the role of phosphorylation in CLOCK-BMAL1 DNA binding is beyond the scope of this manuscript. 6.) “In the section for “Oligonucleotides”, the authors need to add “Biotin” to the first and third primers, just as they did for the mononucleosome assembly primers.”Answer: The requested modification has been made to Materials and Methods. 7.) “Can the authors explain the discrepancies in Y-axis values (DNA binding activity) for Figure 2B vs 2C? The difference in CLOCK (Fig. 2B) and BMAL (Fig. 2C) binding can perhaps be explained by the use of different antibodies. But what about the difference between Figure 2C and Figure 1? The values for Figure 2C seems oddly low, given that both figures are using liver nuclear extracts prepared.”Answer: Figure 2B represents raw counts from a single set of nuclear extracts prepared simultaneously, i.e. one set of mice. While Figure 2C represents independently prepared extracts from several sets of mice. Raw counts across sets of mouse extracts were too variable to be represented in the same graph. Independently prepared extracts were used in Figure 2C and Figure 1, which is why the scales are different. We do not fully understand the reason behind this, but it is an important technical observation. 8.) “Since I am interested in the regulation of clock protein function by post-translational modifications such as phosphorylation, I would be interested to see the authors discuss the possibility of using phosphospecific antibodies (instead of the polyclonal antibodies they list) to detect if specific isoforms of CLOCK and BMAL1 bind preferentially to DNA.”Answer: This is a very interesting suggestion, as using phospho-specific antibodies in the CPDBA could give an initial indication of differential DNA binding affinities across phosphospecies either across circadian time and/or within a population of molecules at a given CT. Perhaps it would be best if the assay could be multiplexed to use phospho-state and non-phospho specific antibodies simultaneously from the same sample.Best Regards"
}
]
}
] | 1
|
https://f1000research.com/articles/6-1316
|
https://f1000research.com/articles/6-1680/v1
|
12 Sep 17
|
{
"type": "Research Article",
"title": "Factors determining access to oral health services among children aged less than 12 years in Peru",
"authors": [
"Diego Azañedo",
"Akram Hernández-Vásquez",
"Mixsi Casas-Bendezú",
"César Gutiérrez",
"Andrés A. Agudelo-Suárez",
"Sandra Cortés",
"Akram Hernández-Vásquez",
"Mixsi Casas-Bendezú",
"César Gutiérrez",
"Andrés A. Agudelo-Suárez",
"Sandra Cortés"
],
"abstract": "Background: Understanding problems of access to oral health services requires knowledge of factors that determine access. This study aimed to evaluate factors that determine access to oral health services among children aged <12 years in Peru between 2014 and 2015. Methods: We performed a secondary data analysis of 71,614 Peruvian children aged <12 years and their caregivers. Data were obtained from the Survey on Demography and Family Health 2014-2015 (Encuesta Demográfica y de Salud Familiar - ENDES). Children’s access to oral health services within the previous 6 months was used as the dependent variable (i.e. Yes/No), and the Andersen and col model was used to select independent variables. Predisposing (e.g., language spoken by tutor or guardian, wealth level, caregivers’ educational level, area of residence, natural region of residence, age, and sex) and enabling factors (e.g. type of health insurance) were considered. Descriptive statistics were calculated, and multivariate analysis was performed using generalized linear models (Poisson family). Results: Of all the children, 51% were males, 56% were aged <5 years, and 62.6% lived in urban areas. The most common type of health insurance was Integral Health Insurance (57.8%), and most respondents were in the first quintile of wealth (31.6%). Regarding caregivers, the most common educational level was high school (43.02%) and the most frequently spoken language was Spanish (88.4%). Univariate analysis revealed that all variables, except sex and primary educational level, were statistically significant. After adjustment, sex, area of residence, and language were insignificant, whereas the remaining variables were statistically significant. Conclusions: Wealth index, caregivers’ education level, natural region of residence, age, and type of health insurance are factors that determine access to oral health services among children aged <12 years in Peru. These factors should be considered when devising strategies to mitigate against inequities in access to oral health services.",
"keywords": [
"Factors Associated",
"Oral Health",
"Access",
"Oral Health Services",
"Children",
"Peru"
],
"content": "Introduction\n\nAccording to the World Health Organization, dental caries is a prevalent chronic disease, with an estimated 60%–90% of school-age children and almost all adults affected in 2012 (https://goo.gl/D8sh9n). Caries and other oral diseases may negatively affect the health and quality of life of people, particularly children1–3. Failing to treat caries might cause tooth loss that can significantly alter chewing, phonation, and occlusion2. Likewise, the presence of caries during childhood can predict the development of caries in adulthood4. High quality and timely preventive dental interventions can reduce the occurrence of caries and avoid complications in later life. However, no recent nationwide studies have analyzed the prevalence of dental caries in Peru.\n\nA national study conducted by the Ministry of Health of Peru in 2001–2002 (https://goo.gl/Zppe36) reported that the occurrence of dental caries was 90.4% of children aged 6–15 years, with 34.4% being diagnosed with dental conditions, such as dental pain or evident dental or periodontal infection, requiring urgent attention. Another study5 conducted on the Social Security Health Insurance in 2013 showed that the prevalence of caries was 79.8% and 90.4% of children aged 3–5 and 6–12 years, respectively. Although no updated nationwide data regarding the prevalence rates of dental caries are available, studies6,7 indicate that the rates remain high (75.9% for children aged 6–7 years and 91.2% for children aged 11–12 years). Thus, there is a need to evaluate and treat dental conditions in Peruvian children and improve the promotion and prevention of oral diseases. Despite this need to improve provision, it should be acknowledged that Peru is a developing country with limitations such as large socioeconomic gaps, fragmented healthcare systems8, limited provision of dental services at health centers, and few professional odontologists9–11. Moreover, cultural factors can affect children’s access to dental services, including their parents’ education and/or knowledge12. Although these factors determine access to oral health services, most factors vary by region, country, and in response to other factors9.\n\nTo tackle the problem of poor access to oral health services in Peru, a better understanding of the factors that determine access is required. This knowledge can be used to develop strategies to improve health inequalities and care provision9,13. Over the recent years, the Survey on Demography and Family Health (Encuesta Demográfica y de Salud Familiar - ENDES) has begun to include data related to oral health. Although its results have been descriptively reported nationwide, no existing studies have assessed whether any of the identified factors positively or negatively influence the oral health of Peruvian children.\n\nWe used the latest oral health data from ENDES (2014–2015) to evaluate factors that determine access to oral health services among children aged <12 years in Peru and to provide current data to empower policy makers when developing strategies to improve oral health in Peru.\n\n\nMethods\n\nWe performed a secondary analysis of data obtained from ENDES 2014 and 2015 (Dataset 1), which was performed by the National Institute of Statistics and Informatics of Peru (Instituto Nacional de Estadística e Informática - INEI). This cross-sectional survey involved self-weighted, stratified, two-stage, and independent probabilistic sampling with nationwide, regional, and urban/rural representation, in accordance with INEI specifications (https://goo.gl/2AvdZ8) (https://goo.gl/jE8Wt3). ENDES aimed to update our knowledge of the demographic and health statuses of mothers or caregivers and their children aged <5 years. Other target populations were fertile women (those aged 15–49 years) and all male and female children aged <12 years (https://goo.gl/87q8mT).\n\nA dependent variable was built to categorize minors into those who had accessed oral health services within the previous 6 months and those who had either accessed services at >6 months ago or those who had no access. Thus, access to an oral health service within the previous 6 months (yes/no) was determined to be the dependent variable.\n\nIndependent variables were contextualized by considering Andersen and col. model of access14 on the basis of predisposing, enabling, and need factors. This model has been widely used for evaluating factors that determine access to health services15. However, we could not include any health need factors because ENDES does not have any suitable variables or proxies for the measurement of the factors.\n\nPredisposing factors. We included the following variables as predisposing factors: language spoken by the caregiver (i.e., Spanish or any other language), quintil of wealth, caregivers’ educational level, area of residence, age, and sex. Wealth was calculated with the standard method used by INEI, i.e., from quintile 1 as the poorest to quintile 5 as the richest, and was defined in ENDES in terms of assets or wealth in the surveyed homes, rather than in terms of income or consumption. In ENDES, data regarding the characteristics of the houses and the availability of certain consumable goods and services directly related to the socioeconomic level were collected. Each house was then given a score, and each resident was assigned a value for the house in which they live; in this way, population quintiles of wellbeing or wealth were created following the methodology reported by Rutstein and Johnson (https://goo.gl/5fFFwz). The caregivers’ educational level was categorized into none/kindergarten, primary, secondary, and higher education. The area of residence was categorized into urban and rural. The natural regions were categorized as follows: the Lima Metropolitan area, rest of the coastline, highlands, and jungle. Age was divided into three ranges (namely 0–2, 3–5, and 6–11 years) according to Technical Health Norm data for the control of growth and development of male and female children aged <5 years, as published by the Ministry of Health of Peru (https://goo.gl/4C3Tc2). The last variable was sex (male/female).\n\nEnabling factors. The type of health insurance was considered to be an enabling factor. The Peruvian health insurance system is structured as follows: 1) public Integral Health Insurance (i.e., SIS, initials in Spanish), which is the only public health insurance in Peru and provides health services to low-income earners via health centers in small communities; 2) Social Security Health Insurance (i.e., EsSalud), which provides health services to employed workers and those depending on that worker, with centers typically located in provincial capitals; 3) Armed Forces and Police Health System for employees of these institutions and those who depend on them; and 4) private insurance for individuals who are able to pay insurance. Since the creation of SIS and the Law for Universal Insurance, 66% of the population receives care in public health centers that are managed by the Ministry of Health16. On the basis of these subsystems, we divided the enabling factor into whether participants had no insurance, SIS insurance, EsSalud insurance, Armed Forces and Police Health System insurance, or private insurance.\n\nThe ENDES database was downloaded (http://iinei.inei.gob.pe/microdatos/) and imported to the Stata statistical software v14.1 (Stata Corporation, College Station, Texas, USA). The sampling patterns were specified on the basis of the strata, expansion, and design factors using the svy command. Categorical variables were described using absolute frequencies, and weighted proportions with 95% confidence intervals were estimated. Because of the high prevalence of access to oral health services, Poisson generalized linear models were used with robust variance, and the log link function was used with the dependent variable. For the adjusted model, we used variables that showed a minimal association (p < 0.2) with access to oral health services, and prevalence ratios (PRs) were reported with 95% confidence intervals, with the assumption that p values of <0.05 were statically significant.\n\nThis study did not require the approval of our ethics committee because it only involved analysis of secondary data obtained from a public and freely accessible source which does not require the identification of participants. That retains participant anonymity.\n\n\nResults\n\nThe flow chart for the study inclusion is shown in Figure 1, and the sociodemographic characteristics of the 71,614 included children are summarized in Table 1. In total, 51% were males, 56% were aged <5 years, and 44% were aged 6–11 years. In addition, 62.6% lived in urban areas and 35.6% lived in highlands. The most common type of health insurance cover was SIS (57.8%), whereas the least common type was private insurance (0.5%). Most children belonged to the first quintile of wealth (31.6%, 26.9%, 18.93%, 13.62%, and 9.75% in the first, second, third, fourth, and fifth quintiles, respectively). The most common education level of caregivers was high school (43.02%) and the least common was none/kindergarten (3.8%). The most frequently spoken language by caregivers was Spanish (88.4%), but a sizable portion spoke other languages (11.6%).\n\nThe figure shows: number of surveyed homes, number of children aged <12 years, exclusions and final sample.\n\nSD, standard deviation.\n\nThe univariate analysis revealed that all independent variables, except sex and primary educational level, were statistically significant. After adjusting the model for all variables, we found that sex, geographical location, and language spoken were insignificant (Table 2). In this model, the probability of access to oral health services increased with age, with the probability being three times higher in the group aged 6–12 years than in the group aged 0–2 years (95% CI, 2.91–3.23). Moreover, children living in highlands had a higher likelihood of access to oral health services (PR, 1.06; 95% CI, 1.01–1.11) than those who living in the Lima Metropolitan area, rest of the coastline area, and jungle, with children in the latter two groups also showing lower probabilities of access to oral health services than those living in the Lima Metropolitan area. Another relevant finding was that children from the first quintile of wealth had lower probabilities of access to oral health services than those from the other quintiles. The probability of access to oral health services tended to increase as the quintile of wealth increased, with the probability being 1.61 times higher in the fifth quintile (95% CI, 1.48–1.74) (Table 2). Caregivers’ higher educational level was also associated with a higher probability of access to oral health services, with the highest probabilities corresponding to children whose caregivers had a higher education level (PR, 1.63; 95% CI, 1.47–1.82). In contrast, the adjusted model indicated that the language spoken by caregivers was statistically associated with the access to oral health services (Table 2).\n\nPoisson regression models were used with robust variance. PR, prevalence ratio.\n\n*Adjusted by all the variables shown in the column.\n\n\nDiscussion\n\nIn this study, we identified several factors that determined access to oral health services. We showed that wealth index, caregivers’ education level, natural region of residence, and age were significant predisposing factors, whereas the type of health insurance was a significant enabling factor. However, we did not assess need factors as determinants of access.\n\nAs expected, wealth influenced the probability of access to oral health services among Peruvian children aged <12 years, which is compatible with the findings in studies of other Latin American countries and other countries worldwide. It is globally established that wealth plays a key role in oral health services, from access as well as prevention of oral disease for maintenance of better hygiene habits17–19. However, universal health insurance, aimed at covering the population living in poverty and extreme poverty and reaching 66% of the population in 2011, should improve the negative impact of the lack of resources on access to health services20. Although this does not appear to be the case universally, programs such as JUNTOS, devised to encourage the Peruvian population to join health programs, appear to be achieving good results in some poor regions in Peru (http://goo.gl/J7LlFJ). Nevertheless, the situation is more precarious when viewed at the national level, with evidence that the potential access to oral health services in Peru is incongruent with the actual access. Although numerous factors likely account for these dissimilar results, they probably include the limited portfolio of dental services in some health subsystems.\n\nThe primary caregivers’ lack of knowledge about oral health could have a negative impact on the oral health of a child21. Consistent with our results, such knowledge correlates to a person’s educational level22. We showed that children whose caregivers had a higher education level were more likely to have accessed oral health services within the 6 months before the survey. In Colombia, where most caregivers had a low educational level, 72.5% of the children had never been examined by an odontologist, although 97.5% had access to a general health social security service23. Another study among children aged 7, 9, and 12 years in Lithuania showed that children whose parents had high educational levels were more likely to be informed and to receive information regarding oral hygiene than those whose parents had a low educational level (73.5% vs. 58.0; p < 0.001). Likewise, children whose parents had high educational levels and sufficient family wealth were 1.34 (95% CI, 1.05–1.71) and 1.71 (95% CI, 1.35–2.16) times more likely to visit a dentist for preventive revision than those whose parents had low educational levels and wealth18.\n\nWe identified that the region with the highest probability of access to oral health services for children was the Peruvian highlands. These findings reinforce those of another study24 where the frequency of access to oral health services was higher in the Peruvian highlands than in coastal or jungle regions. This result corresponds to the regions where the JUNTOS program has been in place the longest according to its performance report (http://goo.gl/9psqai). In these cases, access may be improved because the JUNTOS program provides economic incentives to expectant mothers and those aged <19 years if their family are in extreme poverty. In exchange, participants must commit to using preventive health services, attending growth and development checkups, and engaging in child and adolescent education.\n\nThere was a clear correlation between a child’s age and his or her access to oral health services, with access being the highest among children aged >6 years. This may be because of the greater influence of caregivers’ beliefs, habits, and knowledge regarding oral healthcare for children aged <6 years4,25. This situation is equally apparent in other countries such as India, where 59.08% of children first visited a dentist at the age of 6–12 years, with toothaches typically being the main reason for visitation the dentist (42.04%)26. Moreover, only 8.52% and 32.40% of children in that study had their first dental visit at the ages 0–3 and 3–6 years, respectively26. Such results contradict the current recommendations that state that the first dental visit should occur during the first year of the child.\n\nDuring the analysis of enabling factors, we found that access to oral health services was mediated by the type of health insurance, which is an interesting result because according to the theory of Andersen (the author of the model used in this study)27, a health system is inequitable when enabling factors influence the effective access to oral health services; it is the predisposing and need factors that facilitate an equitable health system. This reinforces the idea that universal health insurance does not, by itself, determine access to health services28,29. Therefore, it is necessary to consider other factors that determine access and to develop social programs that focus on ensuring effective health service use and not merely on achieving a greater coverage30. We must prevent the development of the “inverse care law,” by which people with the greatest need are the least served, and address the concern that health services exposed to market forces have increased inequality31. Although we could not find a study that evaluated the factors that determined access to oral health services for the age group we analyzed, a study32 conducted in Chile, which included all age groups, found that the probability of not receiving dental care was highest for people from lower socioeconomic groups, indigenous ethnic groups, and rural areas, as well as those receiving public health insurance. This result is consistent with the results of the current study.\n\nA principal limitation of this study is that its cross-sectional design precludes establishing causal associations. This is compounded by the use of secondary data from ENDES, which limits the precision and accuracy of the collected data. Another limitation, albeit minor, is that the survey may have included two or more siblings, which prevents us from claiming the independence of data regarding the parent or caregiver of the child. It was also problematic that ENDES lacked suitable variables for assessing the need for access to oral health services and that this may be an area that needs to be changed in future studies of ENDES. The American Association of Pediatric Dentistry recommends semi-annual visits to the dentist, which supports our argument that all participants in the study required greater access to an oral health service.\n\nDespite the study limitations, it should be noted that ENDES was a robustly performed, national study that required all participating surveyors to undergo training and standardization. The data generated in the current study may therefore be relevant to strengthening and improving oral health programs in Peru among children aged <12 years because, to date, there have been few studies that assessed the factors associated with access to oral health services. However, in the future, we recommend prospective studies that use primary data to collect information regarding access to oral health services and its determinants in Peru.\n\nIn conclusion, we showed that wealth index, education level, natural region of residence, age, and type of health insurance determine access to oral health services among children aged <12 years in Peru. When analyzing these results in the light of Andersen and Col’s theoretical model, we concluded that there was inequitable access to oral health services in Peru, which we consider was most likely because of the fragmented and inequitable healthcare system. Therefore, we make several recommendations to public health policy makers that may improve the current situations. First, it is essential that the gaps in oral health treatments offered through each subsystem be closed so that the population has truly equal opportunities. Second, the strategies used to promote and prevent oral health should focus on improving knowledge and demystifying oral health among parents and children, targeting those with the lowest probabilities of access to oral health services (i.e., pre-school children, those in displaced populations, and those whose parents have low educational levels). Third, the reach of social programs such as JUNTOS should be increased to cover regions where access to oral health services is not optimized because such programs appear to be effective in improving access, specifically in regions where the poverty levels are high. We hope that decision makers in Peru consider these recommendations and attempt to improve access to oral health services among children aged <12 years. By targeting this age group, we believe that better oral health outcomes can be achieved in the long term.\n\n\nData availability\n\nDataset 1: ENDES 2014–2015\n\nThe data was obtained from Instituto Nacional de Estadística e Informática (http://iinei.inei.gob.pe/microdatos/) and merged by the authors. 10.5256/f1000research.12474.d17674833",
"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 would like to thank Enago (www.enago.com) for the English language review.\n\n\nReferences\n\nAcharya S, Tandon S: The effect of early childhood caries on the quality of life of children and their parents. Contemp Clin Dent. 2011; 2(2): 98–101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nÇolak H, Dülgergil ÇT, Dalli M, et al.: Early childhood caries update: A review of causes, diagnoses, and treatments. J Nat Sci Biol Med. 2013; 4(1): 29–38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGomes MC, Pinto-Sarmento TC, Costa EM, et al.: Impact of oral health conditions on the quality of life of preschool children and their families: a cross-sectional study. Health Qual Life Outcomes. 2014; 12: 55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTagliaferro EP, Pereira AC, Meneghim Mde C, et al.: Assessment of dental caries predictors in a seven-year longitudinal study. J Public Health Dent. 2006; 66(3): 169–173. PubMed Abstract | Publisher Full Text\n\nOrtiz León F: Perfil epidemiológico de salud bucal en niños atendidos en el seguro social del Perú. Odontol Pediatr. 2014; 13(2): 10. Reference Source\n\nChumpitaz-Durand R, Guezzi-Hernández L: Prevalencia e incidencia de caries a partir de vigilancia epidemiológica realizada a escolares en Chiclayo, Perú. Kiru. 2013; 10(2): 9. Reference Source\n\nMoses-Augusto A: Caries dental asociada al índice de higiene oral simplificado en niños de 6 a 12 años de una institución educativa pública del distrito de Ate-Vitarte en el año 2013. Lima, Perú: Universidad Peruana de Ciencias Aplicadas; 2014. Reference Source\n\nSánchez-Moreno F: [The national health system in Peru]. Rev Peru Med Exp Salud Publica. 2014; 31(4): 747–753. PubMed Abstract\n\nFisher-Owens SA, Soobader MJ, Gansky SA, et al.: Geography matters: state-level variation in children's oral health care access and oral health status. Public Health. 2016; 134: 54–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKandelman D, Arpin S, Baez RJ, et al.: Oral health care systems in developing and developed countries. Periodontol 2000. 2012; 60(1): 98–109. PubMed Abstract | Publisher Full Text\n\nHernández-Vásquez A, Vilcarromero S, Rubilar-González J: [Neglect of oral health in children as a public health problem in Peru]. Rev Peru Med Exp Salud Pública. 2015; 32(3): 604–605. PubMed Abstract\n\nPatrick DL, Lee RS, Nucci M, et al.: Reducing oral health disparities: a focus on social and cultural determinants. BMC Oral Health. 2006; 6 Suppl 1: S4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingh A, Purohit BM: Addressing oral health disparities, inequity in access and workforce issues in a developing country. Int Dent J. 2013; 63(5): 225–229. PubMed Abstract | Publisher Full Text\n\nAndersen RM: Revisiting the behavioral model and access to medical care: does it matter? J Health Soc Behav. 1995; 36(1): 1–10. PubMed Abstract | Publisher Full Text\n\nBabitsch B, Gohl D, von Lengerke T: Re-revisiting Andersen’s Behavioral Model of Health Services Use: a systematic review of studies from 1998–2011. Psychosoc Med. 2012; 9: Doc11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaley SJ, Ponce Terashima J, Hoffman KA, et al.: Barriers to Primary Care in Lima, Peru. World Med Health Policy. 2017; 9(2): 164–185. Publisher Full Text\n\nSafiri S, Kelishadi R, Heshmat R, et al.: Socioeconomic inequality in oral health behavior in Iranian children and adolescents by the Oaxaca-Blinder decomposition method: the CASPIAN- IV study. Int J Equity Health. 2016; 15(1): 143. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaldūnaitė K, Bendoraitienė EA, Slabšinskienė E, et al.: The role of parental education and socioeconomic status in dental caries prevention among Lithuanian children. Medicina (Kaunas). 2014; 50(3): 156–61. PubMed Abstract | Publisher Full Text\n\nRomo-Pinales MR, de Jesús Herrera MI, Bribiesca-García ME, et al.: Caries dental y algunos factores sociales en escolares de Cd. Nezahualcóyotl. Bol Med Hosp Infant Mex. 2005; 62(2): 124–135. Reference Source\n\nYpanaqué-Luyo P, Martins M: Uso de los servicios de salud ambulatorios en la población peruana. Rev Peru Med Exp Salud Publica. 2015; 32(2): 464–470. Publisher Full Text\n\nCastilho AR, Mialhe FL, Barbosa Tde S, et al.: Influence of family environment on children's oral health: a systematic review. J Pediatr (Rio J). 2013; 89(2): 116–123. PubMed Abstract | Publisher Full Text\n\nSingh A, Gambhir RS, Singh S, et al.: Oral health: How much do you know? - A study on knowledge, attitude and practices of patients visiting a North Indian dental school. Eur J Dent. 2014; 8(1): 63–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFranco-Cortez AM, Ramirez-Puerta S, Escobar-Paucar G, et al.: Barreras de acceso a los servicios odontológicos de niños y niñas menores de 6 años pertenecientes a familias desplazadas. CES Odontol. 2010; 23(2): 8. Reference Source\n\nHernández-Vásquez A, Azañedo D, Díaz-Seijas D, et al.: [Access to oral health services in children under twelve years of age in Peru, 2014]. Salud Colect. 2016; 12(3): 429–441. PubMed Abstract | Publisher Full Text\n\nLourenço CB, Saintrain MV, Vieira AP: Child, neglect and oral health. BMC Pediatr. 2013; 13: 188. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeera R, Muthu MS, Phanibabu M, et al.: First dental visit of a child. J Indian Soc Pedod Prev Dent. 2008; 26(Suppl 2): S68–S71. PubMed Abstract\n\nRocha-Buelvas A: Análisis sobre el acceso a los servicios de la salud bucal: un indicador de equidad. Rev Gerenc Polít Salud. 2013; 12(25): 96–112. Reference Source\n\nEdelstein BL, Chinn CH: Update on disparities in oral health and access to dental care for America's children. Acad Pediatr. 2009; 9(6): 415–419. PubMed Abstract | Publisher Full Text\n\nIsmail AI, Sohn W: The impact of universal access to dental care on disparities in caries experience in children. J Am Dent Assoc. 2001; 132(3): 295–303. PubMed Abstract | Publisher Full Text\n\nMouradian WE, Huebner CE, Ramos-Gomez F, et al.: Beyond access: the role of family and community in children's oral health. J Dent Educ. 2007; 71(5): 619–631. PubMed Abstract\n\nSheiham A: Is there an inverse ‘dental’ care law? Br Dent J. 2001; 190(4): 203–206.\n\nDelgado BI, Cornejo-Ovalle M, Jadue HL, et al.: Determinantes sociales y equidad de acceso en la salud dental en Chile. Cient Dent. 2013; 10(2): 8. Reference Source\n\nAzañedo D, Hernández-Vásquez A, Casas-Bendezú M, et al.: Dataset 1: in: Factors determining access to oral health services among children aged less than 12 years in Peru. F1000Research. 2017. Data Source"
}
|
[
{
"id": "27559",
"date": "16 Nov 2017",
"name": "Ana Flávia Granville-Garcia",
"expertise": [
"Reviewer Expertise Epidemiology",
"pediatric dentistry"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle: Suitable for what you propose.\nSummary: Insert level of significance ..\nIntroduction: I inserted data from other countries, to situate the reader on the subject in the world and not only in the country being studied.\nMethod: The study used retrospective data with a sample of considerable size. In these data are not registered the experience of caries? It would be interesting to see the association between caries experience and going to the dentist. Why not use your dental history data for more than six months. Did the trip to the dentist in the last six months, in fact, reflect the problem?\nResults: well described.\nDiscussion: Adequate and associated variables were discussed. I suggest a more consistent conclusion reporting the strategies to be carried out more clearly. In addition, I suggest that perhaps the authors could talk a bit about literacy in oral health that is problematic that comes before all the problems cited in the 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": "28106",
"date": "17 Nov 2017",
"name": "Sonia Constanza Concha Sánchez",
"expertise": [
"Reviewer Expertise Public health",
"epidemiology and dentistry"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would recommend some modifications in the results section. In the second paragraph it should be reported that \"The univariate analysis revealed that all independent variables, except sex, were statistically significant”. After adjusting the model for all variables, we found that sex, geographical location, and language spoken were no significant…”\nOn the other hand, in the last paragraph I suggest referring ... \"In contrast, the adjusted model indicated that the language spoken by caregivers was not statistically associated with the access to oral 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?\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": "30717",
"date": "19 Feb 2018",
"name": "Efigênia Ferreira e Ferreira",
"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 appreciate the opportunity to read this manuscript. It is a population-based study with a well-planned and scientifically rigorous methodology, about access to dental care (dependent variable) for children <12 years old in Peru. It discusses some independent variables, and exposes the absence of dental care, especially for younger children (<5 years old).\nIt uses the theoretical model of Andersen (1995) and points as factors associated with access: age group years, natural region of residency, type of health insurance, quintile of wealth and caregivers' educational level.\nIn my point of view, the manuscript has quality, is a relevant study and is suitable for this journal.\nOnly one suggestion. On page 5, at the end of the first paragraph the authors report: In contrast, the adjusted model indicated that the language spoken by caregivers was statistically associated with the access to oral health services (Table 2). However, this association was not observed in the adjusted model and was not part of the study's findings. I suggest that this statement should be revised.\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/6-1680
|
https://f1000research.com/articles/6-679/v1
|
15 May 17
|
{
"type": "Research Article",
"title": "Transcriptomic analysis of Saccharomyces cerevisiae x Saccharomyces kudriavzevii hybrids during low temperature winemaking",
"authors": [
"Jordi Tronchoni",
"Estéfani García-Ríos",
"Jose Manuel Guillamón",
"Amparo Querol",
"Roberto Pérez-Torrado",
"Jordi Tronchoni",
"Estéfani García-Ríos",
"Jose Manuel Guillamón",
"Amparo Querol"
],
"abstract": "Background: Although Saccharomyces cerevisiae is the most frequently isolated species in wine fermentation, and the most studied species, other species and interspecific hybrids have greatly attracted the interest of researchers in this field in the last few years, given their potential to solve new winemaking industry challenges. S. cerevisiae x S. kudriavzevii hybrids exhibit good fermentative capabilities at low temperatures, and produce wines with smaller alcohol quantities and larger glycerol quantities, which can be very useful to solve challenges in the winemaking industry such as the necessity to enhance the aroma profile. Methods: In this study, we performed a transcriptomic study of S. cerevisiae x S. kudriavzevii hybrids in low temperature winemaking conditions. Results: The results revealed that the hybrids have acquired both fermentative abilities and cold adaptation abilities, attributed to S. cerevisiae and S. kudriavzevii parental species, respectively, showcasing their industrially relevant characteristics. For several key genes, we also studied the contribution to gene expression of each of the alleles of S. cerevisiae and S. kudriavzevii in the S. cerevisiae x S. kudriavzevii hybrids. From the results, it is not clear how important the differential expression of the specific parental alleles is to the phenotype of the hybrids. Conclusions: This study shows that the fermentative abilities of S. cerevisiae x S. kudriavzevii hybrids at low temperatures do not seem to result from differential expression of specific parental alleles of the key genes involved in this phentoype.",
"keywords": [
"Saccharomyces cerevisiae",
"Saccharomyces kudriavzevii",
"hybrids",
"cold stress",
"winemaking"
],
"content": "Introduction\n\nWine yeasts are specialised organisms adapted to restrictive environmental conditions created by human technology. The most frequently isolated species in wine fermentations is Saccharomyces cerevisiae, but other species of the Saccharomyces genus and their interspecific hybrids are also present in the final wine fermentations. These species have attracted significant interest in the last years due to their potential in solving the main challenges the winemaking industry faces, such as the enhancement of aroma. There is a trend in winemaking to decrease fermentation temperatures, which improves the wine aromatic profile. The wine industry has yeasts that are commercialized as cryotolerant S. cerevisiae yeasts, however most of them do not show desirable fermentation performance at low temperatures (10–15°C).\n\nSeveral studies have addressed the yeast cold stress adaptation topic. A transcriptomic analysis using QA23, a commercial S. cerevisiae wine-making strain, during low temperature industrial fermentations showed how the expression profiles at 25°C contrasted significantly with those at 13°C1. In particular, the expression of genes associated with cell growth, cell cycle and maintenance was lower in the exponential phase at 13°C than at 25°C, and those genes activated in the exponential phase of growth at 13°C were essentially involved in environmental stress response2.\n\nPhysiological and enological studies have suggested the potential benefits of the use of S. kudriavzevii in low temperature wine fermentations, and its cryotolerant nature3. In a study comparing transcriptomes of S. kudriavzevii and S. cerevisiae, both species showed up-regulation of genes related to translational machinery, although S. kudriavzevii presented an enhanced response compared to S. cerevisae4. Tronchoni et al.4 postulated that this response could be the result of alterations in the stability of a functional RNA conformation related to a competing structure. This suggests adaptation to cold shock in S. kudriavzevii due to higher ribosome availability and enhanced translation efficiency.\n\nIn cold European regions S. kudriavzevii x S. cerevisiae yeast hybrids are frequently used to produce wines. The enological characterization of several of those S. cerevisiae x S. kudriavzevii hybrids have suggested that the S. cerevisiae genome contributes to ethanol tolerance and elevated fermentative capacity5,6, whereas the S. kudriavzevii genome provides adaptation to low temperatures7,8. S. kudriavzevii produce higher amounts of glycerol during wine fermentations3,5, a characteristic that has been linked to cell survival in fermentations at low temperatures9 and has also been found to be involved in freeze-thawing stress resistance10.\n\nThis work presents a transcriptomic study of S. cerevisiae x S. kudriavzevii hybrids, aimed at deciphering the molecular adaptation of these strains to low temperatures. To perform this study, we used two wine yeast strains of S. cerevisiae x S. kudriavzevii hybrids that are commercialized in the market as cryophilic strains, Lalvin W27 and VIN7. These strains were selected according to previously published data6,11 and showed differences in genomic parental allele composition. Although both of them are allotriploid hybrids, the chromosomes inherited from each parent are different. W27 has lost part of chromosome IV, IX and XV of the S. kudriavzevii genome6, whilst VIN7 has lost chromosomes III and parts of chromosomes IV and VII11,12.\n\n\nMethods\n\nThe yeasts strains included in this study were the strain Lalvin T73 (S. cerevisiae) and the strain IFO1802 (S. kudriavzevii), used as the parental species, and two S. cerevisiae x S. kudriavzevii hybrids, VIN7 and Lalvin W27, isolated from wine in South Africa and Switzerland, respectively. The yeast was grown and maintained in GPY medium (2% glucose, 0.5% peptone, 0.5% yeast extract) and plates (with 2% agar). Wine fermentations were performed in grape juice from the Tempranillo variety at 28°C or 12°C in vessels with 0.45 L of Tempranillo grape must.\n\nEach yeast strain had two independent fermentations and three samples were taken from each independent fermentation. Cells from each yeast strain fermentation were pelleted by centrifugation (4000 rpm/min, 5 min), at 12°C and 28°C. Cells were collected at the beginning of the exponential phase by taking samples two generations after inoculation. The RNA extraction protocol was based on subsequent treatments with phenol-tris, phenol-chloroform (5:1) and chloroform-isoamyl alcohol (24:1), and an ethanol precipitation with sodium acetate13. RNA concentrations and purity were determined using a Nanodrop spectrophotometer ND-1000 (Nanodrop Technologies™, Wilmington, DE). RNA integrity was checked by electrophoresis in agarose gel (1%). 2–4 μg of total RNA from each sample was linearly amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies™, Ca, USA). 2–3 µg of amplified cRNA was used as template for cDNA synthesis. cDNA was marked indirectly using the SuperScript™ Indirect cDNA Labeling System (Invitrogen™, San Diego, CA). Cy3 and Cy5 mono-reactive Dye (Amersham GE Healthcare™, Amersham UK) were used as the fluorophores and dye incorporation was monitored using a Nanodrop spectrophotometer.\n\nA 200 to 300 pmol mixture of the labelled cDNA samples was concentrated (Concentrator Plus, Eppendorf™, Hamburg, Germany). Competitive hybridization was performed on a Yeast 6.4K Array with PCR-amplified ORFs of yeast S288c strain (Microarray Centre, UHN, Toronto, Ontario, Canada) in AHC hybridization chambers (ArrayIt Corporation, CA, USA) at 42°C overnight. Heterologous conditions as stated by Gamero et al.14 were employed to assure the hybridization of the S. kudriavzevii genome. The pre-hybridization solution contained 3X SSC, 0.1% SDS and 0.1 mg/ml BSA; the hybridization solution contained 0.1% SDS, 0.1 mg/ml of salmon DNA and 5X SSC. Microarrays were manually washed with different solutions containing different SSC 20X and SDS 10% concentrations (Sol.1: 0.1% SDS-2X SSC; Sol.2: 0.1% SDS-0.1X SSC; Sol.3: 0.1 SSC; Sol4: 0.01X SSC). The signal intensities of Cy3 and Cy5 were acquired with an Axon GenePix 4100A scanner (Molecular Devices, CA, USA) using GenePix Pro v.6.1 software, at a resolution of 10 µm.\n\nMicroarray data were derived from three independent cDNA hybridization experiments. Background correction was performed with GenePix pro 6.0. Subsequent analyses were performed with the Acuity 4.0 software (Molecular Devices, CA, USA). Individual datasets were normalized to log2 ratio value of 1 and data were filtered to remove spots that were not flagged and manually processed for print tip effect corrections. Only spots data with a minimum of two replicates were considered. Replicates were combined and medians were calculated. The first cut-off was the selection of the genes presenting at least 2-fold log2 ratio values, according to the literature15–17. These genes underwent a “GO term” enrichment analysis using the GO Term Finder tool in the Saccharomyces Genome Database (http://www.yeastgenome.org/). Regarding the statistics, False Discovery Rate (FDR) analysis and a significance level of 99% (p value < 0.01) were applied.\n\nExpression of genes NUG1, LSM8, PDC5, NSR1, GPD1 and GUT2 was investigated by qPCR. Normalization of gene expression was carried out using ACT1 and RDN18-1 as controls since their expression remains constant along fermentation. Primers were designed using Primer-BLAST (NCBI) and S. kudriavzevii and S. cerevisiae gene sequences were deposited in databases (www.ncbi.nlm.nih.gov; GSE90793). Forward and reverse oligonucleotides were synthetized to hybridize to the selected alleles of S. kudriavzevii or S. cerevisiae genes (Table 1). PCR Mastercycler pro (Eppendorf, Germany) confirmed the primer specificity and their annealing temperature. cDNA synthesis and RNA extraction were carried out as previously explained. qPCR runs were done in triplicate in a LightCycler® 480 Real-Time PCR System (Roche, Switzerland) and analyzed using the software from the manufacturer (LightCycler® Software, version 4.0). The relative gene expression was quantified by comparison with ACT1 and RDN18-1 expression, after kinetic PCR efficiency correction.\n\nYeast cells grown overnight in GPY were diluted to 2 × 106 cells/ml the next morning. They were then grown until the mid-log phase (approximately 1 × 107 cells/ml), and 175 μl were inoculated on each GPY plate. A filter (1 cm diameter) with paromomycin (2 μg) was placed on the surface and plates were incubated at 12°C or 28°C until the lawn was formed. The assays were repeated twice.\n\n\nResults\n\nWe carried out micro-fermentations in vessels with 0.45 L of Tempranillo grape must, mimicking previous work conditions4. The time needed for both hybrid strains to finish the fermentation at 28°C was similar, 5 and 6 days for Lalvin W27 and VIN7, respectively (Table 2). In contrast, at low temperature (12°C) the fermentation performance was quite different. W27 behaved more similar to the S. kudriavzevii type strain (11 days), taking 14 days to finish fermentation. Hybrid strain VIN7 performance was more similar to the S. cerevisiae parental strain (21 days) and took 23 days. The differences in fermentation kinetics amongst these strains revealed how allelic differences can determine the behaviour of oenological traits of interest.\n\nTo evaluate changes in the global expression of genes during acclimation to low temperature in the wine fermentation of natural must, sampling was done at the beginning of the exponential phase, two generations after inoculation. RNA from the samples was hybridised against the S288c microarray to study transcriptomic changes. An average of 86% sequence similarity exists between species of S. cerevisiae and S. kudriavzevii4, so we used heterologous hybridisation conditions for the microarrays. We observed that 95% of the total S288c gene spots were hybridised by S. kudriavzevii DNA4. Gene expression of each strain at both temperatures was analysed. Genes that were differentially expressed at 12°C and 28°C at a level that was considered significant were analysed further using the SAM (Significance Analysis of Microarrays) test with an FDR below 5% (Supplementary Table 1).\n\nIn S. cerevisiae x S. kudriavzevii hybrid strain VIN7 at low temperature, 18 genes were up-regulated, while 22 genes were down-regulated. For W27, 20 genes were up-regulated while 3 genes were down-regulated. GO analysis using the GO Term Finder on the Saccharomyces Genome Database was performed to evaluate the GO categories arising from the up- and down-regulated genes in both hybrid strains (Supplementary Table 2). Analysing the up-regulated genes in both strains after applying the GO-module online tool (http://www.lussiergroup.org/GO-Module) for false positives resulted mainly in GO terms related to “magnesium ion binding”, “thiamine pyrophosphate binding”, “branched-chain-2-oxoacid decarboxylase activity” and “pyruvate decarboxylase activity”. The last category appeared, because amongst the up-regulated genes we found the three pyruvate decarboxylases PDC1, PDC5 and PDC6, together with IDP2. The electron transport category was also significantly up-regulated in W27, similar to what has been previously described in S. cerevisiae and S. kudriavzevii4. Amongst other up-regulated GO-terms in W27 is also “amino acid metabolism” with a number of sub-categories, and in “heavy metal binding” in VIN7. These GO-terms were also previously observed in S. kudriavzevii. For the hybrid strain VIN7 the genes down-regulated at 12°C were related to rRNA synthesis, while for W27 no GO term categories were found.\n\nOverall, the most remarkable group of regulated genes (either up or down-regulated) in the hybrid strains fell into the translation machinery efficiency category, as previously shown for S. kudriavzevii compared to S. cerevisiae at 12°C4. When comparing the two strains that showed high fermentation performance at 12°C (S. kudriavzevii IFO1802 and the hybrid W27) to the two strains with low fermentation performance at 12°C (S. cerevisiae T73 and VIN7), the cold adapted strains overexpressed 13 genes, including NUG1, involved in rRNA export18, and chaperones DDR4819 and SRP120 that couple proteasomes to polypeptides emerging from the ribosome (Supplementary Table 1).\n\nAn open question regarding yeast hybrids is the relative contribution of the different parental alleles to the total expression of specific genes. To test the role of the different alleles (S. cerevisiae or S. kudriavzevii) in the better adaptation to cold temperatures, we selected three differentially overexpressed genes in W27 in this work, NUG1, PDC5 and LSM8, and also three S. kudriavzevii cold stress markers described in different studies, NSR1, GPD1 and GUT23,4,21. We observed overexpression of the overlapping dubious ORF YJR023C for LSM8 and assumed that this overexpression was effectively in LSM8. NSR1, LSM8 and NUG1 are related to translation machinery efficiency, whereas GPD1 and GUT2 are related to glycerol metabolism, involved in cold adaptation. GPD1 and GUT2 are also involved in NAD+/NADH balance, with PDC5. In Table 3 we included the genomic configuration, obtained from previous work6,11,12, for the selected genes in each of the two hybrid strains, showing that most genes have at least one copy of each parental allele with the exception of S. kudriavzevii allele losses for NSR1 in VIN7 and for GPD1 and GUT2 in the W27 strain. The results do not suggest a general correlation between the relative contribution of S. kudriavzevii alleles to total gene expression of key genes and the cryotolerance shown by W27.\n\nSc: S. cerevisiae; Sk: S. kudriavzevii. Sc/Sk is the relative expression of Sc alleles divided by relative expression of Sk. nd: not determined; <dl: below detection limit. C: S. cerevisiae allele; K: S. kudriavzevii allele. All values were normalized relative to the W27 LSM8 S. cerevisiae allele. *: Data obtained from previous work6,11,12.\n\nW27 has one copy of the NSR1 S. cerevisiae allele, whereas VIN7 has three copies, but the S. cerevisiae allele expression is higher in the W27 strain. Thus, despite the higher number of total S. cerevisiae allele copies in VIN7, relative gene expression of the S. cerevisiae NSR1 allele is higher in W27, and also total relative expression is higher in W27. One explanation is that the S. kudriavzevii allele in W27 may promote expression of both alleles. NUG1 is another example of gene that does not show correlation between the number of copies and the level of expression. This gene has two copies of the S. kudriavzevii allele in W27 and one in VIN7 but shows higher expression in VIN7 than in W27. No expression was found for GUT2, but GPD1 expression was remarkably higher in the VIN7 strain mainly due to the high S. kudriavzevii allele contribution. There is also a negative correlation between the copy number of the S. cerevisiae allele for the PDC5 gene and gene expression. Despite the similar expression of the S. cerevisiae allele for the PDC5 gene between both strains, we observe that the Sc/Sk ratio is higher in VIN7 than W27, which suggests this gene may have impact on efficiency of cold vinifications. The Sc/Sk ratio is the relative expression of S. cerevisiae alleles divided by the relative expression of S. kudriavzevii alleles.\n\nThe expression of genes related to translation efficiency in the hybrid strains prompted us to study this phenotype. The translation efficiency of the hybrid strains compared to the parental strains at low temperature was analysed by testing their sensitivity to paromomycin, a potent inhibitor of translation22.\n\nAs it can be seen in Figure 1, the S. cerevisiae strain shows a growth inhibition halo at 12°C, whereas no inhibition halo can be seen in the S. kudriavzevii strain at 12°C, confirming that the translation efficiency is not compromised for this yeast at low temperatures. S. kudriavzevii showed growth defects at 28°C, whereas S. cerevisiae remained unaffected. The hybrid strains, on the other hand, clearly show growth defects at both temperatures. This means that 28°C is not an optimal growth temperature for either of these hybrids. An important difference between the hybrid strains is that W27 shows a narrower halo at 12°C compared to 28°C while for the VIN7 strain the situation is the opposite, supporting the idea of W27 being more similar to S. kudriavzevii strains in low temperature conditions.\n\nThe inhibitory effect of the translation inhibitor paromomycin was evaluated by observing the halo diameter generated in the lawns of S. cerevisiae (T73), S. kudriavzevii (CR85) or S. cerevisiae x S. kudriavzevii hybrids (VIN7 and W27) growing in GPY plates at 28°C or 12°C. Representative experiments are shown, and the end of the inhibition halos are indicated with arrows.\n\n\nDiscussion\n\nIn a previous paper we analysed the transcriptomic behaviour of S. cerevisiae and S. kudriavzevii strains under low temperature fermentation conditions. Transcriptomic data showed the differences in gene expression between both species and also highlighted that the translation efficiency under low stress temperatures was higher in S. kudriavzevii than in S. cerevisiae4. In this work we wanted to further investigate the transcriptomics of S. cerevisiae x S. kudriavzevii hybrids at low temperature and compare the translation efficiency to previous data on S. cerevisiae. The results show that hybrids maintain the winemaking abilities classically attributed to S. cerevisiae, with increased expression of fermentation related genes. This explains their advantage in the winemaking environment over other natural species like S. kudriavzevii, which cannot compete with S. cerevisiae even in low temperature conditions7. The hybrids also showed increased expression of genes related to cold adaptation, such as ribosome management genes (NUG1, SRP1), and also displayed paromomycin resistance, which confirmed this adaptation. Resistance to paromomycin is the result of enhanced translation efficiency due to an increased number of ribosomes available to a new round of mRNA translation. In total, these results suggest that hybrids maintain both fermentative and cold adaptation abilities attributed to each parental species. This showcases their industrially relevant characteristics.\n\nIn our attempt to determine the relative contribution of S. cerevisiae and S. kudriavzevii alleles to the total expression of genes in the hybrids, our results showed that allele copy number did not correlate with allele gene expression in the hybrid strains. Although the genomic contribution of S. kudriavzevii provides improved fermentation at colder temperatures, the reason cannot be explained solely by the presence of these allele copy numbers. It must be taken into account that the S. kudriavzevii genomic contribution in these hybrids is smaller than the S. cerevisiae genomic contribution, and probably under the control of S. cerevisiae genomic regulators. The equilibrium acquired between the genomes of S. cerevisiae and S. kudriavzevii in stable hybrid strains is the result of a complex process aiming to improve environmental adaptation, and cannot be explained only by the sum of both genomes.\n\n\nData availability\n\nThe microarray data discussed in this publication can be found in the Gene Expression Omnibus database (NCBI), with accession number GSE90793.",
"appendix": "Author contributions\n\n\n\nAQ and JMG conceived the study. AQ and JMG designed the experiments. JT and EGR carried out the research. JT and RPT prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government and ERDF (European Regional Development Fund) and by grant PROMETEO (project PROMETEOII/2014/042) from Generalitat Valenciana to AQ.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript\n\n\nSupplementary material\n\nSupplementary Table 1. Transcriptomic dataset with genes that were differentially expressed at 12°C and 28°C genes in S. cerevisiae x S. kudriavzevii hybrids (VIN7 and W27) when compared to S. cerevisiae (T73) and S. kudriavzevii (CR85). The genes that were considered significant were analysed further using the SAM (Significance Analysis of Microarrays) test with an FDR below 5%.\n\nClick here to access the data.\n\nSupplementary Table 2. GO analysis of the differentially expressed genes at different temperatures in S. cerevisiae x S. kudriavzevii hybrids (VIN7 and W27). GO analysis using the GO Term Finder on the Saccharomyces Genome Database was performed to evaluate the GO categories arising from the up- and down-regulated genes in both hybrid strains. Analysis of the up-regulated genes in both strains was corrected for false positives after applying the GO-module online tool (http://www.lussiergroup.org/GO-Module).\n\nClick here to access the data.\n\n\nReferences\n\nBeltran G, Novo M, Leberre V, et al.: Integration of transcriptomic and metabolic analyses for understanding the global responses of low-temperature winemaking fermentations. FEMS Yeast Res. 2006; 6(8): 1167–1183. PubMed Abstract | Publisher Full Text\n\nGasch AP, Spellman PT, Kao CM, et al.: Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell. 2000; 11(12): 4241–4257. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOliveira BM, Barrio E, Querol A, et al.: Enhanced enzymatic activity of glycerol-3-phosphate dehydrogenase from the cryophilic Saccharomyces kudriavzevii. PLoS One. 2014; 9(1): e87290. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTronchoni J, Medina V, Guillamón JM, et al.: Transcriptomics of cryophilic Saccharomyces kudriavzevii reveals the key role of gene translation efficiency in cold stress adaptations. BMC Genomics. 2014; 15(1): 432. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArroyo-Lopez FN, Pérez-Torrado R, Querol A, et al.: Modulation of the glycerol and ethanol syntheses in the yeast Saccharomyces kudriavzevii differs from that exhibited by Saccharomyces cerevisiae and their hybrid. Food Microbiol. 2010; 27(5): 628–37. PubMed Abstract | Publisher Full Text\n\nBelloch C, Orlic S, Barrio E, et al.: Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex. Int J Food Microbiol. 2008; 122(1–2): 188–95. PubMed Abstract | Publisher Full Text\n\nArroyo-López FN, Orlić S, Querol A, et al.: Effects of temperature, pH and sugar concentration on the growth parameters of Saccharomyces cerevisiae, S. kudriavzevii and their interspecific hybrid. Int J Food Microbiol. 2009; 131(2–3): 120–7. PubMed Abstract | Publisher Full Text\n\nSalvadó Z, Arroyo-López FN, Guillamón JM, et al.: Temperature adaptation markedly determines evolution within the genus Saccharomyces. Appl Environ Microbiol. 2011; 77(7): 2292–302. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTulha J, Lima A, Lucas C, et al.: Saccharomyces cerevisiae glycerol/H+ symporter Stl1p is essential for cold/near-freeze and freeze stress adaptation. A simple recipe with high biotechnological potential is given. Microb Cell Fact. 2010; 9: 82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIzawa S, Sato M, Yokoigawa K, et al.: Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells. Appl Microbiol Biotechnol. 2004; 66(1): 108–114. PubMed Abstract | Publisher Full Text\n\nPeris D, Lopes CA, Belloch C, et al.: Comparative genomics among Saccharomyces cerevisiae × Saccharomyces kudriavzevii natural hybrid strains isolated from wine and beer reveals different origins. BMC Genomics. 2012; 13: 407. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBorneman AR, Desany BA, Riches D, et al.: The genome sequence of the wine yeast VIN7 reveals an allotriploid hybrid genome with Saccharomyces cerevisiae and Saccharomyces kudriavzevii origins. FEMS Yeast Res. 2012; 12(1): 88–96. PubMed Abstract | Publisher Full Text\n\nGómez-Pastor R, Pérez-Torrado R, Cabiscol E, et al.: Transcriptomic and proteomic insights of the wine yeast biomass propagation process. FEMS Yeast Res. 2010; 10(7): 870–884. PubMed Abstract | Publisher Full Text\n\nGamero A, Belloch C, Ibáñez C, et al.: Molecular analysis of the genes involved in aroma synthesis in the species S. cerevisiae, S. kudriavzevii and S. bayanus var. uvarum in winemaking conditions. PLoS One. 2014; 9(5): e97626. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarks VD, Ho Sui SJ, Erasmus DJ, et al.: Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response. FEMS Yeast Res. 2008; 8(1): 35–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPizarro FJ, Jewett MC, Nielsen J, et al.: Growth temperature exerts differential physiological and transcriptional responses in laboratory and wine strains of Saccharomyces cerevisiae. Appl Environ Microbiol. 2008; 74(20): 6358–68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRossignol T, Dulau L, Julien A, et al.: Genome-wide monitoring of wine yeast gene expression during alcoholic fermentation. Yeast. 2003; 20(16): 1369–85. PubMed Abstract | Publisher Full Text\n\nWu S, Tutuncuoglu B, Yan K, et al.: Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes. Nature. 2016; 534(7605): 133–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSheng S, Schuster SM: Purification and characterization of Saccharomyces cerevisiae DNA damage-responsive protein 48 (DDRP 48). J Biol Chem. 1993; 268(7): 4752–8. PubMed Abstract\n\nHa SW, Ju D, Xie Y: Nuclear import factor Srp1 and its associated protein Sts1 couple ribosome-bound nascent polypeptides to proteasomes for cotranslational degradation. J Biol Chem. 2014; 289(5): 2701–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaget CM, Schwartz JM, Delneri D: Environmental systems biology of cold-tolerant phenotype in Saccharomyces species adapted to grow at different temperatures. Mol Ecol. 2014; 23(21): 5241–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKurata S, Nielsen KH, Mitchell SF, et al.: Ribosome recycling step in yeast cytoplasmic protein synthesis is catalyzed by eEF3 and ATP. Proc Natl Acad Sci U S A. 2010; 107(24): 10854–9. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "22727",
"date": "30 May 2017",
"name": "Matthias Sipiczki",
"expertise": [
"Reviewer Expertise Yeast 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\nThis well-structured and nicely written paper demonstrates that the combinations of certain industrially relevant phenotypic properties of S. cerevisiae and S. kudriavzevii in strains of chimerical genomes comprised of different proportions of their gene pools (referred to as hybrids by the authors) cannot be simply attributed to differential expression of orthologous gene pairs assumed to be involved in the determination of those traits. The transcriptomic experiments were well designed and performed, and correctly interpreted. As genes involved in the general translational machinery were identified among those overexpressed in strains with higher fermentation efficiency at low temperature, the authors compared the sensitivity of the strains to paromomycin, an agent that inhibits translation by binding to rRNA. The S. kudriavzevii laboratory strain (the parents of the chimerical strains are unknown) and the chimerical strain more active at the lower temperature were more resistant to the inhibitor. This finding was interpreted as indicating that their cells had higher numbers of ribosomes (to be inhibited by the drug) because of more active translation. This explanation is quite reasonable, but it should be mentioned in the Discussion that the increased resistance might also be due to mutations that make the RNA molecules of S. kudriavzevii less sensitive to the drug or impair its uptake at lower temperatures. Numerous paromomycin-resistant Saccharomyces mutants have been described in the literature1-4. In Kluyveromyces lactis, a membrane transporter specific for paromomycin was found5. In Leishmania, the major mechanism of paromomycin resistance was found to be due to decreased drug uptake6,7. The possibility that cold-sensitive mutations can also be involved in the different responses to paromomycin should be taken into consideration (and then refuted if the authors have good counterarguments) because all strains show the same growth intensity outside the inhibition zones at 12 °C in Fig. 1.\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? Partly",
"responses": [
{
"c_id": "2740",
"date": "02 Jun 2017",
"name": "Roberto Pérez-Torrado",
"role": "Author Response",
"response": "We appreciate the reviewer comment and consider that it can significantly improve the paper. We have included this paragraph in the discussion to address the point raised by the reviewer: \"We cannot discard that differences in paromomycin resistance are due to mutations in genes unrelated to translation as has been described before23-26. However, this possibility is unlikely since enhanced resistance phenotype at low temperatures has been observed in two different S. kudriavzevii strains4 and, less accused, in the two S. cerevisiae x S. kudriavzevii hybrids. In addition, our previous work have related paromomycin resistance with translation efficiency in these S. kudriavzevii strains by performing 35S-Methionine incorporation assay4\" Also, references suggested by the reviewer were incorporated."
}
]
}
] | 1
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https://f1000research.com/articles/6-679
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https://f1000research.com/articles/6-944/v1
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20 Jun 17
|
{
"type": "Research Note",
"title": "Saccharomyces cerevisiae show low levels of traversal across the human blood brain barrier in vitro",
"authors": [
"Roberto Pérez-Torrado",
"Amparo Querol",
"Amparo Querol"
],
"abstract": "Background: Saccharomyces cerevisiae is generally considered safe, and is involved in the production of many types of foods and dietary supplements. However, some isolates, which are genetically related to strains used in brewing and baking, have shown virulent traits, being able to produce infections in humans, mainly in immunodeficient patients. This can lead to systemic infections in humans. Methods: In this work, we studied S. cerevisiae isolates in an in vitro human blood brain barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans. Results: The results showed that this food related yeast is able to cross the blood brain barrier in vitro. However, in contrast to C. glabrata and C. albicans, S. cerevisiae showed very low levels of traversal. Conclusions: We conclude that using an in vitro human blood brain barrier model with S. cerevisiae can be useful to evaluate the safety of S. cerevisiae strains isolated from foods.",
"keywords": [
"food emerging pathogens",
"Saccharomyces cerevisiae",
"blood brain barrier",
"virulence"
],
"content": "Introduction\n\nSaccharomyces cerevisiae is generally considered safe, and is involved in the production of a variety of foods and dietary supplements. Several types of food and beverage still contain viable yeast cells1–5. However, in the last years human infections with Saccharomyces cerevisiae have increased6–8. Consequently, S. cerevisiae is considered an emerging pathogen9–11. Different parts of the body can be affected in immunocompromised12–15 and healthy patients16–18. The potential virulence of this yeast has been analysed with different methods in vitro19–22 and in vivo23–27, for example by measuring epithelial barrier traversal28. These reports have suggested that certain strains can cause disease and death in murine models. However, the bio-therapeutic agent Ultralevure (S. cerevisiae var. boulardii) and other supplements are consumed in high doses, ranging from 107 to 1010 live yeast cells per day and for long periods.\n\nThe study of yeast virulence includes studying their behaviour when they encounter endothelial barriers. Opportunistic pathogenic yeasts such as C. glabrata and C. albicans are able to pass the intestinal barrier29,30 and generate systemic infections31–33. Also, C. albicans can cross the blood-brain barrier (BBB) to reach the brain34,35. Regarding S. cerevisiae, infections after oral ingestion16 or digestive translocation12,14,36 show that it can reach brain in murine models25. However, few studies have investigated the behaviour of S. cerevisiae when they reach endothelial barriers28.\n\n\nMethods\n\nThe yeast strains are described in Table 1. Strains were propagated in YPD media (1% glucose, 1% BactoPeptone, 0.5% yeast extract) for 24 h at 30°C.\n\nHuman umbilical endothelial cells (HUVECs) (Clonetics®) were grown in minimum essential medium (Earle’s salt, 25 mM HEPES and GlutaMAX™, Invitrogen) supplemented with 10% foetal bovine serum (FBS, Cambrex Bio Science), 1% nonessential amino acids (Invitrogen) and 50 μg mL–1 gentamicin (Invitrogen). The cells were grown in 150 cm2 culture flasks (TPP) at 37°C in a humidified atmosphere of 5% CO2 and 95% air until a confluence. Culture medium was changed every second day.\n\nHUVEC cells (1×105 cells cm−2) were seeded on Transwell® filter inserts (8 μm, Corning Incorporated) in 24-well plates (Corning Incorporated). A volume of 200 μL cell growth medium was added to the apical compartment and 1250 μL to the basolateral compartment. The TEER was measured using the Millicell-ERS Electrical Resistance System (Millipore). The net value of the TEER (Ωcm−2) was corrected for background resistance by subtracting the contribution of the cell-free filter and the medium (110 Ωcm−2). The TEER was measured before the addition of yeasts.\n\n1 μg/mL of fluorescein (Sigma) was added to the media in the apical compartment of the transwell, with or without established HUVEC monolayers, and fluorescence was measured over time in the media of the apical and basolateral compartment. The apparent permeability, Papp, was defined as (Hilgers et al., 1990):\n\nPapp = (ΔAR/Δt))/CD,0 (1)\n\n(ΔAR/Δt) is the rate of drug appearance in the receiver side, S is the surface area of the Transwell (0.33 cm2 for Transwell® inserts (8 μm pore size, Corning) of 6.5-mm insert diameter), and CD,0 is the initial drug concentration in the donor side at time = 0. Values are expressed in cm/s.\n\nHUVEC cells were seeded on Transwell® filter as described above. Yeasts grown overnight at 30°C in YPD were resuspended (106 cells mL–1) in the apical compartment and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. After 12 h, the basolateral compartment medium was replaced. Colony forming units were counted in YPD plate triplicates after two days. Control wells used to evaluate yeast growth showed no significant growth after 12 h.\n\n\nResults\n\nTo establish an in vitro BBB, we used HUVEC monolayers, a methodology that has been widely used37,38. Monolayers were formed in transwells and two different methods were used to determine the robustness, consistency and integrity of the barrier. First, we studied the TEER, indicative of physical separation. After seeding the HUVECs, TEER was measured and we observed increased values over time that were overcoming 450 Ωcm−2, which correlates with the establishment of a monolayer barrier. Second, we studied the monolayer permeability. The value obtained was 1.82±0.13 (10-6 cm/s) on average, which indicates an integral barrier with low permeability39.\n\nTo determine whether S. cerevisiae is able to cross the human BBB, we used an in vitro model of the human BBB with HUVECs40. The number of cells in the basolateral compartment was measured 12 hours after addition of S. cerevisiae, C. albicans and C. glabrata strains to the apical compartment (Figure 1). The results showed that all yeast strains were able to cross the BBB. While elevated number of cells from C. glabrata and C. albicans strains were able to cross the BBB, S. cerevisiae values were low. Furthermore, while the S. cerevisiae control strain W303 showed the lowest levels of yeast transcytosis, the other opportunistic pathogenic strains presented higher levels.\n\nTo compare the different species, the average level of cell transcytosis for all strains of each species was calculated (Figure 2). After 12 h, Candida species showed a high number of cells in the basolateral chamber (4.9–5.7 Log10 units). On the contrary, we observed that S. cerevisiae showed significantly lower levels (1.0–3.3 Log10 units) than the Candida species.\n\nTo perform this assay we established HUVEC monolayers in Transwell® filter inserts in 24 well plates. 24 hours after apical addition of various strains of S. cerevisiae, C. albicans and C. glabrata, yeast cells from the basolateral compartment were incubated on YPD plates and colonies were counted after one day of growth. Values were obtained after plating several dilutions of the basolateral compartment media. Average of three experiments and standard deviation is shown. To determine statistically significant data, Student t-tests were performed in Excel with 0.05 as the p-value.\n\n\nDiscussion\n\nA model for traversal across the BBB in vitro has been used to study behaviour and pathogenicity mechanisms of yeast strains such as C. albicans34,35. Here, we have shown that S. cerevisiae strains are able to cross the BBB. This data is in accordance with previous studies, where S. cerevisiae cells were observed in the brain after systemic infections in murine models25. When comparing to other well-known yeast pathogens such as C. glabrata and C. albicans, none of the S. cerevisiae strains were able to cross the BBB at high levels. Despite S. cerevisiae pathogenicity levels being lower than other opportunistic yeasts, we recommend the potential risk of new S. cerevisiae strains to be evaluated before using them in food production.\n\n\nData availability\n\nDataset 1: Raw data of permeability measurements and cell counts for BBB traversal. DOI, 10.5256/f1000research.11782.d16511341",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government and ERDF (European Regional Development Fund) and by grant PROMETEO (project PROMETEOII/2014/042) from Generalitat Valenciana to AQ.\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\nFröhlich-Wyder MT: 8 – Yeasts in dairy products. In: Yeasts in Food. Beneficial and Detrimental Aspects. Boekhout T, Robert V, Editors. Behr, Hamburg. 2003; 209–237. Publisher Full Text\n\nJacques N, Casaregola S: Safety assessment of dairy microorganisms: the hemiascomycetous yeasts. Int J Food Microbiol. 2008; 126(3): 321–326. PubMed Abstract | Publisher Full Text\n\nDeak T, Beuchat L: Handbook of spoilage yeasts. CRC, Boca Raton; 1996. Reference Source\n\nLoureiro V, Malfeito-Ferreira M: Spoilage yeasts in the wine industry. Int J Food Microbiol. 2003; 86(1–2): 23–50. PubMed Abstract | Publisher Full Text\n\nLoureiro V, Querol A: The prevalence and control of spoilage yeasts in foods and beverages. Trends Food Sci Technol. 1999; 10(11): 356–365. Publisher Full Text\n\nHerbrecht R, Nivoix Y: Saccharomyces cerevisiae fungemia: an adverse effect of Saccharomyces boulardii probiotic administration. Clin Infect Dis. 2005; 40(11): 1635–1637. PubMed Abstract | Publisher Full Text\n\nMontineri A, Lacobello C, Larocca L, et al.: [Saccharomyces cerevisiae fungemia associated with multifocal pneumonia in a patient with alcohol-related hepatic cirrhosis]. Infez Med. 2008; 16(4): 227–229. PubMed Abstract\n\nSwinne D, Nolard N, VAN Rooij P, et al.: Bloodstream yeast infections: a 15-month survey. Epidemiol Infect. 2009; 137(7): 1037–1040. PubMed Abstract | Publisher Full Text\n\nHazen KC: New and emerging yeast pathogens. Clin Microbiol Rev. 1995; 8(4): 462–478. PubMed Abstract | Free Full Text\n\nMurphy A, Kavanagh K: Emergence of Saccharomyces cerevisiae as a human pathogen. Implications for biotechnology Review. Enzyme Microb Technol. 1999; 25(7): 551–557. Publisher Full Text\n\nPontón J, Rüchel R, Clemons KV, et al.: Emerging pathogens. Med Mycol. 2000; 38(Suppl 1): 225–36. PubMed Abstract | Publisher Full Text\n\nEnache-Angoulvant A, Hennequin C: Invasive Saccharomyces infection: a comprehensive review. Clin Infect Dis. 2005; 41(11): 1559–1568. PubMed Abstract | Publisher Full Text\n\nLolis N, Veldekis D, Moraitou H, et al.: Saccharomyces boulardii fungaemia in an intensive care unit patient treated with caspofungin. Crit Care. 2008; 12(2): 414. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuñoz P, Bouza E, Cuenca-Estrella M, et al.: Saccharomyces cerevisiae fungemia: an emerging infectious disease. Clin Infect Dis. 2005; 40(11): 1625–1634. PubMed Abstract | Publisher Full Text\n\nWilliams JS, Mufti GJ, Powell S, et al.: Saccharomyces cerevisiae emboli in an immunocompromised patient with relapsed acute myeloid leukaemia. Clin Exp Dermatol. 2007; 32(4): 395–397. PubMed Abstract | Publisher Full Text\n\nJensen DP, Smith DL: Fever of unknown origin secondary to brewer's yeast ingestion. Arch Intern Med. 1976; 136(3): 332–333. PubMed Abstract | Publisher Full Text\n\nSmith D, Metzgar D, Wills C, et al.: Fatal Saccharomyces cerevisiae aortic graft infection. J Clin Microbiol. 2002; 40(7): 2691–2692. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSobel JD, Schmitt CA, Lynch M, et al.: Emerging problem of vaginitis due to Saccharomyces cerevisiae. Clin Infect Dis. 1993; 16: 93–94.\n\nde Llanos R, Fernández-Espinar MT, Querol A: A comparison of clinical and food Saccharomyces cerevisiae isolates on the basis of potential virulence factors. Antonie van Leeuwenhoek. 2006; 90(3): 221–231. PubMed Abstract | Publisher Full Text\n\nKlingberg TD, Lesnik U, Arneborg N, et al.: Comparison of Saccharomyces cerevisiae strains of clinical and nonclinical origin by molecular typing and determination of putative virulence traits. FEMS Yeast Res. 2008; 8(4): 631–640. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcCusker JH, Clemons KV, Stevens DA, et al.: Saccharomyces cerevisiae virulence phenotype as determined with CD-1 mice is associated with the ability to grow at 42 degrees C and form pseudohyphae. Infect Immun. 1994; 62(12): 5447–5455. PubMed Abstract | Free Full Text\n\nYáñez A, Murciano C, Llopis S, et al.: In vivo and in vitro studies on virulence and host responses to Saccharomyces cerevisiae clinical and non-clinical isolates. Open Mycol J. 2009; 3: 37–47. Publisher Full Text\n\nByron JK, Clemons KV, McCusker JH, et al.: Pathogenicity of Saccharomyces cerevisiae in complement factor five-deficient mice. Infect Immun. 1995; 63(2): 478–485. PubMed Abstract | Free Full Text\n\nClemons KV, McCusker JH, Davis RW, et al.: Comparative pathogenesis of clinical and nonclinical isolates of Saccharomyces cerevisiae. J Infect Dis. 1994; 169(4): 859–867. PubMed Abstract | Publisher Full Text\n\nde Llanos R, Llopis S, Molero G, et al.: In vivo virulence of commercial Saccharomyces cerevisiae strains with pathogenicity-associated phenotypical traits. Int J Food Microbiol. 2011; 144(3): 393–399. PubMed Abstract | Publisher Full Text\n\nMcCullough MJ, Clemons KV, McCusker JH, et al.: Species identification and virulence attributes of Saccharomyces boulardii (nom. Inval.). J Clin Microbiol. 1998; 36(9): 2613–2617. PubMed Abstract | Free Full Text\n\nLlopis S, Querol A, Heyken A, et al.: Transcriptomics in human blood incubation reveals the importance of oxidative stress response in Saccharomyces cerevisiae clinical strains. BMC Genomics. 2012; 13: 419. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPérez-Torrado R, Llopis S, Jespersen L, et al.: Clinical Saccharomyces cerevisiae isolates cannot cross the epithelial barrier in vitro. Int J Food Microbiol. 2012; 157(1): 59–64. PubMed Abstract | Publisher Full Text\n\nLi L, Redding S, Dongari-Bagtzoglou A: Candida glabrata: an emerging oral opportunistic pathogen. J Dent Res. 2007; 86(3): 204–15. PubMed Abstract | Publisher Full Text\n\nNaglik JR, Moyes DL, Wächtler B, et al.: Candida albicans interactions with epithelial cells and mucosal immunity. Microbes Infect. 2011; 13(12–13): 963–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrun S, Dalle F, Saulnier P, et al.: Biological consequences of petite mutations in Candida glabrata. J Antimicrob Chemother. 2005; 56(2): 307–14. PubMed Abstract | Publisher Full Text\n\nDalle F, Wächtler B, L’Ollivier C, et al.: Cellular interactions of Candida albicans with human oral epithelial cells and enterocytes. Cell Microbiol. 2010; 12(2): 248–271. PubMed Abstract | Publisher Full Text\n\nHoarau G, Kerdraon O, Lagree M, et al.: Detection of (1,3)-β-D-glucans in situ in a Candida albicans brain granuloma. J Infect. 2013; 67(6): 622–4. PubMed Abstract | Publisher Full Text\n\nJong AY, Stins MF, Huang SH, et al.: Traversal of Candida albicans across human blood-brain barrier in vitro. Infect Immun. 2001; 69(7): 4536–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJong AY, Chen SH, Stins MF, et al.: Binding of Candida albicans enolase to plasmin(ogen) results in enhanced invasion of human brain microvascular endothelial cells. J Med Microbiol. 2003; 52(Pt 8): 615–22. PubMed Abstract | Publisher Full Text\n\nLherm T, Monet C, Nougière B, et al.: Seven cases of fungemia with Saccharomyces boulardii in critically ill patients. Intensive Care Med. 2002; 28(6): 797–801. PubMed Abstract | Publisher Full Text\n\nLangford D, Hurford R, Hashimoto M, et al.: Signalling crosstalk in FGF2-mediated protection of endothelial cells from HIV-gp120. BMC Neurosci. 2005; 6: 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilhelm I, Fazakas C, Krizbai IA: In vitro models of the blood-brain barrier. Acta Neurobiol Exp (Wars). 2011; 71(1): 113–128. PubMed Abstract\n\nGaillard PJ, de Boer AG: Relationship between permeability status of the blood-brain barrier and in vitro permeability coefficient of a drug. Eur J Pharm Sci. 2000; 12(2): 95–102. PubMed Abstract | Publisher Full Text\n\nSun SW, Zu XY, Tuo QH, et al.: Caveolae and caveolin-1 mediate endocytosis and transcytosis of oxidized low density lipoprotein in endothelial cells. Acta Pharmacol Sin. 2010; 31(10): 1336–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPérez-Torrado R, Querol A: Dataset 1 in: Low levels of in vitro human blood brain barrier traversal of the food related emerging pathogen Saccharomyces cerevisiae. F1000Research. 2017. Data Source"
}
|
[
{
"id": "24594",
"date": "14 Aug 2017",
"name": "Rosa de Llanos",
"expertise": [
"Reviewer Expertise Fungal pathogenesis",
"food microbiology",
"environmental microbiology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction: I would consider to change the sentence “Consequently, S, cerevisiae is considered an emerging pathogen” with “Consequently, S, cerevisiae is considered an emerging pathogen of low virulence”\n\nOrigin of isolation of the yeast strains could be included in Table 1.\n\nMethods: Abbreviation of BBB should be added in the title Ability to cross the blood-brain barrier.\n\nResults: In Figure 1 there are different colours but not information about the meaning of it has been included. In Figure 2, there is a mistake for C. glabrata and C. albicans, they are named as S.glabrata and S.albicans.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "25622",
"date": "01 Sep 2017",
"name": "Felipe H. Santiago-Tirado",
"expertise": [
"Reviewer Expertise Fungal pathogenesis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlthough the article is interesting and the data clear, I believe the authors are overstating the findings. First, HUVECs are not considered a good model for blood-brain barrier anymore. They used to be a favorite one because they are a human cell line, however, they are not of cerebral origin, and deviate considerably from the behavior of cerebral endothelial cells. They could fix this by calling their model an \"endothelial\" monolayer instead, or repeat the experiment using \"real\" BBB cell lines (i.e. hCMEC/D3, which is commercially available). Also, they report the TEER values (which by the way the correct units should be resistance (ohms) times area (cm2) rather than dividing by it) before the start of the experiment, but they should also measure the integrity of the monolayer at the end of the experiment, to rule out that the amount of S. cerevisiae crossing is due to rupture of the monolayer. This assay is also hard to interpret in the absence of a negative control - in fact, S. cerevisiae has been traditionally used as a negative control on this type of assays! Would inert beads also cross? Would any other organisms cross at the same rate? Maybe they can check this by using fluorescent beads and measuring fluorescence on the bottom. Or if easier to do by CFUs, they could add another organism known to not been able to cross and count CFUs. Overall, it is a nice preliminary report, one worth the time pursuing. Considering this was submitted as a Research Note, I believe is appropriate for indexing once they address my comments above.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
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https://f1000research.com/articles/6-944
|
https://f1000research.com/articles/6-1679/v1
|
12 Sep 17
|
{
"type": "Research Article",
"title": "The effect of cognitive-behavioral counseling on anxiety in the mothers of infants in the NICU: A randomized controlled trial",
"authors": [
"Massumeh Koochaki",
"Zohreh Mahmoodi",
"Sara Esmaelzadeh – Saeieh",
"Kourosh Kabir",
"Maryam Tehranizadeh",
"Mahrokh Dolatian",
"Massumeh Koochaki",
"Sara Esmaelzadeh – Saeieh",
"Kourosh Kabir",
"Maryam Tehranizadeh",
"Mahrokh Dolatian"
],
"abstract": "Background: Pressures and tensions in everyone’s life can cause a wide range of mental disorders such as anxiety. One of these tensions is the birth of a baby who requires special care, which can cause personal and social problems for the mother if no appropriate measures are taken to help them. The present study was conducted to determine the effect of cognitive-behavioral counseling on anxiety in the mothers of infants in the Neonatal Intensive Care Unit. Methods: This randomized controlled trial recruited 90 women presenting to Kowsar Hospital in Qazvin in 2016. They were enrolled by convenience sampling and randomly assigned to control and intervention groups. Eight sessions were held for each group. Beck Anxiety Inventory was filled by mothers at the beginning of intervention, at the end of the eighth session and three weeks after the intervention. The data was analyzed by generalized estimating equations (GEE) method. Results: According to the results, maternal anxiety showed no significant differences between the two groups before intervention (p = 0.408 and p = 0.881). Based on GEE test, the mean score of anxiety was significantly different in the two groups (p = 0.026) immediately and three weeks after the intervention in that it was lower in the intervention group. Friedman test results also confirmed the reducing trend of mean score of anxiety in the three stages (p = 0.000). Conclusions: Counseling has a positive effect on reducing the anxiety of mothers of children with special needs, therefore it can be used to improve their condition.",
"keywords": [
"newborns",
"neonatal intensive care unit",
"counseling"
],
"content": "Introduction\n\nAnxiety is one of the most common adjustment disorders in all societies, which has a global prevalence of 14% and a prevalence of 20.8% in Iran1,2.\n\nWomen’s life is made up of stages and events such as menstruation, pregnancy and childbirth and their associated psychological and physical changes, which make women more vulnerable than men in the face of the stresses of life3,4. One of the most important and common causes of stress and anxiety in women during the reproductive years is concern about the health of their fetus and future child4.\n\nThe birth of an infant who requires intensive care is considered a serious crisis in the mother’s life. Uncertainty about survival, future life and consequences, duration of hospitalization and taking care of the child at home are some of the mother's concerns that may lead to confusion, frustration, fear and ultimately anxiety5. If these parents receive no support in coping with these circumstances, they may adopt ineffective coping strategies6, which can affect the quality of the mother’s personal and social life and her ability to take care of the infant and may thus increase her anxiety6–8. Under such circumstances, the mother needs emotional support from her spouse and even the society, and providing this support can diminish her anxiety9. Paying attention to the mother's needs and identifying the factors causing stress in her are necessary to reduce her anxiety10.\n\nThe strategies for coping with and treating this anxiety include pharmacological and non-pharmacological therapies. A wide variety of non-pharmacological interventions are currently available that can be more effective than mere pharmacological therapy; for instance, counseling and anxiety management training11.\n\nCounseling refers to the professional relationship established between a trained counselor and a client. This relationship is built to help the client better grasp her own views of her living environment and to teach her how to achieve her personal goals by choosing tested and meaningful personal strategies and also solve her emotional and interpersonal problems12, which can be performed individually or in a group setting. Group counseling is more effective than individual counseling in critical conditions when mental pressure is accompanied by severe disappointment and the individual finds herself feeling broken, inadequate, helpless and feels fear and failure12. Several studies have investigated the effectiveness of counseling with a cognitive-behavioral approach; for instance, Seyed Gholami et al. found that this method is effective in reducing anxiety sensitivity in divorced women13.\n\nCognitive-behavioral therapy is a combination of cognitive and behavioral approaches to help the patient identify and correct her distorted patterns of thinking and inefficient behaviors and is carried out with regular discussions and carefully-organized behavioral assignments14. In other words, the patient is encouraged in this method to consider the relationship between her negative thoughts and feelings as assumptions that ought to be put to test and to use behaviors resulting from negative thoughts as a benchmark for assessing the validity or accuracy of these thoughts15.\n\nSince midwives are a group of healthcare workers that have an important role in providing counseling and health training not just to women but to entire families and the community, they can play a significant part in the provision of these interventions16. Given the importance of addressing the health of mothers of children requiring special care, the present study was conducted to determine the effect of cognitive-behavioral counseling on anxiety in the mothers of infants in the Neonatal Intensive Care Unit (NICU).\n\n\nMaterials and methods\n\nThe present parallel randomized controlled trial was approved by the Ethics Committee of Alborz University of Medical Sciences with the code abzums.rec.1395.13, dated May 11th 2016, Irct ID: IRCT2016051427728N1 (registered on June 28th 2016). It was conducted on 90 eligible women with infants hospitalized at the NICU of Kowsar Hospital in Qazvin in August – October 2016.\n\nThe sample size was estimated as 42 per group based on the existing literature2,4,6 and the pre- and post-intervention standard deviations of 3.65 and 2.71 in order to reach a mean difference (reduction in anxiety score) of 1.96 with the following specifications and using the sample size equation (N=(z1−α2⁄−z1−β)2(SD1+SD2)2/d2) for comparing two means; the estimated sample size was increased to 45 per group to take account of potential attrition 10%.\n\nThe study inclusion criteria were: 1)Being an Iranian woman, 2) having at least a high school diploma, 3) having had a preterm birth (less than 37 weeks) or low-birth-weight infant (weighing less than 2500 grams) or both with a minimum NICU stay of one month, 4) suffering from anxiety based on Beck's Anxiety Inventory17, 5) no self-reported psychiatric disorders, 6) no use of anxiolytic medications, 7) willingness to participate in the study, 8) no history of adverse life events such as the death of a first-degree relative in the past six months and 9) no history of giving birth to a child with special diseases or severe abnormalities. (Supplementary file 1, Supplementary file 2).\n\nThe tools used in this study included a personal-demographic checklist as well as Beck's Anxiety Inventory (BAI), which contains 21 items scored based on a 4-point Likert scale (with responses including ‘not at all’, ‘mildly’, ‘moderately’ and ‘severely’). BAI is an international scale with a confirmed validity and reliability according to several studies18. This questionnaire was completed by the participants of this study before and immediately after the end of the counseling sessions and also three weeks after the intervention.\n\nThe study began after obtaining the necessary permissions from the Research Council, Ethics Committee and Director of Alborz University of Medical Sciences as well as the director and department head of Kowsar Hospital in and the registration of the project at the Iranian Registry of Clinical Trials (IRCT). For sampling, the researcher (MK) visited the NICU of Kowsar Hospital and selected all the eligible mothers through convenience sampling and briefed them on the study objectives and obtained their written consent for entering the study if they were willing to participate. Permuted block randomization was used to randomly allocate participants into the trial group while maintaining a balance across the groups. Each block had a specified number of randomly-ordered trial assignments, so the mothers were divided into a trial group (anxiety counseling + routine care counseling) and a control group (routine care counseling). Both groups received eight sessions of routine counseling (routine neonatal care), twice per week for four weeks. The subjects discussed in the routine care counseling sessions included:\n\nSession 1: Information about the hospitalized infant, such as the type of disease and the diagnostic and therapy methods used. Session 2: Information about the disease symptoms and consequences. Session 3: Obtaining knowledge and skills about nutrition. Session 4: Obtaining knowledge and skills about moving and positioning. Session 5: Obtaining knowledge and skills about hygiene and infection control. Session 6: Obtaining knowledge and skills about temperature and how to clothe the infant. Session 7: Obtaining knowledge and skills about the infant’s behavior. Session 8: Obtaining knowledge and skills on how to interact with the infant.\n\nIn addition to the routine counseling, the trial group also received anxiety counseling with a cognitive-behavioral approach. It should be noted that the researcher (MK) had received prior training on CBT counseling and participants’ appropriate mental state and their diagnosis of anxiety were confirmed under the supervision of a clinical psychologist. After routine counseling for 50 minutes, the trial group also received anxiety counseling with the following subjects: Session 1: Establishing relationships among the mothers, learning the rules of the group, determining the goals of group therapy and getting feedback. Session 2: The psychological recounting of feelings and thoughts about the birth of their hospitalized infant in the context of a support group, emotional adjustment and release in a supportive environment and help homogenize the group members’ feelings. Session 3: Reviewing the signs of stress and introducing the concept of stress relief for obviating signs of stirred emotions. Session 4: Assessing the effect of cognition and thoughts on stress responses and helping mothers recognize their negative inner self-talk and introducing the importance of coping skills for stress management and assessing how people cope with problems. Session 5: Reviewing stressful self-talk and encouraging the mothers to turn self-talk into effective coping and going over previous stress relief exercises. Session 6: Problem-solving training and extracting a description of the problem from every member of the group. Session 7: Providing alternative solutions, assessing the solutions and using the best one. Session 8: Assessing the efficacy of the solutions used and their readjustment if necessary.\n\nBAI was completed by the mothers at the beginning of the first session, the end of the eighth session and three weeks after the intervention (for the final follow-up).\n\nThe data collected were analyzed in SPSS-19 using the Friedman test and the General Estimating Equations (GEE). Statistical hypotheses were verified at the level of p<0.05.\n\n\nResults\n\nIn the course of the study, nine mothers were excluded from the study, including three from the trial group (one due to her infant’s death and two for failing to complete the sessions) and six from the control group (for failing to complete the sessions). The study thus ended with 81 participants (Supplementary file 1). Dataset 1\n\nGroup: 1=case 2=control\n\nJob: 1= Housekeeper, 2= teacher, 3=Engineer, 5= Employee, 4= other\n\nEducation: 1,2= Associate Degree, 3= BS, 4= MS/PhD\n\nanxA1= Anxiety quetion1 before intervention\n\nanxB1= Anxiety quetion1 Immediately after intervention\n\nanxC1= Anxiety quetion1 Three weeks after intervention\n\nThe results obtained showed no significant differences between the trial and control groups in terms of age, education, occupation and income and the groups were thus matching in terms of these variables (Table 1). Moreover, the groups were not significantly different in terms of the mean score of anxiety before the intervention (p=0.408) and had the same level of anxiety before the interventions begun.\n\np-value <0.05\n\n*:Independent t-test\n\n**:Mann–Whitney U test\n\nA comparison of the mean score of anxiety between the two groups over three measurement phase using the GEE showed a significant difference between them in the trend of changes in anxiety (Table 2). The results showed that the score of anxiety reduced immediately after the intervention compared to before in both groups, and although this score had increased in the three-week follow-up compared to immediately after the intervention, it was still lower than the scores obtained before the intervention.\n\np-value <0.05\n\nThe trend of changes in the score of anxiety in the trial and control groups was analyzed using the Friedman test, which revealed a significant difference over the three measurement occasions and thus suggests the effectiveness of the intervention in the trial group (Table 3).\n\np-value <0.05\n\n\nDiscussion\n\nAnxiety is a highly unpleasant, generalized and often significant feeling of concern that is accompanied by one or several collective feelings and common symptoms, such as feeling discharged, shortness of breath, heart palpitations, headache, perspiration, sudden urinary incontinence and restlessness19. The present findings showed that cognitive-behavioral counseling reduces anxiety in mothers of children in a NICU. No significant differences were observed between the two groups in the mean score of anxiety before the intervention. The GEE showed a significant difference between the two groups in the mean score of anxiety immediately and three weeks after the intervention, as this mean score was lower in the trial group. The Friedman test showed significant differences in the mean score of anxiety in each group before, immediately and three weeks after the intervention. Comparing the mean scores showed a reduction in anxiety. The results of these two tests showed a reduction in the mean level of anxiety in both the trial and control groups; however, the reduction was higher in the trial group. As a result, both routine and cognitive-behavioral counseling can help reduce anxiety levels, but the reduction achieved is greater and more long-lasting with the latter.\n\nThe present findings are consistent with the results of other studies, Dehshiri found cognitive-behavioral interventions to be significantly effective in reducing anxiety and concern in patients with generalized anxiety disorder14. Patients with generalized anxiety disorder lack the ability to regulate their basic emotions, and cognitive-behavioral counseling teaches them techniques to help control the physiological components of their anxiety and modify their inaccurate interpretations of the events around them and thus helps reduce their anxiety; these techniques include diaphragmatic breathing and progressive/mental relaxation14. A study by Danae et al. confirmed the effectiveness of this intervention in reducing anxiety. According to their study, cognitive-behavioral counseling reduces anxiety in patients with migraine headaches by correcting their self-induced negative thoughts (concern about other’s misunderstandings, etc.) and fundamental beliefs and by teaching problem-solving skills18.\n\nOther studies have also shown the effectiveness of cognitive-behavioral counseling in reducing anxiety by identifying inefficient thoughts and correcting them and by teaching diaphragmatic or deep breathing and progressive/mental relaxation techniques as well as problem solving skills20,21.\n\nCognitive-behavioral therapy is based on the assumption that, instead of constructive behavior and appropriate coping responses, anxious people are rather predisposed to the perception of threat and avoidance coping responses, which lead to the persistence of anxiety and concern22. The daily observation of the behaviors of people who react differently to the same situation and the formation of judgments, evaluations, expectations, perceptions and other mental processes concerned with the individual's awareness can help change the individual’s own behavior and treatment in the face of different situations. Cognitive-behavioral therapy helps patients take the most adaptive and reasonable interpretation and adopt behaviors that match their new perspective23.\n\nThe trial group received training on how to be aware of the physiological and emotional signs of anxiety in themselves and learned more about their own behavioral patterns in response to anxiety and about how to perform a relaxation technique with the first signs of anxiety so as to reduce anxiety. The control of self-induced thoughts may be another reason for the reduced anxiety in the trial group. Learning meditative relaxation and cognitive techniques helps the individual deal effectively with her anxiety24.\n\nAs noted earlier, midwives are a group of healthcare workers that have an important role in providing counseling and health training not just to women but to entire families and the community, and they can therefore play a significant part in the provision of these interventions19. Facilitating the provision of this type of counseling to mothers can therefore help reduce their anxiety and improve their quality of life and infant care skills.\n\nAs for the limitations, the participants with their conditions (i.e. have a infants in the NICU), may have affected their response to the questions; however, this point was beyond the researcher's control, even though attempts were made to obviate this limitation by imposing certain inclusion and exclusion criteria and ensuring homogeneity between the two groups.\n\n\nConclusion\n\nThe present findings showed that both routine and cognitive-behavioral counseling can reduce anxiety in mothers with infants in the NICU; however, this reduction was greater and more long-lasting in the cognitive-behavioral counseling group. Given the role of medical personnel, especially midwives, in communicating with mothers and helping them deal with their problems, efforts should be made to create a conducive environment for counseling in order to help reduce anxiety and stress in mothers with special conditions and ultimately improve their quality of life and readiness for taking care of their infants.\n\n\nData availability\n\nDataset 1: Raw data obtained from Beck's Anxiety Inventory (BAI)\n\nGroup: 1=case 2=control\n\nJob: 1= Housekeeper, 2= teacher, 3= Engineer, 5= Employee, 4= other\n\nEducation: 1,2= Associate Degree, 3= BS, 4= MS/PhD\n\nanxA1=Anxiety quetion1 before intervention\n\nanxB1= Anxiety quetion1 Immediately after intervention\n\nanxC1= Anxiety quetion1 Three weeks after intervention\n\n10.5256/f1000research.12539.d17643325\n\n\nEthics and consent\n\nThis study was approved by the Ethics Committee of Alborz University of Medical Sciences with the code abzums.rec.1395.13, dated May 11th 2016, Irct ID: IRCT2016051427728N1 (registration date June 28th 2016). All participants provided written informed consent to participate in the study.",
"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 article is the result of a MSc thesis in Midwifery Counseling. We would like to express our gratitude to the Research Deputy, of Alborz University of Medical Sciences for supporting us and also to the personnel of Kowsar Hospital in Qazvin in 2016 and all the participating mothers for their help in conducting the study.\n\n\nAuthor details\n\nM. Koochaki is an MSc student in Consultation on Midwifery, at Alborz University of Medical Sciences, Karaj, Iran.\n\nZ. Mahmoodi is an Assistant Professor of Non-Communicable Disease Research Center, at Alborz University of Medical Sciences, Karaj, Iran.\n\nS. Esmaelzadeh – Saeieh is an Assistant Professor of Nursing & Midwifery, and a Faculty member of Alborz University of Medical Sciences, Karaj, Iran.\n\nK. Kabir is an Assistant Professor of Community Medicine, at the School of Medicine; and a Faculty member of Alborz University of Medical Sciences, Karaj, Iran.\n\nM. Tehranizadeh is an Assistant Professor and Faculty member of Payame Noor University, Kara, Iran.\n\nM. Dolatian is an Assistant Professor at the Department of Midwifery and Reproductive Health, and a Faculty member of Shahid Beheshti University of Medical Science, Tehran, Iran.\n\n\nReferences\n\nShaban Z, Dolatian M, Shams J, et al.: Post-Traumatic Stress Disorder (PTSD) Following Childbirth: Prevalence and Contributing Factors. Iran Red Crescent Med J. 2013; 15(3): 177–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTowhid B, Reza S, Farhad B, et al.: Prevalence of depression, anxiety and stress disorders in elderly people residing in Khoy, Iran (2014–2015). J Anal Res Clin Med. 2016; 4(2): 122–8. Publisher Full Text\n\nFolkman S: Stress: appraisal and coping. Springer. 2013; 1913–1915. Publisher Full Text\n\nSalari P, Firoozi M, Sahebi A: Study of the Stressors Associated with Pregnancy. Journal of Sabzevar University of Medical Sciences (Persion). 2005; 12(3): 34–40(Persion). Reference Source\n\nGhorbani M, Dolatian M, Shams J, et al.: Compare of post traumatic stress disorder between parents of term and pre term infants. Adv Nurs Midwifery. 2015; 24(86): 9–16. Publisher Full Text\n\nShaw RJ, Bernard RS, Storfer-Isser A, et al.: Parental coping in the neonatal intensive care unit. J Clin Psychol Med Settings. 2013; 20(2): 135–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMahmoodi Z, Karimlou M, Sajjadi H, et al.: Association of Maternal Working Condition with Low Birth Weight: The Social Determinants of Health Approach. Ann Med Health Sci Res. 2015; 5(6): 385–91. PubMed Abstract | Free Full Text\n\nWells A: Cognitive therapy of anxiety disorders: A practice manual and conceptual guide. John Wiley & Sons; 2013. Reference Source\n\nDolatian M, Mahmoodi Z, Alavi-Majd H, et al.: Psychosocial factors in pregnancy and birthweight: Path analysis. J Obstet Gynaecol Res. 2016; 42(7): 822–30. PubMed Abstract | Publisher Full Text\n\nSeyedamini B: Fears, Needs and Nursing Support of Mothers during Their Child's Hospitalization. Iran J Nurs. (2008–5923). 2011; 24(72): 57–66. Reference Source\n\nMoradi Z, Akbarzadeh M, Moradi P, et al.: The effect of acupressure at GB-21 and SP-6 acupoints on anxiety level and maternal-fetal attachment in primiparous women: a randomized controlled clinical trial. Nurs Midwifery Stud. 2014; 3(3): e19948. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeukrug ES: Counseling theory and practice. Cengage Learning; 2010. Reference Source\n\nGholami Farzane S, Jafari A, Ggamari M: Effectiveness of group counseling based on cognitive-behavioral therapy (CBT) on the reduction of the symptoms of anxiety sensitivity of divorced women. Woman & Study of Family. 2013; 5(19): 115–31. Reference Source\n\nDehshiri GR: The Effectiveness of Cognitive-Behavior Therapy on Anxiety and Worry of People with Generalized Anxiety Disorder. J Clin Psychol Med Settings. 2012; 4(2): 19–27.\n\nKhodai S, Kazemi T, Ali Abadi Z: The Effect of Cognitive–Behavioral Group Therapy on Depression and anxiety in Patients with myocardial infarction. Modern Care Journal. 2012; 9(4): 364–70. Reference Source\n\nTaghizadeh Z, Rezaiepour A, Mehran A, et al.: Usage of communication skills by midwives and its relation to clients’ satisfaction. J Hayat. 2007; 12(4): 47–55. Reference Source\n\nFydrich T, Dowdall D, Chambless DL: Reliability and validity of the Beck Anxiety Inventory. J Anxiety Disord. 1992; 6(1): 55–61. Publisher Full Text\n\nDanae Sij Z, Dehghani Firoozabadi M, Sharifzade G: Effects of cognitive-behavioral stress management on depression, anxiety, and pain control in patients with migraine headaches. Modern Care Journal. 2014; 10(3): 157–64. Reference Source\n\nBurkman RT: Berek & Novak’s gynecology. JAMA. 2012; 308(5): 516–7. Publisher Full Text\n\nBorkovec TD, Costello E: Efficacy of applied relaxation and cognitive-behavioral therapy in the treatment of generalized anxiety disorder. J Consult Clin Psychol. 1993; 61(4): 611–9. PubMed Abstract | Publisher Full Text\n\nSchultz DP, Schultz SE: Theories of personality. Cengage Learning; 2016. Reference Source\n\nTwohig MP, Hayes SC, Plumb JC, et al.: A randomized clinical trial of acceptance and commitment therapy versus progressive relaxation training for obsessive-compulsive disorder. J Consult Clin Psychol. 2010; 78(5): 705. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCoon DW, Thompson L, Steffen A, et al.: Anger and depression management: psychoeducational skill training interventions for women caregivers of a relative with dementia. Gerontologist. 2003; 43(5): 678–89. PubMed Abstract | Publisher Full Text\n\nKaramoozian M, Askarizadeh G: Effectiveness of cognitive-behavioral stress management intervention on anxiety and depression during pregnancy. Journal of Kerman University of Medical Sciences. 2013; 20(6): 606–21. Reference Source\n\nKoochaki M, Mahmoodi Z, Esmaelzadeh – Saeieh S, et al.: Dataset 1 in: The effect of cognitive-behavioral counseling on anxiety in the mothers of infants in the NICU: A randomized controlled trial. F1000Research. 2017. Data Source"
}
|
[
{
"id": "25942",
"date": "15 Sep 2017",
"name": "Soheila Pirdadeh Beiranvand",
"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-conducted and written study. I have the following comments:\n\nPlease explain main reasons why you used Cognitive-behavioral therapy for this subject?\n\nIn my opinion, it is better to write ethical code in the end of method not in design.\n\nPlease explain about validity and reliability of questionnaire in paper\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3035",
"date": "15 Sep 2017",
"name": "Zohreh Mahmoodi",
"role": "Author Response",
"response": "Dear Editor ,Thank you for your decision. I answered your questions: Please explain main reasons why you used Cognitive-behavioral therapy for this subject? Answer: Cognitive-behavioral therapy is a combination of cognitive and behavioral approaches to help the patient identify and correct her distorted patterns of thinking and inefficient behaviors and is carried out with regular discussions and carefully-organized behavioral assignments In my opinion, it is better to write ethical code in the end of method not in design. Answer: We did it as the editor Suggested. Please explain about validity and reliability of questionnaire in paper BAI is an international scale with a confirmed validity and reliability according to several studies and we got a reference in Data and measures"
}
]
},
{
"id": "25941",
"date": "15 Sep 2017",
"name": "Katayoun Falahat",
"expertise": [
"Reviewer Expertise Mental helath"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nّFirst of all, Thank you so much to choose me as a reviewer.\n\nThe research topic is new and very interesting. However, reading this article causes some question\nWould you please explain more about GEE? Why do you use this analysis method? I think the description of this method is empty in Data analysis Section.\n\nHas the researcher have a clinical experience to perform CBT in different subjects? How long did each session of CBT take? And how did the CBT session perform? Individually of in a group?\n\nThank you so much again.\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": "3034",
"date": "15 Sep 2017",
"name": "Zohreh Mahmoodi",
"role": "Author Response",
"response": "Dear Editor Thank you for your decision.I answer the question: Would you please explain more about GEE? Why do you use this analysis method? I think the description of this method is empty in Data analysis Section. Answer: GEE is a Generalized Estimating Equations and the Description data is in Table 1 Has the researcher have a clinical experience to perform CBT in different subjects? How long did each session of CBT take? And how did the CBT session perform? Individually of in a group? Answer: Yes the researcher had received prior training on CBT counseling.Both groups received eight sessions of routine counseling (routine neonatal care), twice per week for four weeks and each session was 50 minutesRegards."
}
]
},
{
"id": "25999",
"date": "27 Sep 2017",
"name": "Razieh Bagherzadeh",
"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 appreciate the authors for their initiative. They have selected an interesting topic for study. Certain points are given below so that they can work on them to make it better.\nIn result section of abstract (line 2 of result) authors have reported two P value for non-differences in maternal anxiety between the two groups before intervention. Why two P value?\n\nResult section of abstract (line 4 and 5 of result) does not match with results in Table 2. Please see mean of anxiety score immediately after the intervention in two groups.\n\nIn sample size calculation, how much α and β is considered? Please mentioned.\n\nPlease correct attrition rate (10%.??)\n\nIn Table 1, income per week or month??\n\nIn table 1: why mean rank for Job Status? Mean rank for nominal variable is not correct.\n\nResults (In Table 2 and 3) show reduction of anxiety score in both groups and further reduction in the control group Immediately after the intervention, but in discussion section (paragraph 1) authors have written: \"The GEE showed a significant difference between the two groups in the mean score of anxiety immediately and three weeks after the intervention, as this mean score was lower in the trial group.\" Reported results did not indicate whether the statistically significant mean difference between the two groups is Immediately after the intervention and/or in lasting? Discussion section will be according to results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1679
|
https://f1000research.com/articles/6-40/v1
|
12 Jan 17
|
{
"type": "Research Article",
"title": "Texture analysis of the developing human brain using customization of a knowledge-based system",
"authors": [
"Hugues Gentillon",
"Ludomir Stefańczyk",
"Michał Strzelecki",
"Maria Respondek-Liberska",
"Ludomir Stefańczyk",
"Michał Strzelecki",
"Maria Respondek-Liberska"
],
"abstract": "Background: Pattern recognition software originally designed for geospatial and other technical applications could be trained by physicians and used as texture-analysis tools for evidence-based practice, in order to improve diagnostic imaging examination during pregnancy.\n\nMethods: Various machine-learning techniques and customized datasets were assessed for training of an integrable knowledge-based system (KBS), to determine a hypothetical methodology for texture classification of closely-related anatomical structures in fetal brain magnetic resonance (MR) images. Samples were manually categorized according to the magnetic field of the MRI scanner (i.e. 1.5-tesla (1.5T), 3-tesla (3T)), rotational planes (i.e. coronal, sagittal and axial), and signal weighting (i.e. spin-lattice, spin-spin, relaxation, proton density). In the machine-learning sessions, the operator manually selected relevant regions of interest (ROI) in 1.5/3T MR images. Semi-automatic procedures in MaZda/B11 were performed to determine optimal parameter sets for ROI classification. Four classes were defined: ventricles, thalamus, grey matter, and white matter. Various textures analysis methods were tested. The KBS performed automatic data pre-processing and semi-automatic classification of ROIs.\n\nResults: After testing 3456 ROIs, statistical binary classification revealed that combination of reduction techniques with linear discriminant algorithms (LDA) or nonlinear discriminant algorithms (NDA) yielded the best scoring in terms of sensitivity (both 100%, 95% CI: 99.79-100), specificity (both 100%, 95% CI: 99.79-100) and Fisher coefficient (≈E+4, ≈E+5, respectively).\n\nConclusions: LDA and NDA in MaZda can be useful data mining tools for screening a population of interest subjected to a clinical test.",
"keywords": [
"prenatal",
"fetal brain",
"computer-assisted radiology",
"Mazda",
"b11",
"cybernetics",
"artificial intelligence",
"computational visual cognition"
],
"content": "Introduction\n\nMedicine is not an exact science but an applied, interdisciplinary field1. Therefore, the time to produce physicians specialized in radiology is very long2,3. Moreover medicine is still and will still be evolving in years to come4–6. As cures are being discovered or invented, new diseases become known and mutations surface along with new variants7–10. Trainable knowledge-based systems (KBS) could be an answer to the global shortage of radiologists1,11–14. Besides, there are other major obstacles, which also prevent KBS from being fully functional out of the factory. The body of medical knowledge, known to this date, is gathered and transferred through theoretical and clinical intuition as well as experience1,2,12. Then physicians continue to expand their acquired knowledge with years of evidence-based practice15,16. On top of the challenge is the fact that some conditions may not be symptomatic during medical examination17,18. Hence, medicine is indeed a continuing learning process15,16. That was why we proposed the approach to have the observer (in this case the physicians working in the field) as the direct trainer (the programmer) of a KBS designed as a customizable, conceptual framework. In computer science, a framework is a system which implements the process of abstraction ― i.e. a technique where developers make computer modeling/programming simpler to understand, use and apply. Such a KBS should not be designed as “a hammer to drive a nail” but as an abstraction system with generic functionality ― which can be changed with user-written codes and customized for unlimited applications. The purpose is to enhance human-computer interaction (HCI) in medicine.\n\nThe aforementioned points are introduced especially to show the need for customization in computer-aided-diagnosis (CAD). Pre-programmed CAD systems surely help radiologists and obstetricians,19–21 but they could be better and more useful with some room for customization ― and could even improve the sharing and preservation of diagnostic innovations.\n\nThe aim of this research was to find a customizable software framework (KBS) to mathematically determine an optimal combination of texture analysis methods to differentiate anatomical structures in the developing fetal brain (i.e. regions of interest (ROI)). Why did this experiment focus on normal development of central nervous system (CNS) rather than common conditions affecting fetal organogenesis? When considering fetal intervention to correct an anomaly in utero, the first ethical priority is actually mother safety. Prenatal well-being is clinically second, after evaluation of benefit-risk ratio22–26. In the 1960s, fetal surgery was conceived and introduced in clinical practice27. In spite of the improvement in surgical technology, the number of successfully-treated cases of congenital defects and life expectancy of survivors are still limited28–31. The theoretical procedures are troublesome and have not been investigated enough32. Hence, they are considered as experimental treatments32. Whether invasive or minimally invasive, fetal intervention is not the ultimate solution of this societal conundrum. It is usually reserved for cases of severe fetal anomalies28–32. In recent years, prenatal therapy is gaining popularity in religiously conservative territories particularly where abortion is prohibited33,34. Fetal intervention is recommended for fetuses with mild and non-lethal defects, in order to discourage abortion and continue childbearing. In the country where this research was carried out (i.e. Poland), abortion is, once again, nationally banned ― except in cases of rape, life-threatening pregnancy and childbirth, and grave malformations33–36. Pressures are due to growing pro-life supporters demanding total anti-abortion, child-bearing at all costs, and capital punishment for illegal termination of pregnancy33–36. Nevertheless, Poland neither sanctions nor executes the outlaws of illegal abortion. Despite the absence of penalty, abortion is respectfully performed as permitted by local authority. The aforementioned dilemma justified the usage of fetal MRI (fMRI) in this research. In theory, it was previously hypothesized and documented that the high electromagnetic fields (EMF) used for MR procedures can disrupt the early stage of organogenesis. To this date, the embryotoxic, fetoxic, and teratogenic effects of MRI are not well known. Normally, fMRI is not recommended during the first and second trimester, unless it is absolutely necessary to confirm and/or supplement the diagnosis of fetal anomalies. Legal decision to prescribe abortion to a patient sometimes requires advanced clinical investigation, accompanied by psychological counseling before and after the operations37–41. To this date, ultrasound (US) devices are preferred for obstetrical examinations. With 2D, 3D, and 4D US scans, physicians can effectively and efficiently diagnose the majority of life-threatening conditions affecting mother and fetus42–46. Therefore, ultrasonography (USG) is sufficient for diagnosis of severe malformations affecting abdominal organs. Why was magnetic resonance imaging (MRI) prescribed? Fetal brain is where US devices struggle to produce desirable results. MRI was subsequently performed to rule out severe abnormalities in brain development, which are not visible on sonogram. Magnetic resonance (MR) samples came from fetuses with suspected heart and kidney defects. Disruption of organogenesis in the latter, depending on severity, might affect normal development of the brain. MR samples used in this experiment were visually investigated by specialists, structure-by-structure. No severe malformations were observed. In these cases, MRI studies did not add any further indication to legally fulfill the criteria to terminate pregnancy. The human visual apparatus has its limit. Unfortunately, missed diagnoses do occur. Malformations may not be apparent prior to birth. If fetal defects are suspected, bureaucracy may also restrict access to more advanced testing and healthcare. Consequently, pregnant women are deliberately forced to bear and deliver malformed babies. At the end of the day, physicians may still have to deal with the legal liability for failure to terminate pregnancy41. Unwanted fetuses may become neglected, and foundling is also a growing problem in society47–49 . Congenital brain defects and its impacts on physical and cognitive development may not be detectable until after birth. Fetal outcome and mental retardation can be difficult for a physician to predict. The process requires access to better medical examination, development of more advanced tools and further investigation. That is why the ultimate goal of this feasibility study was to gather knowledge for the practicality of a proposed project seeking to improve diagnostic accuracy and precision, by extending HCI usage in medicine. There are many computer-aided diagnostic tools on the commercial shelves ( — e.g. Radiomics,50–52. Definiens Tissue Phenomics®,53 CAD4TB Diagnostic Software54,55). Sadly, they are primarily designed for pre-loaded applications but not much else.\n\n\nMethods\n\nThis study was approved by the Research Ethics Committee of the Medical University of Lodz (MUL). Written informed consents were obtained from all subjects, as well as perpetual licenses pertaining to copyright and ownership bundle-of-rights of the medical records ― for the purpose of education, research and publication. All experiments were performed in accordance with the relevant institutional and national guidelines, regulations, licenses and approvals ― from, but not limited to MUL, Lodz University of Technology (TUL), Barlicki University Hospital (BUH), Central University Hospital (CUH), Kopernik Hospital, Biegański Specialty Hospital, Polish Mother’s Memorial Hospital & Research Institute of Lodz (ICZMP) and National Health Fund (NFZ). The collective approval-certificate (number RNN/213/13/KE) and the researchproposal were reviewed and endorsed by the senior officers of the Research Ethics Committee.\n\nThis feasibility study was approved by the Bioethics Research Board which regulates experiments carried out at the Medical University of Lodz and affiliated research hospitals in Poland (permit number: RNN/213/13/KE). Due to administrative and logistic delay as well as finding volunteers and expensive cost and availability of MRI56,57, it took us nearly five years to collect the magnetic resonance (MR) samples. The nature of the study was explained to patients in Polish by attending physicians, and individual written informed consent was obtained for this research and its publication. Agreement number: 3/2011 - concluded on December 6, 2011, between the experimenter (Hugues Gentillon), research supervisor (Ludomir Stefańczyk) and rector of Medical University of Lodz (Radzisław Kordek), Faculty of Biomedical Science - Post-graduate research in Diagnostic Imaging and Radiotherapy. Consent for publication of these data was obtained. Furthermore all data from human participants were anonymized as per consent agreement. Informed Bioethics Committee Approval Number: RNN/213/13/KE, JULY 16, 2013. If you have any questions regarding the decision please include the above number and date in your letter. Send correspondence to: THE BIOETHICS COMMITTEE OF THE MEDICAL UNIVERSITY OF LODZ Al. Kościuszki 4, 90-419 Łódź, tel. 0 785 911 596, 42 272-59-05, fax 42 272-59-07.\n\nThough this research shares similarities with a clinical trial, it is “virtual” ― i.e. it is non-interventional. Such medical study does not meet criteria for clinical trial registration58–62. Furthermore the investigational tools were merely used for technical exploration and to measure their feasibility in medical practice ― by using simulation settings. Lastly, the results were not used to alter patients’ therapeutic care and outcome58–62.\n\nAdvance notice to readers. Readers should not expect us to teach the entire science of artificial neural network (ANN) (feedforward neural networks, recurrent neural network, etc.) in just a manuscript. It is not possible. A full introduction is not even possible. Unfamiliar readers are expected to make an effort on their own to read and learn the basic principles of artificial neural network and know the basic terminology. Like a human brain, an ANN can store memories. ANN can also judge based on stored memories and logical rules. To run a naïve trial run, it was ideal to have the ANN in a condition like ‘permanent global amnesia’ – i.e. a phenomenon where a brain is in a state of total blackout and thus cannot judge based on prior memories. The KBS used in this experiement was lacking an automatic memory cleaner and optimizer. Hence, stored memories were manually deleted in the ANN for every trial run.\n\nComputer vision.Under a reciprocal cooperation-agreement between MUL and TUL, we obtained permissionto test a KBS consisting of a custom version of MaZda software package.63,64 It contains algorithms for data classification and visualization. How did it come to us? In 1998, a group of medical scientists, engineers, physicists, mathematicians and others initiated the B11 project at the Cooperation in Science and Technology (COST). The purpose was to develop software frameworks for quantitative analysis of MR scans, to improve medical diagnosis. MaZda, a Delphi/C++ computer program, was originally built in 1996 for applications in mammography. It was later used by COST to complement its MRI software modules (e.g. B11, B21). MaZda 4.6 package was the last official release (B11 version 3.3 included). In this research, we collaborated with modular programmers to further upgrade, separate and amalgamate the functionality and compatibility of MaZda (version 5 RC HG) with other modules. In the MaZda 5 version used in this research, the new features names were introduced to maintain compatibility with WEKA software (www.cs.waikato.ac.nz/ml/weka/, a popular package for data mining, classification and analysis). This way data generated by Mazda may be used as the input to the WEKA. Also, 3D deformable model that are used for 3D volume-of-interest (VOI) generating was introduced (— for details, see the publication by: Piotr M. Szczypiński, Ram D. Sriram, Parupudi V.J. Sriram, D. Nageshwar Reddy, A model of deformable rings for interpretation of wireless capsule endoscopic videos, Medical Image Analysis, Volume 13, Issue 2, April 2009, Pages 312–324). Moreover, to speed up and automate, some commands regarding 3D analysis were introduced. This way one can build a script (that contains e.g. commands for image loading, analysis option loading, performing analyses and storing results) for automatic analysis of a number of 3D data. Finally, this MaZda 5 version was compiled with Embarcadero software environment, which was not an ideal framework. Thus, the inventors decided to return to Builder C++. Currently, the latest version of Mazda being built is qMaZda (described and available at www.eletel.p.lodz.pl/pms/SoftwareQmazda.html).\n\nFigure 1 shows the main steps of texture analysis with MaZda and B11. A key change in this custom version is the integration of MaZda with a variety of computational geometric algorithms from Qhull (e.g. VSCH_1, VSCH_2, VSCH_3, VSCH_4, VSCH_5). For details about 1D, 2D, 3D, and 4D features, see Dataset 7. VSCH network algorithms are located in the ‘Convex Border’ menu.VSCH_1, for example, can be used to identify the strongest discriminant parameter in a large dataset.\n\nOur trial-and-error experiments and texture-analysis software development spanned over five years of research. All the findings shared common denominators. Quantitative brain-tissue segmentation was affected by several factors, such as characteristics of fetuses (e.g. gestational age, shape, normal/abnormal development) and the quality of fMR images (e.g. 1.5/3T, resolution, slice thickness, rotational planes, artifacts, etc). Automatic segmentation of newborn brain MRI has been documented in the literature65. The algorithmic contributions reported so far have achieved limited success, unfortunately. Automatic segmentation of prenatal brain is even more challenging and time-consuming.\n\nUnsupervised segmentation.Once an image is acquired in a readable format (bitmap format (BMP)), the first step is texture segmentation – i.e. partitioning an image into ROIs. B11 can perform unsupervised segmentation and cluster analysis. In some instances, B11 achieved accuracy closed to that of clinicians. However, unsupervised segmentation was not reliable enough for therapeutic use. Fetal brain segmentation with B11 still required extensive expert interaction. In our observations, the key problems were maternal factors, environmental effects, growth variability, randomness of fetal movements and its detrimental effects on image quality. Therefore, automatic segmentation was used for new insight into the possibility of improving supervised segmentation. The steps of unsupervised segmentation are relatively simple: image acquisition and run analysis. ROIs and segment numbers can be manually adjusted. The drawback with B11 segmentation is limitation to 8-bit grayscale BMP. 16-bit DICOM was converted to visually lossless BMP, by dropping least significant bits. Note that B11 identified textures not anatomical structures. The information collected from the unsupervised trials was later used as guidance to manually estimate boundaries of anatomical structures (ROIs) for the supervised trials. The preliminary trials were single-blinded ― i.e. the user knew the characteristics of the ROIs, and the KBS received no hints (no ROI selection). Further information was gathered with a semi-automatic (unblended) segmentation by defining 4 classes (ROIs): thalamus, ventricles, grey matter and white matter. In unsupervised mode, the KBS performs quite well when brain images are from MR examination of the same subjects and same sequences ― but performs poorly when they came from different subjects. The findings were likewise for same sequences of the same patient taken at a different time and MR scanner settings. The challenge was: how do we match macroscopic characteristics with electronic recognition, regardless of MR image shadings? MaZda and B11 are not yet designed to allow user to well define semantic rules and/or import plugins for fully electronic recognition of anatomical structures. Object-based image analysis tools such as eCognition work consistently well for geo-spatial applications (e.g. identification of a river in an image)66,67. In fetal radiology, it is still a challenge to achieve consistent results with automated-pattern recognition of prenatal anatomy. Programming a reliably effective system for such highly sensitive application is feasible but also time-consuming. Such a task would require taking into account all known variations due to pregnancy chronology and fetal developmental, to minimize segmentation errors. MaZda and B11 were originally built for HCI rather than fully-automated applications. Therefore, the best practical methodology, in this research, was for the operator to at least have prior knowledge of human embryogenesis, in order to manually and correctly identify and select fetal ROIs. Often, macroscopic appearance of many brain structures are not well differentiated in the first trimester. Hence, the selected samples were at least 20 weeks of maternal age.\n\nSupervised segmentation. 3-tesla (3T) and 1.5-tesla (1.5T) magnetic resonance (MR) sequences of fetal brain were manually segmented into 3456 ROI. The categories were predefined as followed: ventricles (class 1), thalamus (class 2), white matter (class 3) and grey matter (class 4). The selected samples did not have any brain malformations. As previously mentioned, the anomalies were in the cardiovascular and/or renal systems. The focus of this research was on normal anatomy of fetal brain. Forward processing method ― also known as “supervised segmentation”68 ― was performed as delineated: (1) image acquisition from MR scanner, (2) selection of ROIs with MaZda, (3) image normalization with MaZda (4) feature extraction with MaZda (5) data preprocessing with B11 (6) texture classification with B1163,64,68. The first four steps were done with MaZda and last two steps with B11 (Figure 1). After the preliminary trials, we became interested to learn what needs to be adjusted in order to reduce misclassification of MR images. The unsupervised segmentation revealed that the KBS was very sensitive to greyscale shading, artifacts, and image thickness as well as resolution quality. Thus, we trained the KBS accordingly. 1.5T images were originally encoded as 12-bit lossless JPEG (Joint Photographic Experts Group) format and wrapped in Digital Imaging and Communications in Medicine (DICOM). 1.5T images were transcoded from lossless JPEG to uncompressed DICOM. 3T images were natively uncompressed. 360 parameters were extracted with MaZda (histogram: 9; co-occurrence matrix: 220; run-length matrix: 20; gradient matrix: 5; auto-regression: 5; Haar wavelet: 28; geometry: 73). Parameters’ names are provided in the appendix at the end of this manuscript. We assessed three automated techniques of parameter selection ― i.e. Fisher selection, POE + ACC (classification error probability and average correlation coefficients), MI+PA+F (combination of mutual information, pair analysis and fisher selection). Further details about the mechanics and functionality of these techniques are provided in the manufacturer’s user manual and tutorial guides69. For each technique, we also used the VSCH (vector supported convex hull) module in MaZda to enhance computation and visualization of geometric structures: 1) pre-reduction (before selection) to rule out insignificant parameters, 2) post-reduction (after selection) to identify strongest relevant parameters. All aforementioned parts of the process were computed automatically by the KBS without any manual interference. Data imported [SEL (Schweitzer Engineering Laboratories) to CSV (comma-separated values)] in B11 were preprocessed with PCA, LDA and NDA and classified by means of 1-nearest neighbor (1-NN) and ANN. In machine learning and cognitive science, 1-NN is also known as k-NN, where n=1. It is a “lazy-learning” algorithm, in which new ROIs are locally classified by getting interweaved into the closest cluster in the training set. The rest of the computation is delayed until the end of the classification process. K-NN is implemented in B11 classifiers, as well as in the preprocessing procedures in MaZda69.On the other hand, ANN is a self-organizing algorithm with hidden layers and adjustable number of neurons69–72. It can be used for both supervised and unsupervised classification. Neural classification algorithms are implemented in MaZda/B11. ANN training, for example, is standardized for NDA analysis69,70.― i.e. a type of feedforward-artificial neural network, based on multilayer Perceptron (MLP)69. Nonlinear procedures and classifiers in B11 are MPL algorithms69,70. For optimal performance, two sets of samples are needed: one for training and another one for validation70. The pitfall with this algorithm is its sensitivity to overtraining (too strong memorization)69,70. ANN training time is shorter with standardization. For continuation, training without standardization was carried out, in spite of long processing time. ANN (one-class/n-class) and 1-NN training runs were conducted with different sequences of MR images: T2-weighted (T1), T1 weighted, and proton-density (PD) sequences. N-class training was discontinued due to repeated problems with overtraining and lack of reproducibility in F values and miss-classification errors.\n\nDespite the usage of multi-level, automated selection/reduction techniques, some extracted values still did not match the controlled ROI values. Differentiating thalamus from other thalamic nuclei and grey matter was the key problem. That was when we manually intervened to customize and improve the extracted data. First ROI surface areas were manually increased, in order to limit the number of parameters reporting zero and infinity values. Parameters which couldn’t be correctly computed were manually omitted in the report file. Some pre-processing procedures in both MaZda and B11 couldn’t be performed when the report file contained erroneous values. We accessed MaZda generated report files by changing the extension format from SEL to CSV and then imported them into Excel 2013 for adjustment. Parameters measured with other CAD tools can also be entered in the report files by simply using Microsoft Excel. The edited file can then be imported in B11 to perform texture classification.\n\nAdditional tests were carried out with same ROIs (i.e. thalamus, ventricle, grey matter and white matter) to dramatically improve accuracy and precision of the KBS: it was done with a customized dataset derived from MaZda algorithms, using semi-manual reduction and nearest-neighbor feature selection (see Data availability: Dataset 1–Dataset 2). The training data were used to orient the KBS to recognize what ROIs had the same tissue characteristics, in spite of being originated from different patients or different sequences of the same patients. The training was conducted with combination of two built-in classification tools (i.e. nearest neighbor (NN) and artificial neural network) and four data processing techniques (i.e. RAW: read as written; PCA: principal component analysis, LDA: linear discriminant analysis; NDA: nonlinear discriminant analysis). To measure the KBS sensitivity and specificity, we defined “normal” as \"ROIs with identical tissue\" characteristics and “abnormal” those with different tissue characteristics (Figure 2–Figure 5). Apart from noise and artifacts, we found out that the preliminary results were also affected by the planes (axial, coronal, sagittal) ― which refer to the rotational planes of the spinning MR scanner in relation to the mother, not the fetus. There flows the reason for the classification by rotational planes. In learning mode, we observed a consistent scoring for all the ROIs. Thus the logical and semantic information provided to the KBS was effective. Statistical binary tests (also known as classification function tests) were computed in STATISTICA version 10 to assess the performance of each procedure (combination of preprocessing techniques and classifiers). In medicine, binary scores (TP, FP, TN, FN etc.) are used to determine not just normal and abnormal characteristics but also classification property of an examination.\n\na) specificity scoring: ROI 1 = white matter control, ROI 2 = white matter | ― b) sensitivity scoring: ROI 1: white matter control, ROI 2: thalamic nucleus other than thalamus.\n\na) specificity scoring: ROI 1 = thalamus control, ROI 2 = thalamus | ― b) sensitivity scoring: ROI 1: thalamus control, ROI 2: thalamic nucleus other than thalamus.\n\na) specificity scoring: ROI 1 = ventricle control, ROI 2 = ventricle | ― b) sensitivity scoring: ROI 1: ventricle control, ROI 2: white matter.\n\na) specificity scoring: ROI 1 = grey matter control; ROI 2 = grey matter | ― b) sensitivity scoring: ROI 1: grey matter control, ROI 2: white matter.\n\n\nResults\n\nWith Fisher coefficient (F), we tested for difference between ROIs. It was nearly zero for ROIs which were alike. Therefore, the tissue anatomy was consistently the same among the normal ROI group. In testing mode, misclassification values, as low as 0%, were also recorded, in some trials (Table 1). RAW and PCA did not responded to the training, while LDA and NDA did. We obtained high F values, 100% sensitivity and 100% specificity for LDA and NDA (Table 1–Table 2) ― which means that there was likely a real difference between the normal and the abnormal ROIs.\n\nSn: Sensitivity, Sp: Specificity, CI: Confidence Interval, PPV: Positive Predictive Value, NPV: Negative Predictive Value, PLR: Positive Likelihood Ratio, NLR: Negative Likelihood Ratio, P: Prevalence, F: Fisher Coefficient, RAW: read as written; PCA: principal component analysis, LDA: linear discriminant analysis; NDA: nonlinear discriminant analysis (see Data availability: Dataset 3–Dataset 4).\n\nSn: Sensitivity, Sp: Specificity, CI: Confidence Interval, PPV: Positive Predictive Value, NPV: Negative Predictive Value, PLR: Positive Likelihood Ratio, NLR: Negative Likelihood Ratio, P: Prevalence, F: Fisher Coefficient, RAW: read as written; PCA: principal component analysis, LDA: linear discriminant analysis; NDA: nonlinear discriminant analysis (see Data availability: Dataset 5–Dataset 6).\n\n\nDiscussion\n\nTo this date, no such research has been documented in the literature. The explanation could be derived from the difficulty of finding fetal MRI samples for medical research, as well as the common hindrance to their availability ― i.e. continuing systematic concerns over the theoretical risks of MRI usage during pregnancy, in parallel to the lack of clinical studies and trials assessing such theoretical risks73–76, plus the expensive cost of MRI examination56–57 and the scarcity of customizable CAD tools on the freeware shelves ― just to list a few.\n\nThe majority of the KBS we came across were designed for technical use and not easily customizable. Such programs required paying for marketing company maintenance and for in- house-developed customization service, on top of the annual license fee. Thus this option was not feasible for application in real-world settings, where resources are often sparse ( — e.g. eCognition,66,67 Media Cybernetics,77–78 Radiomics,50–52 Definiens Tissue Phenomics®,53 CAD4TB Diagnostic Software54,55, etc.).\n\nPrevious medical studies done with MaZda include inflammation, brain cancer detection, multiple sclerosis, electrophoresis, etc79–82. Herein, we defined some test samples as “abnormal” ROIs. However, they were, in reality, normal tissue. Not testing directly for a common anomaly doesn’t necessarily mean that there is no real medical application. Though the tests were simulated, the research design was conceived for real-world medical applications83–85. For example, this research design could be used to detect ectopic tissue migration, neurogenesis and neuronal migration (brain function migration as a result of natural process or after injury), metaplasia and interference with brain development. Last but not least, this simulation research followed standards used in clinical trials86.\n\nThe choice of binary classification (sensitivity/specificity) was favored over frequentist inference (p-value) because it provides more information in terms of statistical relevance to medical diagnosis, prognosis and disease prevalence87–89. One key difference between Fisher, POE+ACC, and MI+PA+F is the number of parameters. To perform MI+PA+F, the dataset must contain at least 30 parameters strongly matching its selection-reduction criteria69. Otherwise, the KBS reported an error. In our study, it was a common occurrence when the surface area of a ROI was insufficient to extract 30 parameters meeting the MI+PA+F semantics. RAW and PCA were not so affected by the training process and thus remained very sensitive to minute difference in greyscale shading.\n\nA solution to high misclassification (M) was to exclude some parameters which were very sensitive to post-editing sharpness. In this research, the images were, however, processed without post-editing sharpness because high M was not regarded as a problem. Instead, we used RAW and PCA as reference tests (results before the training of the KBS). On the other hand, LDA and NDA responded well to the training, and M was consistently zero.\n\nDuring the study, we had to obviously clear the KBS memory several times for every trial run. We hope that the software developers will soon implement a more effective and efficient way (e.g. one-click) to clear specific random-access memory (RAM) without closing module(s) or without manually dumping the entire RAM or restarting the computer.\n\nPatients gave consent to perform MRI examination and use of images in research and for the manuscript publication. Nevertheless, this authorization was not enough, as ownership and copyright of medical records are not always exclusively attributed to patients ― and such rights may not be assignable90–92. For the sake of prudence, we had to also seek institutional/research- hospitals’ clearance and approval ― which in turn were then subject to different administrative and logistic factors and regulations. Consequently, it took nearly five years to collect sufficient MRI samples to make this research possible.\n\n\nConclusion\n\nIn brief, the findings show that better results were obtained with LDA and NDA. The observed difference between the two imaging modalities was previously and repeatedly proven to be due to 3T MRI having higher resolution and able to capture more details93–95. Lastly, LDA and NDA could be useful tests for pre-screening ― provided ruling-in/ruling-out semantics are well defined and the KBS is well trained.\n\n\nData and software availability\n\nMaZda Package v5 RC HG available from: http://dx.doi.org/10.17632/dkxyrzwpzs.196.\n\nF1000Research: Dataset 1. 1.5T data, 10.5256/f1000research.10401.d14678297.\n\nF1000Research: Dataset 2. 3T data, 10.5256/f1000research.10401.d14678398.\n\nF1000Research: Dataset 3. 1.5T, 10.5256/f1000research.10401.d14678499.\n\nF1000Research: Dataset 4. 3T, 10.5256/f1000research.10401.d146785100.\n\nF1000Research: Dataset 5. 1.5T, 10.5256/f1000research.10401.d146786101.\n\nF1000Research: Dataset 6. 3T, 10.5256/f1000research.10401.d146787102.\n\nF1000Research: Dataset 7. Parameter list, 10.5256/f1000research.10401.d146788103.",
"appendix": "Author contributions\n\n\n\nHG: experimenter, research designer, and writer; LS: supervisor, sample provider, clinical feedback; MRL: coordinator, sample provider, clinical feedback; MS: software maker, updates, technical feedback.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMedical University of Lodz & Polish Research Committee and affiliated institutions and hospitals; Self-funded; MRI cost was covered by Polish National Health Fund, grants and financial aid from Swedish Ministry of Education and Research/Centrala Studiestödsnämnden and from U.S. Department of Education.\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 gratefully acknowledge prof. Rafal Pawliczak and MUL staff for research coordination, logistics, and administration; prof. 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Reference Source\n\nManlove W, Fowler KJ, Mellnick VM, et al.: Role of MRI in Trauma in the Pregnant Patient. In: MRI of Fetal and Maternal Diseases in Pregnancy. Springer International Publishing, 2016; 491–49. Publisher Full Text\n\nBulas D, Egloff A: Benefits and risks of MRI in pregnancy. Semin Perinatol. 2013; 37(5): 301–304. PubMed Abstract | Publisher Full Text\n\nThompson O, Otigbah C, Nnochiri A, et al.: First trimester maternal serum biochemical markers of aneuploidy in pregnancies with abnormally invasive placentation. BJOG. 2015; 122(10): 1370–1376. PubMed Abstract | Publisher Full Text\n\nNambiar M, Rema T: Cancer in Pregnancy. In: Principles of Critical Care in Obstetrics. Springer India, 2016; 289–29. Publisher Full Text\n\nZhang R, Bo T, Shen L, et al.: Semi-Quantitative Analysis of Brain MR Imaging in 76 Cases of Neonatal Indirect Hyperbilirubinemia. Open J Pediatr. 2016; 6(04): 280–289. Publisher Full Text\n\nLu LN, Qian ZM, Wu KC, et al.: Expression of Iron Transporters and Pathological Hallmarks of Parkinson’s and Alzheimer’s Diseases in the Brain of Young, Adult, and Aged Rats. Mol Neurobiol. 2016; 1–12. PubMed Abstract | Publisher Full Text\n\nBrown AM, Nagala S, McLean MA, et al.: Multi-institutional validation of a novel textural analysis tool for preoperative stratification of suspected thyroid tumors on diffusion-weighted MRI. Magn Reson Med. 2016; 75(4): 1708–1716. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFetit AE, Novak J, Peet A, et al.: Three-dimensional textural features of conventional MRI improve diagnostic classification of childhood brain tumours. NMR Biomed. 2015; 28(9): 1174–1184. PubMed Abstract | Publisher Full Text\n\nSavio S, Hakulinen U, Ryymin P, et al.: Hemispheric asymmetry measured by texture analysis and diffusion tensor imaging in two multiple sclerosis subtypes. Acta Radiol. 2015; 56(7): 844–851. PubMed Abstract\n\nFernandez-Lozano C, Seoane JA, Gestal M, et al.: Texture analysis in gel electrophoresis images using an integrative kernel-based approach. Sci Rep. 2016; 6: 19256. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTun JK, Alinier G, Tang J, et al.: Redefining simulation fidelity for healthcare education. Simul Gaming. 2015; 46(2): 159–174. Publisher Full Text\n\nBeyer-Berjot L, Patel V, Ziprin P, et al.: Enhanced recovery simulation in colorectal surgery: design of virtual online patients. Surg Endosc. 2015; 29(8): 2270–2277. PubMed Abstract | Publisher Full Text\n\nAzarnoush H, Alzhrani G, Winkler-Schwartz A, et al.: Neurosurgical virtual reality simulation metrics to assess psychomotor skills during brain tumor resection. Int J Comput Assist Radiol Surg. 2015; 10(5): 603–618. PubMed Abstract | Publisher Full Text\n\nCheng A, Auerbach M, Hunt EA, et al.: Designing and conducting simulation-based research. Pediatrics. 2014; 133(6): 1091–1101. PubMed Abstract | Publisher Full Text\n\nFleiss JL, Levin B, Paik MC: Statistical Methods for Rates and Proportions, Third Edition John Wiley & Sons; 2013; Ch. 1–2. Publisher Full Text\n\nPetrie A, Sabi C: Medical statistics at a glance. Diagnostic Tools. John Wiley & Sons; 2013; Ch. 38. Reference Source\n\nLeeflang MM, Rutjes AW, Reitsma JB, et al.: Variation of a test’s sensitivity and specificity with disease prevalence. CMAJ. 2013; 185(11): E537–E544. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKokkonen EW, Davis SA, Lin HC, et al.: Use of electronic medical records differs by specialty and office settings. J Am Med Inform Assoc. 2013; 20(e1): e33–e38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDevakumar D, Brotherton H, Halbert J, et al.: Taking ethical photos of children for medical and research purposes in low-resource settings: an exploratory qualitative study. BMC Med Ethics. 2013; 14: 27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMezrich JL, Siegel E: Who Owns the Image? Archiving and Retention Issues in the Digital Age. J Am Coll Radiol. 2014; 11(4): 384–386. PubMed Abstract | Publisher Full Text\n\nvan Wijk DF, Strang AC, Duivenvoorden R, et al.: Increasing spatial resolution of 3T MRI scanning improves reproducibility of carotid arterial wall dimension measurements. MAGMA. 2014; 27(3): 219–226. PubMed Abstract | Publisher Full Text\n\nKim H, Caldairou B, Hwang JW, et al.: Accurate cortical tissue classification on MRI by modeling cortical folding patterns. Hum Brain Mapp. 2015; 36(9): 3563–3574. PubMed Abstract | Publisher Full Text\n\nChu R, Tauhid S, Glanz B, et al.: Whole Brain Volume Measured from 1.5T versus 3T MRI in Healthy Subjects and Patients with Multiple Sclerosis. J Neuroimaging. 2016; 26(1): 62–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGentillon H: MaZda Package v5 RC HG (release candidate: Hugues Gentillon). Mendeley Data. v1; 2016. Data Source\n\nGentillon H, Stefańczyk L, Strzelecki M, et al.: Dataset 1 in: Texture Analysis of the Developing Human Brain Using Customization of a Knowledge-Based System. F1000Research. 2016. Data Source\n\nGentillon H, Stefańczyk L, Strzelecki M, et al.: Dataset 2 in: Texture Analysis of the Developing Human Brain Using Customization of a Knowledge-Based System. F1000Research. 2016. Data Source\n\nGentillon H, Stefańczyk L, Strzelecki M, et al.: Dataset 3 in: Texture Analysis of the Developing Human Brain Using Customization of a Knowledge-Based System. F1000Research. 2016. Data Source\n\nGentillon H, Stefańczyk L, Strzelecki M, et al.: Dataset 4 in: Texture Analysis of the Developing Human Brain Using Customization of a Knowledge-Based System. F1000Research. 2016. Data Source\n\nGentillon H, Stefańczyk L, Strzelecki M, et al.: Dataset 5 in: Texture Analysis of the Developing Human Brain Using Customization of a Knowledge-Based System. F1000Research. 2016. Data Source\n\nGentillon H, Stefańczyk L, Strzelecki M, et al.: Dataset 6 in: Texture Analysis of the Developing Human Brain Using Customization of a Knowledge-Based System. F1000Research. 2016. Data Source\n\nGentillon H, Stefańczyk L, Strzelecki M, et al.: Dataset 7 in: Texture Analysis of the Developing Human Brain Using Customization of a Knowledge-Based System. F1000Research. 2016. Data Source"
}
|
[
{
"id": "19493",
"date": "13 Feb 2017",
"name": "Michael Hanke",
"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 article describes an predominantly explorative analysis of the capabilities of a machine-learning based texture analysis of fetal MR images for the purpose of extracting information of brain structure (ROI labeling/segmentation) with a (semi-)automatic procedure.\n\nMy background is in neuroimaging data analysis, including the application of machine-learning algorithms on such data. Consequently, I cannot provide an expert opinion on the suitability of the proposed analysis strategies for diagnosing brain development abnormalities, and I will focus on the technical aspects of the procedure and its description in the article.\nIn my opinion, the present structure of the manuscript, and chosen balance of the level of detail with which the research is motivated vs. its methodological details are described, are suboptimal for communicating the implications of these findings. In the following I summarize aspects that I consider critical:\n\nObjective and conclusions\nIt is not clear to me what the exact objective of this research was. How good does ROI classification have to be in order to improve the status quo? Are the developed methods feasible enough (computational demands, ability to obtain suitable raw data, ...) to be employed in clinical applications? What exactly is not possible with available solution (quote \"Sadly, they are primarily designed for pre-loaded applications but not much else\").\n\nIt would be very helpful, if the author would provide a concrete example of the segmentation problem they are trying to solve. This could be a figure showing actual data. Figures 2-5 do not provide this information. It is unclear whether those show a schematic depiction of the problem, are actual empirical results -- this uncertainty is compounded by the very short figure captions.\n\nProvided information on methods is insufficient\nI would like to refer to this report http://www.humanbrainmapping.org/COBIDASreport for guidelines on what to report for MRI studies in general.\nIn particular, there is no information provided on how the MR images were obtained, this includes missing information on the type of MR sequence, its parameters, vendor of the equipment, etc.\n\nThere is no information on the nature of the MR image preprocessing. One of the issues with fetal MRI is the impact of unavoidable motion of the fetus during the scan. This aspect is not touched upon in the manuscript.\nAnalysis description assumes familiarity with the MaZda package. Here is an example:\n\n\"360 parameters were extracted with MaZda (histogram: 9; co-occurrence matrix: 220; run-length matrix: 20; gradient matrix: 5; auto-regression: 5; Haar wavelet: 28; geometry: 73). Parameters’ names are provided in the appendix at the end of this manuscript.\"\nDataset 7 contains a plain list of names such as \"GeoUg\" that are uninterpretable without familiarity with the MaZda package (which in turn only runs on outdated windows machine (98,2000,XP, according to the website), and source code is not available).\n\nStructure of manuscript\nEspecially the methods section does not contain typical sections, such as \"MRI acquisition\", \"Participants\", etc. Instead, it has \"Advance notice to readers\" that states that it is impossible to provide an introduction to machine learning. While that may or may not be true, I consider it problematic that the KBS is only described at a conceptual level, while there is extensive space devoted to the development history of MaZda, which seems irrelevant in the context of this study. (Note that the statement: \"ANN is a self-organizing algorithm\" is not true in its generality)\nThe heading levels seem to be off at times. \"Computer vision\" is a subsection of \"Clinical trial registration\".\nIn general the section heading should be more indicative of the content. The is \"Customization\" which reports on adjustments in the original procedure, but also on how files were renamed. The discussion has a section \"Constraints, limitations, and assumptions\" which essentially restates that data acquisitions took several years.\nThe authors should reconsider the components of the manuscript. What is presented as \"datasets\" are actually Excel tables with text content in their cells that are pretty much tables that should go into the manuscript. Proprietary formats are inappropriate for sharing data. Additionally data types of shared data should focus on re-use, i.e. numerical values of result statistics should be shared as such, and not embedded in textual descriptions.\n\nCitations\nThe author often cite several publication in a single context. It was frequently difficult for me to see why particular choices were relevant. For example, in the context of \"Due to administrative and logistic delay as well as finding volunteers and expensive cost and availability of MRI, it took us nearly five years to collect the magnetic resonance (MR) samples.\" Stipp H: Using neuroscience to improve ad impact: How new research tools can\n\nadvance cultural marketing. Journal of Cultural Marketing Strategy. 2016; 1(2):193–202. and a news item on \"Portable MRI developed at Los Alamos\".",
"responses": [
{
"c_id": "2488",
"date": "13 Feb 2017",
"name": "Hugues Gentillon",
"role": "Author Response",
"response": "Thank you for the report. We totally agree with your comments, but the purpose of our paper was to determine a hypothetical methodology for texture classification of closely-related anatomical structures in fetal brain magnetic resonance images."
}
]
},
{
"id": "24724",
"date": "02 Aug 2017",
"name": "Sanjay Kalra",
"expertise": [
"Reviewer Expertise Neuroimaging"
],
"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 study aims to investigate whether texture analysis can be used as an ROI classification tool in fetal MRIs.\nThe authors used data collected from 1.5T and 3T MRI scanners. Commercially provided software Mazda was used to perform texture analysis and B11 was used for classification.\nThere are several concerns regarding this manuscript:\n\nThe exact objectives and aims are not clear, either in the Abstract or Introduction.\n\nIn the Introduction there is too much information on topics which are not relevant to the paper. The dialogue verges into the arenas of ethics, religion, politics, and medical jurisprudence. What is required here, yet missing, is a more focused rationale for the study, and background material on the methods used, namely texture analysis and artificial networks. Thus, I disagree with the stance of the authors’ (as stated in the Methods, “Advance notice to readers”) to not even offer a brief description given that these methods are otherwise quite abstract to the uninformed reader.\n\nThe authors do not provide basic information about their data including the number of subjects, age, gender, etc. The clinical and demographic information should be provided to allow the readers a sense of the size and characteristics of their datasets. Additionally, no information regarding the imaging dataset is provided including image resolution and acquisition parameters.\n\nThe manuscript is hard to follow. The method section is fairly complex and convoluted. The authors should consider removing information from the Methods that does not add value to the manuscript in allowing a third party to replicate the study. Furthermore, results should be removed from the methods section as they should be provided in the Results section.\n\nThe authors used normal tissue to test for classification of potential \"abnormal\" tissue. This is an interesting proof of concept approach; however, to be clinically relevant, a dataset with abnormal anatomy should be tested as well to investigate the power of the texture analysis in differentiating between tissue classes and potential diagnoses.\n\nThe authors should compare texture classification against clinical evaluation and classification of the MRI images to investigate if texture analysis adds any value to the current practices. It is of note that the authors achieved very high classification rates with texture analysis and should be commended on the study as it does seem to be a large project.\n\nConstraints, limitations, and assumptions section: A discussion on the administrative and logistical challenges of this study is not needed and should be removed.\n\nOverall, it is an interesting study. Sections need to be considerably streamlined and focused to the science and methods. More information needs to be added that are crucial in a neuroimaging study. The authors should consider applying texture analysis techniques to anatomically abnormal data and compare their results against human classification.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "2928",
"date": "02 Aug 2017",
"name": "Hugues Gentillon",
"role": "Author Response",
"response": "Thank you for your comments and suggestions. We do not agree with some of your comments and suggestions from a practical (clinical) point of view, for the following reasons below. This article briefly considers existing legislations relevant to protecting patient privacy and clinical data. You stated that \"exact objectives and aims are not clear\" to you. However, you did not explain why, and we feel that some of your comments mentioned are a matter of personal preference. For example, you ask us to publish details about 'patient demographics' , but we feel that this is unnecessary, in this case. We strongly recommend you to read: Hrynaszkiewicz I, Norton ML, Vickers AJ, Altman DG. Preparing raw clinical data for publication: guidance for journal editors, authors, and peer reviewers. BMJ. 2010;340(8):c181."
}
]
}
] | 1
|
https://f1000research.com/articles/6-40
|
https://f1000research.com/articles/6-1235/v1
|
26 Jul 17
|
{
"type": "Research Article",
"title": "A 4-week, lifestyle-integrated, home-based exercise training programme elicits improvements in physical function and lean mass in older men and women: a pilot study",
"authors": [
"Jessica Cegielski",
"Matthew S. Brook",
"Jonathan I. Quinlan",
"Daniel J. Wilkinson",
"Kenneth Smith",
"Philip J. Atherton",
"Bethan E. Phillips",
"Jessica Cegielski",
"Matthew S. Brook",
"Jonathan I. Quinlan",
"Daniel J. Wilkinson",
"Kenneth Smith",
"Philip J. Atherton"
],
"abstract": "Background: Developing alternative exercise programmes that can alleviate certain barriers to exercise such as psychological, environmental or socio-economical barriers, but provide similar physiological benefits e.g. increases in muscle mass and strength, is of grave importance. This pilot study aimed to assess the efficacy of an unsupervised, 4-week, whole-body home-based exercise training (HBET) programme, incorporated into daily living activities, on skeletal muscle mass, power and strength. Methods: Twelve healthy older volunteers (63±3 years, 7 men: 5 women, BMI: 29±1 kg/m²) carried out the 4-week “lifestyle-integrated” HBET of 8 exercises, 3x12 repetitions each, every day. Before and after HBET, a number of physical function tests were carried out: unilateral leg extension 1-RM (one- repetition maximum), MVC (maximal voluntary contraction) leg extension, lower leg muscle power (via Nottingham Power Rig), handgrip strength and SPPBT (short physical performance battery test). A D3-Creatine method was used for assessment of whole-body skeletal muscle mass, and ultrasound was used to measure the quadriceps cross-sectional area (CSA) and vastus lateralis muscle thickness. Results: Four weeks HBET elicited significant (p<0.05) improvements in leg muscle power (276.7±38.5 vs. 323.4±43.4 W), maximal voluntary contraction (60°: 154.2±18.4 vs. 168.8±15.2 Nm, 90°: 152.1±10.5 vs. 159.1±11.4 Nm) and quadriceps CSA (57.5±5.4 vs. 59.0±5.3 cm2), with a trend for an increase in leg strength (1-RM: 45.7±5.9 vs. 49.6±6.0 kg, P=0.08). This was despite there being no significant differences in whole-body skeletal muscle mass, as assessed via D3-Creatine. Conclusions: This study demonstrates that increases in multiple aspects of muscle function can be achieved in older adults with just 4-weeks of “lifestyle-integrated” HBET, with a cost-effective means. This training mode may prove to be a beneficial alternative for maintaining and/or improving muscle mass and function in older adults.",
"keywords": [
"home based",
"exercise",
"muscle mass",
"muscle function"
],
"content": "Introduction\n\nOlder age is associated with the loss of skeletal muscle mass. Termed sarcopenia, age-related muscle wasting is associated with loss of muscle strength (dynapenia), increased morbidity, loss of independence and premature mortality (Rudrappa et al., 2016). There are however proven means by which to offset these detrimental progressive declines. For example, structured and fully-supervised progressive resistance exercise training (RET) has been shown to improve both muscle mass (Phillips et al., 2012) and function (Liu & Latham, 2009).\n\nDespite the established effects of RET on muscle mass and function, compliance is difficult to achieve, due to perceived (and real) socio-economic, psychological and environmental factors (Zlot et al., 2006). Almost all current evidence for the efficacy of RET is based on 2–3 sessions each week, for up to 12 weeks, at a gym or with specialist equipment, reflecting current physical activity guidelines for adults of 2–3 days per week RET for each of the major muscle groups (Bull et al., 2010). However, compliance to physical activity recommendations is poor, with recent data suggesting that they are only achieved by 38% of UK adults (Department of Health, Physical Activity, 2011), a figure that is lesser still in older adults (Jefferis et al., 2014). As a result, home-based exercise training (HBET) programmes have been suggested as an alternative to overcome some of the barriers associated with poor compliance to exercise (Hong et al., 2017; Silveira et al., 2013), whilst also providing a platform to develop the benefits of RET for (pre-) sarcopenic individuals (Maruya et al., 2016).\n\nThe majority of HBET studies to date, have focussed on pre- or rehabilitation for specific clinical groups (Bassett & Prapavessis, 2007; Chang et al., 2017). A small number of studies have specifically investigated the efficacy of HBET on skeletal muscle function in the elderly, although the focus of these studies has mainly been on lower limb exercises to improve walking and balance (Ito et al., 2015; Yamauchi et al., 2009) in relation to fall and fracture prevention. Other studies in older adults have incorporated the use of specialist aids e.g. dumbbells (Plotnikoff et al., 2010), elastic bands (Jette et al., 1999; Ribeiro et al., 2009) or ankle weights (Gardner et al., 2001) to provide resistance for HBET; rather than attempting to incorporate exercise into tasks of daily living, as is done herein. To date, no studies have attempted to investigate the efficacy of incorporating whole-body exercise into activities of daily living on muscle mass and function in older adults. Consequently, in this pilot study we aimed to assess the effects of a 4-week, progressive, whole-body HBET fully integrated into activities of daily living, on muscle mass and function in healthy older individuals. We also aimed to utilise a recently re-introduced (Clark et al., 2014) and underexploited creatine method to estimate whole-body muscle mass in humans.\n\n\nMaterials and methods\n\nTwelve healthy older male and female volunteers (63±3 y, 7 men: 5 women, BMI: 29±1 kg/m2) with no previous chronic disease history were recruited via an advertisement on a local radio station. Before enrolment into the study, all volunteers had an assessment of previous medical history and an ECG to check for any subclinical arrhythmias. The ECG’s were approved by a clinically qualified physician. All volunteers provided written informed consent prior to the start of this study. This study was approved by The University of Nottingham Medical School Ethics Committee (B28102015 SoMS GEM BBC) and conformed to The Declaration of Helsinki.\n\nThe study involved two visits by the study participants, before and after the 4-week HBET (Figure 1), to the University of Nottingham Medical School, at the Royal Derby Hospital. All assessments at these visits were conducted by researchers from the MRC-ARUK Centre for Musculoskeletal Ageing Research. At each visit, volunteers provided a baseline urine sample for the muscle mass assessment by D3-Creatine before undergoing measures of height, weight (Marsden Weighing Group, UK), resting heart rate and blood pressure (Omron M5-1 digital BP monitor, Omron, UK). Each volunteer had the thickness, fascicle length (Lf) and pennation angle (θpen) of their vastus lateralis muscle, and the cross-sectional area (CSA) of their quadriceps measured by ultrasound (Mylab 70, Esaote Biomedica, Genova, Italy), using the protocol described by Franchi et al. (2014). Volunteers then completed a number of muscle function assessments:\n\n• maximal voluntary contraction (MVC) for seated leg extension and flexion using an isokinetic dynamometer (Isocom; Isokinetic Technologies, Eurokinetics, UK),\n\n• leg extension 1-repetition maximum (1-RM; ISO leg extension, Leisure Lines Ltd., GB),\n\n• leg power (Nottingham Power Rig; Bassey & Short, 1990),\n\n• handgrip strength (Grip D 5401; Takei Scientific Instruments Co. Ltd., Japan); and\n\n• a short physical performance battery test (SPPBT; Guralnik et al., 1994).\n\nBL: baseline urine sample; HR: resting heart rate; BP: resting blood pressure; SPPBT: short physical performance battery test; US: leg ultrasound; MVC: maximum voluntary contraction; 1-RM: leg extension 1-repetition maximum; D3-Cr: D3-Creatine for whole-body muscle mass assessment; 48 and 72h represent the time of urine samples post-tracer consumption.\n\nMVC was measured at two knee angles (60 and 90°), whilst isokinetic (eccentric/concentric) contractions were measured over a 70° range (20–90°) at three different speeds (60, 180 and 240°/sec). For both measures, horizontal seated leg extension was set at 0°. Following the muscle function assessments, volunteers ingested 30mg of D3-Creatine dissolved in 100ml H2O for muscle mass assessment, before providing a pooled 24h urine collection and single urine samples at 48 and 72 h post consumption (Clark et al., 2014).\n\nVolunteers were required to complete 3 sets of 12 repetitions across 8 different exercises, every day, for 4 weeks. Each exercise was designed to be easily incorporated into activities of daily living to minimise the time-commitment required (e.g. bicep curls whilst cooking, see Figure 1). In total the exercise programme consisted of: squats, lunges, calf raises, bicep curls, triceps extensions, semi-incline press-ups, oblique twists and deadlifts. Participants were instructed to take 2 seconds to perform each phase (eccentric and concentric) of each exercise and to hold for 2 seconds at the mid-point of each exercise. Progression via increased resistance or time-under-tension was encouraged once the full complement of repetitions could be completed. Compliance to exercise was measured by self-report (see self-report log in Figure 1). An exercise would only be marked as complete if 3 sets of 12 repetitions had been performed within a day.\n\nUsing a creatine-tracer measure of muscle mass (Clark et al., 2014), D3-creatine and D3-creatinine enrichment, plus unlabelled creatine and creatinine in the urine was measured using high-performance liquid chromatography/mass spectrometry (HPLC/MS), as detailed in (Stimpson et al., 2012). Total muscle mass was calculated using the following equation:\n\nTotal muscle mass =(MWUnlabelledMWlabelled)×(Amount of D3−Cr dosed(g)−Amount of D3−Cr excreted (g))(mean steady−state D3−Creatine enrichment ratio÷4.3(g/kg)\n\n(Clark et al., 2014),\n\nwhere MWUnlabelled and MWlabelled represents the molecular weights of both unlabelled and labelled creatine, respectively. The estimated creatine pool size is then divided by 4.3 g/kg, which reflects the concentration of creatine in wet muscle.\n\nDescriptive statistics were carried out on all datasets and checked for a normal distribution using the Shapiro-Wilk normality test. Datasets were analysed using paired Student’s t test to assess the effect of HBET, with GraphPad Prism Software v5 (La Jolla, CA, USA). All data is presented as mean ±SEM. Significance was set at P<0.05.\n\n\nResults\n\nUsing data from the self-report training logs, overall compliance to the HBET was 87.3±3.0% over the 4-week period of training. Adherence to each individual exercise was >86%, with the calf-raises being the most completed exercise (91%). No significant changes were found following HBET for body weight (79.3±4.7 vs. 78.9±4.6 kg), resting heart rate (71.9±3.1 vs. 71.4±3.9 bpm) or blood pressure (systolic: 144.4±3.4 vs. 140.7±4.2, diastolic: 92.2±2.5 vs. 89.0±2.4; average of 3 measurements).\n\nHBET did not elicit significant changes in leg extension 1-RM, handgrip strength or SPPBT; there was however, a trend for an increase in leg extension 1-RM (45.7±5.9 vs. 49.6±6.0 kg, P=0.08; Figure 2A). HBET did elicit significant increases in leg power (276.7±38.5 vs. 323.4±43.4 W, P<0.05; Figure 2B) and MVC at 60° (154.2±18.4 vs. 168.8±15.2 Nm, P<0.05; Figure 2C) and 90° (152.1±10.5 vs. 159.1±11.4 Nm, P<0.05; Figure 2D). There were also significant increases in peak torque recorded at 60°/sec during both seated flexion (74.7±8.3 vs. 80.8±8.6 Nm, P<0.05; Figure 2E) and extension (114.9±10.2 vs. 123.8±9.4 Nm, P<0.01; Figure 2F) after HBET.\n\nA) 1-RM leg extension (45.7±5.9 vs. 49.6±6.0 kg, P=0.08), B) Leg power via Nottingham Power Rig (276.7±38.5 vs. 323.4±43.4 W), C) MVC at 60° (154.2±18.4 vs. 168.8±15.2 Nm), D) MVC at 90° (152.1±10.5 vs. 159.1±11.4 Nm), E) Seated leg flexion at 60°/sec (74.7±8.3 vs. 80.8±8.6 Nm), F) Seated leg extension at 60°/sec (114.9±10.2 vs. 123.8±9.4 Nm). Baseline vs. post-HBET (mean ± SEM), n=12, *=p<0.05; **=p<0.01. MVC: Maximal voluntary contraction.\n\nCSA of the thigh at 50% of thigh length significantly increased (57.5±5.4 vs. 59.0±5.3 cm2, P<0.05; Figure 3A) after HBET. However, there was no significant increase in muscle thickness, fascicle length (Lf) or pennation angle (θpen) of the m. vastus lateralis. There was a trend for an increase in muscle thickness (25.0±1.6 vs. 25.5±1.7 mm, P=0.08; Figure 3B) after HBET.\n\nA) quadriceps cross-sectional area (CSA; 57.5±5.4 vs. 59.0 ± 5.3 cm2); B) m. vastus lateralis (VL) thickness (25.0±1.6 vs. 25.5±1.7 mm, P=0.08); C) whole-body muscle mass, before and after home-based exercise training (HBET) (23.9±2.1 vs. 22.8±2.2 kg). (mean ± SEM), *=p<0.05, baseline vs. post-HBET.\n\nCorresponding with no significant change in body weight, there was no significant difference in whole-body muscle mass as estimated by D3-Creatine (Figure 3C) after HBET (23.9±2.1 vs. 22.8±2.2 kg).\n\n\nDiscussion\n\nIn this pilot study, we show that lifestyle-integrated, whole-body HBET has the potential to meaningfully improve muscle function in older adults. We have shown that this training mode can increase leg power by 16.9% in just 4-weeks. Although lower than the 37% power increase demonstrated in a solely lower-extremity focused HBET programme, this study was conducted over an 8-week period (DeBolt & McCubbin, 2004), suggesting that if the magnitude of increase was to continue at a constant rate, our training mode may elicit similar improvements. Additionally, we demonstrate increases in muscle strength (via MVC at 60°) similar to that observed in response to a 6-week, fully-supervised, gym-based training study (Brook et al., 2016). Furthermore, despite no significant increase, there was a positive trend in the leg extension 1-RM, similar to that seen in an 8-week HBET study by Zion et al. (2003). Thus, while muscle function adaptations to HBET were numerically lesser compared to longer-term structured exercise regimens (some requiring specialist equipment), our data support the notion that just 4-weeks of a ‘lifestyle-integrated’ HBET meaningfully improves muscle function.\n\nDespite these positive results, no changes in handgrip strength were found, a similar result to Nelson et al. (2004), who implemented a 6-month home-based whole-body exercise programme in 70 male and female elderly individuals. This may be due to a lack of exercises specifically targeting the forearm flexors. Indeed, although it is well-established that extant grip strength represents a biomarker of muscle function in older adults (Bassey & Harries, 1993), our data suggests that it is an insensitive biomarker for important functional and mass gains in other muscle groups (e.g. the legs) following certain exercise interventions.\n\nImportantly, increases in thigh CSA (and a trend towards increased muscle thickness), which may partly explain the functional improvements, were observed. Measures of thigh CSA by ultrasound have been positively correlated with those by MRI (Reeves et al., 2004), therefore suggesting (leg) muscle hypertrophy in this study. However, since this occurred in the absence of detectable changes in whole-body muscle mass as measured by D3-Creatine, we speculate i) that preferential hypertrophy of leg muscles occurred, but remained undetectable on a whole-body level; or ii) that measurement of whole-body muscle mass was below the detection threshold for the D3-creatine method.\n\nWe acknowledge limitations to our study design. The use of validated physical activity monitors (e.g. Actiheart, CamNtech, Cambridge, UK) to assess compliance to exercise would have provided greater information on the adoption of, and adherence to our exercise programme. Similarly, the use of interactive technologies (e.g. training applications for tablets/smartphones) for motivation and/or instruction may have improved adherence (Silveira et al., 2013). The lack of improvements in SPPBT were likely a result of our volunteers achieving optimal scores before the intervention.\n\nTo conclude, our findings demonstrate that significant increases in muscle mass, maximal voluntary contraction, maximal power and isokinetic strength can be achieved with just 4-weeks of unsupervised, lifestyle-integrated HBET in older adults. These improvements were achieved via cost-effective means, without the requirement for specialist equipment, facilities or supervision (DeBolt & McCubbin, 2004; Jette et al., 1996; Zion et al., 2003). With high compliance and beneficial physiological effects in only a 4-week period, this study highlights the potential for lifestyle-integrated HBET to improve skeletal muscle ‘health’ in older adults. As increases in muscle mass and strength are positively correlated with overall physical function (Liu & Latham, 2009), this novel training mode may be of particular benefit to older frail/(pre-) sarcopenic adults.\n\n\nData availability\n\nDataset 1: Raw data supporting the findings in this study.\n\nSheet 1: Maximum Voluntary Contraction (MVC) at 60° and 90°, pre- and post 4-weeks of home-based whole-body exercise training.\n\nSheet 2: Seated leg extension measures at 60, 180 and 240 deg/sec pre- and post 4-weeks of home-based whole-body exercise training\n\nSheet 3: Seated leg flexion at 60, 180 and 240 deg/sec pre- and post- 4-weeks of home-based whole-body exercise training\n\nSheet 4: Handgrip strength, single-leg 1-RM leg extension, SPPBT (short physical performance battery tests) and leg muscle power, measured pre- and post- 4-weeks of home-based whole-body exercise training\n\nSheet 5: Participant characteristics. Heart rate (HR), systolic and diastolic blood pressure (BP) and body weight, pre- and post- 4-weeks of whole-body home-based exercise training.\n\nSheet 6: Quadriceps cross-sectional area (CSA), vastus lateralis muscle thickness, fascicle length and pennation angle measured by ultrasound pre- and post- 4-weeks of whole-body home-based exercise training.\n\nSheet 7: Muscle mass measured by using D3-creatine, pre- and post- 4 weeks of whole-body home-based exercise training.\n\nDOI, 10.5256/f1000research.11894.d170288 (Cegielski et al., 2017).",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWe would like to thank MRC-ARUK for providing centre funding (MRC: MR/K00414X/1 and ARUK: 19891) and supporting this work. The stable-isotope analysis in this work was funded by Medical Research Council Confidence in Concept Awards (CiC12019 and CiC2014035). The Abbeyfield Research Foundation, charity number 1167685 (RGS117347) supports PJA, BEP and KS at the University of Nottingham.\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\nBassett SF, Prapavessis H: Home-based physical therapy intervention with adherence-enhancing strategies versus clinic-based management for patients with ankle sprains. Phys Ther. 2007; 87(9): 1132–43. PubMed Abstract | Publisher Full Text\n\nBassey EJ, Short AH: A new method for measuring power output in a single leg extension: feasibility, reliability and validity. Eur J Appl Physiol Occup Physiol. 1990; 60(5): 385–390. PubMed Abstract | Publisher Full Text\n\nBassey EJ, Harries UJ: Normal values for handgrip strength in 920 men and women aged over 65 years, and longitudinal changes over 4 years in 620 survivors. Clin Sci (Lond). 1993; 84(3): 331–337. PubMed Abstract\n\nBrook MS, Wilkinson DJ, Mitchell WK, et al.: Synchronous deficits in cumulative muscle protein synthesis and ribosomal biogenesis underlie age-related anabolic resistance to exercise in humans. J Physiol. 2016; 594(24): 7399–7417. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBull FC, Biddle S, Buchner D, et al.: Physical activity guidelines in the U.K.: Review and Recommendations. School of Sport, Exercise and Health Sciences, Loughborough University, 2010; 1–72. Reference Source\n\nCegielski J, Brook M, Quinlan J, et al.: Dataset 1 in: A 4-week lifestyle-integrated home-based exercise training programme elicits improvements in physical function and lean mass in older men and women: a pilot study. F1000Research. 2017. Data Source\n\nChang CF, Lin KC, Chen WM, et al.: Effects of a Home-Based Resistance Training Program on Recovery From Total Hip Replacement Surgery: Feasibility and Pilot Testing. J Nurs Res. 2017; 25(1): 21–30. PubMed Abstract | Publisher Full Text\n\nClark RV, Walker AC, O'Connor-Semmes RL, et al.: Total body skeletal muscle mass: estimation by creatine (methyl-d3) dilution in humans. J Appl Physiol (1985). 2014; 116(12): 1605–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeBolt LS, McCubbin JA: The Effects of Home-Based Resistance Exercise on Balance, Power, and Mobility in Adults with Multiple Sclerosis. Arch Phys Med Rehabil. 2004; 85(2): 290–297. PubMed Abstract | Publisher Full Text\n\nDepartment of Health, Physical Activity, H.I. and P.: Start Active, Stay Active - A report on physical activity for health from the four home countries’ Chief Medical Officers. 2011. Reference Source\n\nFranchi MV, Atherton PJ, Reeves ND, et al.: Architectural, functional and molecular responses to concentric and eccentric loading in human skeletal muscle. Acta Physiol (Oxf). 2014; 210(3): 642–54. PubMed Abstract | Publisher Full Text\n\nGardner MM, Buchner DM, Robertson MC, et al.: Practical implementation of an exercise-based falls prevention programme. Age Ageing. 2001; 30(1): 77–83. PubMed Abstract | Publisher Full Text\n\nGuralnik JM, Simonsick EM, Ferrucci L, et al.: A short physical performance battery assessing lower extremity function: association with self-reported disability and prediction of mortality and nursing home admission. J Gerontol. 1994; 49(2): M85–94. PubMed Abstract | Publisher Full Text\n\nHong J, Kim J, Kim SW, et al.: Effects of home-based tele-exercise on sarcopenia among community-dwelling elderly adults: Body composition and functional fitness. Exp Gerontol. 2017; 87(Pt A): 33–39. PubMed Abstract | Publisher Full Text\n\nIto S, Hashimoto M, Aduma S, et al.: Effectiveness of locomotion training in a home visit preventive care project: one-group pre-intervention versus post-intervention design study. J Orthop Sci. 2015; 20(6): 1078–1084. PubMed Abstract | Publisher Full Text\n\nJefferis BJ, Sartini C, Lee IM, et al.: Adherence to physical activity guidelines in older adults, using objectively measured physical activity in a population-based study. BMC Public Health. 2014; 14(1): 382. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJette AM, Harris BA, Sleeper L, et al.: A Home-based Exercise Program for Nondisabled Older Adults. J Am Geriatr Soc. 1996; 44(6): 644–649. PubMed Abstract | Publisher Full Text\n\nJette AM, Lachman M, Giorgetti MM, et al.: Exercise--It’s never too late: The strong-for-life program. Am J Public Health. 1999; 89(1): 66–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu CJ, Latham NK: Progressive resistance strength training for improving physical function in older adults. Cochrane Database Syst Rev. 2009; 3(CD002759): 1–227. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaruya K, Asakawa Y, Ishibashi H, et al.: Effect of a simple and adherent home exercise program on the physical function of community dwelling adults sixty years of age and older with pre-sarcopenia or sarcopenia. J Phys Ther Sci. 2016; 28(11): 3183–3188. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNelson ME, Layne JE, Bernstein MJ, et al.: The Effects of Multidimensional Home-Based Exercise on Functional Performance in Elderly People. J Gerontol A Biol Sci Med Sci. 2004; 59(2): 154–60. PubMed Abstract | Publisher Full Text\n\nPhillips B, Williams J, Atherton P, et al.: Resistance exercise training improves age-related declines in leg vascular conductance and rejuvenates acute leg blood flow responses to feeding and exercise. J Appl Physiol (1985). 2012; 112(3): 347–53. PubMed Abstract | Publisher Full Text\n\nPlotnikoff RC, Eves N, Jung M, et al.: Multicomponent, home-based resistance training for obese adults with type 2 diabetes: a randomized controlled trial. Int J Obes (Lond). 2010; 34(12): 1733–41. PubMed Abstract | Publisher Full Text\n\nReeves ND, Maganaris CN, Narici MV: Ultrasonographic assessment of human skeletal muscle size. Eur J Appl Physiol. 2004; 91(1): 116–118. PubMed Abstract | Publisher Full Text\n\nRibeiro F, Teixeira F, Brochado G, et al.: Impact of low cost strength training of dorsi- and plantar flexors on balance and functional mobility in institutionalized elderly people. Geriatr Gerontol Int. 2009; 9(1): 75–80. PubMed Abstract | Publisher Full Text\n\nRudrappa SS, Wilkinson DJ, Greenhaff PL, et al.: Human Skeletal Muscle Disuse Atrophy: Effects on Muscle Protein Synthesis, Breakdown, and Insulin Resistance-A Qualitative Review. Front Physiol. 2016; 7: 361. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSilveira P, van de Langenberg R, van Het Reve E, et al.: Tablet-based strength-balance training to motivate and improve adherence to exercise in independently living older people: a phase II preclinical exploratory trial. J Med Internet Res. 2013; 15(8): e159. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStimpson SA, Turner SM, Clifton LG, et al.: Total-body creatine pool size and skeletal muscle mass determination by creatine-(methyl-D3) dilution in rats. J Appl Physiol (1985). 2012; 112(11): 1940–1948. PubMed Abstract | Publisher Full Text\n\nYamauchi J, Nakayama S, Ishii N: Effects of bodyweight-based exercise training on muscle functions of leg multi-joint movement in elderly individuals. Geriatr Gerontol Int. 2009; 9(3): 262–269. PubMed Abstract | Publisher Full Text\n\nZion AS, De Meersman R, Diamond BE, et al.: A home-based resistance-training program using elastic bands for elderly patients with orthostatic hypotension. Clin Auton Res. 2003; 13(4): 286–292. PubMed Abstract | Publisher Full Text\n\nZlot AI, Librett J, Buchner D, et al.: Environmental, Transportation, Social, and Time Barriers to Physical Activity. J Phys Act Health. 2006; 3(1): 15–21. Publisher Full Text"
}
|
[
{
"id": "24550",
"date": "08 Aug 2017",
"name": "Stuart Gray",
"expertise": [
"Reviewer Expertise Exercise",
"muscle",
"training",
"ageing."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 investigated the effects of HBET on muscle function. This is an important advance as resistance exercise is known to be of benefit to health and yet (due to reasons mentioned in the paper) it is performed by very few people. I have a few comments on this but overall the manuscript is well written and the data clearly presented.\n1) I would like to see a bit more detail on exactly how these exercise were integrated into daily life, e.g. what did you tell the participants to do exactly.\n2) The authors mention that it may be that the changes in whole body muscle mass may have been below the detection level of the d3-creatine method - could the authors elaborate and provide details.\n\n3) May be worth citing https://www.ncbi.nlm.nih.gov/pubmed/249039081 in the discussion about the lack of change in grip strength.\n4) I think it would be useful to highlight in the discussion (as you mentioned briefly when discussing the SPPBT results) that these participants were not that old and were very healthy.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1235
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https://f1000research.com/articles/6-1669/v1
|
08 Sep 17
|
{
"type": "Correspondence",
"title": "Fibromax-based nonalcoholic fatty liver disease in chronic obstructive pulmonary disease patients with obstructive sleep apnea: Methodological considerations",
"authors": [
"Denis Monneret"
],
"abstract": "The relationship between nonalcoholic fatty liver disease (NAFLD) and obstructive sleep apnea (OSA) has been well demonstrated, but remains to be evidenced in chronic obstructive pulmonary disease (COPD). Recently, Viglino et al. (Eur Respir J, 2017) attempted to determine the prevalence of liver fibrosis, steatosis and nonalcoholic steatohepatitis (NASH) in COPD patients, some of whom had OSA, basing the NAFLD diagnostic on three circulating biomarker-based liver scores: the FibroTest, SteatoTest and NashTest, from the Fibromax® panel. Among the main findings, the absence of OSA treatment emerged as independently associated with liver fibrosis and steatosis, when compared to effective treatment. However, besides the low number of treated patients, no polysomnographic respiratory data was provided, making it difficult to differentiate the impact of OSA from that of COPD in NAFLD prevalence. Furthermore, NAFLD diagnosis relied exclusively on circulating biomarker-based liver scores, without histological, imagery or other liver exploratory methods. Therefore, in this article, some methodological points are reminded and discussed, including the choice of OSA measurements, and the significance of ActiTest and AshTest scores from Fibromax® in this pathophysiological context.",
"keywords": [
"nonalcoholic fatty liver disease",
"chronic obstructive pulmonary disease",
"obstructive sleep apnea",
"Fibromax",
"biomarker standardization"
],
"content": "\n\nThe relationship between nonalcoholic fatty liver disease (NAFLD) and obstructive sleep apnea (OSA) has been well demonstrated1–3, but remains to be evidenced in chronic obstructive pulmonary disease (COPD). To this end, using biomarker-based Fibromax® scores, Viglino et al. recently attempted to determine the prevalence of liver fibrosis, steatosis and nonalcoholic steatohepatitis (NASH) in COPD patients, which they found at nearly 61%, 41%, and 37%, respectively4. Interestingly, the absence of OSA treatment emerged as independently associated with liver fibrosis and steatosis, when compared to effective treatment. However, the number of treated patients was low (10 versus 38 untreated), and no polysomnographic respiratory data was provided, making it difficult to differentiate the impact of OSA from that of COPD in NAFLD prevalence. Furthermore, NAFLD diagnosis relied exclusively on circulating biomarker-based liver scores, without histological, imagery or other liver exploratory methods. It is, therefore, the opportunity to remind and discuss some methodological points, especially concerning the choice of OSA measurements, and the significance of ActiTest and AshTest scores from Fibromax® in this pathophysiological context.\n\nRecently, Pépin’s team showed a prevalence of liver steatosis of about 40–45% in moderate-to-severe OSA patients, with nearly 40–60% of patients displaying borderline NASH5. They also showed in obese OSA patients that fibrosis and NAFLD-related lesions, like hepatocyte ballooning and lobular inflammation, were more severe in those with the highest nocturnal oxygen desaturation6. Accordingly, nocturnal time spent at <90% oxygen saturation was independently associated with liver fibrosis in patients with suspicion of OSA7. In the Viglino et al. study, the absence of OSA treatment emerged as an independent factor of liver fibrosis and steatosis, as compared to treatment. Including the absence versus effective OSA treatment in the multivariate model is not the most appropriate criterion, since the authors recently showed that 6–12 weeks of CPAP treatment did not reduce steatosis, NASH or liver fibrosis5. This is in addition to the very low number of treated patients (10 vs 38 untreated). Instead, and according to the increasingly obvious hypothesis of chronic intermittent hypoxia (CIH) on NAFLD, the authors could have chosen the oxygen desaturation index as a criterion (e.g. with a cut-off < or ≥15 events/h), which they elsewhere claimed to be “a good marker of CIH”6. Studies displaying detailed results for both OSA and liver scores are few to date, and do not allow an in-depth analysis of their relationships. Therefore, in such NAFLD/COPD/OSA-related studies, polysomnographic respiratory profiles should be provided, including oxygen desaturation measurements, along with liver scores and detailed biology, for the overall COPD group and for OSA patients (all, treated and untreated), in order to strengthen conclusions and enable comparison with further studies.\n\nViglino et al. focused on the three most appropriate NAFLD scores (i.e. FibroTest, SteatoTest, and NashTest). However, they did not provide ActiTest, another Fibromax® score proposed for the estimation of liver necroinflammatory activity in chronic hepatitis C and B8, which is based on the measurement of the five Fibrotest® parameters plus alanine aminotransferase. Interestingly, ActiTest has been shown as highly accurate for the diagnosis of NASH and steatosis in patients with severe obesity, with notably an excellent negative predictive value for NASH at 96% using a cut-off at 0.299. In another study on patients with suspected NAFLD, ActiTest showed a significant diagnostic value for NASH, which was not shown for FibroTest10. The informative value of ActiTest on the inflammatory component of NAFLD in COPD patients with or without OSA remains questionable; it could therefore be provided and discussed with regards to other inflammatory biomarkers (tumour necrosis factor-α and leptin in the Viglino et al. study).\n\nFurthermore, alcohol consumption at ≥20g/day (women) and ≥30g/day (men) was chosen by the authors as exclusion criteria to discard potential alcoholic steatohepatitis (ASH). However, alcohol consumption may vary over time, even in abstainers or occasional drinkers, and may thus introduce a misclassification bias11. Self-reported alcohol consumption remains subjective and should ideally be evaluated using a reliable and objective measure. In this way, the AshTest score –the fifth of Fibromax® – is proposed for the detection of alcoholic steatohepatitis12, and thus could be provided as a control for non-excessive alcohol consumption in such pathophysiological contexts.\n\nDepending on the score, age, sex and/or weight and height are included in the Fibromax® calculation formulas. Therefore, the multivariate analyses from Viglino et al, which included age, sex and/or BMI as independent variables in addition to the Fibromax® score as a dependent variable may induce multicollinearity, and thus cause imprecise estimates of coefficient values or introduce large prediction errors in the case of extrapolation. Consequently, multicollinearity must be tested in such models, and controlled as much as possible.\n\nViglino et al. did not provide any information about the methods used for Fibromax® parameters. Analytically, standardization allows the reduction of inter-laboratory variability. It is of particular importance for gamma-glutamyl transferase, known for its high inter-method variability, as well as for transaminases, which are measurable with or without pyridoxal 5-posphate as a coenzyme activator13. Fibromax® proteins also need standardization given their weight in score calculation, especially α2-macroglobulin14. Comparison with peer and method groups – through programs of quality control – allows inter-laboratory variation assessment; it is an analytical requirement of the ISO15189 standard for accreditation of medical laboratories, which is a strong guarantee of result reliability15,16. If ISO15189 certified methods are used for Fibromax® assays, it must be mentioned, along with methods and analyzers, in order to strengthen the biomarker component, to make it sufficiently informative to be compared with further studies.\n\nTo conclude, in such studies evaluating NAFLD, based exclusively on combined-biomarker scores without clinical, histological, imagery or other liver exploratory methods, information on assay methods, analyzers, and guarantees of analytical performance are required, which requires a strong collaboration between clinicians and lab practitioners.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe author is grateful to Vincent Fitzpatrick for the English rereading.\n\n\nReferences\n\nMusso G, Cassader M, Olivetti C, et al.: Association of obstructive sleep apnoea with the presence and severity of non-alcoholic fatty liver disease. A systematic review and meta-analysis. Obes Rev. 2013; 14(5): 417–31. PubMed Abstract | Publisher Full Text\n\nPolotsky VY, Patil SP, Savransky V, et al.: Obstructive sleep apnea, insulin resistance, and steatohepatitis in severe obesity. Am J Respir Crit Care Med. 2009; 179(3): 228–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNobili V, Cutrera R, Liccardo D, et al.: Obstructive sleep apnea syndrome affects liver histology and inflammatory cell activation in pediatric nonalcoholic fatty liver disease, regardless of obesity/insulin resistance. Am J Respir Crit Care Med. 2014; 189(1): 66–76. PubMed Abstract | Publisher Full Text\n\nViglino D, Jullian-Desayes I, Minoves M, et al.: Nonalcoholic fatty liver disease in chronic obstructive pulmonary disease. Eur Respir J. 2017; 49(6): pii: 1601923. PubMed Abstract | Publisher Full Text\n\nJullian-Desayes I, Tamisier R, Zarski JP, et al.: Impact of effective versus sham continuous positive airway pressure on liver injury in obstructive sleep apnoea: Data from randomized trials. Respirology. 2016; 21(2): 378–85. PubMed Abstract | Publisher Full Text\n\nAron-Wisnewsky J, Minville C, Tordjman J, et al.: Chronic intermittent hypoxia is a major trigger for non-alcoholic fatty liver disease in morbid obese. J Hepatol. 2012; 56(1): 225–33. PubMed Abstract | Publisher Full Text\n\nMinville C, Hilleret MN, Tamisier R, et al.: Nonalcoholic fatty liver disease, nocturnal hypoxia, and endothelial function in patients with sleep apnea. Chest. 2014; 145(3): 525–33. PubMed Abstract | Publisher Full Text\n\nMorra R, Munteanu M, Imbert-Bismut F, et al.: FibroMAX: towards a new universal biomarker of liver disease? Expert Rev Mol Diagn. 2007; 7(5): 481–90. PubMed Abstract | Publisher Full Text\n\nPoynard T, Lassailly G, Diaz E, et al.: Performance of biomarkers FibroTest, ActiTest, SteatoTest, and NashTest in patients with severe obesity: meta analysis of individual patient data. PLoS One. 2012; 7(3): e30325. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRatziu V, Massard J, Charlotte F, et al.: Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease. BMC Gastroenterol. 2006; 6: 6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRehm J, Irving H, Ye Y, et al.: Are lifetime abstainers the best control group in alcohol epidemiology? On the stability and validity of reported lifetime abstention. Am J Epidemiol. 2008; 168(8): 866–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThabut D, Naveau S, Charlotte F, et al.: The diagnostic value of biomarkers (AshTest) for the prediction of alcoholic steato-hepatitis in patients with chronic alcoholic liver disease. J Hepatol. 2006; 44(6): 1175–85. PubMed Abstract | Publisher Full Text\n\nHalfon P, Imbert-Bismut F, Messous D, et al.: A prospective assessment of the inter-laboratory variability of biochemical markers of fibrosis (FibroTest) and activity (ActiTest) in patients with chronic liver disease. Comp Hepatol. 2002; 1(1): 3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRosenthal-Allieri MA, Peritore ML, Tran A, et al.: Analytical variability of the Fibrotest proteins. Clin Biochem. 2005; 38(5): 473–8. PubMed Abstract | Publisher Full Text\n\nGuzel O, Guner EI: ISO 15189 accreditation: Requirements for quality and competence of medical laboratories, experience of a laboratory I. Clin Biochem. 2009; 42(4–5): 274–8. PubMed Abstract | Publisher Full Text\n\nAbdelWareth LO, Pallinalakam F, Ibrahim F, et al.: Fast Track to Accreditation: An Implementation Review of College of American Pathologists and International Organization for Standardization 15189 Accreditation. Arch Pathol Lab Med. 2017. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "27106",
"date": "27 Nov 2017",
"name": "Valentina Giorgio",
"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 letter is interesting, written in good and fluent English, clearly addressing the limits of a paper attempting to find a link between obstructive sleep apnea (OSA) and Non alcoholic Fatty Liver, in patients with chronic obstructive pulmonory disease (COPD). I agree with Monneret that one of the main limit of this paper, among others, is that it is quite difficult to differentiate the impact of OSA from that of COPD in fatty liver prevalence, because of the common respiratory nature of OSA and COPD and the common negative effect in oxygen saturation. Therefore, although the paper has the merit of pointing out that non alcoholic fatty liver disease can be present -and maybe more often present- in patients with COPD, more polysomnographic respiratory profiles should be provided, including oxygen desaturation measurements.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes",
"responses": []
},
{
"id": "27720",
"date": "27 Nov 2017",
"name": "Omar A Mesarwi",
"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, by Dr. Monneret, presents many very salient points in reference to the article by Viglino et al. I would like to make a minor remark:\nIn section 1, I think the article may benefit from a very brief mention of the biological differences between intermittent and sustained hypoxia - experimentally these exposures yield very different metabolic outcomes, and the mechanisms which underlie these effects (sympathetic activation, oxidative stress, tissue oxygen profiles, etc.) may be quite different in sustained hypoxia as in COPD, versus chronic intermittent hypoxia, as in OSA.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1669
|
https://f1000research.com/articles/6-93/v1
|
31 Jan 17
|
{
"type": "Data Note",
"title": "Prenatal brain MRI samples for development of automatic segmentation, target- recognition and machine-learning algorithms to detect anatomical structures",
"authors": [
"Hugues Gentillon",
"Ludomir Stefańczyk",
"Michał Strzelecki",
"Maria Respondek-Liberska",
"Ludomir Stefańczyk",
"Michał Strzelecki",
"Maria Respondek-Liberska"
],
"abstract": "In this data note, we present a sorted pool of fetal magnetic resonance imaging (MRI) specimens, selected for a project seeking to further develop a computer-vision software called MaZda, originally created for magnetic resonance (MR) image analysis. A link to download the samples is provided in the manuscript herein. This data descriptor further explains how and why these fetal MRI samples were selected. Firstly, thousands of cross-sectional images obtained from fetal MRI scans were processed and sorted semi-manually with other software. We did so because a built-in “samplesort” (sorting algorithm) is missing in MaZda version 5. Additionally, the software is unfortunately lacking effective and efficient algorithms to allow automatic identification and segmentation of anatomical structures in fetal MRI samples. Hence, the finals sorting steps were carried out manually via time-consuming methods — i.e. human- visual detection and classifications by the gestational age of pregnancy and the rotational plane of the MR scanner. Thus the latter correlates with the anatomical plane of the mother, rather than the hypothetical plane used to transect the fetus. In brief, we collated these fetal MRI samples in an effort to facilitate future research and discovery, especially to aid the improvement of MaZda.",
"keywords": [
"fetal MRI",
"automatic segmentation",
"algorithms",
"fetal brain",
"neuroinformatics",
"cybernetics",
"applied artificial intelligence"
],
"content": "Introduction\n\nSample sorting can be useful for clinical research seeking to measure the feasibility of new ideas and to develop new technology. MaZda software (http://www.eletel.p.lodz.pl/programy/mazda/) makers and developers have shown that they are listening to their users, by continuing to release updates and new versions1,2. The samples provided with the manuscript herein were collected and sorted especially for testing upcoming versions of MaZda. The aim is to continue to collaborate with MaZda software engineers in order to code target-recognition semantics and eventually build ideal algorithms for automatic segmentation of prenatal brain. It is important to also note that it is not an easy task to deconstruct the scientific knowledge acquired by radiologists after several hours of practice to master the skills of diagnostic imaging. Moreover, we have recently tested MaZda version 4.6 and version 5.0 and made some recommendations to the software engineers3–5. In reaction to our need, the MaZda team announced an upcoming version called qMaZda, being co-developed with Weka (www.eletel.p.lodz.pl/pms/SoftwareQmazda.html; www.cs.waikato.ac.nz/ml/weka). We are expecting to see some improvements in qMaZda.\n\n\nMethods\n\nThis dataset was created to improve the efficacy of MaZda. Sample collection was approved by the Research Ethics Committee of the Medical University of Lodz (permit number: RNN/213/13/KE). Subjects were informed with a written statement of consent for research and publication. As per agreement, personal information was removed from the original specimens.\n\nIn terms of subject demographics and phenotypes, the background of the patients was consistent with the majority of the Polish population. In 2015, the World Health Organization (WHO) reported 2.68 million neonatal deaths (WHO fact sheet on congenital anomalies, updated September 2016: www.who.int/mediacentre/factsheets/fs370/en/). The estimate of children born with at least one congenital malformation is about 2-3% worldwide (www.who.int/genomics/anomalies/en/Chapter02.pdf). In Poland, the prevalence rate of birth defects was estimated at 52-53 per 1000 live births (http://www.marchofdimes.org/materials/global-report-on-birth-defects-the-hidden-toll-of-dying-and-disabled-children-full-report.pdf). Known birth defects can be detected early in pregnancy using non-invasive and/or invasive techniques6–9. Some can be even treated in-utero10,11. There flows the rationale behind this collation of fetal magnetic resonance imaging (MRI) data to improve the efficacy of MaZda.\n\nThe enrolled subjects underwent MRI examination for the purpose of investigating suspected congenital, obstetrical and placental anomalies that could not be detected by routine ultrasound and genetic amniocentesis. Volunteers who donated fetal MRI samples to create this dataset were in need of fetal, obstetrical or placental care. The criteria for inclusion and exclusion were as follows: 1) 1.5T or 3T MRI; 2) all three anatomical planes were scanned (axial, coronal, sagittal); 3) individual cross-sectional images are “usable” — i.e. not heavily degraded by noise and artifacts, as well as motion blur, due to uncontrollable movement of fetal head; 4) visible fetal brain with no significant malformation; 5) thalamus, grey matter, white matter, and ventricles are also visible (Figure 1). The request to collect MRI scans was sent long after MRI examination was performed. Hence, MRI examination was not prescribed for the purpose of creating this dataset. After looking at 1358 MRI scans in two teleradiology databases, we manually selected 6 patients who had undergone 1.5T MRI examination at Barlicki University Hospital (Łódź, Poland) and 11 patients who had undergone 3T MRI examination at Polish Mother’s Memorial Hospital- Research Institute (Łódź, Poland).\n\nFetal magnetic resonance imaging studies were extracted from compact discs and sorted by gestational age of pregnancy and anatomical plane of the mother.\n\nElectro-radiology technicians performed fetal MRI as per details provided on the prescription and hospital regulations12. Hence, we did not have control of MR scanners settings, for example. The technicians stored the MRIs on compact discs (CD). By default, MaZda version 4.6 and 5 are lacking an automatic samplesort (sorting algorithm) to extract images and arrange them into folders and subfolders. An option was to create a plugin especially written for MaZda. Due to time consumption, we used other software to carry out image acquisition (Micro Dicom 0.9, Dimensions 2, Sante Dicom 4, Photoshop CS6 64-bit Extended)5. For the extraction of Digital Imaging and Communications in Medicine (DICOM) data, we used the 64-bit portable version of Micro Dicom 0.9.1. Most CDs could be accessed with Micro Dicom or Photoshop CS6 64-bit Extended. We used the rescue feature in Sante Dicom 4 to recover the data and export them in DICOM format. Micro Dicom was the preferred samplesort because it also allowed image selection, batch-conversion and export of DICOM files as 32-bit BMP. Sorting of the sample by MR strength (3T, 1.5T) was carried out with Dimensions 2. Clinical arrangements of the sample by gestational age of pregnancy and anatomical plane of the mother were carried out manually. The primary goal was to select images that had clearly identifiable anatomical regions such as grey matter, white matter, ventricles and thalamus. It was not possible to complete the task with MaZda version 5 package as the available algorithms were lacking automatic segmentation to detect anatomical structures of fetal brain. Additional details about in-depth sorting as well as MRI specifications and file formats are provided in the methods of the cited article5.\n\n\nEthics and informed consent\n\nPermission to collect samples was approved by the Research Ethics Committee of the Medical University of Lodz. Written informed consent was obtained from all subjects (permit number: RNN/213/13/KE).\n\n\nData and software availability\n\nDataset 1: Fetal MRI data. 1.5/3T samples were manually sorted by gestational age and anatomical plane. Format: 32-bit BMP. doi, 10.5256/f1000research.10723.d15029613\n\nMaZda Package v5 RC HG available from: http://dx.doi.org/10.17632/dkxyrzwpzs.1\n\nMicro Dicom 0.9: www.microdicom.com/downloads.html\n\nDimensions 2: www.skwire.dcmembers.com/wb/pages/software/dimensions-2-folders.php\n\nSante Dicom 4: www.santesoft.com/downloads.html\n\nPhotoshop CS6 64-bit Extended: https://helpx.adobe.com/photoshop/using/dicom-files.html",
"appendix": "Author contributions\n\n\n\nHG conceived, designed, and wrote this data descriptor; LS and MRL contributed to sample collection and sorting. LS, MRL and MS helped with the description and the clinical arrangement of the data. MS helped with editing the research notes, coordinated with the software engineers to get technical feedback, and provided the latest updates. All authors were involved in the revision process and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMedical University of Lodz & Polish Research Committee and affiliated institutions and hospitals; Self-funded; MRI cost was covered by Polish National Health Fund, grants and financial aid from Swedish Ministry of Education and Research/Centrala Studiestödsnämnden and from U.S. Department of Education.\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 gratefully acknowledge prof. Rafał Pawliczak and MUL staff for research coordination, logistics, and administration; prof. Paweł Liberski and MUL neuropathology department for counsels with funds to cover MRI expenses; prof. Ludomir Stefańczyk and Barlicki hospital staff for sample supply and clinical feedback; prof. Maria Respondek- Liberska and Matki Polki Hospital for sample supply and clinical feedback ― as well as prof. Tadeusz Biegański, ICZMP director; prof. Michał Strzelecki and TUL staff for providing MaZda software and technical feedback.\n\n\nReferences\n\nSzczypiński PM, Strzelecki M, Materka A, et al.: MaZda--a software package for image texture analysis. Comput Methods Programs Biomed. 2009; 94(1): 66–76. PubMed Abstract | Publisher Full Text\n\nSzczypiński PM, Sriram RD, Sriram PV, et al.: A model of deformable rings for interpretation of wireless capsule endoscopic videos. Med Image Anal. 2009; 13(2): 312–324. PubMed Abstract | Publisher Full Text\n\nGentillon H, Stefańczyk L, Strzelecki M, et al.: Parameter set for computer-assisted texture analysis of fetal brain. BMC Res Notes. 2016; 9(1): 496. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGentillon H, Stefańczyk L, Strzelecki M, et al.: Texture analysis of the developing human brain using customization of a knowledge-based system [version 1; referees: awaiting peer review]. F1000Res. 2017; 6: 40. Publisher Full Text\n\nGentillon H: Artificial Systems Can Complement Human Vision in Medical Imaging. Int J Sci Basic Appl Res (IJSBAR). 2016; 25(3): 259–271. Reference Source\n\nSonigo PC, Rypens FF, Carteret M, et al.: MR imaging of fetal cerebral anomalies. Pediatr Radiol. 1998; 28(4): 212–222. PubMed Abstract | Publisher Full Text\n\nGuillemette-Artur P, Besnard M, Eyrolle-Guignot D, et al.: Prenatal brain MRI of fetuses with Zika virus infection. Pediatr Radiol. 2016; 46(7): 1032–1039. PubMed Abstract | Publisher Full Text\n\nDriggers RW, Ho CY, Korhonen EM, et al.: Zika Virus infection with Prolonged Maternal Viremia and Fetal Brain Abnormalities. N Engl J Med. 2016; 374(22): 2142–2151. PubMed Abstract | Publisher Full Text\n\nPontabry J, Rousseau F, Studholme C, et al.: A discriminative feature selection approach for shape analysis: Application to fetal brain cortical folding. Med Image Anal. 2017; 35: 313–326. PubMed Abstract | Publisher Full Text\n\nKitagawa H, Pringle KC: Fetal surgery: a critical review. Pediatr Surg Int. 2017; 1–13. PubMed Abstract | Publisher Full Text\n\nFan D, Wu S, Wang R, et al.: Successfully treated congenital cystic adenomatoid malformation by open fetal surgery: A care-compliant case report of a 5-year follow-up and review of the literature. Medicine (Baltimore). 2017; 96(2): e5865. PubMed Abstract | Publisher Full Text\n\nMalicki J: 275. Does the electro-radiology, the new university specialty created a career opportunity for radiation technologists? Rep Pract Oncol Radiother. 2003; 8(Supplement 2): S325. Publisher Full Text\n\nGentillon H, Stefańczyk L, Strzelecki M, et al.: Dataset 1 in: Prenatal brain MRI samples for development of automatic segmentation, target- recognition and machine-learning algorithms to detect anatomical structures. F1000Research. 2017. Data Source"
}
|
[
{
"id": "22563",
"date": "31 May 2017",
"name": "Ivana Išgum",
"expertise": [
"Reviewer Expertise Medical image analysis"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTo my understanding this manuscript provides a description of a data repository containing fetal MR scans and a description of an analysis software package. This is a very nice idea. However, the manuscript is currently not clearly written which hampers reading and understanding of the paper. In my opinion the purpose of the paper needs to be clarified. Description of the data repository can be more concise, and description of collected images could be more specific. It is good that patient inclusion is provided but it would be nice to know how many images acquired with what protocol are collected, as well as the patient characteristics.\nFurthermore, exact purpose of the software is currently unclear. Reading descriptions available at provided links provides some clarification but it would be much better if this manuscript is self-contained.\nThe manuscript contains many specific terms that are to the best of my knowledge not generally known and hence need to be introduced and explained to allow understanding of the work. For example, the introduction starts with “sample sorting” which is not introduced. In the same paragraph, the authors mention “target recognition semantics” but it is not known what is meant by this.\nThe authors mention that personal information has been removed according to agreement, but it is not clear what agreement.\nIt would be important to describe in the manuscript if and how scientific community can benefit from this image database and the described software.\nThe title does not reflect the content of the paper well. I would advise to change it accordingly.\n\nIs the rationale for creating the dataset(s) clearly described? No\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? No\n\nAre the datasets clearly presented in a useable and accessible format? No",
"responses": [
{
"c_id": "2743",
"date": "02 Jun 2017",
"name": "Hugues Gentillon",
"role": "Author Response",
"response": "Thank you for your comments and suggestions. On F1000Research’s policies, it states that ‘Data Notes are brief descriptions of scientific datasets that include details of why and how the data were created; they do not include any analyses or conclusion’, and we feel that some of your comments mentioned are a matter of personal preference. For example, you request us to publish information about 'patient characteristics'. It is unnecessary."
}
]
},
{
"id": "23366",
"date": "03 Jul 2017",
"name": "Feng Shi",
"expertise": [
"Reviewer Expertise Early brain development"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors proposed a sample sorting method for fetal MR images. Below are several suggestions to potentially improve the clarity of the paper.\n\nThe Introduction stated that the goal of this work is to further improve the MaZda software. The authors may consider enlarging its audience size by introducing how this data could benefit other researchers in the fetal research community.\n\nThe sample sorting seems totally manual, which may be more efficient with the help of some machine learning algorithms.\n\nExperiments could be added to evaluate the performance/correctness of the sample sorting process.\n\nSome details of the data itself could be useful for readers, such as the final data number, demographic information. The dataset 1 for downloading seem only contain 3 subjects.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\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? No",
"responses": [
{
"c_id": "2852",
"date": "05 Jul 2017",
"name": "Hugues Gentillon",
"role": "Author Response",
"response": "Thank you for your comments and suggestions. Again, on F1000Research’s policies, it states that ‘Data Notes are brief descriptions of scientific datasets that include details of why and how the data were created; they do not include any analyses or conclusion’, and we feel that some of your comments mentioned are a matter of personal preference. For example, you ask us to publish experimental analysis and information about 'patient demographics' but we feel that this is unnecessary, in this case. We do not agree with some of your comments and suggestions from a practical (clinical) point of view, for the following reasons below. The main goal of the Mazda software is to evaluate signal-to-noise ratio. In fact, cerebral fluid should be homogeneous in both 1.5T and 3T MR images, regardless of patient demographics (Africans, Europeans, Asians, etc.). If so it is a plus for MaZda and the method, as well as a good reference for MR image quality. It is also a good start to assess the maturation of fetal brain in MR. This process is a fact."
}
]
}
] | 1
|
https://f1000research.com/articles/6-93
|
https://f1000research.com/articles/6-1657/v1
|
07 Sep 17
|
{
"type": "Research Article",
"title": "Single-incision laparoscopic surgery in gynecologic surgery: a single-institutional experience from Saudi Arabia",
"authors": [
"Kareemah Salamah",
"Mohammed Abuzaid",
"Ahmed Abu-Zaid",
"Kareemah Salamah",
"Mohammed Abuzaid"
],
"abstract": "Background: Laparoscopy is rapidly replacing laparotomy in the field of gynecologic surgery. Generally, there are limited data concerning the utility of single-incision laparoscopic surgery (SILS) in gynecologic surgery. Specifically, in Saudi Arabia, a third-world country, data are further limited; only one related study has been conducted so far. The purpose of this study is to retrospectively report our single-institutional experience of SILS in terms of feasibility, safety and perioperative outcomes in the management of various gynecologic conditions. Methods: The study took place at the Women’s Specialized Hospital, King Fahad Medical City, Riyadh, Saudi Arabia. From January 2012 to May 2016, all gynecologic patients who underwent SILS procedures were analyzed for pre-, intra- and post-operative details. SILS was performed using a single multi-port trocar and standard laparoscopic instruments. Results: A total of 54 patients underwent 66 SILS procedures. The median age and body mass index (BMI) were 36 years and 28.2 kg/m2, respectively. Fourteen patients (26%) had ≥ 1 previous abdominal and/or pelvic surgeries. Twenty-four patients (44.4%) were nulliparous. The three most commonly performed SILS procedures were unilateral salpingo-oophorectomy (45.5%) and unilateral ovarian cystectomy (27.3%) and adhesiolysis (6.1%). The median operative time, estimated blood loss and hospital stay were 74 min, 50 ml and 1 day, respectively. Three patients required conversion to laparotomy, as follows: unidentified non-stopping bleeding source (n=1) and endometriosis stage IV resulting in difficult dissection (n=2). One patient developed post-operative incisional hernia that was treated surgically. The median patients’ post-operative pain (according to Wong-Baker FACES Foundation pain rating scale) within 4 hours was 2. At 4-week post-operatively, the median wound scar length (measured at outpatient clinic) was 2 cm. Conclusions: SILS is feasible, safe and associated with acceptable clinical and surgical outcomes.",
"keywords": [
"Laparoscopic single-site surgery",
"single-incision laparoscopic surgery",
"minimally invasive surgery",
"Gynecologic oncology",
"Saudi Arabia"
],
"content": "Introduction\n\nMinimally invasive surgery (laparoscopy) is rapidly replacing laparotomy in the field of gynecologic surgery1. As opposed to laparotomy, laparoscopy provides plentiful benefits. Such benefits comprise: reduced postoperative pain, faster recovery to previous performance status, shorter hospital stay, better cosmesis, lower cost, reduced morbidity/mortality and overall improved surgical outcomes2–4.\n\nOne of the most notable advances in laparoscopy is the introduction of single-incision laparoscopic surgery (SILS). In contrast to the conventional laparoscopy that is regularly executed by using a total of three to five small incisions (5–20 mm each), SILS is performed by using a single small incision of the umbilicus to completely accomplish the laparoscopic surgical procedures1. SILS has been demonstrated in retrospective and prospective studies to be feasible, safe and reproducible in managing various gynecologic conditions ranging from simple procedures (for example, adnexectomy)1,3,5–9 to highly complicated ones (for example, hysterectomy, complex pelvic masses and lymphadenectomy)10–20.\n\nGenerally, there are limited data concerning the utility of SILS in gynecologic surgery21. Specifically, in Saudi Arabia, a third-world country, data are further limited; only one study about SILS in gynecologic surgery has been conducted so far22.\n\nThe purpose of this study is to retrospectively report our single-institutional experience of SILS (feasibility, safety, and clinical/surgical outcomes) in the management of various gynecologic conditions.\n\n\nMethods\n\nThe study protocol was approved by the Institutional Review Board (IRB) of King Fahad Medical City [ID: 15-477].\n\nThis was a retrospective study from January 2012 to May 2016 which took place at the Women’s Specialized Hospital, King Fahad Medical City, Riyadh, Saudi Arabia — a tertiary healthcare institution. At our institution, as of January 2012, SILS has been the standard management option for gynecologic patients who met the inclusion criteria, namely: age less than 90 years, body mass index (BMI) less than 50.0 kg/m2, acceptable preoperative performance status and laboratory profile, technically resectable early stage adnexal or endometrial lesions, and signed written informed consent by patients after being well-informed about the risks/benefits of SILS.\n\nFrom January 2012 to May 2016, the medical records of all gynecologic patients who underwent SILS procedures for gynecologic conditions were retrospectively analyzed for pre-, intra- and post-operative details. Pre-operative details comprised: age, BMI, previous abdominal and/or pelvic surgeries, concomitant co-morbidities and parity. Intra-operative details comprised: type of procedures performed, conversion to conventional laparotomy, operative time (OT), estimated blood loss (EBL), lesion size and intra-operative morbidity/mortality. OT was defined as the interval from the initial umbilical skin incision to its closure. Post-operative details comprised: lesion pathology, patients’ self-reported recommendation (yes/no) of SILS to others, hospital stay, and post-SILS wound size, morbidity, mortality and pain. The post-SILS wound size was measured at 4 weeks at the outpatient clinic. Post-SILS morbidity/mortality were defined as any surgery-related complications/death up to 24 weeks postoperatively. The self-reported scores for post-operative pain were documented within 4 hours postoperatively by the operating surgeon/ward nurse using the Wong-Baker FACES Foundation pain rating scale.\n\nAll SILS procedures were primarily performed by a single surgeon from Section of Gynecologic Oncology, Department of Obstetrics and Gynecology. Procedures were performed under general anesthesia. Patients were placed in supine/lithotomy position, prepped and draped according to the hospital protocol. A 15 mm transverse intra-umbilical incision was made and skin edges were grabbed with Allis clamps. Afterwards blunt dissection was performed to create a 15 mm opening into the peritoneum. Then, Medtronic SILSTM device (Medtronic, Minnesota, USA) was introduced into the peritoneal cavity using packing forceps. The device has one gas inlet and 3-access ports (two 5-mm ports and one 12-mm port). Then, pneumoperitoneum was accomplished using carbon dioxide (CO2) through the gas inlet valve. Various laparoscopic instruments (rigid 0-degree and prebent) were used as deemed appropriate by the operating surgeon to avoid instrumental clashing and improve the operation field. The resected specimens were removed using the 12-mm port and endobag to allow for intact removal of the surgical specimen. The rectus sheath was closed with number 0 maxon sutures and skin closed with 3-0 vicryl sutures. In case of failure of SILS, the procedure was converted to either conventional laparoscopy or laparotomy.\n\nAll patients were followed up for at least 6 months post-operatively at the outpatient clinic.\n\nThe descriptive raw data are reported in Dataset 1. Data were analyzed with Microsoft Excel 2013. Whenever possible, data were presented as percentages, median ± standard deviation (SD) and range values.\n\n\nResults\n\nA total of 54 patients underwent SILS procedures. Characteristics of the patients are depicted in Table 1. The median age ± SD was 36 ± 16.9 years (range: 15–88) whereas the median BMI ± SD was 28.2 ± 6.1 kg/m2 (range: 18.9–44.1). A total of 14 patients (26%) had ≥ 1 previous abdominal and/or pelvic surgeries. Regarding parity, 24 (44%), 2 (4%) and 28 (52%) patients were nulliparous (para 0), primiparous (para 1) and multiparous (para 2+), respectively.\n\nThe intra-operative details of SILS are portrayed in Table 2. A sum of 66 SILS procedures were carried out. A total of three patients required conversion to laparotomy. The three most frequently performed SILS procedures were unilateral ovarian salpingo-oophorectomy (45.5%), unilateral ovarian cystectomy (n=27.3%) and adhesiolysis (n=6.1%). The median size of resected lesions ± SD was 12 ± 8.6 cm (range: 1.3–50). The median OT and EBL ± SD were 74 ± 39.4 min (range: 40–200) and 50 ± 271.7 ml (range: 20–2000), respectively. None of the patients experienced intra-operative death.\n\nThe post-operative details of SILS are shown in Table 3. There were 44 (81.5%) and 10 (18.5%) benign and malignant lesions, respectively. The median patients’ self-reported scores for postoperative pain was 2 ± 1.5 (range: 0–6). The median hospital stay was 1 ± 0.7 days (range: 1–4). At 4-week post-operatively, the median length of the wound scar (measured at the outpatient clinic) was 2 ± 0.4 cm (range: 1.5–2.5). During 6-month follow-up, one patient developed postoperative complication (incisional hernia) that was managed surgically.\n\n*Wong-Baker FACES Foundation pain rating scale\n\n\nDiscussion\n\nSILS is one of the cutting-edge developments in the field of minimally invasive surgery1. However, there are insufficient data concerning the feasibility, safety and perioperative outcomes of SILS in gynecologic surgery in Saudi Arabia.\n\nTo the best of our knowledge, this is the second ever study in Saudi Arabia to report the single-institutional experience of SILS in gynecologic surgery. Moreover, it is among the first studies originating from third-world countries that examined the efficacy of SILS in gynecologic surgery. Our study demonstrated that SILS is feasible, safe and associated with acceptable clinical and surgical outcomes. Nearly all patients (98.1%) agreed to recommend SILS to other patients.\n\nThe only study of SILS in Saudi Arabia was reported by Al-Badawi et al.22. They reported their single-center experience of SILS in the management of benign salpingo-ovarian pathologies. They had 80 patients and a total of 104 performed procedures. They concluded that SILS in the management of benign salpingo-ovarian conditions was generally feasible, potentially safe, and associated with satisfactory operative and postoperative outcomes. As opposed to the Al-Badawi et al. study that included only adnexectomy procedures22, our study included far more complex (non-adnexectomy) procedures, such as: SILS laparoscopic-assisted vaginal hysterectomy (LAVH, n=3), and SILS total laparoscopic hysterectomy (TLH, n=3). Several studies showed that SILS approach can be used to successfully perform both TLH and LAVH procedures10–20.\n\nIn our study, 3 patients required conversion to laparotomy for several reasons: unidentified non-stopping bleeding source (n=1) and endometriosis stage IV resulting in difficult dissection (n=2). Patient safety should never be recklessly put in danger at any given time. Therefore, conversion from SILS to laparotomy —whenever at the best interests of patients— is more significant than mere cosmetic concerns. Also, such conversion from SILS to laparotomy should not be prematurely judged as a surgical failure or an incompetence of the operating surgeon to perform SILS procedures.\n\nPatients with high BMI are regarded as a challenging group for SILS procedures due to the anticipated high intraperitoneal fat contents which can cause port access difficulties and garble the field visualization23. It has been recommended that patients with BMI less than 28 kg/m2 are regarded as suitable patients for SILS24. In our study, more than 50% of patients had BMI of more than 28 kg/m2 and none experienced eventful surgical courses.\n\nMoreover, patients with previous abdominal and/or pelvic surgeries are regarded as a challenging group for SILS procedures due to the anticipated dense surgery-related adhesions and difficult dissection23. In our study, around 11.1% of patients had 2 or more previous surgeries; however, none of them required conversion to laparotomy.\n\nFurthermore, port-site metastasis in malignant gynecologic oncology is sometimes worrisome to both patients and laparoscopists. However, the frequency of port-site metastasis following laparoscopic procedures in women with malignant gynecologic pathology is very low (less than 1%)25. Numerous studies showed feasibility, safety and reproducibility of the SILS for management of precancerous pathologies and select early-stage ovarian and uterine malignancies10,11,13. Thus, it can be concluded that SILS should not be avoided in the management of patients with early-stage gynecologic malignancy. In our study, 10 patients had malignant pathologies and successfully underwent SILS procedures without proof of port-site metastasis at a median follow up of 29 months (range: 23–37).\n\nIn brief, apposite selection of patients for SILS is of great significance. According to our study, our recommendations for patients who may be eligible candidates for SILS procedures comprise: BMI less than 40 kg/m2, less than 3 previous surgeries (irrespective of laparoscopic and/or laparotomic), presence of native umbilicus and early-stage gynecologic malignancy.\n\nSILS is not without its technical challenges which are well-documented in literature. Such challenges generally comprise: the limited triangulation and retraction capacities as well as the camera/laparoscopic instruments conflict that can distort proper surgical field exposure26. Measures to prevail these downsides are continuously in progress and comprise the introduction of flexible and prebent laparoscopic instruments of different lengths22.\n\nSimilar to conventional laparoscopy, SILS is a surgical procedure that necessitates a great deal of fine-motor hand dexterity. Two studies documented that around 10 to 15 procedures were required to perform SILS adroitly which was reflected on reduced operating time10,27. Many measures have been advocated to improve the learning curve, for example: watching live/recorded procedures, virtual simulation and hands-on practice on animals26,28. There are serious ongoing efforts to implement the above-mentioned measures at our institution.\n\nOur future research directions include: 1) the learning curve of SILS at our institution, and 2) comparison between SILS and conventional laparoscopy with regard to management of various gynecologic pathologies.\n\nOur study has several limitations and comprise: retrospective study design, lack of control group (conventional laparoscopic), relatively small sample size, single-institutional experience and patients subjective self-reported scores for postoperative pain.\n\n\nConclusion\n\nThis is the second single-institutional experience of SILS in gynecologic surgery from Saudi Arabia. Our study demonstrated that SILS is feasible, safe and associated with acceptable clinical and surgical outcomes. SILS is an operator-dependent procedure and requires advanced surgical training.\n\n\nData availability\n\nDescriptive Raw Data- Pre-operative, intra-operative and post-operative details\n\n10.5256/f1000research.12545.d17645329",
"appendix": "Competing interests\n\n\n\nAuthors declare no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBradford LS, Boruta DM: Laparoendoscopic single-site surgery in gynecology: a review of the literature, tools, and techniques. Obstet Gynecol Surv. 2013; 68(4): 295–304. PubMed Abstract | Publisher Full Text\n\nMedeiros LR, Rosa DD, Bozzetti MC, et al.: Laparoscopy versus laparotomy for benign ovarian tumour. Cochrane Database Syst Rev. 2009; (2): Cd004751. PubMed Abstract | Publisher Full Text\n\nJung YW, Kim SW, Kim YT: Recent advances of robotic surgery and single port laparoscopy in gynecologic oncology. J Gynecol Oncol. 2009; 20(3): 137–144. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee IO, Yoon JW, Chung D, et al.: A comparison of clinical and surgical outcomes between laparo-endoscopic single-site surgery and traditional multiport laparoscopic surgery for adnexal tumors. Obstet Gynecol Sci. 2014; 57(5): 386–392. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFader AN, Cohen S, Escobar PF, et al.: Laparoendoscopic single-site surgery in gynecology. Curr Opin Obstet Gynecol. 2010; 22(4): 331–338. PubMed Abstract\n\nYoon BS, Park H, Seong SJ, et al.: Single-port laparoscopic salpingectomy for the surgical treatment of ectopic pregnancy. J Minim Invasive Gynecol. 2010; 17(1): 26–29. PubMed Abstract | Publisher Full Text\n\nFagotti A, Fanfani F, Marocco F, et al.: Laparoendoscopic single-site surgery (LESS) for ovarian cyst enucleation: report of first 3 cases. Fertil Steril. 2009; 92(3): 1168.e13–6. PubMed Abstract | Publisher Full Text\n\nKim TJ, Lee YY, Kim MJ, et al.: Single port access laparoscopic adnexal surgery. J Minim Invasive Gynecol. 2009; 16(5): 612–615. PubMed Abstract | Publisher Full Text\n\nMereu L, Angioni S, Melis GB, et al.: Single access laparoscopy for adnexal pathologies using a novel reusable port and curved instruments. Int J Gynaecol Obstet. 2010; 109(1): 78–80. PubMed Abstract | Publisher Full Text\n\nFader AN, Rojas-Espaillat L, Ibeanu O, et al.: Laparoendoscopic single-site surgery (LESS) in gynecology: a multi-institutional evaluation. Am J Obstet Gynecol. 2010; 203(5): 501.e1–6. PubMed Abstract | Publisher Full Text\n\nFader AN, Escobar PF: Laparoendoscopic single-site surgery (LESS) in gynecologic oncology: technique and initial report. Gynecol Oncol. 2009; 114(2): 157–161. PubMed Abstract | Publisher Full Text\n\nLee YY, Kim TJ, Kim CJ, et al.: Single-port access laparoscopic-assisted vaginal hysterectomy: a novel method with a wound retractor and a glove. J Minim Invasive Gynecol. 2009; 16(4): 450–453. PubMed Abstract | Publisher Full Text\n\nYim GW, Jung YW, Paek J, et al.: Transumbilical single-port access versus conventional total laparoscopic hysterectomy: surgical outcomes. Am J Obstet Gynecol. 2010; 203(1): 26.e1–6. PubMed Abstract | Publisher Full Text\n\nFanfani F, Fagotti A, Scambia G: Laparoendoscopic single-site surgery for total hysterectomy. Int J Gynaecol Obstet. 2010; 109(1): 76–77. PubMed Abstract | Publisher Full Text\n\nJung YW, Kim YT, Lee DW, et al.: The feasibility of scarless single-port transumbilical total laparoscopic hysterectomy: initial clinical experience. Surg Endosc. 2010; 24(7): 1686–1692. PubMed Abstract | Publisher Full Text\n\nLangebrekke A, Qvigstad E: Total laparoscopic hysterectomy with single-port access without vaginal surgery. J Minim Invasive Gynecol. 2009; 16(5): 609–611. PubMed Abstract | Publisher Full Text\n\nYoon G, Kim TJ, Lee YY, et al.: Single-port access subtotal hysterectomy with transcervical morcellation: a pilot study. J Minim Invasive Gynecol. 2010; 17(1): 78–81. PubMed Abstract | Publisher Full Text\n\nEscobar PF, Fader AN, Rasool N, et al.: Single-port laparoscopic pelvic and para-aortic lymph node sampling or lymphadenectomy: development of a technique and instrumentation. Int J Gynecol Cancer. 2010; 20(7): 1268–1273. PubMed Abstract | Publisher Full Text\n\nKim TJ, Lee YY, Cha HH, et al.: Single-port-access laparoscopic-assisted vaginal hysterectomy versus conventional laparoscopic-assisted vaginal hysterectomy: a comparison of perioperative outcomes. Surg Endosc. 2010; 24(9): 2248–2252. PubMed Abstract | Publisher Full Text\n\nChen YJ, Wang PH, Ocampo EJ, et al.: Single-port compared with conventional laparoscopic-assisted vaginal hysterectomy: a randomized controlled trial. Obstet Gynecol. 2011; 117(4): 906–912. PubMed Abstract | Publisher Full Text\n\nUppal S, Frumovitz M, Escobar P, et al.: Laparoendoscopic single-site surgery in gynecology: review of literature and available technology. J Minim Invasive Gynecol. 2011; 18(1): 12–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAl-Badawi IA, AlOmar O, Albadawi N, et al.: Single-port laparoscopic surgery for benign salpingo-ovarian pathology: a single-center experience from Saudi Arabia. Ann Saudi Med. 2016; 36(1): 64–69. PubMed Abstract | Publisher Full Text\n\nChern BS, Lakhotia S, Khoo CK, et al.: Single incision laparoscopic surgery in gynecology: Evolution, current trends, and future perspectives. Gynecology and Minimally Invasive Therapy. 2012; 1(1): 9–18. Publisher Full Text\n\nRoss SB, Clark CW, Morton CA, et al.: Access for laparoendoscopic single site surgery. Diagn Ther Endosc. 2010; 2010: 943091. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZivanovic O, Sonoda Y, Diaz JP, et al.: The rate of port-site metastases after 2251 laparoscopic procedures in women with underlying malignant disease. Gynecol Oncol. 2008; 111(3): 431–437. PubMed Abstract | Publisher Full Text\n\nAhmed I, Paraskeva P: A clinical review of single-incision laparoscopic surgery. Surgeon. 2011; 9(6): 341–351. PubMed Abstract | Publisher Full Text\n\nEscobar PF, Starks DC, Fader AN, et al.: Single-port risk-reducing salpingo-oophorectomy with and without hysterectomy: surgical outcomes and learning curve analysis. Gynecol Oncol. 2010; 119(1): 43–47. PubMed Abstract | Publisher Full Text\n\nBehnia-Willison F, Foroughinia L, Sina M, et al.: Single incision laparoscopic surgery (SILS) in gynaecology: feasibility and operative outcomes. Aust N Z J Obstet Gynaecol. 2012; 52(4): 366–370. PubMed Abstract | Publisher Full Text\n\nSalamah K, Abuzaid M, Abu-Zaid A: Dataset 1 in: Single-Incision Laparoscopic Surgery in Gynecologic Surgery: A Single-Institutional Experience from Saudi Arabia. F1000Research. 2017. Data Source"
}
|
[
{
"id": "25813",
"date": "18 Sep 2017",
"name": "Ahmed Nazer",
"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\nSmall descriptive study about SILS. Based on the presets data, it is difficult to comment about the safety of this approach for TLH or LAVH as the number is very small and there is no informations about the indications of the hysterectomies.\nOverall promising results and worth to publish .\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": "25796",
"date": "21 Sep 2017",
"name": "Sarfraz Ahmad",
"expertise": [
"Reviewer Expertise Gynecologic oncology research & teachings"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWith great interest I read the paper by Salamah et al. The authors reported their single-center experience of single-incision laparoscopic surgery (SILS) in the field of gynecologic surgery from a tertiary healthcare center in Saudi Arabia. The topic is of great significance since this study is only the second ever from Saudi Arabia, and will add valuable data to the limited regional peer-reviewed literature.\n\nIntroduction: The authors introduced the topic appropriately, defined terminology of SILS, and highlighted the significance of the study thereby clearly mentioned the objectives of study.\n\nMethods: The ethical approval for the study was obtained. Appropriate descriptions of the study design/setting, research subjects and surgical procedure are provided. The study was descriptive, retrospective, and one-group of patients. Only simple statistical tests were carried out, i.e., calculations of numbers/percentages, SDs and ranges.\n\nResults: Overall, the results section reads well reporting the major findings. Results were presented in simple tables and divided into pre-, intra- and post-operative categories. There are some repetitions between the text and the tables which makes easier to understand the findings. In Table 2, perhaps the authors should correct the “Type of Surgery” data: i.e., Laparoscopy=51, Laparotomy=3 (instead of 3 and 18, respectively) and appropriate percentages.\n\nDiscussion: It is succinct and properly reflecting on the study’s main findings without unnecessary details. The significance of study was re-highlighted. Brief comparisons with the the existing peer-reviewed literature were made with regards to aspects pertaining to the pre-operative details such as BMI and previous surgery. Most importantly, the study findings were directly compared-and-contrasted to the only study from Saudi Arabia (by Al-Badawi et al. 2016) which is a plausible and appreciated move. Study’s future directions and limitations are adequately pinpointed.\n\nConclusion: Reads very well and reflects on the study’s main conclusions.\n\nOverall Comments: The manuscript is very well-written in terms of English language, grammar, word counts, clarity, logical sequence, citations, and appropriate references cited. The strong points of paper are: 1) being the second in Saudi Arabia, which highlights the significance of the study to warrant additional peer-reviewed publication based on scientific soundness, despite no novelty; and 2) the study is of great importance in terms of regional interests as it enriches the scarce limited peer-reviewed literature on the topic. The study weakness mainly revolve around the methodology part as this is a mere descriptive retrospective study without a control-group and detailed statistical calculations, which makes it a “less” strong evidence; however, the study limitations are duly acknowledged by the authors. I highly recommend the authors to pursue future studies proposed in their manuscript, as these directions are going to have more impact and contribute substantially to the body of existing of peer-reviewed literature regionally and internationally. More strong-evidence-based study design should be considered too, such as prospective and controlled-study designs. A cost-effective study comparing conventional vs. SILS in gynecologic surgery indications may be an interesting topic, especially from a third-world country with relatively constrained healthcare economy.\nFinal Remarks and Decision: According to the scope/policy of the F1000 Research Open-Access journal, the study is “scientifically sound” and “suitable” irrespective of the perceived level of novelty. Therefore, I approve the study for publication in its current draft.\n\nConflict of Interest: I report no conflict of interest pertaining to this manuscript.\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/6-1657
|
https://f1000research.com/articles/6-1656/v1
|
07 Sep 17
|
{
"type": "Research Article",
"title": "Phenotypic and genotypic detection of carbapenemase enzymes producing gram-negative bacilli isolated from patients in Khartoum State",
"authors": [
"R.A. Dahab",
"Alamin Mohamed Ibrahim",
"Hisham N. Altayb",
"Alamin Mohamed Ibrahim",
"Hisham N. Altayb"
],
"abstract": "Background: Carbapenems are used as antibiotics of last resort for treating infections due to multidrug-resistant Gram-negative bacilli, but emergence of Carbapenem resistant Gram-negative bacilli have been reported due to the production of Carbapenemase enzymes that significantly limits treatment options for life-threatening infections. Objective: This study aimed to detect Carbapenem resistant Gram-negative bacilli from patients attended to different hospitals in Khartoum state and to detect Carbapenemase enzymes production by phenotypic and genotypic methods. Methods: A hospital based cross sectional study was conducted in Khartoum state in the period from February to August 2016. Hundred and forty nine Gram-negative bacilli bacteria were isolated from different clinical specimens. Blood agar, Chromogenic agar media, MacConkey agar, XLD mediaandstandard biochemical tests were used for isolation and identification of Gram-negative bacilli from different samples. Standard antimicrobial susceptibility testing to Carbapenem antibiotic was performed for all isolates, then detection of Carbapenemase enzymes production for the resistant isolates was performed using Modified Hodge Test and PCR. Results: Hundred and forty nine Gram-negative bacilli were isolated from 147 different clinical specimens. The most predominant Gram-negative bacilli isolates was E.coli (54.4%), followed by Klebsiella species (29.5%). More than fifty percent of the isolates were Carbapenem resistant. Fifty six percent of the resistant isolates were positive by Modified Hodge Test. By using PCR, 17.3% of resistant organisms were harbored blaOXA48 gene, and 6.7% harbored blaIMP gene. E.coli was the most bacteria that harbored the blaoxa48 followed by Klebsiella species. blaIMP gene was harbored only by E.coli. Conclusion: The percentage of resistance to Carbapenems due to production of Carbapenemase enzymes is very high in Sudan.BlaOXA48 gene is more predominant than blaIMP in this study.",
"keywords": [
"carbapenem resistance",
"carbapenemase enzymes",
"gram-negative bacilli",
"Modified Hodge Test",
"PCR",
"bla IMP gene",
"bla OXA48"
],
"content": "Introduction\n\nCarbapenems are beta-lactam antibiotics often used as last resort antibiotics for treating infections caused by multidrug resistant Gram-negative bacilli, since they have the broadest spectra among all beta-lactams1,2. carbapenemases (also known as carbapenem hydrolyzing enzymes) represent the most versatile family of beta-lactamases3. Beta-lactamases can be classified according to their functional and molecular properties. Functional classification divides beta-lactamases into four major functional groups (1, 2, 3, and 4), whereas in molecular classification Ambler and others have classified beta-lactamases into four classes (A–D) based on amino acid sequence. In addition, based on the active site of the enzyme, they classified beta-lactamases into serine-beta-lactamases (class A,C and D) and metallo-beta-lactamases (class B)3. https://www.microrao.com/micronotes/pg/carbapenemases.pdf\n\nOxacillinases (OXAs) (class D), so called because of their ability to hydrolyze Oxacillin, are classified according to their hydrolysis spectrum. Broad-spectrum OXA enzymes are able to hydrolyze carbapenems. This family is plasmid encoded and they have been identified mainly in Enterobacteriaceae and Pseudomonas aeruginosa. Currently, there are 239 OXA enzymes, of which at least 37 are carbapenemases and at least 9 are extended spectrum beta-lactamases. The first OXA enzyme with carbapenemase activity was detected in 1985 in an Acinetobacter baumanii isolate from Scotland, and OXA-48 cluster is the most important among class D carbapenemases3,4. https://www.microrao.com/micronotes/pg/carbapenemases.pdf\n\nIMP (for “active on Imipenem”) a metallo-beta-lactamase (class B), was first detected in 1990 in P. aeruginosa isolate from Japan, and then reported in Serratia marcescens isolate in Japan in 1991, IMP-2 was observed in Italy in A.baumannii. Currently there are 37 known IMP types which are most commonly seen in P. aeruginosa and A.baumannii isolates, but have also been reported in most Enterobacteriaceae members5. https://www.microrao.com/micronotes/pg/carbapenemases.pdf\n\nOne of the risk factors of acquiring carbapenem resistant bacteria is the increased consumption of carbapenems . Many patients are most likely affected by carbapenem resistant bacteria, including those who have poor immunity, prolonged hospitalization, admission to ICU, an indwelling medical device, and multiple exposures to different antibiotics. Also the increased frequency of international travel for work, leisure and migration contributes to their spread, and most of the carbapenemases are found on transferable plasmids, which are highly transmissible6. https://www.safetyandquality.gov.au/wp-content/uploads/2013/12/MRGN-Guide-Enterobacteriaceae-PDF-1.89MB.pdf\n\nThe prevalence of carbapenemase producing gram negative bacteria is increasingly reported in Sudan and Africa. Manenzhe et al. (2014) reported about 83 studies conducted in Africa which showed that the prevalence of carbapenemase producer isolates in hospital settings ranged from 2.3% to 67.7% in North Africa and from 9% to 60% in sub-Saharan Africa. Oxacillinases especially blaOXA48 was the most predominant in the whole country7.\n\nIn Sudan there are limited reports of carbapenemase producers, therefore the present study was performed8. This study aimed to detect carbapenem resistant Gram-negative bacilli (using conventional biochemical tests and chromogenic agar media), determine the antimicrobial susceptibility pattern of the isolates against carbapenems, detect carbapenemase enzyme production for the resistant isolates (using phenotypic methods, Modified Hodge Test), and detect blaOXA 48 and blaIMP genes for carbapenem resistant isolates (using PCR, genotypic methods).\n\n\nMethods\n\nThis was cross sectional laboratory based study, conducted in Khartoum state in the period from February to August 2016. Ethical clearance was obtained from SUMASRI (University of Medical Sciences and Technology Sudanese Institute of Medical and Scientific Research) Institutional Review Board (SIRB); (IRB No: 00008867), which ensures that all ethical considerations for conducting the research in a way that protects patient’s confidentiality and privacy are followed. Informed consent was obtained from the hospital laboratories (laboratory manager) after providing them with the ethical clearance to collect samples during routine procedures from the microbiology laboratories. Participants’ privacy and confidentially was protected for all samples; personal information was not of great value in the current study and was thus not taken.\n\nOne hundred and forty nine Gram-negative bacilli were isolated from 147 different clinical specimens that were collected from patients attending different hospitals in Khartoum state. Blood agar, MacConkey agar, CLED media, and XLD media were used for primary plating depending on the type of clinical specimens, cultures were examined macroscopically for colonial morphology, and Gram stain was performed from suspected colonies. All Gram-negative bacilli isolates were selected then subcultured on MacConkey agar media for purity and further identification tests, and incubated at 37° overnight. Chromogenic agar media and different standard biochemical reactions, including oxidase, Kligler Iron Agar (KIA), urease production, citrate utilization, indole production, and motility tests were performed for their identification (all media mentioned above were obtained from LAB M, UK, except chromogenic media which was obtained from Laboratories Flow Media, Sudan)9.\n\nAntimicrobial susceptibility testing to carbapenem antibiotic was performed for all Gram-negative bacilli isolates using the disc diffusion method, according to the Clinical Laboratory Standards Institute Guidelines9. Bacterial colonies were suspended in sterile normal saline and compared with McFarland standard and cultured in Muller-Hinton agar media (media were obtained from Pronadisa Laboratories Conda., Spain) using a sterile cotton swab. After overnight incubation at 37°C, zone of inhibition was measured and the reading was compared to the sheet provided by manufacturer9.\n\nGram-negative isolates showing resistant zones to Meropenem were tested for carbapenemase enzyme production using MHT and PCR.\n\nMHT was used to detect carbapenemase enzyme production10. McFarland 0.5 dilution of E.coli (ATCC 25922) was prepared in 5 ml of sterile saline. The suspension was diluted to 1:10 by adding 0.5 ml of the 0.5 McFarland (Oxoid, UK) to 4.5 ml of sterile saline. A lawn of the 1:10 dilution of E.coli was streaked onto a Mueller Hinton agar plate using a sterile cotton swab. A 10 μg Meropenem susceptibility disk (Hi media, India) was placed on the center of the test area. The tested bacilli was streaked in a straight line from the edge of the disk to the edge of the plate. The plate was incubated overnight at 37°C in ambient air (Figure 1)11.\n\nClover leaf like indentation from E. coli ATCC showing positive result.\n\nPositive MHT: Shown by clover leaf-like indentation of E. coli susceptible strains growing along the tested organism growth streak within the disk diffusion zone (Figure 1)12.\n\nNegative MHT: No growth of E. coli along the tested organism growth streak within the disc diffusion zone12.\n\nDNA extraction. DNA extraction for all resistant isolates was done by boiling method from fresh bacterial cultures within 24 hours, in which bacteria are in the logarithmic growth phase, which is the most suitable phase for bacterial DNA extraction according to the following protocol: Cells of interest were suspended in sterile normal saline and pelleted (106 to 107) by centrifugation at 4.000 rpm for 5 minutes using a labelled 1.5 ml safe-lock tube. The pellet was resuspended in 100 μl of phosphate buffer saline. The tubes were placed at 95°C for 15 minutes then centrifuged at >10.000 rpm for 5 minutes to pellet the cellular debris. Then the supernatant (lysate) was transferred into a labelled 1.5 ml safe-lock. The lysate was stored at -20 to 4°C. https://www.dkfz.de/gpcf/fileadmin/ccontrol/lysate_Protocol_DKFZ.pdf\n\nPCR. A multiplex conventional PCR was designed to detect blaIMP and blaOXA 48 using specific primers for each. The primers were obtained from Macrogen Company, Korea (Table 1).\n\nThe amplification of DNA was performed using a TC-312 PCR machine (TECHNE, UK). For amplification, 17 μl of distilled water were added to ready manufactured premix solution (Intron biotechnology, Korea) and mixed well. Then 0.5 μl from forward primer and 0.5 μl from reverse primer of both blaIMP and blaOXA48 genes were added. Then 2 μl of DNA was added to the mixture. For both genes, the cycling conditions were: initial denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 53°C for 1 minute, and elongation at 72°C for 1 minute. The cycles were repeated 35 times, with a final extension step at 72°C for 5 minutes13. PCR product (5μl) were analyzed by gel electrophoresis in 1.0% agarose stained with ethidium bromide. The results were photographed under ultraviolet light machine (Transillumnator; Uvite, UK) to detect the specific amplified product by comparing it with 100 base pairs standard DNA ladder (Figure 2)14.\n\nLane 1, DNA ladder 100bp; Lane 2, negative control; Lane 3, typical band size of 238 bp corresponding to the molecular size of blaOXA48 gene; Lanes 4–7, negative samples.\n\nData obtained were analysed using descriptive statistics and chi-square tests performed using SPSS version 20.0, to check the statistical significance and Excel 2013. The p-value that considered significant was < 0.05.\n\n\nResults\n\nOne hundred forty seven different clinical specimens were collected from patients attending different hospitals in Khartoum state. One hundred forty nine Gram-negative bacilli were isolated. Urine specimens were the most frequent specimen, comprising 104 out of 147 clinical specimens; 106 Gram negative bacilli were isolated from the 104 urine specimens.\n\nThe isolated Gram-negative bacilli comprised of 81(54.4%) E.coli, 44 (29.5%) Klebsiella species, 17(11.4%) Proteus species, 6(4.0%) Pseudomonas species and 1(0.7%) Enterobacter species (Table 2).\n\ncarbapenem (Meropenem) susceptibility testing using disc diffusion method showed that 75 (50.3%) of Gram-negative rods isolates were Meropenem resistant, 57 (38.3%) were Meropenem sensitive, and 17 (11.4%) were Meropenem intermediate (Table 3).\n\nRegarding carbapenem (Meropenem) resistance, the most resistant organism was E. coli, which constituted 45 out of 75 resistant bacteria (30.2%), followed by Klebsiella species 15.4% (23 isolates), Proteus species 2.7% (4 isolates), Pseudomonas species 1.3% (2 isolates), and Enterobacter species 0.7% (1 isolate) (Table 3).\n\ncarbapenemase production by both MHT and PCR constituted 42 isolates (56%) of the total carbapenem resistant isolates.\n\nMHT was performed for the 75 resistant isolates, 42 isolates (56%) were positive and the other 33 (44%) were negative for carbapenemase enzyme production by MHT (Table 4).\n\nConventional PCR assay for detection of blaOXA 48 and blaIMP genes was performed for all 75 resistant isolates. 17.3% of the resistant Gram-negative isolates were positive for blaOXA 48 gene, while 6.7% of them were positive for blaIMP gene; both constituted 24% carbapenemase producing bacteria using PCR (Table 5).\n\ncarbapenemase production constituted 42 isolates (56%) of all resistant isolates by both MHT and PCR; some were positive by both methods, some were positive by MHT only, and others were positive by PCR only.\n\nRegarding MHT, E.coli was the most carbapenemase producer among the resistant isolates (it constituted 36% of the 56% resistant isolates) followed by Klebsiella species (it constituted 16% of the 56% resistant isolates). Pseudomonas species and Enterobacter species constituted 2.7% and 1.3%, respectively (Table 4).\n\nRegarding blaOXA48 gene production, E.coli was the organism that harboured the blaOXA48 gene the most, which constituted 12.0% of the 17.3% blaOXA48 gene positive result, followed by Klebsiella species (4%), and Pseudomonas species (1.3%). Proteus species and Enterobacter species were negative for blaOXA48 gene. blaIMP gene was positive only for E.coli (constituted the whole percentage of resistant bacteria that harbored blaIMP gene). None of the other types of resistant Gram-negative isolates harboured the blaIMP gene (Table 5).\n\n\nDiscussion\n\nIn this study E.coli was the most predominant organism among the isolated Gram-negative bacteria followed by Klebsiella species. This result agreed with the study done by Hayajneh, et al. in Jordan 2011–2013, and disagreed with the study done by Mataseje, et al. in Canada 2009–2010, in which Pseudomonas species was the commonest isolate14,15. This may be due to the difference in sample size and study area.\n\nThe resistance to carbapenem antibiotics in this study was 50.3%, which is not in agreement with a previous study conducted in India by Henkhoneng Mate, et al., the study conducted by Hayajneh, et al. in Jordan, and the study conducted by Gladstone et al., which showed that resistance to carbapenem antibiotics was 30%, 1.6%, and 12.2% respectively2,13,16. This may be due to the uncontrolled and misuse of antibiotics including broad spectrum antibiotics in our study area (Sudan)17.\n\nUrine samples had the maximum number of carbapenem resistant isolates, which was similar to the study conducted by Henkhoneng Mate, et al in India2.\n\ncarbapenemase production constituted 42 isolates (56%) of the total carbapenem resistant isolates, which disagreed with the study conducted in Tanzania 2013. That study showed that 35% of the resistant isolates were carbapenemase producers; this may be to the difference in the methods used for enzyme detection, since the previous study used only genotypic methods, while the present study used both phenotypic and genotypic methods14. A study conducted in India 2015 by Panduragan, et al reported that 62% of isolates produced carbapenemases, found using both phenotypic and genotypic methods, which is agreed with the current study, which reported a near percentage (56%)12.\n\nIn this study, the most carbapenemase producing organism was E. coli, which disagrees with a previous study done by Mushi et al, who reported that K. pneumoniae was the most predominant carbapenemase producer14.\n\nRegarding MHT, 50.6% of the total carbapenem resistant isolates were positive for carbapenemase production. This was not in agreement with a study conducted in India 2015 by Pandarangan, et al, who reported lower percentage (30.5%), and also disagreed with the study conducted by Henkhoneng Mate, et al in the same country, which reported a higher percentage (60.4%)2,12. This may be due to the difference in study area, which may affect the types and percentage of carbapenemase enzymes according to their spread.\n\nRegarding blaOXA48 gene, 17.3% of the isolates harboured this gene, which disagreed with a previous study done by Memish, et al in Saudi Arabia 2015. This previous study showed a higher percentage (61.3%) of isolates with the blaOXA48 gene, which may be due to the different study area18.\n\nblaIMP gene was harboured by 6.7% of the resistant isolates, which also disagreed with the study done in Tanzania (2013) and Thailand (2008). These studies showed that 21.6% and 15.4% of the resistant isolates, respectively, harboured the blaIMP gene14,19. In addition, a study performed by Nasr El-din in Egypt 2014 revealed that the blaIMP gene was absent from isolates20. This may be due to the different study area, the difference in sample size and the five different PCR assays that were performed in the study done in Tanzania, thus increasing the sensitivity and specificity compared to the present study.\n\nIn this study, blaOXA 48 gene was the most predominant carbapenemase harboured by the resistant isolates. This is in agreement with the study performed by Memish et al in Saudi Arabia 201518. However, our results disagreed with the study performed in Tanzania 2013, which showed that blaIMP gene was the most predominant14. This may be due to the difference in sample size and the five different PCR assays that were performed in this study.\n\n\nConclusion\n\nThis study concludes that the percentage of resistance to carbapenems in Khartoum state, Sudan, is very high and must be taken in consideration by the Sudanese government, and any large organization that can help in the control and prevention of carbapenemases, such as the World Health Organisation. E. coli, followed by Klebsiella species, were the most carbapenem resistant organisms and the largest carbapenemase producers. MHT is a simple method that can detect many types of carbapenemase enzymes, but it cannot detect some types of and does not specify the type of enzymes produced. In contrast, PCR is more sensitive, rapid and specific method for detection of specific types of carbapenemase enzymes. Additionally in the present study, the percentage of Gram-negative bacilli that produce blaOXA48 gene was more than those producing blaIMP; however, one of the isolates harboured both blaOXA48 and blaIMP genes. In this study, some Gram-negative bacilli were positive for carbapenemase enzyme production by both MHT and PCR, some were negative by MHT and positive by PCR, and some of them were positive by MHT and negative by PCR (this study did not detect all carbapenemase genes using PCR; thus those positive by MHT but negative by PCR may process other type of carbapenemase enzymes rather than OXA48 and IMP. Although this may also be because the gene is not expressed).\n\n\nRecommendations\n\nA larger sample size should be tested to cover a wider range of isolates.\n\nOther specific tests for detection of carbapenemase enzymes should be used, such as EDTA disc synergy test, MDI, RDS, MBL E test, more primers should be used to detect most types of carbapenemase enzymes using PCR, and different types of PCR assays should be coupled with each other to increase the sensitivity for enzyme detection.\n\nDetection of carbapenemase producers should be introduced as routine tests in microbiology labs for rapid detection of resistant isolates and to control their spread, especially for newly admitted patients to the hospitals.\n\nPrevention and control programs of carbapenem resistant Gram-negative bacteria should be performed to prevent the spread of carbapenemase producers, which includes appropriate use of antimicrobials and facility-level prevention strategies, as recommended by the CDC.\n\nDataset 1: Raw data for the 147 specimens analysed showing the results of the genotypic and phenotypic tests. The file contains the same data in Excel and SPSS formats. DOI, 10.5256/f1000research.12432.d17637721.\n\nDataset 2: Pictures related to the methods used in the current study (in zipped file): ‘Standard antimicrobial susceptibility testing’, ‘Modified Hodge Test (MHT)’ and ‘Molecular detection of blaIMP and blaOXA 48 genes’. DOI, 10.5256/f1000research.12432.d17637822.",
"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\nI would like to offer my special thanks to Miss Maha Baballah for her assistance especially in sample collection. Also Dr. Abd Elhakam Hassan Ibrahim for his strong assistance in keeping my progress in the practical work (especially MHT). My grateful thanks are also extended to Dr. Asim Halfawi for his contribution to the analysis of the data.\n\nMy great appreciation and thanks are extended to those who helped me during my practical work; Eman Eshag, Tahani Mursal, Roaa Malik, Samah Abdallah, Reham Abdelrahman, and Mogadam Bahr Eldeen who helped me a lot in susceptibility testing and DNA extraction. Khansa Esam who helped me in sample collection and culture technique, Hyam Abdelrahman and Aymen Jalal Eldeen who assisted in the software. I would like to thank the lab staff of Sudan University of Sciences and Technology for giving me the chance to perform the PCR assay in their laboratory and assisting me.\n\n\nReferences\n\nJeon JH, Lee JH, Lee JJ, et al.: Structural basis for carbapenem-hydrolyzing mechanisms of carbapenemases conferring antibiotic resistance. Int J Mol Sci. 2015; 16(5): 9654–9692. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMate PH, Devi KS, Devi KM, et al.: Prevalence of carbapenem resistance among Gram-negative bacteria in a tertiary care hospital in north-east India. IOSR Journal of Dental and Medical Sciences. 2014; 13(12): 56–60. Publisher Full Text\n\nQueenan AM, Bush K: Carbapenemases: the versatile beta-lactamases. Clin Microbiol Rev. 2007; 20(3): 440–58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSahuquillo-Arce JM, Hernández-Cabezas A, Yarad-Awad F, et al.: Carbapenemases: a worldwide threat to antimicrobial therapy. World J Pharmacol. 2015; 4(1): 75–95. Publisher Full Text\n\nDjahmi N, Dunyach-Remy C, Pantel A, et al.: Epidemiology of carbapenemase-producing Enterobacteriaceae and Acinetobacter baumannii in Mediterranean countries. Biomed Res Int. 2014; 2014: 305784. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEFSA Panel on Biological Hazards: Scientific opinion on carbapenem resistance in food animal ecosystems. European food safety authority journal. 2013; 11(12): 3501. Publisher Full Text\n\nManenzhe RI, Zar JH, Nicol MP, et al.: The spread of carbapenemase-producing bacteria in Africa: a systematic review. J Antimicrob Chemother. 2014; 70(1): 23–40. PubMed Abstract | Publisher Full Text\n\nSatir SB, Elkhalifa AI, Ali MA, et al.: Detection of Carbepenem Resistance Genes among Selected Gram Negative Bacteria Isolated from Patients in -Khartoum State, Sudan. Clin Microbiol. An open access journal. 2016; 5(6): 266. Publisher Full Text\n\nCollee JG, Fraser AG, Marmion BP, et al.: Practical medical microbiology. 14thed. New Delhi (India): Elsevier, 2006.\n\nSolank R, Vanjari L, Subramanian S, et al.: Comparative Evaluation of Multiplex PCR and Routine Laboratory Phenotypic Methods for Detection of Carbapenemases among Gram Negative Bacilli. J Clin Diagn Res. 2014; 8(12): DC23–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmjad A, Mirza Ia, Abbasi S, et al.: Modified hodge test: a simple and effective test for detection of carbapenemase production. Iran J Microbiol. 2011; 3(4): 189–193. PubMed Abstract | Free Full Text\n\nPandurangan S, BegumEsak S, Narayanasamy A: Phenotypic detection methods of carbapenemase production in Enterobacteriaceae. Int J Curr Microbiol App Sci. 2015; 4(6): 547–52. Reference Source\n\nHayajneh WA, Hajj A, Hulliel F, et al.: Susceptibility trends and molecular characterization of Gram-negative bacilli associated with urinary tract and intra-abdominal infections in Jordan and Lebanon: SMART 2011–2013. Int J Infect Dis. 2015; 35: 56–61. PubMed Abstract | Publisher Full Text\n\nMushi MF, Mshana SE, Imirzalioglu C, et al.: Carbapenemase genes among multidrug resistant gram negative clinical isolates from a tertiary hospital in Mwanza, Tanzania. Biomed Res Int. 2014; 2014: 303104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMataseje LF, Bryce E, Roscoe D, et al.: Carbapenem-resistant Gram-negative bacilli in Canada 2009-10: results from the Canadian Nosocomial Infection Surveillance Program (CNISP). J Antimicrob Chemother. 2012; 67(6): 1359–67. PubMed Abstract | Publisher Full Text\n\nGladstone P, Rajendran P, Brahmadathan KN: Incidence of carbapenem resistant nonfermenting gram negative bacilli from patients with respiratory infections in the intensive care units. Indian J Med Microbiol. 2005; 23(3): 189–191. PubMed Abstract | Publisher Full Text\n\nElhag KM: Diversification of antibiotics as a means to control antimicrobial resistance and improve treatment options in Sudan. Sudan Med J. 2013; 49(3): 128–35. Publisher Full Text\n\nMemish ZA, Assiri A, Almasri M, et al.: Molecular characterization of carbapenemase production among Gram-negative bacteria in Saudi Arabia. Microb Drug Resist. 2015; 21(3): 307–314. PubMed Abstract | Publisher Full Text\n\nNiumsup PR, Boonkerd N, Tansawai U, et al.: Carbapenem-resistant Acinetobacter baumannii producing OXA-23 in Thailand. Jpn J Infect Dis. 2009; 62(2): 152–4. PubMed Abstract\n\nFattouh M, El-din AN: Emergence of carbapenem-resistant Acinetobacter baumannii in the intensive care unit in Sohag university hospital, Egypt. Int J Curr Microbiol App Sci. 2014; 3(4): 732–744. Reference Source\n\nDahab RA, Ibrahim AM, Altayb HN: Dataset 1 in: Phenotypic and Genotypic Detection of Carbapenemase Enzymes Producing Gram-negative Bacilli Isolated from Patients in Khartoum State. F1000Research. 2017. Data Source\n\nDahab RA, Ibrahim AM, Altayb HN: Dataset 2 in: Phenotypic and Genotypic Detection of Carbapenemase Enzymes Producing Gram-negative Bacilli Isolated from Patients in Khartoum State. F1000Research. 2017. Data Source"
}
|
[
{
"id": "78127",
"date": "16 Feb 2021",
"name": "Elsa De La Cadena",
"expertise": [
"Reviewer Expertise Bacterial resistance in Gram negative."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 investigation, which provides relevant information. But it needs to have a better methodological design.\nEnterobacteriaceaes are currently called Enterobacterales. Nowadays, Enterobacteriaceaes is a limited group of species within the Enterobacterales. It is suggested to change the term.\nThey use Modified Hodge Test. Currently, THM is not the recommended method to identify carbapenemases. It has very low sensitivity for metalloenzymes. Should not be performed on Pseudomonas\nTo identify resistance to carbapenems it is better to use ertapenem in Enterobacterales. It is the first carbapenem to rise when there is a resistance mechanism, and it could still be sensitive or intermediate to meropenem.\nThe ideal would have been to search for NDM, VIM and KPC that have been previously described in African countries and could be the cause of resistance. Due to this, it is wrong to speak of the percentage of carbapenemases if the presence of other carbapenemases is not ruled out.\nThe discussion needs to be improved, it is a bit repetitive.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "7592",
"date": "24 Dec 2021",
"name": "Reem Dahab",
"role": "Author Response",
"response": "Greetings, I am the author of this paper Reem Dahab. The research was done in 2016. The MHT was from the most useful method, easiest and cheapest one. So this was one of my goals which concluded that It is simple to detect many carbapenemases, but it can't detect all. This conclusion is written on the thesis. I couldn't write detailed method and conclusion here and I couldn't write my recommendations as well because at the time of writing I had to adhere to a word count. I know its poor sensitivity regarding some families, but my research is about just 2 genes according to my budget which were confirmed by PCR. Regarding the ertapenem, I read many research articles that compare the best means to check the carbapenem resistance, all said meropenem and this is why I used it according to previous studies. You said I should engage with research on VIM, NDM, KPC which are previously described in Africa. I already saw the previous studies before I chose the genes, I wish to do it all but lacked funding. So I chose the most abundant resistance genes in Africa at that time. I didn't include the KPC because I already tried it and I had a problem with the primer so I excluded it. I said the percentage of carbapenemases because those were the most abundant ones in Africa, as well as any other percentage written were related to the 2 genes detected. I will improve my discussion in my new papers with my respect. There will be a new version soon, as the word count is no longer a matter this will include more details adhering to the journal policies. Best Regards, Reem Dahad"
}
]
},
{
"id": "78128",
"date": "05 Mar 2021",
"name": "Tanzina Nusrat",
"expertise": [
"Reviewer Expertise Microbiology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhat is the study population?\n\nHow many gram positive bacteria did you find, how did you separate them? Why is there no method for that?\n\nThere is no adds ratio, please add the odds ratio?\n\nWhy is the infection rate so high? Briefly describe the study limitation.\n\nWhy didn't you chose the other genes?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "7591",
"date": "24 Dec 2021",
"name": "Reem Dahab",
"role": "Author Response",
"response": "Greetings, I am Reem Dahab the author of this paper. I am very glad that you reviewed my paper. I would like to answer your questions. What is the study population? The study population are the patients attended to different hospitals in Khartoum state having bacterial infections in the period from February to August 2016. How many gram positive bacteria did you find, how did you separate them? Why is there no method for that? I didn't mention them because my research concerns gram negatives, the method mentioned above in detail is for isolation of gram negative bacilli from gram positive ones. Culture media were used to identify gram negative from gram positive ones according to the clinical specimen type for primary culture then gram staining was performed to differentiate between gram negatives and gram positives. Chromogenic agar culture was performed. Subculturing to purify gram negative isolates were done in blood agar and MacConkey agar media, then , biochemical tests were used for differentiation. At this time of submission I was limited to a word count for this paper, but there is a whole thesis written in detail especially methods and results, from it I wrote this small paper. There is no adds ratio, please add the odds ratio? Can you clarify this questions because the statistics were done by specialist. Why is the infection rate so high? Briefly describe the study limitation. The sample size is small, due to the budget limitations because as the research was self-funded and I was a masters degree student. This paper is taken from a complimentary thesis. The infection rate is very high in our country Sudan as no preventive methods are followed as well as the over the counter use of antibiotics. There are many causes included here, but unfortunately this is the fact, regarding a large amount of research in Sudan. Why didn't you chose the other genes? I wanted to cover all genes to find if there is an increased rate from the previous studies in our country and Africa, but according to my budget it was very expensive so I reviewed the most abundant genes in Africa and in our region. I checked each carbapenemase family the most important for me as per previous studies. I will improve my discussion in my new papers with my respect. There will be a new version soon, as the word count is no longer a matter this will include more details adhering to the journal policies. With my respect, I am grateful. Best Regards, Reem Dahab"
}
]
}
] | 1
|
https://f1000research.com/articles/6-1656
|
https://f1000research.com/articles/6-1650/v1
|
06 Sep 17
|
{
"type": "Research Article",
"title": "Treatment of chronic telogen effluvium with oral minoxidil: A retrospective study",
"authors": [
"Eshini Perera",
"Rodney Sinclair",
"Eshini Perera"
],
"abstract": "Background: Chronic telogen effluvium (CTE) may be primary or secondary to various causes, including drug reaction, nutritional deficiency and female pattern hair loss (FPHL). Oral minoxidil stimulates hair growth, and topical minoxidil is used in the treatment of FPHL and male androgenetic alopecia. minoxidil has not been used to treat CTE. This study aimed to assess the treatment of CTE with once daily oral minoxidil. Methods: Women with a diagnosis of CTE based on >6 month history of increased telogen hair shedding, no visible mid frontal scalp hair loss (Sinclair stage 1) and no hair follicle miniaturization on scalp biopsy were treated with once daily oral minoxidil. Hair shedding scores (HSS) at baseline, 6 and 12 months were analysed using the Wilcoxon rank sum test for pair-wise comparisons. Results: Thirty-six women were treated with oral minoxidil (range, 0.25-2.5 mg) daily for 6 months. Mean age was 46.9 years (range 20-83), HSS at baseline was 5.64, and duration of diagnosis was 6.55 years (range 1-27). There was a reduction in mean HSS scores from baseline to 6 months of 1.7 (p<0.001) and baseline to 12 months of 2.58 (p<0.001). Five women who described trichodynia at baseline, noted improvement or resolution within 3 months. Mean change in blood pressure was minus 0.5 mmHg systolic and plus 2.1 mmHg diastolic. Two patients developed transient postural dizziness that resolved with continued treatment. One patient developed ankle oedema. Thirteen women developed facial hypertrichosis. For 6 patients this was mild and did not require treatment; 4 had waxing of their upper lip or forehead; 3 had laser hair removal. No patients developed any haematological abnormality. All 36 women completed 12 months of treatment. Conclusions: Once daily oral minoxidil appears to reduce hair shedding in CTE. Placebo controlled studies are recommended to further assess this response.",
"keywords": [
"androgenic",
"androgenetic",
"alopecia",
"hair loss",
"shedding",
"baldness"
],
"content": "Introduction\n\nHair shedding severity can be scored using a visual analogue scale1. For women with long hair, a shedding severity of 1, 2 or 3 is considered normal; severity 4 is borderline; while a shedding severity of 5 or 6 is excessive (Figure 1). Visual inspection of shed hair bulbs will determine if the hairs shed are being lost during the anagen phase of the hair cycle or the telogen phase. This examination of the bulb provides an important clue to the aetiology of the hair loss2.\n\nTelogen effluvium is a non-scarring alopecia characterised by excessive shedding of telogen club hair diffusely from the scalp. It generally begins 8–12 weeks after a triggering event, such as pregnancy, major illness or complicated surgery, and resolves within 3–6 months. Once resolved, self-limiting telogen effluvium can be retrospectively diagnosed as acute telogen effluvium3. Telogen shedding that persists beyond 6 months is called chronic telogen effluvium (CTE)4. CTE may be primary or secondary to a range of triggers, including androgenetic alopecia (AGA), nutritional deficiency, endocrinopathy, connective tissue disease or drug induced5. The aetiology of primary CTE is unknown4. Mathematical modelling of CTE implicates a reduction in anagen duration variance in the pathogenesis6 and not due to an overall reduction in anagen duration, which is a feature of AGA7. The natural history is for continued hair shedding over many years. There may be some seasonal variation in the intensity of hair shedding8. Long-term follow up studies of women with primary CTE9 and histomorphometric and immunohistochemical examination of scalp biopsies in patients with both female pattern hair loss (FPHL) and CTE have confirmed that primary CTE is not a prodrome to AGA10. Nevertheless some women with longstanding CTE will develop con-incidental AGA as they age11.\n\nScores vary between 1 (minimal shedding) to 6 (copious hair shedding)12.\n\nPrimary CTE most commonly occurs suddenly in females between 30 and 50 years of age. Additional clinical features commonly seen in primary CTE include bi-temporal recession of the anterior hairline, a reduction in the thickness of their ponytail diameter4 and trichodynia13. Widening of the central part line suggests AGA, and is not a feature of primary CTE (Figure 2).\n\nOther than identification and treatment of a triggering event, such as hypothyroidism, there is no known treatment for acute or chronic TE14. Treatments commonly used for AGA, such as finasteride, cyproterone acetate, spironolactone and flutamide, do not work in TE5.\n\nTopical minoxidil has been used for over 30 years to treat a variety of hair loss conditions, including AGA. For some years in our clinic we have also used minoxidil orally to treat men and women with androgenetic hair loss, who are either intolerant to topical minoxidil or do not like the look or feel or minoxidil in their hair15. As Minoxidil is only available in Australia as a 10 mg tablet (Loniten), we compounded minoxidil extemporaneously in various doses ranging from 0.25mg to 2.5mg. More recently, a case report demonstrated that low dose oral minoxidil was useful in treating a female with chemotherapy-induced alopecia with oral minoxidil16. There have been no reports or studies that examine the use of oral minoxidil in CTE. Our understanding of the pathogenesis of CTE and the mechanism of action of minoxidil on hair growth suggest it should work in CTE patients, and we have found that women with AGA who are unresponsive to topical minoxidil often respond to oral minoxidil at our clinic. This retrospective study examines the use of low-dose oral minoxidil in women diagnosed with CTE.\n\n\nMethods\n\nExamination of patient records for this retrospective chart review was conducted at Sinclair Dermatology practice in Melbourne, Australia. As this was a retrospective review of patient charts, it did not require prior approval from our local institutional ethics committee. The data are the property of Sinclair Dermatology and access was approved by the company. Records between Jan 2012 and Oct 2015 were extracted. Inclusion criteria included female patients with a hair shedding score (HSS)12 of 4–6 without visible mid frontal scalp hair loss (Sinclair stage 1)17 and no hair follicle miniaturization on scalp biopsy. Patients were excluded if they were using other treatments for hair loss whilst taking oral minoxidil. Data pertaining to dosage of oral minoxidil, previous treatments, including topical minoxidil use, blood pressure, side effects, hypertrichosis and trichodynia were extracted from the records. Patient subjective responses were based on a HSS score (Figure 1) and were recorded at each consultation. The HSS scores prior to starting oral minoxidil, and scores at 6 and 12 months were extracted for analysis.\n\nData were analysed using Matlab R2014b statistical software. Difference in HSS at baseline, 6 and 12 months were analysed using the Wilcoxon rank sum test for pair-wise comparisons. Differences in blood pressure at baseline and 6 months were also analysed using Wilcoxon rank sum test. Relationship between outcomes (HSS score at 6 and 12 months) and individual patient specific variables, including age, topical minoxidil, duration of disease, dosage of minoxidil and HSS score at baseline were analysed using a generalised linear regression model.\n\n\nResults\n\nThirty-six patients with CTE, who were prescribed oral minoxidil, were included in this analysis. The mean age was 46.9 years (range 20–83 years) and the dosage of oral minoxidil used varied between 0.25 and 2.5 mg with most patients being administered 1 mg. Mean baseline HSS was 5.64. Mean HSS scores improved at 6 and 12 months at 3.9 and 3.05, respectively (Figure 3). There was a reduction in mean HSS scores from baseline to 6 months of 1.7 (p<0.001) and a reduction in mean HSS scores from baseline to 12 months of 2.58 (p<0.001). Similarly, a mean reduction of 0.89 in HSS scores was noted between 6 months and 12 months (p =0.003). Correlation between the duration of disease and previous topical minoxidil with HSS scores at 6 months (R2 < 0.22) and 12 months (R2 < 0.11) were weak. Eleven patients had previously used 5% topical minoxidil. Of these patients the mean change in the HSS score was higher, although not statistically significant compared to patients who did not use topical minoxidil: mean reductions in HSS score for patients who had previously used and not used topical minoxidil were 2 and 1.56 (p= 0.22) at six months and 3.18 and 2.32 at 12 months (p=0.11). The HSS score improved in 31 patients after 6 months; in 4 patients the HSS score remained the same; and in 1 patient the score increased at the 6-month mark before improving, compared to baseline, at the 12-month mark. After 12 months the HSS score remained equal or improved from baseline in all but 3 patients.\n\nThere were no significant differences between blood pressure at baseline and 6 months (p>0.05). The lowest blood pressure recorded was 90/70. All other patients in the study had a blood pressure above 100/70. Mean change in blood pressure was minus 0.5 mmHg systolic and plus 2.1 mmHg diastolic. Two patients developed transient postural dizziness that resolved with continued treatment. One patient developed ankle oedema. Fourteen women developed facial hypertrichosis. For 6 women this was mild and did not require treatment; 4 patients waxed their upper lip or forehead; and 3 patients had laser hair removal. No patients developed any blood test abnormality. Five women described trichodynia at baseline and all noted improvement or resolution within 3 months.\n\n\nDiscussion\n\nCTE is the persistent shedding of telogen hairs diffusely from the scalp, which is associated with bitemporal recession without widening of the central part. CTE was described as a primary idiopathic disease entity by Whiting in 19964. The prognosis for women with CTE is uncertain and women do not go bald8. Rather, the disease has a fluctuating course and the condition may continue for many years. The goal of this retrospective study were to review the use of oral minoxidil in CTE with respect to the response in HSS score and safety.\n\nMinoxidil is a piperidinopyrimidine derivative and a potent arteriolar vasodilator5. Studies examining oral minoxidil demonstrated more rapid and extensive hair growth, compared to topical treatment, in patients with alopecia areata12. A recent study examined the use of oral minoxidil in combination with spironolactone in 100 women with FPHL15. The study demonstrated a reduction of hair loss and hair shedding at 6 and 12 months. Dosages of 0.25 mg of oral minoxidil were used in that study.\n\nUse of oral minoxidil for hair loss has not been extensively reported in the literature to date. Currently, the only suggested treatment for CTE is topical Minoxidil, however results are variable and often disappointing. One study demonstrated an improvement in 55.2% of patients studied, using 5% topical minoxidil for men and 5% topical minoxidil with 50 mg of cyproterone acetate for women14. 25.2% of patients had a moderate response to treatment14.\n\nAll the patients in this study demonstrated an improvement at either the 6 month or 12 month mark, with 33 patients improved from baseline at the 12 month mark.\n\n\nAbbreviations\n\nFPHL: female pattern hair loss, AGA: androgenetic alopecia, CTE: chronic telogen effluvium, HSS: Hair shedding score.\n\n\nData availability\n\nDataset 1: Raw data collected during the study. DOI, 10.5256/f1000research.11775.d17641818.",
"appendix": "Competing interests\n\n\n\nRodney Sinclair is director of Samson Clinical Pty Ltd, owner of PCT Patent Application PCT/AU2015/050682, Entitled: Detection and treatment of excessive hair shedding. The invention relates to a method of treating telogen effluvium in a subject by administering to a subject an oral dose of minoxidil.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSinclair R: Hair shedding in women: how much is too much? Br J Dermatol. 2015; 173(3): 846–8. PubMed Abstract | Publisher Full Text\n\nSinclair R: Diffuse hair loss. Int J Dermatol. 1999; 38 Suppl 1: 8–18. PubMed Abstract | Publisher Full Text\n\nHarrison S, Sinclair R: Telogen effluvium. Clin Exp Dermatol. 2002; 27(5): 389–5. PubMed Abstract | Publisher Full Text\n\nWhiting DA: Chronic telogen effluvium: increased scalp hair shedding in middle-aged women. J Am Acad Dermatol. 1996; 35(6): 899–906. PubMed Abstract | Publisher Full Text\n\nMessenger A, De Berker D, Sinclair R: Disorders of Hair. In: Burns, Breathnach, Cox and Griffiths. Rook’s Textbook of Dermatology. Eighth Edition. Blackwell Publishing. Oxford. 2010; 63.1–63.100. Publisher Full Text\n\nGilmore S, Sinclair R: Chronic telogen effluvium is due to a reduction in the variance of anagen duration. Australas J Dermatol. 2010; 51(3): 163–7. PubMed Abstract | Publisher Full Text\n\nSinclair R: Male pattern androgenetic alopecia. BMJ. 1998; 317(7162): 865–869. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSinclair R: Chronic telogen effluvium: a study of 5 patients over 7 years. J Am Acad Dermatol. 2005; 52(2 Suppl 1): 12–6. PubMed Abstract | Publisher Full Text\n\nBittencourt C, Ferraro DA, Soares TC, et al.: Chronic telogen effluvium and female pattern hair loss are separate and distinct forms of alopecia: a histomorphometric and immunohistochemical analysis. Clin Exp Dermatol. 2014; 39(8): 868–73. PubMed Abstract | Publisher Full Text\n\nWhiting DA: Chronic telogen effluvium. Dermatol Clin. 1996; 14(4): 723–31. PubMed Abstract | Publisher Full Text\n\nSinclair R: Chronic telogen effluvium or early androgenetic alopecia? Int J Dermatol. 2004; 43(11): 842–843. PubMed Abstract | Publisher Full Text\n\nFiedler-Weiss VC, Rumsfield J, Buys CM, et al.: Evaluation of oral minoxidil in the treatment of alopecia areata. Arch Dermatol. 1987; 123(11): 1488–90. PubMed Abstract | Publisher Full Text\n\nKivanç-Altunay I, Savaş C, Gökdemir G, et al.: The presence of trichodynia in patients with telogen effluvium and androgenetic alopecia. Int Dermatol. 2003; 42(9): 691–693. PubMed Abstract | Publisher Full Text\n\nGarcia-Hernandez MJ, Camacho FM: Chronic telogen effluvium: incidence, clinical and biochemical features, and treatment. Arch Dermatol. 1999; 135(9): 1123–4. PubMed Abstract\n\nSinclair R: Treatment of female pattern hair loss with oral minoxidil. Austral J Dermatol. 2016; 57(Suppl 1): 27. Reference Source\n\nYang X, Thai KE: Treatment of permanent chemotherapy-induced alopecia with low dose oral minoxidil. Australas J Dermatol. 2015. PubMed Abstract | Publisher Full Text\n\nSinclair R, Patel M, Dawson TL Jr, et al.: Hair loss in women: medical and cosmetic approaches to increase scalp hair fullness. Br J Dermatol. 2011; 165(Suppl 3): 12–18. PubMed Abstract | Publisher Full Text\n\nSinclair R, Perera E: Dataset 1 in: Treatment of Chronic Telogen Effluvium with Oral Minoxidil. F1000Research. 2017. Data Source"
}
|
[
{
"id": "25750",
"date": "21 Sep 2017",
"name": "Victoria M.L. Jolliffe",
"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 piece of work which contributes to the management of an one challenging but common clinical scenario and additional therapeutic interventions which may be of benefit are to be applauded.\nThe study is well designed and results robust. There are a couple of further issues which I would like to see addressed or clarified in the text.\nAddress the limitations of a retrospective review.\n\nDid the patients have blood work up to exclude any coincidental compounding factors causing effluvium e.g. thyroid/ vit D deficiencies? It would be helpful to know this and indeed see whether is this was the case there was any effect on treatment response in the presence of abnormal blood workup. If they did not have blood work up this should be clearly stated.\n\nI assume all the patients included had undergone a scalp biopsy.\n\nThe dose used was not standardised. This should be mentioned again in the discussion/ limitations of this piece of work and it would be good to know whether side effects - or indeed efficacy - were related to the higher doses used or no difference found.\nThese are small details on an otherwise really valuable piece of work which will be be very valuable to those practising in the Hair Disorders clinical 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? 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": "26886",
"date": "06 Nov 2017",
"name": "Alex J. Chamberlain",
"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 useful study, limited somewhat by the retrospective nature and patient numbers. CTE is a challenging condition to manage and this offers another valuable and seemingly safe treatment option.\n\nI would be interested to know if the authors have a dose preference given their experience for treating CTE. Do they wish to speculate on the mechanism of action of oral minoxidil in hair loss?\n\nIt may be nice to see some of the scalp photographs of the treatment subjects.\n\nDoes the visual shedding scale measure DAILY shedding?\n\nClearly a larger study with a standardised dose and a control arm would strengthen the recommendation for the treatment.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1650
|
https://f1000research.com/articles/6-1285/v1
|
31 Jul 17
|
{
"type": "Research Article",
"title": "Exploring differential evolution for inverse QSAR analysis",
"authors": [
"Tomoyuki Miyao",
"Kimito Funatsu",
"Jürgen Bajorath",
"Tomoyuki Miyao",
"Kimito Funatsu"
],
"abstract": "Inverse quantitative structure-activity relationship (QSAR) modeling encompasses the generation of compound structures from values of descriptors corresponding to high activity predicted with a given QSAR model. Structure generation proceeds from descriptor coordinates optimized for activity prediction. Herein, we concentrate on the first phase of the inverse QSAR process and introduce a new methodology for coordinate optimization, termed differential evolution (DE), that originated from computer science and engineering. Using simulation and compound activity data, we demonstrate that DE in combination with support vector regression (SVR) yields effective and robust predictions of optimized coordinates satisfying model constraints and requirements. For different compound activity classes, optimized coordinates are obtained that exclusively map to regions of high activity in feature space, represent novel positions for structure generation, and are chemically meaningful.",
"keywords": [
"Chemical space",
"active compounds",
"differential evolution",
"support vector regression",
"virtual screening",
"inverse QSAR"
],
"content": "Introduction\n\nInverse quantitative structure-activity relationship (QSAR) analysis aims to identify values of descriptors used to generate a QSAR model that corresponds to high activity, and build structures of active compounds from these values1–4. The inverse QSAR process is challenging since numerical signatures of activity, if they can be determined, must be re-translated into viable chemical structures and active compounds, a task falling into the area of de novo compound design5–7. A predominant approach to inverse QSAR is the use of multiple linear regression (MLR) models to construct chemical graphs that correspond to an MLR equation1–4. Given this equation, a desired y (activity) value constrains relationships between descriptor settings. These constraints make it possible to derive vertex degree or edge sequences, from which chemical graphs might be constructed. For instance, specialized descriptors have been introduced for inverse QSAR on the basis of MLR equations and algorithms for constructing chemical graphs from these descriptors8–11. So far only few inverse QSAR studies have employed methods other than MLR. For example, it was attempted to construct chemical graphs from the centroid of activity of a set of compounds in Hilbert space defined by a kernel function12. In this case, a pre-image approximation algorithm was used to obtain coordinates in descriptor space and construct chemical graphs from these descriptor coordinates. Alternatively, inverse QSAR was divided into a two-stage process by separating the derivation of preferred descriptor values for a desired activity from the chemical graph construction phase13–15. Descriptor information corresponding to a given y value was represented via probability density functions, and regression analysis was performed using Gaussian mixture models in combination with cluster-wise MLR14. Subsequently, chemical graphs satisfying a set of descriptor values, or ranges of descriptor values, were generated by assembling ring systems and atom fragments with monotonically changing descriptors14. Following this approach, descriptor values must increase when adding an atom, ring system, or other structural fragment to a growing chemical graph. Applying Gaussian mixture models and cluster-wise MLR makes it possible to focus on the applicability domain14,15 of the underlying models.\n\nThe two-stage inverse QSAR process is conceptually based on an important premise adopted from conventional (forward) QSAR, i.e., the higher a predicted activity value is, the more desirable a chemical structure becomes. In two-stage inverse QSAR, this conjecture challenges the descriptor value generation phase because value combinations are ultimately desired that correspond to higher predicted activity than exhibited by any currently available training or test compound. In other words, descriptor settings should be optimized for predicted activity. For this purpose, the use of Gaussian mixture models and cluster-wise MLR left considerable room for improvement, due to its multi-parametric nature and tendency of overfitting if training data were organized into large number of clusters14.\n\nIn this work, the descriptor optimization challenge of two-stage inverse QSAR has been specifically addressed. We emphasize that the chemical graph construction phase of inverse QSAR is not subject of this work and beyond its scope. Rather, our focal point has been the development of a new methodology for optimizing descriptor settings with respect to higher than observed compound activity, as a prerequisite for candidate structure generation. Therefore, an evolutionary approach is introduced to identify descriptor coordinates that correspond to the highest predicted activity within the applicability domain of a given QSAR model. The methodology and results of proof-of-concept studies are presented in the following.\n\n\nMethods\n\nInverse QSAR depends on the derivation of descriptor coordinates for a given model and data set. The goal of the methodology presented herein is finding desirable coordinates in a pre-defined descriptor space (x space) on the basis of a regression function f(x) representing a QSAR model. Confining the search to the applicability domain (AD) of the model translates this task into a constrained optimization problem (COP). The concept of the optimization is illustrated in Figure 1. Newly derived coordinates should be more desirable with respect to pre-defined evaluation criteria than any other data point used to construct the regression model. Accordingly, COP is formulated as follows:\n\nA regression function f(x) fits training data to determine new coordinates in descriptor space. Optimized coordinates based on f(x) fall inside the training data range but yield a higher y value than any other data point.\n\nMinimize f(x)\n\nwhere x ∈ Rd, f: Rd → R is the function to be optimized, gj: Rd → R is the j-th inequality function, and hj: Rd → R is the j-th equality function. The i-th component of x: xi falls into the range [li, ui].\n\nFor the purpose of our analysis, the following assignments are made:\n\nx: descriptors;\n\n–f(x): QSAR model;\n\n–g1(x): AD model;\n\ngk(x) (k = 2, …, j), h(x): constraints for descriptors;\n\nli, ui: lower and upper bounds of i-th descriptor.\n\nConstraints are applied to descriptors to ensure meaningful value ranges. For example, if the ‘number of heavy atoms’ (xp) and ‘number of hydrogen bond acceptors’ (xq) are selected descriptors, a value of five for the former and six for the latter would be impossible for any given data point (compound). Therefore, in order to prevent such settings, an inequality constraint is required and applied: xq – xp ≤ 0.\n\nFor addressing COP, the differential evolution (DE) algorithm originally introduced by Stone and Price16 is investigated herein, which has so far not been considered in inverse QSAR. However, given the conceptual simplicity and computational efficiency of DE, the algorithm has been successfully applied to solve optimization problems in other areas of science and engineering, for example, in scheduling of flow shops17. In addition, for deriving a COP solution efficiently, ε differential evolution (εDE) was introduced by Takahama et al. as an extension of DE18, illustrated in Figure 2. A candidate vector v for next generation (also called mutant vector) is derived on the basis of three randomly selected vectors:\n\n\n\nThe steps involved in evolutionary optimization are outlined. First, a candidate v is obtained from three randomly selected individuals by a differential operation. Second, a crossover operation is applied to an individual xi and the candidate. Third, the evaluation step involves ε level comparison of v and xi and results in the next individual.\n\nwhere xr1, xr2, xr3 are different vectors from the current generation and F represents a scale parameter for the difference vector. If the i-th component of v: vi falls outside the range [li, ui], v is updated as follows:4\n\n\n\nAn exponential crossover operation with probability-based crossover points is applied to v (the probability is called CR). Either xi or v is selected as xi+1, the individual for the next generation, following ε level comparison of the corresponding vectors.\n\nFor prioritizing candidates, given constraints and the optimized function are taken into account. The constraint violation Φ(x) is defined as follows:\n\n\n\nwhere Φ(x) represents the degree of violation, with p set to one. The ε level comparison determines the order between two sets of pair (f(x), Φ(x)):\n\nwhere t represents a generation in DE. As a decreasing function of t, ε determines the tolerance of constraint violation and ε(t) is determined as follows:16\n\n\n\nwhere xθ is the top θ-th individual, Tc determines the generation in which ε(t) becomes zero, and cp the convergence speed. During the optimization, Φ(x) gradually outweighs f(x). In the initial stages of the optimization ε(t) settings enable the selection of diverse candidates but convergence of the algorithm is determined by Φ(x) becoming zero. Accordingly, Tc was set to one herein.\n\nFor εDE optimization, any regression function can be employed. In this study, support vector regression (SVR)19 with ν parameter was applied and the AD was defined by one-class support vector machine (OCSVM)20 classification with ν parameter. This parameter ranges from zero to one and defines the upper bound of the fraction of margin error and lower bound of the fraction of support vectors. AD consists of regions where the output of OCSVM is greater than or equal to zero. For both SVR and OCSVM, the radial basis function (RBF) kernel: k(xi, xj) = (−γ||xi − xj||2) was used. A hyper parameter set {C, ν, γ} for ν-SVR was determined by cross validation of training data on the basis of Q2. For OCSVM model construction, γ was set to maximize the variance of the Gram matrix consisting of the kernel function of the training data21 and ν was set to 0.01.\n\nData points on a (x1, x2) plane were randomly generated for x1: [-2 3], x2: [-1, 4] to yield 50 training and 20 test instances. The corresponding y values were calculated using Mishra’s bird function (https://mpra.ub.uni-muenchen.de/2718/) adding a Gaussian error with a mean of zero and variance of one, defined as:\n\n\n\nThree independent trials were carried out with different random number generators. Training and test data sets were plotted on the output domain of the bird function with color-coded true y values (Figure 3).\n\nIn three independent trials, simulation data sets were generated. For each trial, training (black dots) and test (blue squares) data are shown with true y values produced by the bird function f(x1, x2).\n\nFrom ChEMBL22 (version 22), compound data sets were selected using the following criteria: Maximal assay confidence score of ‘9’, interaction relationship type ‘D’(direct), activity standard unit ‘nM’, activity standard type ‘Ki’, and activity standard relation ‘=’. When multiple Ki values were available for a compound, their geometric mean was calculated to yield its final potency value, provided all measurements fell into the same order of magnitude (otherwise, the compound was discarded). In-house implementation of substructure filters for pan assay interference compounds (PAINS)23 and other reactive molecules were applied to eliminate compounds with potential chemical liabilities. Filtering was not critical for modeling, but active compounds with sound chemical structures were desired. From qualifying data sets, nine activity classes were randomly selected, as summarized on Table 1.\n\nNine compound activity classes were taken from ChEMBL (version 22). For each activity class, the target ID (TID), number of compounds (CPDs) for which descriptor values were obtained, and number of descriptors following variable selection are reported.\n\nFor each compound, 45 descriptors were initially calculated using RDKit (http://www.rdkit.org). These descriptors included constitutional descriptors (e.g., MW, number of rings, number of rotatable bonds), topological descriptors (e.g., Chi and Kier indices24) and partial charge descriptors based on chemical graph’s topology (i.e., maximum of Gasteiger/Marsali partial charges25). From correlated pairs of descriptors exceeding a correlation coefficient of 0.9, only one was chosen. For each activity class, the final number of descriptors (variables) is reported in Table 1. Compounds from each class were randomly divided into equally sized training and test data sets.\n\nTo test the ability of virtual screening (VS) to identify new active compounds from optimized coordinates, ChEMBL (version 22) was used as a screening source. From 1,414,176 unique compounds passing the substructure filters, training molecules used for modeling of each activity class were removed. All remaining ChEMBL compounds provided a large screening source for VS. For screening compounds, descriptors were calculated as described above and the compounds falling inside the AD of each class-specific model were preselected. Active compounds from each activity class not used for training represented true-positive test instances, regardless of their potency values. The calculation of descriptors for more than 1.4 million screening compounds was computationally demanding and exceeded the requirements for coordinate optimization.\n\nFor ChEMBL screening compounds including test instances, two VS ranking were generated. First, Euclidean distances to optimized coordinates were calculated. In this case, compound potency was not considered for ranking. Second, pKi values were predicted for all screening compounds using the class-specific SVR models. The latter calculations were carried out to determine if true positives were highly ranked on the basis of activity predictions. The area under the receiver operating characteristic curves (AUC) was calculated as an evaluation criterion.\n\n\nAnalysis protocol\n\nTwo proof-of-concept studies were carried out, one using simulation data, the other compounds and their activities. For simulation data, AD and regression models were constructed with the training data from each trial. Training data range scaling within the interval [-1,1] was applied prior to model building. For the SVR models, preferred parameter settings were determined using 10-fold cross validation. Coordinate optimization was carried using individual training data points. Optimized coordinates were evaluated on the basis of true y and maximal training data y values.\n\nThe same protocol for coordinate optimization was applied to each compound activity class. Furthermore, for hyper-parameter optimization of SVR, five-fold cross validation was carried out. For εDE, predicted pKi values falling into the AD of each model were used to ensure that optimized coordinates were proximal to compound coordinates, as assessed by distance calculations. Furthermore, optimized coordinates were projected on principal component analysis (PCA) maps of the x space formed by the first and second PC.\n\nThe following εDE parameter settings were applied: Number of iterations, 1,000 and 10,000 for simulation and compounds data sets, respectively; F, 0.5; Tc,1; p, 1; CR, 0.9 for all data sets. An initial population was obtained using 50 vectors of training instances for simulation data and 511 to 1,193 vectors for training compounds, depending on the size of the compound data sets.\n\nFinally, the ability of distance- and SVR-based VS to predict new active compounds was analyzed. Although de novo structure generation was beyond the scope of our investigation, VS might be considered as an alternative way to identify novel active compounds, which was thus examined in the context of our study.\n\n\nImplementation\n\nAll SVR models and ADs were constructed with Scikit-learn26 0.18.1 using Python. εDE was implemented in C++. Descriptors were calculated using RDKit interfaced with Python.\n\nAll selected compound entries were standardized by removal of ions and solvent molecules and structure regularization, according to the OEChem toolkit (v1.7.7; OpenEye Scientific Software, Inc. Santa Fe, NM, USA).\n\n\nResults and Discussion\n\nOptimization of descriptor coordinates for preferred values of a given model is a central aspect of the two-stage inverse QSAR process, for which currently only approximate solutions exist. Therefore, a more accurate methodology for coordinate optimization is highly desirable, as investigated herein. The evaluation of εDE as an optimization method for this critical task was inspired by previous results obtained for other types of optimization problems where this approach displayed better performance than alternative evolutionary methods, such as genetic algorithms or particle swarm optimization27–29. Moreover, ε-based lexicological comparison of individual feature vectors makes this algorithm straightforward to apply to problems where several constraints must be balanced, as is the case in inverse QSAR. Studies on simulation and compound data sets were designed to evaluate whether εDE was capable of effectively optimizing coordinates on the basis of a regression function.\n\nFor initial proof-of-concept, εDE-based search for optimized coordinates was carried out using simulation data generated as described above.\n\nFor the three simulation data sets, SVR models were built yielding optimized parameters {γ, ν, C} of {4, 0.25, 2}, {2, 0.25, 1}, {1, 0.125, 16}, respectively. For all OCSVM models, γ was 1. As reported in Table 2, these SVR models accounted for the output of the bird function in a statistically meaningful way.\n\nFor each trial, model performance was assessed on the basis of R2 and root mean square error (RMSE) values for training and test data sets.\n\nFigure 4 shows the different prediction surfaces of the SVR models for the three trial sets. The surfaces of set one and two were overall similar, whereas the surface of set three differed from the others. In each case, however, individual vectors converged at a single point (Table 3) and optimized coordinates were located in regions of highest predicted y values (Figure 4). In set one, for which the SVR model overall best accounted for the bird function, a training data point was found adjacent to the optimized coordinates, which slightly exceeded the largest predicted y value (Table 3).\n\nFor each of three independent trials, optimized coordinates (red squares) and training data (black dots) were mapped on the SVR prediction surface.\n\nFor each simulation data trial, y values predicted by the SVR model are reported. For training data, the largest measured y value is given in parentheses. “Domain” is defined by x1 and x2 with a resolution of 0.005. AD refers to the applicability domain of the OCSVM model. For optimized coordinates, the result of the bird function is given in parentheses as the true y value. (i.e., the y value without error).\n\nFor set two, no training data were mapped to local maxima of the bird function, which resulted in a difficult regression scenario. The maximal measured y value in the training data was 29.46 and for optimized coordinates (1.51, 3.16), the predicted y value was 31.14, also slightly exceeding the largest measured y value. However, the predicted value was much smaller than the corresponding true y value of 48.09 (Table 3), due to the inherent regression limitation.\n\nFor set three, the maximal true y value within the domain was 56.18 at (-1.59, 0.06). In this case, several training data points were mapped to regions of y values into which optimized coordinates fell (Figure 4), leading to an extrapolative over-prediction of the corresponding y value of 71.94. However, this over-prediction was correctly balanced when the AD of the model was considered instead, leading to a value of 59.40 and a predicted y value for the optimized coordinates of 59.41 (Table 3).\n\nDespite typical regression limitations highlighted by findings for set two and three, the results obtained for simulation data indicated the potential of εDE for predicting optimized coordinates. Importantly, all solutions converged to single vectors representing novel points in simulation data space with large predicted y values falling inside the AD. Taken together, these results indicated the principal potential of εDE for coordinate optimization on the basis of SVR modeling.\n\nNext εDE optimization was applied to different compound activity classes. In each case, SVR models were derived, optimized coordinates determined, and activity values predicted.\n\nFor each compound class, optimized coordinates yielded larger predicted pKi values than any training or test set compound (Table 4), consistent with the methodological strategy. Optimized coordinates fell inside the AD of each model and were proximal to several active compounds. Nearest neighbors of optimized coordinates were mostly predicted to be highly active (Table 4), indicating the presence of smooth prediction surfaces in the vicinity of optimized coordinates.\n\nFor optimized coordinates, the predicted pKi value and the output of the OCSVM model for the applicability domain (AD) are reported. For training and test instances, the predicted pKi value and scaled distance from optimized coordinates are given for the nearest neighbor (NN).\n\nPrediction surfaces were further characterized graphically by systematically comparing predicted pKi values of compounds and calculated distances to optimized coordinates. Figure 5 shows the results for two exemplary activity classes, and Figure S1 shows the results for all classes. For set 51 (5-HT1a receptor ligands) in Figure 5, many highly active compounds were located proximal to the optimized coordinates, indicating that these coordinates fell into a well-populated region of activity-relevant chemical space. For set 194 (factor X inhibitors), training and test compounds tended to exhibit higher predicted pKi with decreasing distance to the optimized coordinates, hence delineating regions of activity progression, which are relevant for compound optimization and exploitation of optimized coordinates.\n\nFor two exemplary activity classes, predicted pKi values are related to the scaled distance of the corresponding compounds to the optimized coordinates. Training data (cyan squares), test data (magenta squares), and optimized coordinates (green circle) are shown.\n\nData set compounds and optimized coordinates were also projected onto PCA plots of descriptor space (Figure 6,Figure S2). These projections revealed that optimized coordinates were central to activity class regions in feature space. Furthermore, Figure 7 shows structures of the three nearest neighbors of the optimized coordinates for sets 51 and 194. In both cases, these compounds were structural analogs. Hence, similarity in feature space corresponded to close structural relationships. Consequently, this would also apply to structure generation from optimized coordinates, which would result in additional analog(s), consistent with the principles of QSAR and inverse QSAR.\n\nFor the two activity classes from Figure 5, training data (cyan squares), test data (magenta squares), and optimized coordinates (green circle) were projected on a principal component (PC) plot derived from training data. For PC1 and PC2, contributions to data set variance are reported in %.\n\nFor the two activity classes in Figure 5 and Figure 6, structures of the three nearest neighbors of optimized coordinates are shown and their ChEMBL IDs, scaled distances to the optimized coordinates and predicted pKi values, are reported.\n\nChEMBL compounds were screened relative to optimized coordinates from the nine activity classes and Euclidian distances were determined. Furthermore, pKi values were predicted for screening compounds falling into the AD of each class-specific SVR model. The VS calculations ultimately led to alternative distance- and potency-based compound rankings. Table 5 reports screening compound statistics and VS results. For each activity class, screening compounds contained large numbers (497–1152) of true positive test instances. Distance-based VS yielded AUC values of at least 0.6 for five of nine activity classes, with a maximum of 0.76. For the four remaining classes, essentially random predictions were observed. Potency-based VS produced AUC values of greater than 0.6 for seven of nine classes, including values above 0.7 for three classes and a maximum of 0.77. Thus, potency-based predictions led to slightly better compound rankings than distance-based VS relative to optimized coordinates, but prediction accuracy was overall moderate at best. Moreover, the true positive ratio among the 30 top-ranked compounds was generally very low for both distance- and potency-based VS. Figure 8 shows exemplary potency prediction landscapes including optimized coordinates as a reference. Figure S3 shows these representations for all activity classes. Highly potent compounds proximal to optimized coordinates were predicted for several activity classes. However, most true positives were not separated from the bulk of ChEMBL screening compounds on the basis of potency predictions. Overall the ability of VS calculations to identify novel active compounds and separate them from false positives was only limited. Thus, although de novo structure generation from optimized coordinates is challenging, it would be difficult to replace the structure generation step in two-stage inverse QSAR with standard VS calculations. However, despite limited prediction accuracy, the VS calculations provided support for the chemical relevance of optimized coordinates because for each activity class, at least few true positives were among top-scoring screening compounds and proximal to optimized coordinates.\n\nFor the two activity classes from Figure 5, predicted pKi values are plotted against the scaled distance of corresponding compounds to the optimized coordinates. ChEMBL compounds (gray squares) and test compounds according to Figure 5 (magenta squares) falling inside the applicability domain are shown. Optimized coordinates are displayed as a green circle.\n\nCompound (CPD) statistics and VS results for distance-based compound rankings relative to optimized coordinates and potency-based rankings are reported.\n\n\nConclusions\n\nThe optimization of coordinates in feature space for high activity values predicted with a regression model is a central task in two-stage inverse QSAR. For this multi-constraint optimization problem, no generally applicable approach is currently available. The evaluation of differential evolution for coordinate optimization, as reported herein, was motivated by the successful application of this algorithm in areas of science other than chemistry. The study has provided proof-of-concept evidence that εDE is suitable for generating optimized coordinates in given feature spaces. For different compound classes, consistent predictions were obtained for εDE in combination with SVR, displaying robust convergence behavior and yielding optimized coordinates that not only met statistical and data set requirements, but were also chemically relevant, as indicated by compound mapping and distance- or potency-based VS calculations. However, due to limited prediction accuracy, distance-based VS relative to optimized coordinates would not be suitable to replace the de novo structure generation step in inverse QSAR, at least not on the basis of our reference calculations. Regardless, encouraging results were obtained for coordinate optimization. Taken together, the findings reported herein indicate that εDE optimization has the potential to further advance inverse QSAR analysis.\n\n\nData availability\n\nThe data sets used in this study are freely available in ChEMBL via the identifiers reported in the manuscript.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe project leading to this report has received funding (for TM) from the Japan Society for the Promotion of Science (JSPS) under the JSPS KAKENHI Grant Number 16J05325.\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\nTM is a Fellow of the Japan Society for the Promotion of Science.\n\n\nSupplementary material\n\nSupplementary Figures S1-S3: Prediction surfaces and PCA projections for different activity classes and prediction surfaces for the ChEMBL screening data set, respectively. Click here to access the data.\n\n\nReferences\n\nKier LB, Hall LH, Frazer JW: Design of Molecules from Quantitative Structure-Activity Relationship Models. 1. Information Transfer between Path and Vertex Degree Counts. J Chem Inf Comput Sci. 1993; 33(1): 143–147. Publisher Full Text\n\nHall LH, Kier LB, Frazer JW: Design of Molecules from Quantitative Structure-Activity Relationship Models. 2. Derivation and Proof of Information Transfer Relating Equations. J Chem Inf Comput Sci. 1993; 33(1): 148–152. Publisher Full Text\n\nSkvortsova MI, Baskin II, Slovokhotova OL, et al.: Inverse Problem in QSAR/QSPR Studies for the Case of Topological Indexes Characterizing Molecular Shape (Kier Indices). J Chem Inf Comput Sci. 1993; 33(4): 630–634. Publisher Full Text\n\nSkvortsova MI, Fedyaev KS, Palyulin VA, et al.: Inverse Structure-Property Relationship Problem for the Case of a Correlation Equation Containing the Hosoya Index. Dokl Chem. 2001; 379(1–3): 191–195. Publisher Full Text\n\nSchneider G, Baringhaus KH: De Novo Design: From Models to Molecules. In: De novo Molecular Design. Wiley-VCH Verlag GmbH & Co. KGaA Weinheim Germany; 2013; 1–55. Publisher Full Text\n\nSpeck-Planche A, Cordeiro MN: Fragment-based in silico modeling of multi-target inhibitors against breast cancer-related proteins. Mol Divers. 2017; 1–13. PubMed Abstract | Publisher Full Text\n\nSpeck-Planche A, Dias Soeiro Cordeiro MN: Speeding up Early Drug Discovery in Antiviral Research: A Fragment-Based in Silico Approach for the Design of Virtual Anti-Hepatitis C Leads. ACS Comb Sci. 2017. PubMed Abstract | Publisher Full Text\n\nFaulon JL, Visco DP Jr, Pophale RS: The signature molecular descriptor. 1. Using extended valence sequences in QSAR and QSPR studies. J Chem Inf Comput Sci. 2003; 43(3): 707–720. PubMed Abstract | Publisher Full Text\n\nFaulon JL, Churchwell CJ, Visco DP Jr: The signature molecular descriptor. 2. Enumerating molecules from their extended valence sequences. J Chem Inf Comput Sci. 2003; 43(3): 721–734. PubMed Abstract | Publisher Full Text\n\nChurchwell CJ, Rintoul MD, Martin S, et al.: The signature molecular descriptor. 3. Inverse-quantitative structure-activity relationship of ICAM-1 inhibitory peptides. J Mol Graph Model. 2004; 22(4): 263–273. PubMed Abstract | Publisher Full Text\n\nWeis DC, Faulon JL, LeBorne RC, et al.: The Signature Molecular Descriptor. 5. The Design of Hydrofluoroether Foam Blowing Agents Using Inverse-QSAR. Ind Eng Chem Res. 2005; 44(23): 8883–8891. Publisher Full Text\n\nWong WW, Burkowski FJ: A constructive approach for discovering new drug leads: Using a kernel methodology for the inverse-QSAR problem. J Cheminform. 2009; 1: 4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiyao T, Kaneko H, Funatsu K: Ring-System-Based Exhaustive Structure Generation for Inverse-QSPR/QSAR. Mol Inform. 2014; 33(11–12): 764–778. PubMed Abstract | Publisher Full Text\n\nMiyao T, Kaneko H, Funatsu K: Inverse QSPR/QSAR Analysis for Chemical Structure Generation (from y to x). J Chem Inf Model. 2016; 56(2): 286–299. PubMed Abstract | Publisher Full Text\n\nMiyao T, Kaneko H, Funatsu K: Ring system-based chemical graph generation for de novo molecular design. J Comput Aided Mol Des. 2016; 30(5): 425–446. PubMed Abstract | Publisher Full Text\n\nStorn R, Price K: Differential Evolution – A Simple and Efficient Heuristic for Global Optimization over Continuous Spaces. J Global Optim. 1997; 11(4): 341–359. Publisher Full Text\n\nOnwubolu G, Davendra D: Scheduling Flow Shops Using Differential Evolution Algorithm. Eur J Oper Res. 2006; 171(2): 674–692. Publisher Full Text\n\nTakahama T, Sakai S: Constrained Optimization by the ε Constrained Differential Evolution with Gradient-Based Mutation and Feasible Elites. In: 2006 IEEE International Conference on Evolutionary Computation. IEEE; 2006; 1–8. Publisher Full Text\n\nSmola AJ, Schölkopf B: A Tutorial on Support Vector Regression. Stat Comput. 2004; 14(3): 199–222. Publisher Full Text\n\nSchölkopf B, Platt JC, Shawe-Taylor J, et al.: Estimating the Support of a High-Dimensional Distribution. Neural Comput. 2001; 13(7): 1443–1471. PubMed Abstract | Publisher Full Text\n\nTang Y, Guo W, Gao J: Efficient Model Selection for Support Vector Machine with Gaussian Kernel Function. In: 2009 IEEE Symposium on Computational Intelligence and Data Mining; IEEE, 2009; 40–45. Publisher Full Text\n\nBento AP, Gaulton A, Hersey A, et al.: The ChEMBL bioactivity database: an update. Nucleic Acids Res. 2014; 42(Database issue): D1083–D1090. PubMed Abstract | Publisher Full Text | Free 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\nHall LH, Kier LB: The Molecular Connectivity Chi Indexes and Kappa Shape Indexes in Structure-Property Modeling. John Wiley & Sons, Inc.; 2007; 2: 367–422. Publisher Full Text\n\nGasteiger J, Marsili M: Iterative Partial Equalization of Orbital Electronegativity - a Rapid Access to Atomic Charges. Tetrahedron. 1980; 36(22): 3219–3228. Publisher Full Text\n\nPedregosa F, Varoquaux G, Gramfort A, et al.: Scikit-Learn: Machine Learning in Python. J Mach Learn Res. 2011; 12: 2825–2830. Reference Source\n\nDong X, Liu S, Tao T, et al.: A Comparative Study of Differential Evolution and Genetic Algorithms for Optimizing the Design of Water Distribution Systems. J Zhejiang Univ Sci A. 2012; 13(9): 674–686. Publisher Full Text\n\nTušar T, Filipič B: Differential Evolution versus Genetic Algorithms in Multiobjective Optimization. In: Evolutionary Multi-Criterion Optimization. Springer Berlin Heidelberg: Berlin, Heidelberg; 2007; 257–271. Publisher Full Text\n\nIwan M, Akmeliawati R, Faisal T, et al.: Performance Comparison of Differential Evolution and Particle Swarm Optimization in Constrained Optimization. Procedia Eng. 2012; 41: 1323–1328. Publisher Full Text"
}
|
[
{
"id": "24665",
"date": "09 Aug 2017",
"name": "Hans Matter",
"expertise": [
"Reviewer Expertise Molecular modelling",
"drug design"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting contribution by Bajorath et al. addresses an important part of the inverse QSAR problem towards automated generation of structures with high activity from QSAR models. Inverse-QSAR, while intellectually appealing, does not find significant applications in modelling in the pharmaceutical industry. Main limitations are linked to de-novo structure generation due to issues with synthetic accessibility. Another hurdle is the challenge to identify the optimal descriptor setting for a model. The authors focus on this latter point, namely a novel and accurate approach for coordinate optimization. They demonstrate how to generate optimal descriptor coordinates under certain constraints like model applicability domain and meaningful descriptor values. A convincing validation study using virtual screening in ChEMBL 22 is also presented.\nThe report title and abstract cover the content well. The chemoinformatics approach is well conducted and clearly described. The results are presented in a clear and interesting way and capture the interest of F1000 readers. The authors might also want to mention, whether software tools and subroutines from their study are available. Therefore this contribution is an essential view on interpretation of QSAR models and should be indexed in its present form.\nSome points could be addressed to highlight further aspects of their work.\n\nHow does such an optimization approach handle typical types of variables from real-life models, e.g. two-level variables, variables with small or no SD?\n\nOften model analysis should not result in a single solution, but multiple related structures. Could the optimization approach find multiple descriptor regions to offer options for monitoring secondary properties (e.g. solubility)?\n\nIt might be illustrative for one ChEMBL target to systematically generate analogs for potent leads close to the descriptor optimum by applying simple MedChem transformations and check, whether some analogs come closer to the optimum in descriptor space. Chemical changes are minimal here and one could access their impact to the descriptor optimum.\n\nThe moderate VS success using QSAR models might suggest a non-optimal approach to define the applicability domain. Some details on the AD definition and the descriptor space might be useful. Do the authors expect that a more strict AD definition might produce reliable results? What does this mean for de-novo structure generation as second step?\n\nIs it possible to apply such a concept for multi-parameter optimization, e.g. multiple QSAR models combined for predicting compound profiles / selectivity / druglikeness?\n\nMinor point: Drawings of chemical structures in figure 7 need to be checked.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "24700",
"date": "25 Aug 2017",
"name": "Brian Goldman",
"expertise": [
"Reviewer Expertise machine learning",
"cheminformatics",
"algorithms"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an extremely well written submission by Bajorath et. al detailing an important aspect of the inverse QSAR problem, namely the generation of a feature vector optimizing the output of a QSAR equation. An interesting and novel component of the work is that constrained optimization is utilized ensuring generated solutions lie within the domain of applicability of the QSAR model. This paper is primarily of theoretical interest in that due to inherent limitations with inverse-QSAR the technique is rarely adopted as a means of drug discovery. The primary reason for the lack of adoption revolving around the de-novo design of compounds matching optimized descriptor values.\n\nThe introduction to the paper covers the relevant literature but could be made stronger by discussing the recent work of Gómez-Bombarelli et. al.1 who use generative models to approach the inverse-QSAR problem. Their work concerns building and optimizing QSAR equations in the latent space of an autoencoder and subsequently decoding optimized points into molecular structures. Although the work by Gómez-Bombarelli is preliminary, it addresses both QSAR optimization and structure generation and would most likely be interesting to readers of the current paper.\n\nAn aspect of the current technique that could be discussed in the paper is the generation of a family of solutions. Currently, the presented technique produces one optimized feature vector. It would strengthen the paper if the authors discussed how a family of diverse feature vectors (structures) could be evolved using the presented methodology.\n\nOverall, the paper in its current form is more than acceptable. The methodology is clearly delineated and sufficiently supported by the included results.\n\nMinor points:\nFigure 3: it would be informative to highlight the optimal point with a particular glyph (perhaps a red star?)\n\nFigure 7: Chemical structures look suspect and should be checked\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "24662",
"date": "29 Aug 2017",
"name": "W. Patrick Walters",
"expertise": [
"Reviewer Expertise computational chemistry",
"cheminformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes a new method for approaching one aspect of the inverse QSAR problem. As the authors point out, the inverse QSAR problem can be divided into two steps.\n\nThe generation of a set of coordinates, in some multidimensional space, that corresponds to one or more optimal compounds.\n\nThe generation of molecular graphs that would produce chemical structures with descriptor values corresponding to these coordinates.\n\nThis paper focuses on the first problem and does not address the second.\n\nThe paper is well written and the topic will be of interest to computational chemists working in both industry and academia. The methodology is described well and an individual with some QSAR expertise should be able to reproduce this work. However, in the interest of making it easier to reproduce the work described here, and making the method more widely available, it would be useful for the authors to make a reference implementation available. On a similar note, the authors mention that the ChEMBL datasets are available from the original source. As a service to those readers who would like to reproduce the work, it would be useful if the authors provided the specific datasets used in this paper as a download. As part of this download, the authors could also include a list of specific descriptors used and annotate which compounds were rejected due to PAINS filters.\n\nThe definition of the applicability domain used in this paper could be expanded. It would be useful for the authors to provide a specific worked example demonstrating how the applicability domain is defined for one of the ChEMBL examples described in the paper. This example could be expanded to demonstrate how a set of optimal coordinates is defined.\n\nOne other beneficial addition to this paper would be a comparison with established methods. The authors provide the results of virtual screening based on their method but do not provide a comparison with commonly used techniques. One way to do this would be to provide a simple comparison with an SVM model for activity calculated based on the same descriptions. A comparison with activity calculated based on nearest neighbors in a simple principal component space could also be provided.\n\nA few specific comments:\nIt is unclear what the RMSE value is in Table 2. This is on an arbitrary dataset, how should RMSE be interpreted?\n\nThere appear to be a number of errors in the chemical structures in Figure 7.\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/6-1285
|
https://f1000research.com/articles/6-12/v1
|
05 Jan 17
|
{
"type": "Method Article",
"title": "Supporting evidence-based analysis for modified risk tobacco products through a toxicology data-sharing infrastructure",
"authors": [
"Stéphanie Boué",
"Thomas Exner",
"Samik Ghosh",
"Vincenzo Belcastro",
"Joh Dokler",
"David Page",
"Akash Boda",
"Filipe Bonjour",
"Barry Hardy",
"Patrick Vanscheeuwijck",
"Julia Hoeng",
"Manuel Peitsch",
"Stéphanie Boué",
"Thomas Exner",
"Samik Ghosh",
"Vincenzo Belcastro",
"Joh Dokler",
"David Page",
"Akash Boda",
"Filipe Bonjour",
"Barry Hardy",
"Patrick Vanscheeuwijck",
"Manuel Peitsch"
],
"abstract": "The US FDA defines modified risk tobacco products (MRTPs) as products that aim to reduce harm or the risk of tobacco-related disease associated with commercially marketed tobacco products. Establishing a product’s potential as an MRTP requires scientific substantiation including toxicity studies and measures of disease risk relative to those of cigarette smoking. Best practices encourage verification of the data from such studies through sharing and open standards. Building on the experience gained from the OpenTox project, a proof-of-concept database and website (INTERVALS) has been developed to share results from both in vivo inhalation studies and in vitro studies conducted by Philip Morris International R&D to assess candidate MRTPs. As datasets are often generated by diverse methods and standards, they need to be traceable, curated, and the methods used well described so that knowledge can be gained using data science principles and tools. The data-management framework described here accounts for the latest standards of data sharing and research reproducibility. Curated data and methods descriptions have been prepared in ISA-Tab format and stored in a database accessible via a search portal on the INTERVALS website. The portal allows users to browse the data by study or mechanism (e.g., inflammation, oxidative stress) and obtain information relevant to study design, methods, and the most important results. Given the successful development of the initial infrastructure, the goal is to grow this initiative and establish a public repository for 21st-century preclinical systems toxicology MRTP assessment data and results that supports open data principles.",
"keywords": [
"Systems toxicology",
"Data sharing",
"Harm reduction",
"Open data",
"Database",
"Website"
],
"content": "Introduction\n\nSmoking is addictive and causes a number of serious diseases, including cardiovascular disease (heart disease), lung cancer, and chronic obstructive pulmonary disease (emphysema, chronic bronchitis)1. In addition to initiatives encouraging prevention and cessation of smoking, harm reduction for smokers may be achieved through the development of novel tobacco products that have the potential to reduce the risk of harm compared to continued cigarette smoking.\n\nThe U.S. Family Smoking Prevention and Tobacco Control Act defines a modified risk tobacco product (MRTP) as any that is ‘sold or distributed to reduce the harm or risk of tobacco-related disease associated with commercially marketed tobacco products’2.\n\nPhilip Morris International (PMI) is developing a portfolio of potential MRTPs to address a wide range of adult smokers’ preferences, preserving as much of the possible taste, sensory experience, nicotine delivery profile, and ritual characteristics of cigarettes, while significantly reducing or eliminating the formation of harmful and potentially harmful constituents (HPHC)3. Nonclinical and clinical studies are conducted to assess the risk associated to those products and the full set of data from the relevant scientific studies will be evaluated to determine whether they substantiate reduced exposure or risk.\n\nInsofar as nonclinical laboratory studies (safety and toxicity studies) may provide evidence regarding the relative toxicities of MRTPs, it is proposed that they are to be carried out according to a quality management system (proposed GLP Quality System4). Building quality into study planning, using methods that have been validated, executed by trained personnel in adequate facilities, and with proper data management and processing practices are the essential components of such a system and are a first step in ensuring data quality, reproducibility and reliability.\n\nThe adoption of MRTPs, and thereby their potential public health benefits depend on product acceptance among existing adult smokers and their actual performance in terms of reduction in risk compared to continued smoking, which in turn shall be based on robust and multi-disciplinary scientific substantiation. Ensuring that the underlying evidence and results are openly shared, in a similar way as has been proposed by the European Food Safety Authority for example5, can encourage replication of the studies and increase confidence in the findings. Indeed, several studies have shown that much peer-reviewed scientific literature is difficult to reproduce for reasons such as inadequate documentation of methods and datasets and insufficient sharing of data and methods with the community6–11. Concerns on reproducibility of science have lead to recent calls for a shift to better practices12,13. For example, not only is it crucial that the science is right, i.e. ensuring that the study is blinded, that experiments are repeated, reagents validated, and the analyses appropriate, but it is also important that all results are shown, including negative and positive controls. Equally, for any scientific result on MRTPs it is important that a consistent, science-based regulatory framework is used for identification of innovative alternative products that could significantly reduce the risk of tobacco related disease and death caused by cigarette smoking14. Processes that encourage transparent sharing of data in a way that allows easy review and understanding will facilitate objective evaluation of the evidence.\n\nTo complement the classical peer-review system in the evaluation of the scientific evidence, several initiatives, such as CASP15, BioCreAtIvE16, DREAM17, and sbv IMPROVER18 leverage the scientific community to verify methods of protein structure prediction, information extraction, gene network inference, and systems biology, respectively19. In order for the crowd to be able to review methods and/or data, it is important to prepare those in a form that is easily understandable and usable, and to collect all of the relevant information in one place. Therefore, we developed a database and associated webportal to collect relevant information on studies assessing candidate MRTPs.\n\nThe emerging field of systems toxicology aims to develop integrated frameworks for the prediction and quantification of substance-related toxicity. Systems toxicology is broader than a simple attempt to understand the impacts of exposure at a pathway level; it is an interdisciplinary, integrated approach that depends on data produced by rapidly developing omics technologies, such as transcriptomics, metabolomics, and proteomics20, which complement more traditional toxicity endpoints. The objective is to generate more comprehensive impact overviews by combining complex biological network models with quantitative measurements of impacted pathways at all levels of biochemical and biological organization21,22 to facilitate better-informed decision making as compared with traditional safety assessment alone.\n\nThe National Research Council, commissioned by the US EPA, developed a vision for 21st-century toxicity testing23 characterized by a shift in focus away from traditional toxicity testing and toward the exploration of human signaling pathways whose perturbation by biologically active substances or their metabolites causes adverse health effects24,25.\n\nQuantitative systems toxicology involves mining omics data and functional endpoints for identification of potential adverse outcome pathways (AOPs) and their component events and event relationships. AOPs, as defined by the OECD26, are simplified pragmatic frameworks, which are linear in nature and connect a single molecular initiating event (MIE) to a single adverse outcome (AO) by means of non-branching, and directional sequences of key events (KEs). Supporting evidence for AOPs is arranged according to three levels of information, namely “Biological Plausibility” of the KEs (most important), “Biological Essentiality” of the KEs and “Quantitative Evidence” of the KERs (least important). Building and quantifying AOPs requires multiscale integration of all available and relevant -omics datasets, mining of supporting knowledge, and predictive algorithms that quantify AOPs and their evolutionary and genetic diversity27.\n\nParameters that facilitate reliable quantitative prediction of toxicity and risk are also required. Multicellular and tissue simulation modeling can predict injury and repair of the tissue architecture and are parameterized by molecular models and biological assays (Figure 1). Such a systems toxicology approach has been used successfully in in vitro and in vivo studies28–32 to assess prototypic MRTPs in the context of an integrated scientific assessment program3.\n\nTo understand the effect and mode of action of chemicals or drugs on Human, different studies can be conducted. Epidemiology will provide the final evidence but requires long periods of observation. Phenotypic observations may be obtained at the individual level from biopsies or tissue collection. Animal studies can provide surrogate information in a controlled setup and allow the collection of various tissues and fluids. Alternatively, new in vitro methods are developed to provide information on toxicity and pathways of toxicity. It is possible to obtain organ-tissue level information from macroscopic observation of tissues, but also to understand cellular level or even molecular level by mining data from –omics profilings using modeled knowledge and dedicated algorithms.\n\nPredictive toxicology (i.e., 21st century toxicology) is an active field that is transitioning to a mechanistic, evidence-driven science. Large international programs (e.g., Tox21 and ToxCast in the United States, EU-ToxRisk and SEURAT-1 in Europe, and TGGates in Japan – for more information, please refer to Table 1) aim to develop new biological methods and generate large datasets to probe pathways and mechanisms of toxicity that are relevant to human and environmental health. These endeavors generate increasingly complex datasets for the scientific community to analyze in the development of new hypotheses, predictive models, and integrated testing strategies. These datasets, which encompass multi-omics data, in vitro/in vivo assays, and in silico toxicity prediction and modeling applied to environmental and human hazardous substances, are organized into diverse repositories (a noncomprehensive list of which is given in Table 1). Many large parent database portals and projects host or link to child databases that are available to toxicologists, regulatory agencies (such as the EPA and FDA), and the general scientific community. In addition to these initiatives, a plethora of specialized databases (e.g., ChEBI, OCHEM, and PubChem – for more information, please refer to Table 1) cover individual topics from properties of chemical compounds to biochemical assays that assess physicochemical properties.\n\nThe table highlights sources of information on in vivo chemical inhalation and individual in vitro chemical toxicity. The type of data available and user accessibility (e.g., open source vs licensing) have also been highlighted. While a number of databases and portals are still active, a few of them are no longer maintained. Green color in the “Inh” column means that the resource contains inhalation data.\n\nFor example, OpenTox (www.opentox.net)33 was started as a project of the European Commission’s Seventh Framework Program: HEALTH-2007-1.3-3; it compiles specifications, standards, and tools for the integration of data, algorithms, and models from various public and confidential toxicological sources. It was designed as an open framework for the generation and validation of computer models of toxic effects, libraries for the development and seamless integration of new algorithms, and scientifically sound validation routines. After the end of the initial R&D project in 2011, OpenTox evolved into a practical community resource, extending to all aspects of risk assessment, including experimental design, data management, biological data analysis, modeling, AOP development, and regulatory issues. Finally, this resulted in the foundation of the OpenTox Association (http://www.opentox.net/the-opentox-association) in 2015. This initiative integrates knowledge, processes, and people from many different fields, including toxicology, biology, chemistry, bio- and cheminformatics, and computer science, by organizing community interactions (e.g., working groups, workshops, scientific meetings, and hackathons in the United States, Europe, and Asia). Additionally, OpenTox members are involved in major research projects, such as SEURAT-1: Towards the Replacement of in vivo Repeated Dose Systemic Toxicology Testing (www.seurat-1.eu) and its follow-up project EU-ToxRisk (www.eu-toxrisk.eu).\n\nTo facilitate better-informed decision making in risk assessment, knowledge integration may include evidence from in vivo, in vitro or in silico methods; biology, chemistry, or engineering; and human-health- or environment-oriented research. There are growing opportunities to base knowledge integration and sharing on a combination of emerging concepts and frameworks. Such frameworks require a clearly defined ontological and knowledge basis, and all applications need to employ sound, reproducible scientific bases and good practices in terms of experimental characterization, data organization, and concept description34. One challenge in knowledge integration is that in many areas of predictive toxicology and safety assessment, scientific knowledge is generated not only by existing methods accepted by regulators, but also by a growing number of alternative research methods and initiatives, for which the data and their structures may be less well defined. Hence, as indicated by Gary Miller in his editorial, “Data Sharing in Toxicology: Beyond Show and Tell”35, the quality of the necessary infrastructure for harmonized data sharing has lagged far behind that of the actual data. To facilitate verification of research conclusions, data need to be organized and managed carefully and traceably, processed with a variety of workflows and analysis techniques, and shared with the community for scrutiny and further analysis so that they ultimately generate knowledge. Therefore, data integration, meta-analysis, and the interaction of data and predictive models with existing knowledge frameworks (e.g., AOPs describing the sequence of key events leading to stress, repair, or toxicity) are becoming increasingly important.\n\nWhile several public data resources, as identified in this section, have been developed to provide systematic access to multidimensional systems toxicology data, the ever-increasing disaggregation of data, information, and publications throughout various channels (e.g., blogs, public health news, journals, and key opinion leaders in specific fields) make it challenging for researchers to filter, pursue, and focus on relevant knowledge sources. Thus, a single cloud-based dashboard that aggregates, assimilates, mines, and prioritizes data and information according to relevance could play a central role in enabling an open, data-driven, evidence-based platform for 21st-century toxicology studies. Such a tool may also facilitate identification of key opinion leaders and experts who could perform in-depth reviews of specific data and/or results.\n\nDespite the availability of much information in fields related to systems toxicology, few databases provide integrated toxicological evidence for respiratory analysis/assessment (e.g., for study of in vivo chemical inhalation). Databases such as ACToR36, the Comparative Toxicogenomics Database (CTD)37, and CEBS38 do contain some independent studies focused on inhalation-associated chemical toxicities, but presently, corporations and others interested in inhalation toxicology, who are focused on assessment and mitigation of toxicity associated with inhalation of substance constituents, have no central “go-to” repository. Therefore, approaches to utilize already-present data in reproducible analyses or extract relevant conclusions for specific investigations have been limited by poor coordination and crosslinking and the lack of integrated, harmonized, open access and availability of data.\n\nCollaborative aspects of the systems toxicology approach can be founded on projects such as sbv IMPROVER39, which verifies techniques in computational biology using crowd sourcing to facilitate analysis and understanding of large, complex datasets.\n\nIn this paper, we describe emerging data practices we have developed to support a robust, reproducible predictive toxicology/safety assessment applicable to inhalation science in the context of novel and alternative tobacco products. We also describe a proof-of-concept implementation of a data-sharing infrastructure as the underlying foundation of a knowledge-sharing portal on novel tobacco and alternative products. Here we do not focus on the quality framework in which studies are performed, but emphasis is placed on sharing of information on protocols, and raw and processed data in a standardized way.\n\n\nMethods\n\nA database and searchable web portal (INTERVALS) have been developed as proof-of-concept for data sharing in systems toxicology. They include results from in vivo inhalation studies and in vitro studies conducted by PMI R&D to assess candidate MRTPs (Figure 2). The website and underlying database can be accessed at https://systox.sbvimprover.com/ and should allow the scientific community to easily retrieve relevant and usable information relevant to MRTPs from a single place and with similar standards (described below).\n\nExperiments are planned and protocols as well as metadata are recorded for each of the data entry and curated in ISA-Tab files. They all are imported into a common database that supports defined ontologies. Raw data can be exported from this database and processed with different scripts and/or software to generate analyses results, some of which are usually shared in a publication. All of the results can be saved into the database and the data and results can be accessed through an API to be browsed on and downloaded from the website named INTERVALS. The website also keeps track of publications associated with the studies.\n\nThe data modeling described below adopted the latest standards of data sharing and reproducible research. Therefore, the INTERVALS vision underscores the importance of a central repository for toxicological inhalation data and encourages sharing of information and expertise across the scientific and regulatory communities to foster reproducibility in predictive toxicology and risk assessment.\n\nThe workflow development and principles of data preparation and database infrastructure largely reused open computing resources and standards developed within OpenTox, both for designed programs and associated community engagement. Particularly, OpenTox’s engineering design as an interoperable distributed framework of components, that interact via well-defined application programming interfaces (APIs) and web services, provided a strong technical base for the extensible integration of diverse software components and resources. OpenTox includes services for data integration, model development, validation, and reporting that can satisfy scientific, community, and regulatory requirements for sustainable extension of data management, validation, and regulation.\n\nAnother integral part of the portal’s vision is assimilation and mining of data for identification of scientifically relevant information and identification of key experts to facilitate and validate reviews and analytics. In this paper, we focus on the data science practices developed to support verification of conclusions derived from systems toxicology studies, illustrated by a case study example.\n\nFor proof-of-concept, a number of datasets from assessment studies conducted by PMI R&D on prototype or candidate MRTPs were prepared and integrated into the platform. Two examples are detailed below to exemplify the new data-management and sharing philosophy for large in vivo and in vitro studies. To learn more about these studies’ results, please refer to the respective publications, as only short descriptions are given below.\n\n\n\n1) Chronic obstructive pulmonary disease (COPD) progression in response to chronic exposure to cigarette smoke (CS) or a prototype MRTP (pMRTP) (i.e., the C57BL6-pMRTP-SW dataset)31. Cigarette smoking is a cause of COPD. Thus in the assessment of MRTPs it is of interest to understand to what extent the risk of COPD may be reduced in comparison to exposure to cigarette smoke. Using a systems toxicology approach in a model of COPD (C57BL/6 mice), the potential of such a pMRTP to reduce health risk was assessed. The study investigated physiological endpoints in parallel with the transcriptomics, lipidomics, and proteomics profiles of mice exposed to CS from a reference cigarette (3R4F) or a pMRTP aerosol for up to 7 months. In addition to the control (fresh air-exposed) group, the study also included a cessation group and one that switched to the pMRTP after 2 months of 3R4F exposure to evaluate the potential risk reduction of switching to pMRTP compared with continuous 3R4F exposure; those results were benchmarked to cessation.\n\n2) In vitro assessment of the effects of acute exposure to the aerosol of a candidate MRTP, the Tobacco Heating System version 2.2 (THS2.2), on organotypic acute human buccal or nasal tissue cultures (i.e., the organotypic buccal and nasal datasets, respectively)40,41. Recently developed three-dimensional (3-D) organotypic buccal and nasal epithelial culture models offer physiologically robust systems to study the effects of inhalation exposure. The studies investigated the relevance and applicability of in vitro human 3-D buccal and nasal epithelial culture models to toxicological assessment of inhalation exposure. Biological impacts were assessed following exposure to aerosol generated from THS2.2 (as compared with CS from reference cigarette 3R4F) using an in vitro human 3-D buccal or nasal epithelial culture. The experiments were repeated with multiple applications of the aerosol or CS to obtain reproducible measurements or reliable observations of molecular and cellular changes following exposure. Aligned with the 3Rs strategy (i.e., replacement, refinement, and reduction) and the Vision and Strategy of Toxicity Testing in the 21st Century42, a systems toxicology approach found that at all tested concentrations, 3R4F CS had considerably greater impacts than THS2.2 aerosol in terms of cytotoxicity, tissue morphological alterations, secretion of proinflammatory mediators, impaired ciliary function, and perturbation of transcriptomes and miRNA expression profiles40,41.\n\nWe propose to follow the best practice of requiring all data uploaded to the community portal and the supporting data repository to have well-documented protocols describing the methods followed to generate and process data developed within the ToxBank project. In the current version, summary protocols and key steps in data production and processing are included in the ISA-Tab files. Future development of the database and site will allow for protocol versioning. When a new protocol has been developed, documented, and reviewed, it will be uploaded to the data repository by the investigator following guidelines on the content and organization of the protocol description. The protocol will be loaded through the portal’s upload interface, where additional information associated with the protocol, including a protocol summary and identification of the protocol’s owner and authors. In addition, keywords from our supporting ontology are assigned to support the search function. The protocols will be visible and downloadable on a dedicated set of pages in INTERVALS.\n\nFollowing the recent dataset preparation work in ToxBank supporting SEURAT-1 (and its successor, the EU-ToxRisk program), a strategy proposal on data presentation was prepared and shared with the OpenTox data working group43. This proposal was further expanded based on use cases and datasets from PMI and additional community inputs and experiences from other projects (e.g., ToxCast, Tox21, and TGGates). It incorporates ISA-Tab files to describe experiments, data production, processing, associated metadata, and the use of defined ontologies.\n\nImportantly, data interoperability and submission to regulatory agencies requires conformance to strict data standards (e.g., for FDA submission, refer to the guidance for submission of electronic data44). Protocols, metadata, and data files have been prepared to follow the FAIR principles (i.e. Findability, Accessability, Interoperability, and Reusability)45 to the extent possible. This implied specific rules during data curation, as well as specific design for dataset retrieval in the search tool described below.\n\nSustainable dataset storage requires not only a defined data format but (even more importantly) well-organized, annotated metadata on the experimental setup. The ISA-Tab standard was created for this purpose and has already been used in projects like the ToxBank infrastructure of SEURAT-1 (www.toxbank.net) and diXa (http://www.dixa-fp7.eu/); an extended version, ISA-Tab nano, was used in the eNanoMapper project (www.enanomapper.net).\n\nThe ISA-Tab format46 is a standardized, general-purpose framework for the collection and communication of complex metadata that consists of three types of tables: the Investigation, Study, and Assay tabs (I-, S-, and A-tabs, respectively). The I-tab summarizes general information on the complete investigation, all studies, and all assays, including people involved in the investigation, related publications, and short protocol descriptions. Additionally, it relates the A-tabs to the S-tabs. The S-tab contains information on the study subjects, their characteristics, and any treatments applied. Finally, the A-tab describes the smallest complete unit of experimentation that produces data associated with a subject.\n\nThe ISA-Tab specification has a somewhat different definition of study and assay compared with their use in normal lab settings: an ISA-Tab investigation corresponds to a complete experimental design, often called the study design in practice. Under the ISA-Tab specification, a study deals with the in vivo or in vitro sample and an experimental assay conducted to investigate a specific endpoint, such as transcriptomics. To circumvent possible confusion caused by this contradictory use, we place “ISA-Tab” in front of the terms “study” and “assay” if they are used according to the ISA-Tab definition. The advantage of ISA-Tab is that its generality imparts the flexibility to provide metadata for almost any experimental setup. However, ISA-Tab files from different groups or projects might look very different, even if all files are consistent with the ISA-Tab standard, because of the metadata’s undefined structure. This applies not only to the specific metadata included in the files but also to the splitting between the S- and A-tabs.\n\nInitiatives such as the ToxBank project have attempted to standardize the ISA-Tab format for toxicological applications (i.e., ISA-Tab investigations). So far, the focus has been on relatively small studies with only one or a few endpoints, and the files have been created after study completion, usually by people involved only in parts of the study (or not directly involved at all). For studies like the examples here, which have more complex designs, including multiple tissues and endpoints, this approach is complex, time-consuming, and error-prone, requiring the ISA-Tab files’ creator to consolidate experimenter input and validate the files. Therefore, herein, we propose a new ISA-Tab scheme that follows the data-production workflow and combines data production and documentation into a single step by the researcher, who is the expert on the dataset and experimental parameters.\n\nInstead of one ISA-Tab instance documenting the complete study, a hierarchical structure of interlinked ISA-Tab files was created to follow the study’s experimental steps (Figure 3). The steps covered in specific ISA-Tab instances can be handled flexibly according to tasks performed by different labs, sites or collaborators, even before the full study is completed. New endpoints can be added easily, and the files can be updated if additional information (e.g., publications) becomes available.\n\nThe schema depicts the theoretical splitting strategy of data and metadata from two different studies into ISA-Tab files. The highest level will describe all subjects or samples analyzed in a study. Then, for each endpoint, a file describes the data production step, and links out to a raw data file. Another file will describe data processing steps and link out to processed data files. It is also possible that the two steps are combined into a single file. Eventually, analysis and data modelling could consider data from multiple studies.\n\nThe resulting interconnected ISA-Tab instances were hierarchized into different levels. The highest, most upstream one is the system (SY) level, which describes the main subjects under investigation (i.e., the animals or tissue cultures and their treatment by chemicals for in vivo and in vitro studies, respectively). The next layers describe data acquisition and analysis. For endpoints with various options to derive processed data from raw data, splitting the experiment description into data production (DP) and processing (PR) sets of ISA-Tab instances documented different processing options as independent assays. Layers 4 and 5 were reserved for modeling and validation; these can combine information from different ISA-Tab instances from layers 1–3. To document interconnections between files, the upstream ISA-Tab instance is referenced in the S-tab of the downstream ISA-Tab instance. This ISA-Tab splitting approach is illustrated in Figure 3, and its applicability is demonstrated with a specific example below.\n\nThe C57BL6-pMRTP-SW and organotypic studies were imported according to the above novel concept, resulting in separate ISA-Tab instances for the different endpoints, which can then be used as templates for additional studies.\n\nThe complete C57BL6-pMRTP-SW study was performed on the same population of mice, which were exposed to reference CS, the aerosol from a pMRTP, or fresh air. Exposure conditions were summarized in the SY-level ISA-Tab instance, whereas body-weight measurement was covered in an A-level ISA-Tab instance. Data production for the different endpoints was described in the second level, and some endpoints had third-level instances for raw data processing. The complete structure of the ISA-Tab instances is presented in Figure 4.\n\nA. Switching study concept and study design and setup. B. ISA-Tab splitting strategy of endpoints. The data production (DP) and processing (PR) instances describe the experimental setup and processing steps, from raw to processed data. Transcriptomics processing is separated by tissue, resulting in individual PR ISA-Tab instances. C. A more complicated lipidomics scheme was necessary because the experiment was performed independently for different groups of lipids, and hence separate DP instances were used for each mass spectrometry platform/set of methods. Processing was then performed per tissue, resulting in separate PR instances for blood, right lung, and liver. For simplicity, data files are not depicted here.\n\nIn the following paragraphs, we present the requirements for specific endpoints that were considered during ISA-Tab development. Endpoints without such requirements are not listed but are included in Figure 4.\n\nTranscriptomics: Gene expression was measured in four different tissues, each of which was covered as a separate A-tab in the DP instance. Because processing differed between the four tissues, four separate PR ISA-Tab instances were created.\n\nLipidomics: The exact metabolite-profiling procedure is dependent on the lipids analyzed. Therefore, one DP ISA-Tab instance was created for each group of lipids, which each had three A-tabs for the different tissues for which lipidomics data were available. Similar to the integration of transcriptomics data, the processing was done on a per-tissue basis. The PR ISA-Tab instances incorporate information from all six DP instances, all of which were accordingly referenced as upstream ISA-Tab instances.\n\nProteomics: Protein-expression profiling data were measured using three different, separately assembled experimental approaches. For 2D gel electrophoresis and iTRAQ, data preparation and processing were split between two ISA-Tab instances so that they could be used as templates for future studies, in which they could facilitate any necessary alternative processing options. Proteomics data acquired using Zeptomark reverse protein arrays were described in a single instance that combined data production and processing.\n\nBronchial alveolar lavage fluid (BALF): Cell count measurement and multi-analyte profiling performed on BALF were treated as two A-tab assays in one DP instance.\n\nHistology and histomorphometry: Histology and histomorphometry measurements were covered in two A-tab entries in the same DP instance.\n\nThe organotypic nasal in vitro study included a range of functional and molecular endpoints: adenylate kinase assay as a proxy for cytotoxicity assessment, cytochrome P450 activity, profiling of proinflammatory mediators (MAP), ciliary beating frequency measurement, histological analysis, and molecular endpoints (mRNA and miRNA). The overall study design and ISA-Tab splitting strategy are illustrated in Figure 5.\n\nA. Study design and setup. B. Measurement type per insert. For each condition (test item type and concentration), a set of up to seven inserts was used to measure endpoints at different post-exposure times. Longitudinal measurements were conducted for CBF and CYP1A1/1B1 activity. For other endpoints, a new insert was used for each post-exposure time point. C. ISA-Tab splitting strategy of endpoints data production and processing across ISA-Tab files. Raw and Processed data files are illustrated with green and orange backgrounds, respectively.\n\nEven if the use of multiple ISA-Tab instances is convenient for data input, a central source of information on each specific endpoint for further analysis, validation, and prediction is desired. Therefore, the SY, DP, and PR instances were compressed into a single Microsoft Excel file per endpoint. Because this file format facilitates the inclusion of multiple sheets per spreadsheet file, the structure of split, interlinked ISA-Tab instances can be maintained.\n\nTemplates for data and metadata for different endpoints do not necessarily define standard file formats that everyone has to follow strictly. Efforts to define such standards often face the challenge that resulting formats are not sufficiently flexible to keep up with new developments in a dramatically changing field like in vitro/in silico toxicology and are thus limited to specific applications. For example, the SEND format (https://www.cdisc.org/standards/foundational/send), developed by the Clinical Data Interchange Standards Consortium (CDISC) and advocated by the FDA as standard file format, was designed for regulatory reporting. However the controlled terminology included in SEND does not offer the required flexibility to support reporting of systems toxicology data. The inclusion of nonstandard data and controlled terminology therein is very complicated, and extensions to the standard require the approval of and can only be integrated by CDISC. The ISA approach tackles the harmonization and interoperability problems in another way. Even if the S- and A-tabs are only defined as momentarily required, the tabular form, content, and order of the columns can be freely chosen, and data sharing is possible, because ontologies are used in the metadata’s annotation. Users and computational tools can understand the data associated with specific entries by searching for words in this controlled terminology. Unfortunately, no ontology covers everything from sample preparation to experimental setup and endpoint readouts. While defining the ISA-Tab templates, the ontology terminology for metadata also had to be selected. This involved selections between publicly available ontologies (e.g., the ones available through BioPortal (http://bioportal.bioontology.org)) and defining metadata without the use of ontology (e.g., additional terminology could be defined and included in a new ontology/metadata). For example, a new ontology had to be created to cover terms relevant to studies on cigarette smoke47. There is no single correct decision, and as ISA-Tab is a relatively new standard, no consensus has been established on templates, optimal representation, or hierarchy; this area of emerging data science practice is supported by discussions within the OpenTox Working Group (http://www.opentox.net/wgsmainpage). To support these activities, we provide the ontologies selected to describe the above-mentioned datasets and describe recently published ontologies of interest in Supplementary File 1. Additionally, we list three ontologies whose use is discouraged because they are less well defined than (or are amalgamations of) the other entries. In the future, this ontology collection will be extended to satisfy additional experimental needs. The development and incorporation of ontology supports the creation of a robust knowledge infrastructure to achieve semantic interoperability and the associated benefits of reliability, evidence integration, and accuracy of reasoning. A lookup service to find entries in these ontologies and automatically add the resulting terms to the ISA-Tab files during template creation is currently being developed. This tool will also be able to assign multiple ontological entries to a specific term; this is needed because of the parallel development of overlapping ontologies with slightly different words for the same object.\n\nCurrently, data collection and generation of the ISA-Tab files are performed manually, with rudimentary automation using Excel. To guarantee the datasets’ quality and accuracy, multiple iterations of a checking cycle involving the researcher, modelers, and data managers are conducted. Even if manual data curation will still be necessary for final quality checks, automation of this process is ongoing. Although this will include formal checks of files’ correctness with respect to the ISA-Tab standard (as already performed, e.g., by ISACreator), equipment data, log files, already-available databases, and computational infrastructure will be interfaced to provide the needed information at least partially. This will reduce the effort needed for quality control, because it facilitates the avoidance of copy-and-paste errors.\n\nThe first step in this direction is the development of a data input and management application consisting of several validated web forms and lists with filtering/searching features connected to the uploaded datasets. The input forms are separated into two sections for management of facet terms and the dataset; both sections run in the context of the database management environment, which incorporates tools for user/access token management and access logging.\n\nThe main benefits of the data input management application are:\n\n\n\n• centralized management of dataset information;\n\n• prevention of problems associated with parallel work/versions;\n\n• controlled vocabularies for facets;\n\n• prevention of filename mismatches and other errors;\n\n• history/log files of addition and modification; and\n\n• automatic backups.\n\nThe data repository provides data storage and retrieval according to the OpenTox specification (http://opentox.org/dev/apis/api-1.2). It is implemented as a client–server architecture wherein the server exposes an API to which clients connect to search and retrieve data. The data repository contains implementations of the following open source technologies (see Figure 6): Elasticsearch (https://www.elastic.co/) as a dataset metadata store that provides search/faceting, PostgreSQL (http://www.postgresql.org/) as a store of administrative/access data, Django framework (https://www.djangoproject.com/) as an HTTP web server to provide dataset management, and search-request validation and processing, and JSON as the underlying data-transfer format.\n\nThe two OpenTox HTTP REST-compliant endpoints of the API are search and data retrieval (Figure 7). The search endpoint has full text search (usually found in data-retrieval services) and faceted search facilities. Faceted search allows users to explore the data collection by applying multiple filters whose values are selected from predefined categories (facets) assigned to the datasets. The facets used to classify the present project’s data can be extended easily in the future, but they presently include study, study type (in vivo/in vitro/clinical), mechanism, exposure, organism, system, tissue, and endpoint. At each filtering step, users are presented with the number of datasets currently filtered. We determined that faceted search is an effective extension to the usual full-text search approach, as it provides users with not only data retrieval but also quick, user-friendly data exploration. The data endpoint returns requested datasets that include either raw or processed data and are enriched with additional metadata information stored in ISA-Tab files. For convenience, each dataset is served as a ZIP file that includes all the mentioned parts.\n\nUsers can filter the datasets by Organism, Study, Mechanism, Tissue/Organ, and Endpoint Type. A toggle switch provides a choice between downloading raw and processed data.\n\n\nDiscussion and outlook\n\nWithin the scope of this proof-of-concept definition and implementation phase, we identified and clarified data requirements and developed a common framework for preparing relevant datasets to share with the community. The support of ISA-Tab by a data infrastructure that is interoperable with OpenTox and partner resources such as Garuda48 represents a high-quality and sustainable data-science solution, extensible beyond the presently demonstrated application.\n\nBesides data access and sharing, our goal is to present different stages of processed data, so that users can distill the raw data to strengthen their examinations. The data were prepared according to high-quality data-science methods and can be analyzed rigorously by biologists and computational biologists. Physicians and pathologists may need more refined and processed data for their consumption following methods of evidence-based medicine49 that were recently extended to toxicology50. Biologists can process and analyze data and publish results; those results can then be used translationally by medical scientists, who can interpret the evidence further for clinical use.\n\nThe goal for advancement of alternative testing methods (e.g., those pursued by the SEURAT-1 and EU-ToxRisk programs and supported by OpenTox and ToxBank) is the development of a stronger scientific framework for assessment of systemic toxicity, which could lead to the reduction/replacement of many expensive chronic animal experiments. To achieve this challenging goal, we need to perform case studies to integrate heterogeneous evidence from in silico, in vitro, and in vivo sources to support verification and validation of new methods. The preparation and sharing of dense, high-quality datasets—as described in this paper—is expected to facilitate effective review. In the following sections, we will describe additional relevant topics, which will be priorities in our further extension of the data infrastructure and webportal utilization for in-depth peer review.\n\nIn order to interpret large scale-omics data, filter the signal from the noise and lead to actionable insights, researchers need to focus on the biological small data - data which leads to meaningful information and contributes to knowledge about living systems. To extract meaningful information and knowledge from data, researchers need to use a diverse set of computational tools, algorithms, database and analytical services. Various computational approaches have been developed to study biological systems at the level of genes, transcripts, proteins, metabolites to cells, tissues organ and whole body modeling. Most analyses require the use of multiple database, tools and software in different contexts, and more often than not, it is not possible to define the set of tools and their sequence of connections a priori. The Garuda platform is an open and community-driven platform providing a framework to connect, discover and navigate through different applications named gadgets, databases and services in biology and medicine48,51. The strength of Garuda resides in the language-agnostic build: APIs allow to connect software written in any programming language as gadgets. Moreover, the dashboard allows to explore all available gadgets through the gateway. The Garuda platform enables users to access the data from INTERVALS and to analyze and visualize it through customized gadgets on a customized dashboard accessible from the INTERVALS platform.\n\nIn the near future, a combination of in silico methods, including toxicokinetics modeling, could be used for mechanistic extrapolation of in vitro data and background knowledge to human in vivo risk assessment according to cross-applicability and/or AOPs. For example, the strategy could integrate evidence from distributed OpenTox resources into AOPs and Risk21-based risk assessments52. Starting with harmonized data that are accessible from interoperable services, such as the one described here, a variety of analyses and visualization procedures may be applied. On the bases of such analyses and the knowledge collected in AOPs (Figure 8), the weight of evidence supporting risk assessment and integrated testing strategies could be increased, as already successfully demonstrated by Jaworska and colleagues.43,53 in identification of potential skin sensitizers.\n\nThe schematic includes biological assays to test the molecular initiating event and specific key events on different levels, which could be combined into a bulk of evidence supporting risk assessment or integrated testing strategies. The in vivo tests (orange) should be increasingly replaced by a combination of in vitro assays and in silico tools (green) to reduce animal testing according to the 3Rs principle26.\n\nIt would be particularly attractive to move between different chemical or endpoint spaces using biological signatures. Further, these methods and tools are transferable to other problems of societal concern (e.g., health/safety assessment of new products, safety biomarker discovery, air-pollution risk management, nanotechnology innovation, toxic-dust exposure, and green chemistry). Although some of these goals and activities may be challenging, we suggest that promotion of interdisciplinary data-science practices into an evidentiary framework can significantly advance the development of such alternative methods and engage support for and community involvement with the motivations of 21st-century toxicology.\n\nThe present infrastructure was developed with a research perspective in mind. The collected metadata in the ISA-Tab files represent the information needed to recapitulate the findings of the corresponding scientific publications and perform additional analyses. For regulatory purposes, additional and different information would be needed. For example, animals or samples excluded from analysis would need to be reported. Additionally, file formats like SEND and OECD harmonized templates (HT) require descriptions of file formats, which are not included in the ISA-Tab standard. Defined templates for data and metadata are only available for a limited number of endpoints, even though work is in progress to increase the scope of SEND format to for example in vitro testing. The focus of SEND files is on the data and its annotation using controlled terminology. ISA-Tab files focus more on the protocols used and metadata, and one could imagine combining both standards for a full description of the data, metadata, and protocols. Therefore, we are presently investigating extensions to the ISA-Tab templates and data infrastructure to facilitate reporting of metadata and file generation in the needed formats (whether SEND, OECD HT, or any other emerging reporting standard) on the fly.\n\nIn addition to data integration, validation, and sharing to facilitate cross-study analysis, it is important to assimilate, mine and filter relevant data and facilitate expert reviews on multiple channels of information. As illustrated schematically in Figure 9, data accumulated and assimilated from multiple sources beyond experimental data and publications can be integrated and mined through machine-learning and text-mining algorithms to identify and visualize scientifically relevant information and nominate experts for reviews. In the future, such an intelligent knowledge-mining system with cloud-based visualization and a search interface will power this systems toxicology platform.\n\n\nConclusions\n\nOur reported data-management method employs the latest standards of data sharing and reproducible research. The data and methods curated and prepared in ISA-Tab format, fit for review by scientists, are stored in a database that can be accessed via a search portal on the website. The portal allows browsing data and information related to study design, materials and methods, and key results by either study or mechanism (e.g., inflammation or oxidative stress). A future update is planned to provide analytics for scientists and regulators to explore the data.\n\nThe platform is also intended to foster collaboration in the development of assays for the assessment of the future potential MRTPs. Therefore, protocols are described in detail, and future update of the platform will allow versioning and commenting of protocols.\n\nGiven the successful development of the initial infrastructure, our goal is to grow this initiative into a public repository for 21st-century preclinical systems toxicology assessment data for MRTPs following the Morven Core Principles14, as described above.\n\nWe hope that the infrastructure reported in this paper sparks interest and encourages other industries and institutions producing data relevant to MRTPs to submit their data to this repository for the benefit of the entire community.\n\n\nData availability\n\nThe data can be browsed and downloaded from the website https://systox.sbvimprover.com/.",
"appendix": "Author contributions\n\n\n\nSB, TE, VB, JD, FB, DP and BH conceived and built the platform and web portal. SG conceived and managed the building of the system for knowledge mining and visualization. JH, PV and MCP conceived the idea and managed the project. AB reviewed existing resources and built Table 1. All authors wrote the manuscript text and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nAll authors are employees of Philip Morris International, Douglas Connect, or SBX Corporation and performed this work under a joint research collaboration funded by Philip Morris International.\n\n\nGrant information\n\nThe research described in this article was funded by Philip Morris International.\n\n\nAcknowledgements\n\nThe authors thank Anouk Ertan and Laure Cannesson for their help in project management and manuscript preparation. The authors also thank William Hayes, Kelly McCann and Mike Maria who contributed to the development of the online platform.\n\n\nSupplementary material\n\nSupplementary File 1: List of ontologies used in the ISA-Tab files describing in vivo and in vitro studies.\n\nClick here to access the data.\n\n\nReferences\n\nNational Center for Chronic Disease Prevention and Health Promotion (US) Office on Smoking and Health: The Health Consequences of Smoking—50 Years of Progress: A Report of the Surgeon General.Reports of the Surgeon General. 2014. PubMed Abstract\n\nFamily Smoking Prevention and Tobacco Control Act. Public Law No. 111–131; 2009. Reference Source\n\nSmith MR, Clark B, Lüdicke F, et al.: Evaluation of the Tobacco Heating System 2.2. Part 1: Description of the system and the scientific assessment program. Regul Toxicol Pharmacol. 2016; 81(Suppl 2): S17–S26. PubMed Abstract | Publisher Full Text\n\nFoods and Drugs Administration: 81 FR 58341 - Good laboratory Practice for Nonclinical Laboratory Studies.2016. Reference Source\n\nRabesandratana T: REGULATORY SCIENCE. Europe's food watchdog embraces transparency. Science. 2015; 350(6259): 368. PubMed Abstract | Publisher Full Text\n\nBegley CG, Ioannidis JP: Reproducibility in science: improving the standard for basic and preclinical research. Circ Res. 2015; 116(1): 116–126. PubMed Abstract | Publisher Full Text\n\nCouchman JR: Peer review and reproducibility. Crisis or time for course correction? J Histochem Cytochem. 2014; 62(1): 9–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDrubin DG: Great science inspires us to tackle the issue of data reproducibility. Mol Biol Cell. 2015; 26(21): 3679–3680. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrye SV, Arkin MR, Arrowsmith CH, et al.: Tackling reproducibility in academic preclinical drug discovery. Nat Rev Drug Discov. 2015; 14(11): 733–734. PubMed Abstract | Publisher Full Text\n\nGaudart J, Huiart L, Milligan PJ, et al.: Reproducibility issues in science, is P value really the only answer? Proc Natl Acad Sci U S A. 2014; 111(19): E1934. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIorns E, Chong C: New forms of checks and balances are needed to improve research integrity [version 1; referees: 2 approved, 1 not approved]. F1000Res. 2014; 3: 119. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNature Editorial: Announcement: Reducing our irreproducibility. Nature. 2013; 496: 398. Publisher Full Text\n\nMcNutt M: Journals unite for reproducibility. Science. 2014; 346(6210): 679. PubMed Abstract | Publisher Full Text\n\nMorven Dialogues: Core Principles Concerning the Implementation of Effective and Workable Tobacco, Nicotine, and Alternative Products Policies for Reducing Disease and Death from Tobacco Use.2015. Reference Source\n\nKryshtafovych A, Moult J, Bales P, et al.: Challenging the state of the art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10. Proteins. 2014; 82(Suppl 2): 26–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHirschman L, Yeh A, Blaschke C, et al.: Overview of BioCreAtIvE: critical assessment of information extraction for biology. BMC Bioinformatics. 2005; 6(Suppl 1): S1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStolovitzky G, Monroe D, Califano A: Dialogue on reverse-engineering assessment and methods: the DREAM of high-throughput pathway inference. Ann N Y Acad Sci. 2007; 1115: 1–22. PubMed Abstract | Publisher Full Text\n\nMeyer P, Alexopoulos LG, Bonk T, et al.: Verification of systems biology research in the age of collaborative competition. Nat Biotechnol. 2011; 29(9): 811–5. PubMed Abstract | Publisher Full Text\n\nSaez-Rodriguez J, Costello JC, Friend SH, et al.: Crowdsourcing biomedical research: leveraging communities as innovation engines. Nat Rev Genet. 2016; 17(8): 470–486. PubMed Abstract | Publisher Full Text\n\nMajeed S, Frentzel S, Wagner S, et al.: Characterization of the Vitrocell® 24/48 in vitro aerosol exposure system using mainstream cigarette smoke. Chem Cent J. 2014; 8(1): 62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSturla SJ, Boobis AR, FitzGerald RE, et al.: Systems toxicology: from basic research to risk assessment. Chem Res Toxicol. 2014; 27(3): 314–329. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoeng J, Deehan R, Pratt D, et al.: A network-based approach to quantifying the impact of biologically active substances. Drug Discov Today. 2012; 17(9–10): 413–418. PubMed Abstract | Publisher Full Text\n\nNational Research Council: Toxicity Testing in the 21st Century: A Vision and a Strategy.2007. Reference Source\n\nHartung T: Lessons learned from alternative methods and their validation for a new toxicology in the 21st century. J Toxicol Environ Health B Crit Rev. 2010; 13(2–4): 277–290. PubMed Abstract | Publisher Full Text\n\nKrewski D, Acosta D Jr, Andersen M, et al.: Toxicity testing in the 21st century: a vision and a strategy. J Toxicol Environ Health B Crit Rev. 2010; 13(2–4): 51–138. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrganisation for Economic Co-operation and Development: Guidance document on developing and assessing adverse outcome pathways. Series on Testing and Assessment. 2013; 184. Reference Source\n\nHorvat T, Landesmann B, Lostia A, et al.: Adverse outcome pathway development from protein alkylation to liver fibrosis. Arch Toxicol. 2016; 1–21. PubMed Abstract | Publisher Full Text\n\nAnsari S, Baumer K, Boué S, et al.: Comprehensive systems biology analysis of a 7-month cigarette smoke inhalation study in C57BL/6 mice. Sci Data. 2016; 3: 150077. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKogel U, Schlage WK, Martin F, et al.: A 28-day rat inhalation study with an integrated molecular toxicology endpoint demonstrates reduced exposure effects for a prototypic modified risk tobacco product compared with conventional cigarettes. Food Chem Toxicol. 2014; 68: 204–217. PubMed Abstract | Publisher Full Text\n\nKogel U, Gonzalez Suarez I, Xiang Y, et al.: Biological impact of cigarette smoke compared to an aerosol produced from a prototypic modified risk tobacco product on normal human bronchial epithelial cells. Toxicol In Vitro. 2015; 29(8): 2102–2115. PubMed Abstract | Publisher Full Text\n\nPhillips B, Veljkovic E, Peck MJ, et al.: A 7-month cigarette smoke inhalation study in C57BL/6 mice demonstrates reduced lung inflammation and emphysema following smoking cessation or aerosol exposure from a prototypic modified risk tobacco product. Food Chem Toxicol. 2015; 80: 328–345. PubMed Abstract | Publisher Full Text\n\nHoeng J, Talikka M, Martin F, et al.: Case study: the role of mechanistic network models in systems toxicology. Drug Discov Today. 2014; 19(2): 183–192. PubMed Abstract | Publisher Full Text\n\nHardy B, Douglas N, Helma C, et al.: Collaborative development of predictive toxicology applications. J Cheminform. 2010; 2(1): 7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHardy B, Apic G, Carthew P, et al.: Toxicology ontology perspectives. ALTEX. 2012; 29(2): 139–156. PubMed Abstract | Publisher Full Text\n\nMiller GW: Data sharing in toxicology: beyond show and tell. Toxicol Sci. 2015; 143(1): 3–5. PubMed Abstract | Publisher Full Text\n\nJudson R, Richard A, Dix DJ, et al.: The toxicity data landscape for environmental chemicals. Environ Health Perspect. 2009; 117(5): 685–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavis AP, Murphy CG, Saraceni-Richards CA, et al.: Comparative Toxicogenomics Database: a knowledgebase and discovery tool for chemical-gene-disease networks. Nucleic Acids Res. 2009; 37(Database issue): D786–792. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLea IA, Gong H, Paleja A, et al.: CEBS: a comprehensive annotated database of toxicological data. Nucleic Acids Res. 2016; pii: gkw1077. PubMed Abstract | Publisher Full Text\n\nMeyer P, Alexopoulos LG, Bonk T, et al.: Verification of systems biology research in the age of collaborative competition. Nat Biotechnol. 2011; 29(9): 811–815. PubMed Abstract | Publisher Full Text\n\nIskandar AR, Mathis C, Martin F, et al.: 3-D nasal cultures: Systems toxicological assessment of a candidate modified-risk tobacco product. ALTEX. 2016. In press. PubMed Abstract | Publisher Full Text\n\nZanetti F, Sewer A, Mathis C, et al.: Systems Toxicology Assessment of the Biological Impact of a Candidate Modified Risk Tobacco Product on Human Organotypic Oral Epithelial Cultures. Chem Res Toxicol. 2016; 29(8): 1252–69. In press. PubMed Abstract | Publisher Full Text\n\nAdeleye Y, Andersen M, Clewell R, et al.: Implementing Toxicity Testing in the 21st Century (TT21C): Making safety decisions using toxicity pathways, and progress in a prototype risk assessment. Toxicology. 2015; 332: 102–111. PubMed Abstract | Publisher Full Text\n\nJaworska J, Hoffmann S: Integrated Testing Strategy (ITS) - Opportunities to better use existing data and guide future testing in toxicology. ALTEX. 2010; 27(4): 231–242. PubMed Abstract | Publisher Full Text\n\nFood and Drug Administration F: Guidance for Industry.2012.\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSansone SA, Rocca-Serra P, Brandizi M, et al.: The first RSBI (ISA-TAB) workshop: \"can a simple format work for complex studies?\". OMICS. 2008; 12(2): 143–149. PubMed Abstract | Publisher Full Text\n\nYounesi E, Ansari S, Guendel M, et al.: CSEO - the Cigarette Smoke Exposure Ontology. J Biomed Semantics. 2014; 5: 31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGhosh S, Matsuoka Y, Asai Y, et al.: Software for systems biology: from tools to integrated platforms. Nat Rev Genet. 2011; 12(12): 821–832. PubMed Abstract | Publisher Full Text\n\nEddy DM: Evidence-based medicine: a unified approach. Health Aff (Millwood). 2005; 24(1): 9–17. PubMed Abstract | Publisher Full Text\n\nStephens ML, Andersen M, Becker RA, et al.: Evidence-based toxicology for the 21st century: opportunities and challenges. ALTEX. 2013; 30(1): 74–103. PubMed Abstract | Publisher Full Text\n\nGhosh S, Matsuoka Y, Asai Y, et al.: Toward an integrated software platform for systems pharmacology. Biopharm Drug Dispos. 2013; 34(9): 508–526. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDoe JE, Lander DR, Doerrer NG, et al.: Use of the RISK21 roadmap and matrix: human health risk assessment of the use of a pyrethroid in bed netting. Crit Rev Toxicol. 2016; 46(1): 54–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJaworska JS, Natsch A, Ryan C, et al.: Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment: a decision support system for quantitative weight of evidence and adaptive testing strategy. Arch Toxicol. 2015; 89(12): 2355–2383. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "21168",
"date": "22 Mar 2017",
"name": "Winston A. Hide",
"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\nBoué et al. have built a toxicology data-sharing infrastructure for assessing modified risk tobacco products (MRTPs). They have described used best practice guidelines\n\nOn the INTERVALS website, the authors have exhaustively described the methods of each experiment under the “studies” section. At the end of the page is a results section where a huge amount of data is presented under many tabs which need to be selected in order to see the data. While the description of the methods is immensely important, the focus should surely be around the individual chemical being analysed or at least the results of the study. For example, there is no easy way to observe all the results for THS2.2. I would suggest either removing the use of tabs or, better yet, moving all results to their own section on the website (without the use of tabs) under the name of the MRTP so that all the results are easy to browse with links back to the methods used. This would enable the user to easily examine their MRTP of interest.\n\nAgain on the “data” section, where the data is available to download, the authors have focused filtering by methods rather than filtering by MRTP. If someone wants to assess the potential of THS2.2, they would want to view all the results for this chemical. Secondly, if possible, a feature enabling the user to download multiple datasets (instead of clicking on 37 links for THS2.2) would be incredibly useful.\n\nThe authors have highlighted the importance of the searchability of the data yet they have constrained their own search engine by the terms listed on the “data” section. Currently, you cannot search for any information contained within the metadata. The authors should think about including this in the future.\n\nThe use of “pMRTP” is very confusing and badly named. Especially given that the website glossary definition of it is “See MRTP definition” which mentions nothing of pMRTP. I understand that the original publication on “pMRTP” uses this name but it’s very unclear if this is a chemical/method of heating. If named such, is it an accepted MRTP? As far as I am aware, the FDA has not approved any MRTPs. On the website, the “pMRTP - 7-month systems toxicology inhalation / cessation study in C57BL6 mice” study also fails to mention what it is. A clearer definition is needed on all pages of the website and for the first mention in the paper.\n\nThe authors have turned their ISA-TAB files to excel format. This is prone to excel-specific errors, for example, turning number entries into dates. Why not use the actual ISA-TAB format?\n\nPage 10: “Therefore, herein, we propose a new ISA-Tab scheme that follows the data-production workflow and combines data production and documentation into a single step by the researcher, who is the expert on the dataset and experimental parameters.”\nThe authors have proposed this solution to the fact that creating ISA-TAB files is “complex, time-consuming, and error-prone”. This doesn’t appear to solve the issue fully. How do the authors expect to convince researchers to fill out ISA-TAB files as the experiment is ongoing? ISA-TAB files are complex and each researcher needs training on how to complete these correctly. While the authors have described how to complete ISA-TAB files, it is not easy to perform. They have also suggested ontologies to use but not the standardised terms to use within these ontologies. Each researcher might use different terms. Are the authors going to check over all the ISA-TAB files themselves? When the authors mention “extensions to the ISA-TAB templates”, are they going to provide a web template which creates the ISA-TAB files for the user? Additionally, the suggested ontologies should also be listed on the website.\nPage 14: “The first step in this direction is the development of a data input and management application consisting of several validated web forms and lists with filtering/searching features connected to the uploaded datasets”.\nPlease describe how these were validated. Where are the web forms located on the website? Are these for premade ISA-TAB files or for creating the ISA-TAB files? Please explain more.\n\nWho can add data to the website? There is no option for new members to join, or upload data, will this change in the future?\n\nThere are no publication references on the “data” section of the website. If possible, it would be better to include this.\n\nThere are no obvious explanations of the headers within the downloadable data yet a number of abbreviations are used. Please think about changing this as soon as possible.",
"responses": [
{
"c_id": "2989",
"date": "05 Sep 2017",
"name": "Stephanie Boue",
"role": "Author Response",
"response": "1. On the INTERVALS website, the authors have exhaustively described the methods of each experiment under the “studies” section. At the end of the page is a results section where a huge amount of data is presented under many tabs which need to be selected in order to see the data. While the description of the methods is immensely important, the focus should surely be around the individual chemical being analysed or at least the results of the study. For example, there is no easy way to observe all the results for THS2.2. I would suggest either removing the use of tabs or, better yet, moving all results to their own section on the website (without the use of tabs) under the name of the MRTP so that all the results are easy to browse with links back to the methods used. This would enable the user to easily examine their MRTP of interest.Author’s response: Thanks for the comment, which is very relevant, and would become even more so as the platform grows and the number of studies increases. Since the publication of the first version of this manuscript, we have updated the prototype platform and are building a new platform that will enable easier access to all relevant pieces of information and should replace the current prototype, at the same URL, by the end of the year.A major update is the focus on the test item that has been added to the studies section. It is now easier to find all studies related to a specific test item. Results are, in the prototype, still in tabs, but we have taken the comment into account for the building of the new platform and will add tags and facet search to ease retrieval of results and protocols of interest. 2. Again on the “data” section, where the data is available to download, the authors have focused filtering by methods rather than filtering by MRTP. If someone wants to assess the potential of THS2.2, they would want to view all the results for this chemical. Secondly, if possible, a feature enabling the user to download multiple datasets (instead of clicking on 37 links for THS2.2) would be incredibly useful.Author’s response: Thanks for the comment. Again, this change will be addressed in the new platform. We are also working on the addition of analytical tools directly on the platform to ease data visualization and analysis. 3. The authors have highlighted the importance of the searchability of the data yet they have constrained their own search engine by the terms listed on the “data” section. Currently, you cannot search for any information contained within the metadata. The authors should think about including this in the future.Author’s response: Thanks for the very valuable comment. Again, this change will be addressed in the new platform. In addition to the facets that will be available for easy filtering, the user will have the option to search for text in a number of fields of the datasets. 4. The use of “pMRTP” is very confusing and badly named. Especially given that the website glossary definition of it is “See MRTP definition” which mentions nothing of pMRTP. I understand that the original publication on “pMRTP” uses this name but it’s very unclear if this is a chemical/method of heating. If named such, is it an accepted MRTP? As far as I am aware, the FDA has not approved any MRTPs. On the website, the “pMRTP - 7-month systems toxicology inhalation / cessation study in C57BL6 mice” study also fails to mention what it is. A clearer definition is needed on all pages of the website and for the first mention in the paper.Author’s response: Thanks for the comment. MRTP stands for modified risk tobacco product, as defined in the abstract and introduction of the publication. It is the term chosen by the FDA to refer to products ‘sold or distributed to reduce the harm or risk of tobacco-related disease associated with commercially marketed tobacco products’. The study mentioned pMRTP where the p stands for prototype, as at the time when it was assessed, the product was not sold nor distributed.Moreover, we have updated the website to refer to reduced risk products (RRPs) instead as this is the term we use to refer to products that present, are likely to present, or have the potential to present less risk of harm to smokers who switch to these products versus continued smoking. We have a range of RRPs in various stages of development, scientific assessment and commercialization. Because our RRPs do not burn tobacco, they produce far lower quantities of harmful and potentially harmful compounds than found in cigarette smoke. We have included the definition in the glossary and ensured that the definition is easily accessible when the term is encountered initially on the website (pop-up with definition on mouse-over).5. The authors have turned their ISA-TAB files to excel format. This is prone to excel-specific errors, for example, turning number entries into dates. Why not use the actual ISA-TAB format?Author’s response: Thanks for the comment. We have decided to split the ISA-TAB files by endpoint and stage of the study (starting material, data production, data processing…) to allow easier recording of data and metadata as the experiment is performed by the scientists and technicians in charge. The splitting of the ISA-TAB files by endpoint also allows easier interpretation and reuse of data by others, but it results in a large number of tabs, which would all be separated files when using the CSV format as proposed in the ISA-TAB standard. Using the multiple tab format of Excel allows for the grouping of all tabs belonging to a specific endpoint and, in this way, easier data handling for the end user. Additionally, by the careful preparation of the data files in Excel, we were able to avoid all Excel-specific errors occurring during the automatic conversion into the XLS format during opening CSV files in Excel, a problem correctly stressed by the reviewer. Since Excel is very often used in the domain, we hope to help avoiding these errors by directly providing XLS file, which can be easily concerted to CSV files if needed.6. Page 10:“Therefore, herein, we propose a new ISA-Tab scheme that follows the data-production workflow and combines data production and documentation into a single step by the researcher, who is the expert on the dataset and experimental parameters.”The authors have proposed this solution to the fact that creating ISA-TAB files is “complex, time-consuming, and error-prone”. This doesn’t appear to solve the issue fully. How do the authors expect to convince researchers to fill out ISA-TAB files as the experiment is ongoing? ISA-TAB files are complex and each researcher needs training on how to complete these correctly. While the authors have described how to complete ISA-TAB files, it is not easy to perform.They have also suggested ontologies to use but not the standardised terms to use within these ontologies. Each researcher might use different terms. Are the authors going to check over all the ISA-TAB files themselves? When the authors mention “extensions to the ISA-TAB templates”, are they going to provide a web template which creates the ISA-TAB files for the user? Additionally, the suggested ontologies should also be listed on the website.Page 14:“The first step in this direction is the development of a data input and management application consisting of several validated web forms and lists with filtering/searching features connected to the uploaded datasets”.Please describe how these were validated. Where are the web forms located on the website? Are these for premade ISA-TAB files or for creating the ISA-TAB files? Please explain more.Author’s response: We apologize that the wording here was not clear enough. The application only facilitates the upload of pre-generated ISA-TAB files and the assignment of facets for the search/browse features. This is now clearly described in the manuscript. We also removed the somewhat misleading “validated” in front of “web forms”.The list of ontologies we recommend can be found in this publication as supplementary Excel file 1 and will be available from the platform once the data upload will be available for non-PMI users.7. Who can add data to the website? There is no option for new members to join, or upload data, will this change in the future?Author’s response: Thanks for the very valuable comment. This publication describes indeed the prototype and ideas important for the sharing of data. We are currently building a new platform that will allow scientists who experimented on potential RRPs to share their protocols, their data and results. The platform with necessary features should be launched by the end of the year. 8. There are no publication references on the “data” section of the website. If possible, it would be better to include this.Author’s response: Thanks for the comment. It is not fully clear which references should be added to the data section of the website. References important for specific studies can be found in the study pages. Those important for the protocols are found in the respective material and methods sections.We acknowledge it would be great to have a single ID associated to datasets so they can be cited on the platform and are evaluating this possibility. 9. There are no obvious explanations of the headers within the downloadable data yet a number of abbreviations are used. Please think about changing this as soon as possible.Author’s response: Thanks for the comment. We have, to some extent, reworked the files so they are more self-explanatory. Schema files and/or readme files will be added consistently to the dataset archives by the end of the year (on the new platform)."
}
]
},
{
"id": "24812",
"date": "14 Aug 2017",
"name": "Katharine Briggs",
"expertise": [
"Reviewer Expertise Data sharing"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHere are my comments on this article:\nPage 3: “Concerns on reproducibility of science have lead to recent calls for a shift to better practices” – correct to ‘led’ Page 3: “Building and quantifying AOPs requires multiscale integration of all available and relevant -omics datasets,” – drop the ‘-omics’ Page 7: “The table highlights sources of information on in vivo chemical inhalation and individual in vitro chemical toxicity. The type of data available and user accessibility (e.g., open source vs licensing) have also been highlighted.” – User accessibility is not provided in all cases so I suggest adding ‘where known’ to caption Page 8: “and allapplications need to employ sound, reproducible scientific bases and good practices” – please correct to ‘and all applications’ and ‘basis’ Page 9: “Experiments are planned and protocols as well as metadata are recorded for each of the data entry and curated in ISA-Tab files.” – Consider rewording of the Figure 2 caption as meaning is unclear Page 9: “…on organotypic acute human buccal or nasal tissue cultures (i.e., the organotypic buccal and nasal datasets, respectively) …three dimensional (3-D) organotypic buccal and nasal epithelial culture models …in vitro human 3-D buccal or nasal epithelial culture …in vitro human 3-D buccal or nasal epithelial culture” – Consider rewording point 2) as text is repetitive Page 14: “Although this will include formal checks of files’ correctness with respect to the ISA-Tab standard (as already performed, e.g., by ISACreator), equipment data, log files, already-available databases, and computational infrastructure will be interfaced to provide the needed information at least partially.” – Consider rewording as meaning is unclear Page 15: “researchers need to focus on the biological small data” – What is meant by biological small data? Conclusion: “the platform will allow versioning and commenting of protocols” – clarification is needed on the term protocol; is this study protocols or data analysis protocols? Page 9: “Figure 9. Concepts of an intelligent, knowledge mining and visualization platform for systems toxicology.” – Clarification needed on the link between Figure 9 and the contents of this article Page 18: Reference Source – the link to reference 14 is not working Page 19: “Meyer P, Alexopoulos LG, Bonk T, et al.: Verification of systems biology research in the age of collaborative competition. Nat Biotechnol. 2011; 29(9): 811–5.” – Reference 39 is a duplicate of reference 18\n\nIn the Supplementary data:\nThe MESH & LOINC entries are missing a description “A vocabulary for clinical care, translational and basic research, and public information and administrative activities.” - … and basic research, public information and administrative activities. “Mainly because of the emerging need of systems toxicology to controlled vocabularies and also the lack of suitable ontologies for this domain, the CSEO prepares the ground for integrative systems-based research in the exposure science.” - …emerging need of systems toxicology for controlled vocabularies… in exposure science. “Metrical units for use in conjunction with PATO” – What is PATO? “ A structured classification of chemical compounds of biological relevance.” – Remove space before A “The set of standardized ontologies used to define the domain-specific knowledge are found in Table 1” – Where is the Table 1 referred to? “Ontology developed to harmonize the toxicology datasets from the pharmaceutical industry made available in the eTox project and allow for comparative data-mining across multiple databases to test hypotheses” - …harmonize the nonclinical toxicology datasets… and allows comparative… multiple datasets to test hypotheses\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": "2988",
"date": "05 Sep 2017",
"name": "Stephanie Boue",
"role": "Author Response",
"response": "Page 3: “Concerns on reproducibility of science have lead to recent calls for a shift to better practices” – correct to ‘led’ Author’s response: Thanks for the comment, this has been corrected Page 3: “Building and quantifying AOPs requires multiscale integration of all available and relevant -omics datasets,” – drop the ‘-omics’ Author’s response: Thanks for the comment, this has been corrected Page 7: “The table highlights sources of information on in vivo chemical inhalation and individual in vitro chemical toxicity. The type of data available and user accessibility (e.g., open source vs licensing) have also been highlighted.” – User accessibility is not provided in all cases so I suggest adding ‘where known’ to caption Author’s response: Thanks for the comment, this has been added Page 8: “and allapplications need to employ sound, reproducible scientific bases and good practices” – please correct to ‘and all applications’ and ‘basis’ Author’s response: Thanks for the comment. The original word seems fine, we will make sure the justification in the final document doesn’t impair reading. Page 9: “Experiments are planned and protocols as well as metadata are recorded for each of the data entry and curated in ISA-Tab files.” – Consider rewording of the Figure 2 caption as meaning is unclear Author’s response: Thanks for the comment. The sentence has been modified and hopefully clearer now. Page 9: “…on organotypic acute human buccal or nasal tissue cultures (i.e., the organotypic buccal and nasal datasets, respectively) …three dimensional (3-D) organotypic buccal and nasal epithelial culture models …in vitro human 3-D buccal or nasal epithelial culture …in vitro human 3-D buccal or nasal epithelial culture” – Consider rewording point 2) as text is repetitive Author’s response: Thanks for the comment. The paragraph has been modified. Page 14: “Although this will include formal checks of files’ correctness with respect to the ISA-Tab standard (as already performed, e.g., by ISACreator), equipment data, log files, already-available databases, and computational infrastructure will be interfaced to provide the needed information at least partially.” – Consider rewording as meaning is unclear Author’s response: Thanks for the comment. The paragraph has been modified. Page 15: “researchers need to focus on the biological small data” – What is meant by biological small data? Author’s response: Thanks for the comment. We have tried to explain this concept, namely that on itself the biological data may seem insignificant, but when analyzed in the right context and with the right tools contributes meaningful information. Conclusion: “the platform will allow versioning and commenting of protocols” – clarification is needed on the term protocol; is this study protocols or data analysis protocols? Author’s response: Thanks for the comment. Any protocol on the platform, from study setup to data generation and analysis will be versioned and open for commenting. We explicated this in the text. Page 9: “Figure 9. Concepts of an intelligent, knowledge mining and visualization platform for systems toxicology.”– Clarification needed on the link between Figure 9 and the contents of this article Author’s response: The authors thank the reviewer for the point raised. The section related to Fig.9 lays out the future application of the data platform outlined in this phase, wherein the ability to mine the data and correlate with existing data and knowledge will play a critical role in generating valuable insights from the data. Thus, the section outlined our vision and plan to further enhance the data platform with an intelligent system which automatically integrates and mines the multi-dimensional data through machine-learning and text-mining algorithms to identify and visualize scientifically relevant information and nominate experts for reviews. The relevant section has been updated to clarify and elucidate the role of the intelligent system, built on top of the data platform, as depicted in Fig. 9. Page 18: Reference Source – the link to reference 14 is not working Author’s response: Thanks for the comment. The link has been changed. Page 19: “Meyer P, Alexopoulos LG, Bonk T, et al.: Verification of systems biology research in the age of collaborative competition. Nat Biotechnol. 2011; 29(9): 811–5.” – Reference 39 is a duplicate of reference 18 Author’s response: Thanks for the comment. The duplicated reference has been removed. In the Supplementary data: The MESH & LOINC entries are missing a description Author’s response: Thanks for the comment. A description has been added. “A vocabulary for clinical care, translational and basic research, and public information and administrative activities.” - … and basic research, public information and administrative activities. “Mainly because of the emerging need of systems toxicology to controlled vocabularies and also the lack of suitable ontologies for this domain, the CSEO prepares the ground for integrative systems-based research in the exposure science.” - …emerging need of systems toxicology for controlled vocabularies… in exposure science. Author’s response: Thanks for the comment, this has been corrected. “Metrical units for use in conjunction with PATO” – What is PATO? Author’s response: Thanks for the comment, the acronym was explained (Phenotype And Trait Ontology) “ A structured classification of chemical compounds of biological relevance.” – Remove space before A Author’s response: Thanks for the comment, this has been corrected. “The set of standardized ontologies used to define the domain-specific knowledge are found in Table 1” – Where is the Table 1 referred to? Author’s response: Thanks for the comment, this has been corrected. “Ontology developed to harmonize the toxicology datasets from the pharmaceutical industry made available in the eTox project and allow for comparative data-mining across multiple databases to test hypotheses” - …harmonize the nonclinical toxicology datasets… and allows comparative… multiple datasets to test hypotheses Author’s response: Thanks for the comment, this has been corrected."
}
]
}
] | 1
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https://f1000research.com/articles/6-12
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https://f1000research.com/articles/6-1636/v1
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04 Sep 17
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{
"type": "Research Article",
"title": "Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy",
"authors": [
"Chris D. Balak",
"Jesse M. Hunter",
"Mary E. Ahearn",
"David Wiley",
"Gennaro D'urso",
"Lisa Baumbach-Reardon",
"Chris D. Balak",
"Jesse M. Hunter",
"Mary E. Ahearn",
"David Wiley",
"Gennaro D'urso"
],
"abstract": "Background: X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 (UBA1). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the UBA1 gene, which contains the active adenylation domain (AAD). Methods: In this study, our group characterized the three known missense variants in vitro. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions in vitro to determine possible biochemical effects of the missense variants. Results: Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. Conclusions: Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1’s enzymatic activity with Ubiquitin, to other roles such as altering UBA1 mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important in vivo binding partners. These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations’ biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.",
"keywords": [
"X-linked spinal muscular atrophy",
"SMAX2",
"UBA1",
"ubiquitination",
"ubiquitin proteasome system",
"disease mechanisms"
],
"content": "Introduction\n\nSpinal muscular atrophies (SMA) are a group of genetic disorders characterized by the degeneration of lower motor neurons, resulting in moderate to severe muscle wasting and weakness (Cifuentes-Diaz et al., 2002; Wee et al., 2010). The majority of documented cases are the result of genetic deletion events in the Survival of Motor Neuron 1, Telomeric (SMN1) gene, which plays a crucial role in snRNP biogenesis and is key in mRNA processing and metabolism. This results in SMN1s deficiency in cells and in particular motor neurons (Cifuentes-Diaz et al., 2002; Wee et al., 2010; Woo et al., 2017). In 2008, a second gene was discovered as the cause of a rare, X-linked infantile form of SMA (XL-SMA) only affecting males. This more severe form results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 (UBA1) gene, whose protein product is the pinnacle enzyme in the ubiquitin proteasome system (UPS). This X-linked form of SMA presents very similarly to the classical Type 1 SMA (Werdnig-Hoffmann disease) phenotype, including profound muscle weakness, hypotonia, muscle atrophy, anterior horn cell loss, evidence of denervation by electromyogram (EMG), as well as neurogenic atrophy by muscle biopsy. However, additional phenotypic features of XL-SMA can include congenital hypotonia, multiple congenital contractures (arthrogryposis) +/- bone fractures, myopathic facies, undescended testes and early mortality by the end of the infancy stage (Ramser et al., 2008).\n\nIn cells, the rapid disassembly and recycling of proteins is equally as important as their synthesis, and essential for maintaining cellular and protein homeostasis. Studies on global protein turnover rates have produced average protein half-lives of only 24–48 hours in mammals (Cambridge et al., 2011; Toyama et al., 2013) and breakdowns in this homeostasis give rise to a broad range of disorders including a large subset of neurodegenerative disorders (Hetz & Glimcher, 2011). The cell’s principle proteolytic mechanism responsible for this assembly and disassembly is the ubiquitin-proteasome system (UPS). The Uba1 enzyme initiates the UPS cascade by activating the small protein Ubiquitin which is used in large part as a molecular “death tag” for target proteins. Additionally, Uba1 has shown to be involved in other essential roles such as regulation of cell cycle progression (Joo et al., 2007) and neuron development and function (Rinetti & Schweizer, 2010).\n\nUbiquitination, or the post-translational modification of attaching ubiquitin molecules to target proteins, is the ultimate goal for the ubiquitin-proteasome proteolytic pathway leading up to direct degradation by the 26s proteasome. It consists of three main steps; each with their own key enzymes, and has been described in detail in several studies (Cohen-Kaplan et al., 2016; Dohmen et al., 1995; Glickman & Ciechanover, 2002). Uba1 sits at the pinnacle of the UPS ubiquitination cascade, initiating a series of complex and well-regulated steps that are common to all known processes involving Ubiquitin conjugation (Haas & Rose, 1982; Haas et al., 1982). The first step is the activation of ubiquitin by Uba1. Free Ubiquitin in the cell is adenylated by Uba1 in the active adenylation domain (AAD) at the expense of ATP. This forms a tightly bound ubiquitin adenylate consisting of a high-energy bond between the C-terminal carboxylate of the ubiquitin and AMP, which is then immediately attacked by the catalytically active Cys632 and transferred to the second catalytic cysteine domain (SCCD) forming a Uba1-Cys632 thioester bonded to Ubiquitin’s last amino acid Lys76. Ubiquitin activation is complete when a second ubiquitin is adenylated and loaded in the AAD forming the doubly-loaded, ternary Uba1 complex consisting of a Uba1~Ubiquitin thioester and primed Ubiquitin adenylate. The second step is the conjugation of Ubiquitin to an E2 enzyme. This takes place via a transthioesterification reaction between the catalytic cysteine of Uba1 and a catalytic cysteine on one of the 35 human E2 enzymes. The final step is the ligation of the now E2-bound Ubiquitin to its target protein. This is accomplished in conjunction with one of hundreds of human E3 ligase enzymes, which recognize their own set(s) of target proteins and complete ubiquitin ligation by generally catalyzing an isopeptide bond between a lysine of the target substrate and the C-terminal Gly76 of Ubiquitin. This E1-E2-E3 process is typically repeated until four Ubiquitin molecules are linked to the substrate via Lys48 residues, which is the signal used by the 26S proteasome to recognize and degrade the attached protein.\n\nUba1 is a highly conserved protein in all eukaryotes from both a sequence and functional aspect (Schäfer et al., 2014). Knockout of this gene is embryonic lethal in lower eukaryotic species (Kulkarni & Smith, 2008) and is presumed likewise in humans. Furthermore, UBA1 has a high intolerance to sequence variation. No homozygous or hemizygous loss-of-function (LoF) mutations exist in the genome Aggregation Database (gnomAD), the largest reference sequencing database containing over 123,000 exomes and 15,500 whole genomes highly enriched for healthy individuals. Missenses variants are equally rare in the general population, with UBA1’s 3000+ exonic bps only having three common coding variants with a global frequency of 1% or higher (Lek et al., 2016). UBA1 has two transcripts that result in two known isoforms in humans, Uba1a (118kDa) and Uba1b (110kDa), with the shorter isoform only lacking the first 40 amino acids containing a nuclear localization signal. Further mention of UBA1 in this manuscript will refer to the longest isoform including the NLS. Uba1 is made up of several domains including inactive and active adenylation domains (IAD, AAD), a Ubiquitin fold domain (UFD) as well as first and second catalytic cysteine half domains (FCCH, SCCH). An absolutely critical residue for Uba1’s adenylation activity is found in the ATP-Mg2+ binding site of the AAD at position 576, and mutation to an unreactive alanine residue results in near null rates of adenylation (Tokgöz et al., 2006). The equally important cysteine residue at 632 forms the covalent thioester bond with ubiquitin after adenylation.\n\nFour closely clustered mutations have been shown to cause infantile X-linked SMA (XL-SMA) (Dlamini et al., 2013; Dressman et al., 2007; Ramser et al., 2008). All four mutations, three missense and one synonymous, lie in exon 15 - the active adenylation domain (AAD) - of the UBA1 gene. The three missense mutations, p.M539I, p.S547G and p.E557V all lie closely together in the AAD. A fourth recurrent synonymous mutation, Uba1a c.1731C>T p.N577N, also lies within this region. This c.1731C>T variant is thought to be a methylation site and driving the recurrence of the C to T transition. It is anticipated that these mutations do not completely eliminate Uba1 function as this would almost certainly result in early embryonic lethality. The crystal structure of human Uba1 has yet to be determined, however the well-conserved homolog S. cerevisiae Uba1 has been resolved. The functional domains can be inferred as specific functions of Uba1 and have been confirmed in these animal systems. Uba1’s strict conservation throughout eukaryotes, specifically mammals, provides evidence these domains and functions of Uba1 are consistent in Homo sapiens.\n\n\nMaterials and methods\n\nHuman-recombinant (HR) Ubiquitin, HR Fluorescein-labeled Ubiquitin and all HR E2 enzymes were purchased from Boston Biochem. Restriction enzymes used for primary mutant colony selection were purchased from New England Biolabs or Life Technologies. Purification reagents inorganic pyrophosphate (PPi), adenosine monophosphate (AMP) and Ubiquitin-agarose utilized during Uba1 purification processes were purchased from Sigma-Aldrich. Assay reagents purine nucleoside phosphorylase (PNP), pyrophosphatase and 7-methylthioguanosine (MesG) were components of the EnzChek pyrophosphate assay kit derived from the coupled assay developed by Wilson and Aldrich. Additional assay components hydroxylamine, adenosine triphosphate (ATP) and iodoacetamide were purchased from Sigma-Aldrich.\n\nPlasmids (pENTR-D-TOPO) from Invitrogen’s Gateway system containing the coding nuclear form of the Homo Sapiens wild type (WT) UBA1 and XL-SMA missense mutant forms p.M539I and p.S547G were generously provided by Dr. Gennaro D’Urso from the University of Miami Miller School of Medicine. Wild type and mutant UBA1 coding sequences were excised from pENTR-D-TOPO and cloned into vector pDEST17 with a His-Patch thioredoxin tag in frame. Two additional mutants, the third and final XL-SMA missense variant p.E557V and an adenylation-handicapped control mutant p.D576A (Tokgöz et al., 2006), were generated from the pDEST17 Uba1 WT plasmid clones via the site-directed mutagenesis method of Liu et al (Liu & Naismith, 2008). Mutagenic forward and reverse primer sequences synthesized by Integrated DNA Technologies for the p.E557V mutant were 5’ GGTCCTGACACGGTGCGCATCTATGATGAC and 5’ GTCATCATAGATGCGCACCGTGTCAGGACC, respectively. For variant p.D576A, forward 5’ CCAATGCCCTGGCCAACGTGGATGCC and reverse 5’ GGCATCCACGTTGGCCAGGGCATTGG primers were used. After plating overnight, single colonies were selected for initial mutation screening by mutation-specific restriction enzymes overlapping mutagenesis sites. The pDEST17-Uba1aD576A mutant gained an additional HpaI (GTT’AAC) restriction site while pDEST17-Uba1aE557V gained an additional FspAI (rTGC’GCAy) restriction site. These plasmids were then propagated in NEB5α Escherichia coli cells and purified using either Zymo Research or Qiagen plasmid prep kits. Lastly, all positive mutant colonies were confirmed by Sanger sequencing at Arizona State University Biodesign Institute.\n\nPlasmids pDEST17-Uba1-WT, pDEST17-Uba1M539I, pDEST17-Uba1S547G, pDEST17-Uba1E557V and pDEST17-Uba1D576A were transformed into Rosetta 2(DE3) competent cells (Novagen). Single colonies were selected and grown in 750mL of LB containing both 100 mg/ml Ampicillin and Chloramphenicol at 37°C. At OD600≈ 0.8-0.9, expression of Uba1 protein was induced with 0.5mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16°C for 12 hrs. All of the following was performed at 4°C or on ice unless otherwise noted. Following induction, cells were collected by centrifugation (17000g) for 10mins. Cells were lysed via sonication with ice-cold 50mM HEPES + 150mM NaCl (pH8.0) (Buffer A) plus 0.5mM DTT, 0.1% TX-100 and protease inhibitor cocktail and subsequently centrifuged at 17,000xg for 30 minutes. Lysates were then combined with Mg2+ and ATP (Sigma) to final concentrations of 10mM and 2mM, respectively. These were immediately added to Ubiquitin-linked agarose bead (Sigma) columns at room temperature. Columns were then washed with 20 bed volumes of Buffer A. Elution of Uba1 enzyme was performed with three 10-minute column incubations containing 1mL Buffer A plus 2mM AMP and PPi. For Uba1D576A, affinity purification was unsuccessful as anticipated. Therefore, the inserted 6xHisTag was used in combination with Cobalt beads for purification in the same method as above, except 10mM and 200mM Imidazole were used for washes and elution respectively. All elutions were then combined and dialyzed three times at 4°C against 3 Liters of Buffer A plus 0.5mM DTT over a period of 18 hours. All phosphate buffers/reagents were avoided to eliminate chances of trace PPi and Pi contamination in subsequent assays.\n\nContinuous adenylation activity of wild type and mutant forms of the Uba1 enzyme were measured using the coupled hydroxamate-MesG method reported by Wilson and Aldrich (Wilson & Aldrich, 2010). The overall adenylation–acylation reaction of Uba1 generates one molecule of PPi at the expense of ATP per ubiquitin conjugated to the enzyme. Pyrophosphatase then hydrolyzes PPi into two inorganic phosphates, which are then coupled to phosphorolysis of 7-methylthioguanosine (MesG), catalyzed by the enzyme purine nucleoside PNP, and generating a chromophoric guanine derivative. Reactions totaling 150μl were set up in 96-well half-area UVStar plates (Greiner) containing 100nM WT or mutant Uba1 enzyme, 50 mM HEPES pH 8.0, 150mM hydroxylamine, 0.1U purine nucleoside phosphorylase, 0.15U pyrophosphatase, 200μM MESG and varying concentrations of Ubiquitin and MgATP. Uba1 concentrations were determined by either the Bradford BCA method or Pierce 660nm assay. Reactions were initiated at 37°C with either Ubiquitin or MgATP, at indicated concentrations. Cleavage of MesG into the chromophoric product, 7-methylguanine, was monitored at 360nm on a Synergy Max plate reader (BioTek). All buffers and reagents were phosphate and pyrophosphate free. Working stocks of hydroxylamine were prepared fresh daily by combining 4M hydroxylamine, 7M NaOH and dH2O on ice in a 2:1:1 ratio respectively. Hydroxylamine and NaOH stocks were stored at 4°C for at least 1 month, however freshly prepared stocks of these reagents were later made every 2 months due to the degradation of these reagents over time.\n\nThioester bond formation rates of WT and mutant Uba1 and Ubiquitin were measured by fluorescent (Fl) SDS-PAGE time-course assays containing fluorescein-labeled Ubiquitin. Reactions containing 50mM HEPES (pH8.0), 5mM ATP, 25mM MgCl2, and 300nM Uba1 were preheated to 37°C. Reactions were started by the addition of Fluorescein conjugated Ubiquitin (UbFl) to a final concentration of 3μM. At the indicated time points, 18.75μL aliquots were removed from master reaction and quenched in 6.25μl of 4X Laemmli loading buffer (Bio-Rad) containing 100mM β-mercaptoethanol (BME). It is important to note that disulfide bonds are reduced by BME but thiolester bonds are not. Reactions were heated to 95°C for 10 min and then run on Stain-Free Any-kD SDS-PAGE gels (Biorad) and imaged using a light engine (UVP) for 480nM excitation and 520nm emission filters on a UVP imaging system (UVP). Gels were then crosslinked by exposure to UV light for 2 minutes then washed 3X for 5 min. in deionized water and imaged using a UV Transilluminator with a SYBR Gold filter to measure total protein. Transthioesterification rates of Ub from Uba1 to wild type E2 enzyme UBE2E1 (formerly UbcH6) were measured in a similar assay. 50mM HEPES (pH8.0), 5mM ATP, 25mM MgCl2, 200nM Uba1, and 1.5μM UbcH6 were preheated to 37°C. Reactions were started by the addition of UbFl to a final concentration of 5μM. At the indicated time points, 14μL aliquots were removed from master reaction and quenched in 14μl of 2X Laemmli loading buffer (Bio-Rad) containing 50mM BME. Gels were imaged as above and bands were quantitated using ImageJ software (v. 1.48, (Schneider et al., 2012). Total pixel intensity was measured for each band and background levels were subtracted and lane loads were normalized to total Uba1 protein.\n\n\nResults\n\nTo estimate the degree of sequence conservation in Uba1 and help elicit critical domain sequences surrounding the altered residues in patients with XL-SMA, the complete amino acid sequences of Uba1 enzymes were aligned and analyzed from a wide range of species using the Multalin program 5.4.1 (Corpet, 1988). Saccharomyces cerevisiae (yeast), Danio rerio (zebrafish) and five well-studied mammalian Uba1 Multalin aligments are shown in Figure 1A. Despite the wide range of species aligned, the conservation was 100% identical in three of four variants. The fourth p.S547G variant diverges only at the level of yeast and zebrafish. This lends evidence of the requirement that amino acids spanning the ADD remain unchanged to maintain proper enzyme function, and it is important to note mammalian conservation was 100% among all species aligned in this analysis. A diagram of the various inferred domains of Uba1 and the clustering of XL-SMA mutations is show in Figure 1B.\n\n(a) XL-SMA variant residues show strict amino acid sequence conservation in nearly all eukaryotes from yeast to modern man. (b) Schematic of the inferred domains of Uba1. Pathogenic variants and key residues are labeled. IAD = inactive adenylation domain, FCCH = first catalytic cysteine half domain, 4HB = 4 helix bundle, AAD = active adenylation domain, SCCH = second catalytic half domain. UFD = Ub fold domain. Known pathogenic variants marked with asterisk. Critical Adenylation and Thiolester residues are underlined. (c) 3D protein modeling of S. cerevisiae Uba1 using Jmol modeling software. XL-SMA variants labeled in red, thiolestered Ub in yellow and non-covalently bound Ub-adenylate in blue. This figure has been reproduced and modified from Protein Data Bank public domain content (PDB ID: 4NNJ) under original permission from Schäfer et al. (2014). “Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules.” Acta Crystallogr D Biol Crystallogr 70(Pt 5): 1311–1320.\n\nThe crystal structure for human Uba1 has yet to be determined, however in 2014 Schäfer et al. successfully resolved the crystal structure of Uba1 loaded with two Ubiquitin molecules and AMP (fully loaded) from S. cerevisiae with a resolution of 2.4Å. Using the 3D protein-modeling software Jmol (Bowlin et al., 2013), the three missense variants were highlighted to give spatial insights to possible effects of the variants (Figure 1C). The p.M539I, p.S547G, p.E557V and p.N577 mutations (p.M505, p.A515, p.E525 and p.N545 in S. cerevisiae, respectively) all lie on the surface-exposed residues of the AAD.\n\nPurification of solely full-length, catalytically active XL-SMA forms of Uba1 was necessary for reliable downstream experiments. We employed an affinity purification method first employed by Haas et al. for use in human erythrocytes, and modified it for use with Rosetta 2 (DE3) Escherichia coli competent cells for human protein over-expression. The method of purification relies solely on the catalytic ability of the Uba1 to perform adenylation and thioester bond formation with Ubiquitin. Therefore, highly purified, full-length and active Uba1 was obtained after stringent washing. Uba1 WT and XL-SMA variants were all successfully purified in this matter (Figure 2). Yield was very similar and consistent for p.M539I, p.S547G, and WT Uba1 over numerous purifications. However, p.E557V Uba1 consistently resulted in a lower yield. Due to the Tokgoz et al. Uba1 p.D576A variant’s adenylation ability being reduced to virtually zero, this variant did not bind to the Ubiquitin-agarose column. Therefore, the 6xHisTag attached near the N-terminus of our Uba1 was used in combination with Cobalt Resin HisPur affinity beads (Thermo Fisher) for capture and subsequently eluted with 200mM imidazole.\n\nSDS-PAGE image of the purification of Uba1. Bands at ~118kDa indicate full length, active WT, p.M539I, p.S547G, p.E557V, Uba1 as purified by a thiolester-linkage capture column. As predicted, the p.D576A did not bind to the Ub-agarose. The p.D576A was purified by Co2+ affinity column as seen in the far right lane.\n\nTo test if XL-SMA missense variants alter the catalytic adenylation activity of Uba1, each missense variant was assayed in vitro against wild-type Uba1 using a novel kinetic assay adapted from Wilson et al. (Wilson & Aldrich, 2010). Under saturating conditions of MgATP and Ubiquitin (2mM and 100μM, respectively) the adenylation activities of wild-type Uba1 and XL-SMA variants were assayed over a range of Uba1 concentrations, from 0–250nM. Adenylation-specific activity was measured by cleavage of ATP by Uba1, and the resulting PPi coupled to a colorimetric side reaction measuring at 360nm (see materials and methods). Missense variants p.M539I and p.S547G showed no statistically-reduced adenylation activity, despite their locations in the well-conserved AAD, while variant p.E557V showed a moderate decrease in activity (Figure 3 and Table 1). The adenylation-crippling p.D576A variant was also generated in parallel with the XL-SMA variants, and included in the activity assay in order to serve as a negative control to validate the assay’s data. As predicted, variant p.D576A had essentially no detectable adenylation activity in the assay in relation to any of the other assays variants.\n\nKinetic assays were run in triplicate under saturating conditions (Mg2+ (10mM), ATP (2mM), Ub (100µM)) at 37°C. Wild-type (WT) and XL-SMA variants were assayed with a range of Uba1 amounts in 50mM HEPES buffer pH 8.0. Linear regression of WT slope compared to each variant shows statistical significance for p.M539I (*P = 0.028) and p.E557V (***P<0.0001).\n\nDespite the lack of significant changes in adenylation activity of the XL-SMA missense variants with respect to Uba1 concentrations for the p.M539I and p.S547G variants, further assays were carried out in an attempt to tease out any other significant differences under different assay conditions that might occur in the cell. To this end, varying concentrations of both ATP and Ubiquitin were assayed in separate experiments in triplicate (Figure 4). Under saturating ATP concentrations (2mM), the concentration of Ub was varied between 100µM – 8.8µM. Michaelis–Menten kinetics did not reveal significant differences between the different Uba1 variants (Figure 4 and Table 2). Similarly, under saturating Ub concentrations (100µM), ATP concentration was varied from 2mM down to 3.2nM. Only the p.E557V variant Uba1 had increased Km with 95% confidence intervals that did not overlap with WT Uba1, suggesting this variant has reduced affinity for ATP (Figure 4 and Table 2).\n\nKinetic assays (150µL) were run in duplicate or triplicate with 100nM WT Uba1 and XL-SMA variants in 50mM HEPES buffer at 37°C to determine initial linear rates from the first 10–20 minutes of the reaction. A) Michaelis-Menten graph of Uba1 adenylation activity as a function of Ub concentration with saturating ATP(2000nM). B) Michaelis-Menten graph of Uba1 adenylation activity as a function of ATP concentration with saturating Ub (100µM). Data was normalized to control reactions and graphed in GraphPad Prism. Km and Vmax values as well as 95% confidence intervals were also calculated in GraphPad Prism (see Table 2). Only p.E557V Km (ATP) did not have overlapping confidence intervals with WT values.\n\nNumerous previous studies have shown the importance of Uba1’s Cys632, and its role in attacking the primed Ubiquitin-adenylate in the adenylation domain forming the thioester bond (Haas & Rose, 1982; Haas et al., 1982). With the catalytic adenylation activity of Uba1a showing no significant differences between wild type and XL-SMA mutants, the subsequent thioester bond formation reaction was investigated in order to determine if the mutations had any effect on the ability of Uba1’s catalytic cysteine to properly attack the Ubiquitin adenylate. To measure the covalent conjugation of Uba1’s Cys632 to Ubiquitin’s C-terminus Lys76 a fluorescently-labelled Ubiquitin time course assay was carried out (Figure 5). Unfortunately, a time dependent, ATP-independent accumulation of fluorescence was found at the molecular weight of unmodified Uba1 in addition to the predicted ATP-dependent accumulation of fluorescence at a slightly higher-shifted molecular weight (data not shown). The interference from the ATP-independent accumulation of fluorescence made it impossible to determine Uba1-UbFl thiolester bond formation. We suspect the fluorescence accumulation without a shift in molecular weight was due to unreacted free fluorescein from the UbFl. While our thioester fluorescence assay’s data proved inconclusive, our purification of Uba1 active protein on Ub-agarose columns demonstrated clearly that all Uba1 enzymes tested, with the exception of the p.D576A mutant, readily form thiolester bonds. Interestingly, over many purification experiments, the yield of the p.E557V mutant Uba1 was consistently lower.\n\nWT and mutant forms of Uba1 were incubated with Ub and Ube2e1 in the presence of ATP and Mg2+ for the indicated time. Reactions were quenched with Laemli buffer, separated by SDS-PAGE and quantitated with ImageJ software. A) Gel images are representative of total protein images from three independent experiments. Inset for visualization of Uba1-S-Ub complex gel shift. Note the lack of E2-S-Ub formation in the p.D576A mutant control. B) Quantitation of the E2-S-Ub gel shift bands. Y-axis values are integrated Ube2e1-Ub band densities (in millions) normalized to total Uba1 band densities. Error bars represent the SEM. Symbols indicate statistical significance (#P<0.0001, **P<0.01, *P<0.05) compared to WT using 2-way ANOVA.\n\nIn order to elicit any other type of enzymatic effect(s) the XL-SMA missense mutations might cause, we expanded the study into the final function performed by Uba1: the transfer of the activated Ubiquitin to a downstream E2 enzyme (transthioesterification).\n\nAfter testing a kit of various E2 enzymes (Boston BioChem, data not shown), the Ubiquitin Conjugating Enzyme E2E 1 (UBE2E1, previously UBCH6) was chosen for an extended time course assay due to its well-studied function, properties, and adequate band separation between Ubiquitin dimers/trimers on SDS-PAGE. Again, with this assay, we found an ATP-independent accumulation of fluorescence at a molecular weight consistent with unmodified Ube2e1 (data not shown). We also found an ATP-dependent accumulation of fluorescent Ube2e1 consistent with Ubiquitin modification. Since the MW of Ube2e1-UbFl is significantly larger, we were able to accurately quantify the fluorescence of this gel shifted band (Figure 5). Consistent with all of our previous findings, the p.M539I and p.S547G retained virtually all activity, though at the latest time point, p.S547G did reach statistical significance for a minute decrease in activity. The p.E557V rapidly reached a statistically-significant difference with less than half of wild-type activity.\n\n\nDiscussion\n\nX-linked spinal muscular atrophy is caused by rare disease-causing variants in UBA1. To date, one recurrent synonymous and three single-family missense mutations have been identified. These rare variants all reside in a 54 base-pair hotspot of the 3,174 nucleotide UBA1 mRNA. To elucidate the biological dysfunction of Uba1 in XL-SMA, we performed novel in vitro biochemical assays on wild-type and pathogenic XL-SMA variants.\n\nThe p.M539I, p.S547G and p.E557V missense XL-SMA variants all lie in the catalytic AAD domain of Uba1. It has been demonstrated the C-terminus tail of Ubiquitin must be brought together tightly with ATP in a narrow pocket, positioned in the correct orientation in order to be efficiently attacked by the catalytic cysteine in the adjacent SCCH domain (Lee & Schindelin, 2008; Tokgöz et al., 2006). To determine whether UBA1 missense mutations affect this sensitive biochemical activity in vitro, we developed and performed a novel assay measuring Uba1 adenylation activity. The adenylation rates of Uba1 measured by our assay were somewhat slower in comparison to rates measured by others (Tokgöz et al., 2006; Wee et al., 2000). However, our assay was designed to specifically isolate the adenylation activity of Uba1 in a kinetic assay by substituting Ub with a strong acceptor, hydroxylamine, thus separating the MgATP binding and adenylation half reaction away from binding of Ubiquitin and thioesterification half reaction. All other reported studies do not separate the half reactions and thus cannot accurately distinguish between altered adenylation and altered thioesterification. Using our novel assay, we measured the effect of alterations on the adenylation half reaction independently. Moreover, an endpoint that our rates strictly agree with others’ more sensitive assays was not an overriding goal in the novel assay, but to obtain catalytic rates between the XL-SMA variants relative to each other and wild type under the same conditions. Nevertheless, these differences noted above in assay conditions and reagents likely contributed to differences in rates and/or endpoints values between the current study and others.\n\nSince all known XL-SMA missense variants lie in the AAD, we reasonably hypothesized these result in altered adenylation activity of Uba1. We expected a significant change, but not a complete elimination of enzymatic activity as complete loss of Uba1 function is inconsistent with life and cell survival at any level. To our surprise only 1 of 3 variants, p.E557V, had any significant reduction in enzymatic activity. It is a possibility the reduction is a result from the residue’s close proximity to the critical adenylating p.D576 residue which is why no significant change in activity was seen in the other missense variants located further upstream. Similarly, investigation of the transthioesterification from Uba1 to E2 enzymes, namely to Ube2e1, only p.E557V showed an apparent significant loss of transthioesterification signal compared to WT. This decrease in transthioesterification would be expected since the reduction in adenylation bottlenecks the reaction. However, in the presence of Ubiquitin, transthioesterification cannot be separated from adenylation and thioester half reactions, therefore additional dysfunction of the transthioesterification step cannot be ruled out.\n\nIn contrast to the p.E557V, our data suggest the p.M539I, and p.S547G missense variants in XL-SMA lead to negligible changes in relative enzymatic activity of Uba1 in vitro. These two variants showed no statistically significant effect, positive or negative, on Uba1’s adenylation activity of Ubiquitin in vitro. This discovery is somewhat in contrast with Tokgöz et al. (2006) previous findings of two adenylation-sensitive mutations in the ADD, specifically exon 15. Asp576 and Lys528 proved to be essential to the affinity of MgATP. Furthermore, substitutions to unreactive amino acids proved catalytically detrimental and effectively eliminating adenylation ability in vitro. However, given the complexity of protein domain folding and interaction currently one cannot accurately predict how each amino acid change could alter function. Vital for our studies, this virtually adenylation-dead p.D576A variant was generated and utilized as an optimal negative control for our assay’s own validation; and importantly showed no detectable activity levels in all assays.\n\nAs the p.M539I and the p.S547G mutations do not result in significant loss of in vitro catalytic activity, these observations suggest a different mechanism of pathology such as aberrant splicing, as is the suspected cause of disease associated with the c.1731C>T variant. Splice site and splice element blinding algorithms do suggest possible changes to splicing (Lenski et al., 2005). Another possibility is that p.M539I and the p.S547G mutations disrupt interactions with key binding partners. Allen et al. found that mutations in gigaxonin disrupt its interaction and binding with Uba1 (Allen et al., 2005). Gigaxonin (GAN) is a class of BTB-Kelch proteins critical for specificity of the UPS. Mutations in GAN and several other BTB-Kelch proteins result in neuromuscular diseases with striking overlap with XL-SMA. This evidence demands that the effect of missense mutations on binding to gigaxonin and other BTB-kelch proteins be further explored.\n\nThe discovery of UBA1 as the primary gene affected in XL-SMA was a major step forward in understanding the XL-SMA disease phenotype. Our results suggest a range of pathological effects of these rare variants in UBA1.\n\nIt is very interesting that the same conundrum is faced in our understanding the pathophysiology of autosomal recessive SMA, in which germ-line deletion of a highly important, ubiquitously expressed gene (SMN1), is also confined to targeted lower motor neuron destruction. It has been suggested that UBA1 can rescue the SMN1 phenotype in a murine model of autosomal recessive SMA (Powis et al., 2016). It will be of great interest in future investigations to better define the biological interactions of SMN1 and UBA1; to see, if in fact, perturbations in SMN1 protein are also involved in pathogenesis of XL-SMA.\n\n\nData availability\n\nF1000Research: Dataset 1. Dataset for Figure 2. Expression and purification of Uba1, 10.5256/f1000research.11878.d174721 (Balak et al., 2017a)\n\nF1000Research: Dataset 2. Dataset for Figure 3. Uba1 adenylation activity plotted as a function of Uba1 enzyme amount, 10.5256/f1000research.11878.d174722 (Balak et al., 2017b)\n\nF1000Research: Dataset 3. Data for Figure 4. Uba1 adenylation activity as a function of Ub or ATP concentration, 10.5256/f1000research.11878.d174723 (Balak et al., 2017c)\n\nF1000Research: Dataset 4. Data for Figure 5. Uba1 transthioesteration of Ube2e1, 10.5256/f1000research.11878.d174724 (Balak et al., 2017d)",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by a research grant (MDA186435) from the Muscular Dystrophy Association of America, Arizona Department of Health Services Arizona Biomedical Research Commission (ADHS16-110501), and the Translational Genomics Research Institute Bridge Funding. CDB was supported, in part, by a fellowship from Freeport-McMoRan Copper and Gold Foundation, and the Helios Foundation.\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\nAllen E, Ding J, Wang W, et al.: Gigaxonin-controlled degradation of MAP1B light chain is critical to neuronal survival. Nature. 2005; 438(7065): 224–228. PubMed Abstract | Publisher Full Text\n\nBalak CD, Hunter JM, Ahearn ME, et al.: Dataset 1 in: Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy. F1000Research. 2017a. Data Source\n\nBalak CD, Hunter JM, Ahearn ME, et al.: Dataset 2 in: Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy. F1000Research. 2017b. Data Source\n\nBalak CD, Hunter JM, Ahearn ME, et al.: Dataset 3 in: Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy. F1000Research. 2017c. Data Source\n\nBalak CD, Hunter JM, Ahearn ME, et al.: Dataset 4 in: Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy. F1000Research. 2017d. Data Source\n\nBowlin KM, Embree LJ, Garry MG, et al.: Kbtbd5 is regulated by MyoD and restricted to the myogenic lineage. Differentiation. 2013; 86(4–5): 184–191. PubMed Abstract | Publisher Full Text\n\nCambridge SB, Gnad F, Nguyen C, et al.: Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover. J Proteome Res. 2011; 10(12): 5275–5284. PubMed Abstract | Publisher Full Text\n\nCifuentes-Diaz C, Frugier T, Melki J: Spinal muscular atrophy. Semin Pediatr Neurol. 2002; 9(2): 145–150. PubMed Abstract | Publisher Full Text\n\nCohen-Kaplan V, Livneh I, Avni N, et al.: The ubiquitin-proteasome system and autophagy: Coordinated and independent activities. Int J Biochem Cell Biol. 2016; 79: 403–418. PubMed Abstract | Publisher Full Text\n\nCorpet F: Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988; 16(22): 10881–10890. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDlamini N, Josifova DJ, Paine SM, et al.: Clinical and neuropathological features of X-linked spinal muscular atrophy (SMAX2) associated with a novel mutation in the UBA1 gene. Neuromuscul Disord. 2013; 23(5): 391–398. PubMed Abstract | Publisher Full Text\n\nDohmen RJ, Stappen R, McGrath JP, et al.: An essential yeast gene encoding a homolog of ubiquitin-activating enzyme. J Biol Chem. 1995; 270(30): 18099–18109. PubMed Abstract | Publisher Full Text\n\nDressman D, Ahearn ME, Yariz KO, et al.: X-linked infantile spinal muscular atrophy: clinical definition and molecular mapping. Genet Med. 2007; 9(1): 52–60. PubMed Abstract | Publisher Full Text\n\nGlickman MH, Ciechanover A: The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev. 2002; 82(2): 373–428. PubMed Abstract | Publisher Full Text\n\nHaas AL, Rose IA: The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis. J Biol Chem. 1982; 257(17): 10329–10337. PubMed Abstract\n\nHaas AL, Warms JV, Hershko A, et al.: Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation. J Biol Chem. 1982; 257(5): 2543–2548. PubMed Abstract\n\nHetz C, Glimcher LH: Protein homeostasis networks in physiology and disease. Curr Opin Cell Biol. 2011; 23(2): 123–125. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJoo HY, Zhai L, Yang C, et al.: Regulation of cell cycle progression and gene expression by H2A deubiquitination. Nature. 2007; 449(7165): 1068–1072. PubMed Abstract | Publisher Full Text\n\nKulkarni M, Smith HE: E1 ubiquitin-activating enzyme UBA-1 plays multiple roles throughout C. elegans development. PLoS Genet. 2008; 4(7): e1000131. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee I, Schindelin H: Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes. Cell. 2008; 134(2): 268–278. PubMed Abstract | Publisher Full Text\n\nLek M, Karczewski KJ, Minikel EV, et al.: Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016; 536(7616): 285–291. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLenski C, Erkelenz S, Ramser J, et al.: Synonymous Mutations in the X-linked Disease Genes UBA1 and HADH2 Affect Binding of the Splicing Regulatory Proteins SRSF2, SRSF6 and hnRNP F/H. JSM Genet Genomics. 2015; 2(1): 1007. Reference Source\n\nLiu H, Naismith JH: An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 2008; 8: 91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPowis RA, Karyka E, Boyd P, et al.: Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy. JCI Insight. 2016; 1(11): e87908. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRamser J, Ahearn ME, Lenski C, et al.: Rare missense and synonymous variants in UBE1 are associated with X-linked infantile spinal muscular atrophy. Am J Hum Genet. 2008; 82(1): 188–193. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRinetti GV, Schweizer FE: Ubiquitination acutely regulates presynaptic neurotransmitter release in mammalian neurons. J Neurosci. 2010; 30(9): 3157–3166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchäfer A, Kuhn M, Schindelin H: Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules. Acta Crystallogr D Biol Crystallogr. 2014; 70(Pt 5): 1311–1320. PubMed Abstract | Publisher Full Text\n\nSchneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(7): 671–675. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTokgöz Z, Bohnsack RN, Haas AL: Pleiotropic effects of ATP.Mg2+ binding in the catalytic cycle of ubiquitin-activating enzyme. J Biol Chem. 2006; 281(21): 14729–14737. PubMed Abstract | Publisher Full Text\n\nToyama BH, Savas JN, Park SK, et al.: Identification of long-lived proteins reveals exceptional stability of essential cellular structures. Cell. 2013; 154(5): 971–982. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWee CD, Kong L, Sumner CJ: The genetics of spinal muscular atrophies. Curr Opin Neurol. 2010; 23(5): 450–458. PubMed Abstract | Publisher Full Text\n\nWee KE, Lai Z, Auger KR, et al.: Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate. J Protein Chem. 2000; 19(6): 489–498. PubMed Abstract | Publisher Full Text\n\nWilson DJ, Aldrich CC: A continuous kinetic assay for adenylation enzyme activity and inhibition. Anal Biochem. 2010; 404(1): 56–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWoo CJ, Maier VK, Davey R, et al.: Gene activation of SMN by selective disruption of lncRNA-mediated recruitment of PRC2 for the treatment of spinal muscular atrophy. Proc Natl Acad Sci U S A. 2017; 114(8): E1509–E1518. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "25643",
"date": "19 Sep 2017",
"name": "Barrington Burnett",
"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\nBalak et al investigate the effect of one recurrent synonymous and three single-family missense mutations in the UBA1 gene, which this group and others have previously linked to X-linked spinal muscular atrophy. UBA1 is the first enzyme in the ubiquitin proteasome system which is responsible for tagging and degrading most cellular soluble proteins. Using in vitro assays, the authors investigated Uba1 adenylation, thioester, and transthioesterification reactions to determine possible biochemical effects of the missense variants. One hypothesis is that the disease causing UBA1 mutations result in loss of ubiquitin activation function. The authors present compelling data that ubiquitin activation and the transferred of the charged ubiquitin to an ubiquitin conjugating enzyme, E2, is not disrupted by the patient-derived mutations. Together the biochemical assays suggest that the mechanism of action may be alternative splicing of the UBA1 mutant gene product or selective ubiquitin transfer unto a yet to be determined E2 enzyme. Identifying the putative splice variant(s) or the aberrant E2 interaction and the downstream targets could shed light on why UBA1 mutations lead to X-linked SMA.\nThis is a well written and elegantly done biochemical study.\n\nMinor concern: It is unclear why the first letter in ubiquitin is capitalized throughout the manuscript.\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": "25639",
"date": "25 Sep 2017",
"name": "Christine Beattie",
"expertise": [
"Reviewer Expertise motoneuron biology",
"spinal muscular atrophy"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 examines the functional properties of missense mutations in Ubiquitin-Like Modifier Activating Enzyme 1 (UBA1) found in patients with X-linked spinal muscular atrophy (XL-SMA). These rare mutations occur in the active adenylation domain of UBA1. To better understand how these mutations could be causing this motoneuron disease, the authors asked whether these mutations affected functional properties of this enzyme specifically UBA1 adenylation, thioester, and transthioesterification reactions in vitro.\nThe data support that only the E557V mutation caused a significant decrease in UBA1 activity. The data were consistent from the different assays that analyzed UBA1’s ability to adenylate ubiquitin and transfer of the activated ubiquitin to a downstream E2 enzyme. However, this decrease was not complete and the other missense mutations did not show defects in UBA1 function in adenylation and downstream activities, thus the authors conclude that these functions of UBA1 do not contribute to the disease phenotype.\n\nIn at least two assays the E557V mutations had decreased enzymatic activity. Does this correlate with the severity of the disease? That is, do patients with the E557V mutations exhibit earlier disease onset or more severe disease phenotypes?\n\nFigure 3. In the graph D576A is mis labeled as C576A\n\nFigure 3: What statistical test was used for Fig. 3?\n\nFigure 5: What posthoc analysis was used after the ANOVA?\n\nFigure 4: It is not possible to see the UBA1-S-Ub complex gel shift in the upper gel. It would be beneficial to show them at higher magnification for at least one time point.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": []
}
] | 1
|
https://f1000research.com/articles/6-1636
|
https://f1000research.com/articles/5-1592/v1
|
06 Jul 16
|
{
"type": "Case Report",
"title": "Case Report: Application of whole exome sequencing for accurate diagnosis of rare syndromes of mineralocorticoid excess",
"authors": [
"Ranjit Narayanan",
"Shamsudheen Karuthedath Vellarikkal",
"Rijith Jayarajan",
"Ankit Verma",
"Vishal Dixit",
"Vinod Scaria",
"Sridhar Sivasubbu",
"Shamsudheen Karuthedath Vellarikkal",
"Rijith Jayarajan",
"Ankit Verma",
"Vishal Dixit"
],
"abstract": "Syndromes of mineralocorticoid excess (SME) are closely related clinical manifestations occurring within a specific set of diseases. Overlapping clinical manifestations of such syndromes often create a dilemma in accurate diagnosis, which is crucial for disease surveillance and management especially in rare genetic disorders. Here we demonstrate the use of whole exome sequencing (WES) for accurate diagnosis of rare SME and report that p.R337C variation in the HSD11B2 gene causes progressive apparent mineralocorticoid excess (AME) syndrome in a South Indian family of Mappila origin.",
"keywords": [
"whole exome sequencing",
"mineralocorticoid excess"
],
"content": "Abbreviations\n\nSME: Syndromes of mineralocorticoid excess; AME: Apparent Mineralocorticoid Excess; CAH:17α-hydroxylase: Congenital adrenal hyperplasia due to 17α-hydroxylase deficiency, CAH: 11β-hydroxylase deficiency: Congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; CYP17A1: cytochrome P450, family 17, subfamily A, polypeptide 1; CYP11B1: cytochrome P450, family 11, subfamily B, polypeptide 1, HSD11B2: hydroxysteroid (11β) dehydrogenase 2, ENaC; the epithelial sodium channel subunit genes, WNK1: WNK lysine deficient protein kinase 1, WNK4; WNK lysine deficient protein kinase 1; KLHL3: kelch-like family member 3, CUL3: cullin 3; SPAK: type III secretion system chaperone; MCR/NR3C2: Nuclear Receptor Subfamily 3, Group C, Member 2; WES: Whole exome sequencing.\n\n\nReport\n\nSyndromes of mineralocorticoid excess (SME) are a group of syndromes characterized by an abnormal activation of the amiloride-sensitive sodium channels in the distal tubules of the kidney resulting in an abnormal salt balance. The symptoms are characteristically an abnormality in salt balance (due to an over activation of the channels mediated through the mineralocorticoid receptor), water retention, hypokalemia, low renin levels and hypertension1–3. Based on the clinical presentation, differential diagnosis would include Liddle syndrome, Geller syndrome, Gordon syndrome, Apparent Mineralocorticoid Excess (AME) and other milder variants such as CAH:17α-hydroxylase and CAH:11β-hydroxylase deficiency. Mutations in various genes including CYP17A1, CYP11B1, HSD11B2, ENaC, WNK1, WNK4, KLHL3, CUL3, SPAK and MCR/NR3C2 may result in clinical features that resemble SME1–3. Due to the large diversity and overlapping clinical features, the accurate diagnosis is highly reliant on the genetic characterization. The need to screen a large number of variants and genes that could cause disease, using conventional approaches such as capillary sequencing is tedious, time consuming and often expensive. Whole exome sequencing (WES) has emerged as an alternative strategy in such clinical settings4,5.\n\nFive siblings of a third degree consanguineous family (Figure 1A) were evaluated at the Department of Nephrology, KMCT Medical College Hospital, Kerala, India. They were aged between 14 and 30 years. All but the youngest of the five siblings were hypertensive. On evaluation, four of the siblings had significant hypokalemia without significant acidosis or alkalosis but with ultrasound evidence of medullary nephrocalcinosis (Figure-1A). The youngest sibling, aged 14 years, did not have hypokalemia, acid base abnormality or renal dysfunction but exhibited overt medullary nephrocalcinosis on ultrasound. Two of the older siblings had concentric left ventricular hypertrophy and renal dysfunction with mild proteinuria. Plasma renin and aldosterone levels were evaluated in the two male siblings and were found to be low (Plasma renin activity 0.14 ng/ml/hr and Plasma Aldosterone 28 pg/ml (sitting upright position) in the elder brother and Plasma renin activity 0.1 ng/ml/hr and Plasma aldosterone 6.5 pg/ml in the younger sibling). Both of them required multiple antihypertensive drugs (Amlodipine 10 mg and Metoprolol Extended Release 50 mg daily) in addition to spironolactone (50 mg daily) for control of blood pressure while the others were well controlled on spironolactone (50 mg daily) alone. There was no past history of similar phenotypes in the parents. A provisional diagnosis of AME was made in view of the clinical picture and inheritance pattern. The 24-hour urinary cortisol to cortisone ratio estimation was not done due to unavailability of the test.\n\n(A) Family pedigree marked with progressive phenotypes. (B) Secondary structure of HSD11B2 marked with major domains and the p.R337C amino acid position. (C) Capillary sequencing chromatogram representation of p.R337C variation in the family; arrow and asterisks marks depict the variation loci and affected individuals respectively.\n\nWhole blood was obtained from the parents and the affected members was obtained after informed consent. 50 ng of the isolated high quality DNA was used to prepare library and exome capture using Nextera Rapid Capture Expanded Exome kit and Sequencing was performed on Illumina Hiseq 2500 sequencer using v3 reagents (Illumina Inc, USA) to generate over 49.48 million paired end reads of 101bp. The variation finding, annotation and prioritisation were performed as previously described5 and revealed the presence of homozygous variation p.R337C in HSD11B2 gene (Figure-1B) annotated to be pathogenic in ClinVar6 and predicted to be deleterious using PROVEAN7. Human cell line studies have demonstrated that p.R337C mutation leads to the low activity of HSD11B2 and causes low-renin hypertension thus resulting in AME8. The variant was further validated using Sanger sequencing of the amplicons, confirming the diagnosis (Figure-1C).\n\nAME is a rare heterogeneous low renin retention SME disorder that manifests with severe juvenile hypertension, hypokalemic alkalosis, low birth weight, failure to thrive, poor growth, and in many cases nephrocalcinosis caused by homozygous and compound heterozygous mutations in the HSD11B2 gene. HSD11B2 oxidises the steroid hormone cortisol to inactive cortisone and mutations in this gene result in a high circulating level of cortisol, and further illegitimate activation of the mineralocorticoid receptor, outcompeting aldosterone and causing activation of the downstream pathways and a clinical presentation of AME symptoms.\n\nIn summary, we used WES to characterize and diagnose a family with an extremely rare clinical presentation. p.R337 loci of 11HSDB2 is widely reported to be associated with AME across the world and has been reported in Zoroastrians from India and Iran (Compound heterozygous for p.R337H and Δp.Y338, age of onset from 8 months), in Persian (p.R337C, age of onset from 4 years) and in Japanese (p.R337H and Δp.Y338, age of onset from 2 years) populations9,10. The age of onset for the disease manifestation due to p.R337C mutation has been previously described to be as early as 4 years. Our family, though having the p.R337C mutation, did not present with clinical symptoms in early childhood but exhibits the progressive AME phenotypes with increasing age (Figure 1A) with the older individuals presenting more severe clinical manifestations including renal impairment. Patients with identical homozygous mutations from different families have been described to have varying degrees of severity in clinical and biochemical features4,9. In fact, homozygous mutations in HSD11B2 have been described where the patient has only mild low renin hypertension without other features of AME10. Early diagnosis and prompt institution of salt restriction and spironolactone in these patients can prevent secondary organ damage. Our report also serves to highlight the utility of WES as a tool for diagnosing rare genetic diseases even where biochemical characterization is unavailable.\n\n\nPatient consent\n\nWritten informed consent for publication of these data were obtained from the patients.\n\n\nData availability\n\nThe raw whole exome sequence are available at the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra), accession number SRR3546815.",
"appendix": "Author contributions\n\n\n\nClinical workup: RN; WES data generation, analysis, and validation: SKV, RJ, AV, VD; supervision and mentorship: VS and SS; All authors contributed important intellectual contents during manuscript drafting and accepts accountability for the overall work.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAuthors acknowledge funding from the Council of Scientific and Industrial Research (CSIR), India through Grant No. BSC0212 (Wellness genomics project) granted to SS and VS.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgement\n\nAuthors acknowledge help and support from the GUaRDIAN Consortium. RN acknowledges Dr. Moumita Barua from the Dept. of Medicine, Toronto General Hospital, University of Toronto and Prof. Martin Russell Pollak from Beth Israel Deaconess Medical Center at Harvard Medical School for their input and help during initial workup.\n\n\nReferences\n\nGarovic VD, Hilliard AA, Turner ST: Monogenic forms of low-renin hypertension. Nat Clin Pract Nephrol. 2006; 2(11): 624–30. PubMed Abstract | Publisher Full Text\n\nMelcescu E, Phillips J, Moll G, et al.: 11Beta-hydroxylase deficiency and other syndromes of mineralocorticoid excess as a rare cause of endocrine hypertension. Horm Metab Res. 2012; 44(12): 867–78. PubMed Abstract | Publisher Full Text\n\nMelcescu E, Koch CA: Syndromes of Mineralocorticoid Excess. In Koch CA, Chrousos GP (eds). Endocrine Hypertension: Underlying Mechanisms and Therapy, Contemporary Endocrinology. Humana press: New York City, NYC, USA, 2003; 33–50. Publisher Full Text\n\nMadrigal I, Alvarez-Mora MI, Karlberg O, et al.: Efficient application of next-generation sequencing for the diagnosis of rare genetic syndromes. J Clin Pathol. 2014; 67(12): 1099–103. PubMed Abstract | Publisher Full Text\n\nGupta A, Sharma YK, Vellarikkal SK, et al.: Whole-exome sequencing solves diagnostic dilemma in a rare case of sporadic acrokeratosis verruciformis. J Eur Acad Dermatol Venereol. 2016; 30(4): 695–7. PubMed Abstract | Publisher Full Text\n\nLandrum MJ, Lee JM, Riley GR, et al.: ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 2014; 42(Database issue): D980–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChoi Y, Chan AP: PROVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics. 2015; 31(16): 2745–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nObeyesekere VR, Ferrari P, Andrews RK, et al.: The R337C mutation generates a high Km 11 beta-hydroxysteroid dehydrogenase type II enzyme in a family with apparent mineralocorticoid excess. J Clin Endocrinol Metab. 1995; 80(11): 3381–3383. PubMed Abstract | Publisher Full Text\n\nDave-Sharma S, Wilson RC, Harbison MD, et al.: Examination of genotype and phenotype relationships in 14 patients with apparent mineralocorticoid excess. J Clin Endocrinol Metab. 1998; 83(7): 2244–2254. PubMed Abstract | Publisher Full Text\n\nWilson RC, Dave-Sharma S, Wei JQ, et al.: A genetic defect resulting in mild low-renin hypertension. Proc Natl Acad Sci USA. 1998; 95(17): 10200–5. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "16258",
"date": "03 Oct 2016",
"name": "Parag Tamhankar",
"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\nPlease could you add an introduction about syndrome of apparent mineralocorticoid excess, for example;\nSyndrome of apparent mineralocorticoid excess (MIM#218030) is a rare cause of juvenile hypertension occuring due to homozygous or compound heterozygous mutations in HSD11B2 gene (MIM*614232). The gene encodes an NAD+-dependent enzyme viz corticosteroid 11-β-dehydrogenase isozyme 2, which is expressed in aldosterone-selective epithelial tissues such as the kidney, colon, salivary and sweat glands. This enzyme is responsible for oxidizes the glucocorticoid cortisol to the inactive metabolite cortisone. This protective mechanism is necessary because cortisol circulates at 100-1000-fold higher concentrations than aldosterone, and binds with equal affinity to the mineralocorticoid receptor, thereby out-competing aldosterone in cells that do not produce HSD11B2. In patients with SME the enzyme deficiency allows mineralocorticoid receptors to be occupied by cortisol leading to hypertension.\n\nI believe that more details on clinical features should be provided, such as:\nclinical presentation and history. Draw three generation pedigree. detailed anthropometry, examination findings of the patients. For eg. was there any low birth weight, short stature, polydipsia, polyuria? what were the baseline blood pressure levels in the patients when hypertension was detected? was fundus examination done to detect retinopathy? were the parents hypertensive? give the detailed serum electrolyte levels, detailed 2ECHO report. Give normal ranges of plasma renin, aldosterone levels. clinical photos if relevant or uploading ultrasound kidney scans\n\nMutation: Please give the nucleotide change, exon number of the mutation identified in the patients with the correct Ensembl transcript ID. Please specify as the mechanism of loss of function of the mutation in patients with mutations at position p.335-339 is reduced protein stability due to rapid protein degradation at the proteasome, rather than reduced catalysis.\nGrammar changes: I would begin the words aldosterone, amlodipine, metoprolol with lower case letters when in the midst of a sentence.\nTypo: I think you should correct the following sentence - p.R337 loci of 11HSDB2 to- “The p.R337 residue of 11-β-dehydrogenase isozyme 2 enzyme is a recognised mutation site. The mutation p.R337C has been previously identified in a family from Iran with three affected children.”",
"responses": [
{
"c_id": "2924",
"date": "04 Sep 2017",
"name": "Vinod Scaria",
"role": "Author Response",
"response": "Please could you add an introduction about syndrome of apparent mineralocorticoid excess, for example;Syndrome of apparent mineralocorticoid excess (MIM#218030) is a rare cause of juvenile hypertension occurring due to homozygous or compound heterozygous mutations in HSD11B2 gene (MIM*614232). The gene encodes an NAD+-dependent enzyme viz corticosteroid 11-β-dehydrogenase isozyme 2, which is expressed in aldosterone-selective epithelial tissues such as the kidney, colon, salivary and sweat glands. This enzyme is responsible for oxidizes the glucocorticoid cortisol to the inactive metabolite cortisone. This protective mechanism is necessary because cortisol circulates at 100-1000-fold higher concentrations than aldosterone, and binds with equal affinity to the mineralocorticoid receptor, thereby out-competing aldosterone in cells that do not produce HSD11B2. In patients with SME the enzyme deficiency allows mineralocorticoid receptors to be occupied by cortisol leading to hypertension. We thank reviewers for the comments. Clarifications are as follows: Clarification: The suggested changes in introduction section are made. 1. Clinical presentation and history. Draw three generation pedigree.Clarification 1: The suggestions have been incorporated. A two generation pedigree is provided. Information about the 3rd generation was not available.2. detailed anthropometry, examination findings of the patients. For eg. was there any low birth weight, short stature, polydipsia, polyuria?Clarification 2: There was no h/o low birth weight, polydipsia or polyuria. All developmental milestones were normally attained in all siblings.3. what were the baseline blood pressure levels in the patients when hypertension was detected?Clarification 3: Included in the case history section4. was fundus examination done to detect retinopathy?Clarification 4: No5. were the parents hypertensive?Clarification 5: No6. give the detailed serum electrolyte levels, detailed 2ECHO report. Give normal ranges of plasma renin, aldosterone levels.Clarification 6: Included in the case history section.7. clinical photos if relevantClarification 7: None8. or uploading ultrasound kidney scansClarification 8: The ultrasound scan is attached separately. It may not be of great quality, however shows medullary nephrocalcinosis (see this link).9. Mutation: Please give the nucleotide change, exon number of the mutation identified in the patients with the correct Ensembl transcript ID. Please specify as the mechanism of loss of function of the mutation in patients with mutations at position p.335-339 is reduced protein stability due to rapid protein degradation at the proteasome, rather than reduced catalysis.Clarification 9: Suggestions have been incorporated.10. Grammar changes: I would begin the words aldosterone, amlodipine, metoprolol with lower case letters when in the midst of a sentence.Clarification 10: Suggestions have been incorporated.11. Typo: I think you should correct the following sentence - p.R337 loci of 11HSDB2 to- “The p.R337 residue of 11-β-dehydrogenase isozyme 2 enzyme is a recognised mutation site. The mutation p.R337C has been previously identified in a family from Iran with three affected children.”Clarification 11: Suggestions have been incorporated."
}
]
},
{
"id": "15885",
"date": "09 Jan 2017",
"name": "Shitij Arora",
"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 very interesting study. It highlights the increasing use of WES in diagnosing rare diseases. The authors confirm the findings of ES with Sangers and sequence both the parent and the proband. The manuscript needs editing and rearrangement of text. The paragraph on SME and AME needs/can probably be put together as the introduction. Minor grammar corrections.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-1592
|
https://f1000research.com/articles/6-1634/v1
|
04 Sep 17
|
{
"type": "Research Article",
"title": "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",
"authors": [
"James Galipeau",
"Kelly D. Cobey",
"Virginia Barbour",
"Patricia Baskin",
"Sally Bell-Syer",
"Jonathan Deeks",
"Paul Garner",
"Larissa Shamseer",
"Straus Sharon",
"Peter Tugwell",
"Margaret Winker",
"David Moher",
"James Galipeau",
"Kelly D. Cobey",
"Virginia Barbour",
"Patricia Baskin",
"Sally Bell-Syer",
"Jonathan Deeks",
"Paul Garner",
"Larissa Shamseer",
"Straus Sharon",
"Peter Tugwell",
"Margaret Winker"
],
"abstract": "Background: Scientific editors (i.e., those who make decisions on the content and policies of a journal) have a central role in the editorial process at biomedical journals. However, very little is known about the training needs of these editors or what competencies are required to perform effectively in this role. Methods: We conducted a survey of perceptions and training needs among scientific editors from major editorial organizations around the world, followed by a modified Delphi process in which we invited the same scientific editors to rate the importance of competency-related statements obtained from a previous scoping review. Results: A total of 148 participants completed the survey of perceptions and training needs. At least 80% of participants agreed on six of the 38 skill and expertise-related statements presented to them as being important or very important to their role as scientific editors. At least 80% agreed on three of the 38 statements as necessary skills they perceived themselves as possessing (well or very well). The top five items on participants’ list of top training needs were training in statistics, research methods, publication ethics, recruiting and dealing with peer reviewers, and indexing of journals. The three rounds of the Delphi were completed by 83, 83, and 73 participants, respectively, which ultimately produced a list of 23 “highly rated” competency-related statements and another 86 “included” items. Conclusion: Both the survey and the modified Delphi process will be critical for understanding knowledge and training gaps among scientific editors when designing curriculum around core competencies in the future.",
"keywords": [
"scientific editor",
"core competencies",
"biomedical",
"journal",
"training needs",
"Delphi"
],
"content": "Background\n\nThe Declaration of Helsinki (2013) asks editors to ensure that the quality of what they publish is of the highest quality possible1. However, very little is known about the training needs of scientific editors (i.e., those who make decisions on the content and policies of a journal) or what competencies are required to meet these standards. Presently, a large portion of scientific editors’ learning is informal, often learned on the job through mentoring2. While formal training opportunities for scientific editors do exist in the form of fellowships and intensive courses, these opportunities are limited to a very small number of editors annually. In addition, there may be variations across training opportunities, which are not evidence-based, owing to the lack of any consensus or evidence on the knowledge, skills, and abilities that editors should possess to be competent in their job.\n\nWhile there is no shortage of published literature on the role of scientific editors, most of this takes the form of opinion-based editorials; very few recommendations are evidence-based. For example, a 2015 systematic review of training in writing for scholarly publication, journal editing, and peer review found no studies of formalized training programs for journal editors3. While a 2016 scoping review from our group4 found 25 research-based publications relating to scientific editors, the majority were surveys on a wide variety of topics relating to scientific editors. The same study found 136 published articles, of which 133 were non-research based editorials relating to various aspects of scientific editorship. An associated environmental scan also found an additional 35 documents that were not published in scientific journals, of which 18 were produced by journals, while nine were from associations and societies, six from organizations providing guidance to editors, and two from publishers.\n\nIn 1999, the World Association of Medical Editors conducted a global survey of journal editors regarding the characteristics of their respective journals. This survey included one item related to scientific editor training: of the 269 respondents, 75% said they wanted training for newly appointed editors. Other surveys have echoed this opinion as well2,5,6; however to our knowledge, there have been no dedicated attempts to formally assess the training needs of scientific editors of academic journals. We are also unaware of any large scale collaborative effort to determine the competencies that are required for the role of scientific editor. There are a number of examples of editorial organizations, publishers, and individuals7 who have put forth their opinions on what makes a good scientific editor, yet there lacks clarity on how these ideas were derived, how they relate to each other, or whether they are universal. Additionally, the perceptions and training needs of scientific editors are not well represented in the literature.\n\nThe objective of this research was to better understand the training needs and perceptions of competence of scientific editors of journals. We also sought to solicit editors’ opinions on the importance of particular knowledge, skills, abilities, and characteristics to carrying out their editorial duties.\n\n\nMethods\n\nThe research presented here is the second step of a larger program that our team is carrying out to develop a universal, minimum set of core competencies for scientific editors of biomedical journals. In our first project, we carried out a scoping review of the published research literature and an environmental scan of non-research-based materials to identify competency-related statements found online and in previous research4. In the current project described here, we aimed to solicit the perspective of editors worldwide and have them narrow and refine the number of potential core competencies related to their position. In the third and final step of the process, we brought international experts together to decide on a final set of core competencies, publication of which is forthcoming.\n\nThe current research comprised a survey of scientific editors to understand their perceptions of their role as an editor and to identify any training needs, followed by a modified Delphi process whereby editors rated the importance of 200+ competency-related statements obtained from the aforementioned previous scoping review (describing knowledge, skills, and abilities associated with the role of scientific editor)4. Surveying to gauge training needs and to gather consensus are common tools for creating a competency-based core curriculum in the biomedical field8–12. The study was approved by the Ottawa Health Science Network Research Ethics Board.\n\nWe approached current or former scientific editors of journals, defined as editors who make decisions on the content and policies of a journal – including editors-in-chief and associate/academic editors. Recruitment advertisements were sent to editorial organizations and groups having a large scientific editor membership from around the world. These organizations forwarded the advertisement about the survey of editors to their members through a distribution list email or an announcement on a listserv or message board. The organizations are listed in Box 1:\n\nWe developed an online-based assessment of editors’ perceptions and training needs that was anonymous and self-administered (Supplementary File S1). The questionnaire was developed based on data collected in our previous scoping review4, as well as with input from our research team, comprising scientific editors, representatives from publishing houses, educational experts, and specialists in publication science. Questions were designed to broadly cover major areas associated with the scientific editor role, including editors’ knowledge, expertise, skills, and experience. The questionnaire was not validated; however, it was piloted among five experienced scientific editors of biomedical journals and subsequently revised based on their feedback. The revised questionnaire was uploaded to SurveyMonkey (www.surveymonkey.com) and the survey URL was sent to the editorial organizations for distribution.\n\nThe survey contained 19 demographic questions relating to participants’ age, sex, education level, job title, editorial experience, the journal they edit, and their editorial training experience. The perceptions of respondents were also examined in four areas: Participants indicated in the first two instances how important they thought a series of competency-related statements was to the scientific editor role, and, in the latter two instances, how much they thought they possessed these same competencies. Response options on a 1–7 Likert scale were provided (as indicated):\n\n1. The degree to which participants perceive that expertise-related items are important to their job as editor (18 items) (Response options: Not Important to Very Important)\n\n2. The degree to which participants perceive that particular skills and experience are important to their job as editor (20 items) (Response options: Not Important to Very Important)\n\n3. The degree to which participants perceive they possess particular expertise-related items related to their job as editor (same 18 items as #1) (Response options: Not Much to Very Much)\n\n4. The degree to which participants perceive they perform particular skills and possess particular experience related to their job as editor (same 20 items as #2) (Not Well/Not Much to Very Well/Very Much)\n\nFinally, participants were asked to create a ranked list of their top 10 training needs (from #1 being the most important to #10 being least important). Participants were also asked if they would be willing to participate in a Delphi process to rate the importance of a much larger and more detailed list of competency-related statements for scientific editors of biomedical journals. If so, they were asked to provide their email addresses and were included in the list of potential participants for the modified Delphi process.\n\nWe carried out a three-round modified Delphi process in order to rate and refine the list of potential competencies derived in a previous scoping review we conducted4. A Delphi process typically involves experts and takes place in the form of iterations or “rounds” (normally 2 to 4) in which data is collected anonymously, often on-line, and then fed back to the group in an aggregated and de-identified way, along with individual participants’ comments13. In the current project, we modified the Delphi in several ways: First, we solicited the involvement of any scientific editors of biomedical journals, not only experts, as is normally the case with a Delphi process. Next, we did not require all participants to be involved in all rounds of the modified Delphi. In addition, the number of items included in the modified Delphi was much greater than a Delphi would generally include. Finally, for the sake of efficiency (due to the large number of items), in the third round we did not ask participants to re-rate items that had reached consensus for inclusion or exclusion in Round 2.\n\nInterested Phase 1 participants were invited to participate in the online modified Delphi, which was also administered via SurveyMonkey. For each Delphi round, an invitation was sent to the entire list of potential participants, regardless of whether they responded in the previous round.\n\nRound 1. Participants were asked to rate each competency-related statement on a 5-point Likert scale, from 1 (Not at all Important) to 5 (Absolutely Essential). At the end of each section, an open text box was provided for participants to include comments relating to items in that section if desired. Participants were also asked to name any potential competencies that were not included in the Delphi. Participants were reminded that all competency-related statements exclusively related to the position of Editor-in-Chief had been intentionally removed from the Delphi, as was the case for the scoping review on which the Delphi was built.\n\nRound 2. Phase 1 participants were invited to participate in Round 2 of the Delphi, regardless of whether they completed Round 1. Along with the email invitation, they were provided the mean score for each of the items from Round 1, the participant’s own score for each of the items, if relevant, and collated (de-identified) comments from the text boxes. Participants were then asked to re-rate and provide their rationale for disagreement only for those items for which they disagreed with the mean score from Round 1. Any new competency-related statements arising from the final question in Round 1 were included for rating in the Round 2 survey. Participants were asked to rate these new items in the same way that items had been rated in Round 1, that is, on a 5-point Likert scale from 1 (Not at all Important) to 5 (Absolutely Essential).\n\nRound 3. Phase 1 participants were invited by email to participate in Round 3 of the Delphi, regardless of whether they completed Round 1 and/or Round 2. Attached to the e-mail was a document listing the Round 1 and Round 2 average scores for all items and participants’ comments from previous rounds. Items that did not reach consensus for inclusion or exclusion in Round 2 were highlighted in yellow. Participants were asked to re-rate these highlighted items in Round 3 using a 3-point Likert scale from 1 (Less Important) to 3 (Essential). The shift to a 3-point Likert scale was aimed at simplifying the process for participants by limiting the number of options to the manner in which the ratings of each competency statement would be analysed (i.e., consensus on a ‘1’ would mean the item was excluded and consensus on a 3 would mean the item was included).\n\nTo establish inclusion and exclusion criteria for both the survey of editors and the modified Delphi, we pre-specified the consensus level at 80% of respondents. This decision was based on the use of an 80% cut-off rate in previous Delphi studies in healthcare14 and education15.\n\nThe data on the 76 perception-related survey questions were summarized by calculating mean scores for each of the questions. These scores were then classified as reaching consensus for inclusion (≥6 on a 7-point Likert scale) or exclusion (≤3 on a 7-point Likert scale), for use in a future consensus meeting. The ranked top 10 lists of training needs were collated by one author (JG) by regrouping similar statements and these groupings were then verified by another author (KDC).\n\nIn Round 1 of the Delphi, means were calculated for each of the items and consensus for inclusion (≥4 on a 5-point Likert scale) or exclusion (≤2 on a 5-point Likert scale) was determined. In Round 2, means were calculated for each of the items from Round 1 and consensus for inclusion and exclusion was updated. For any new items suggested by participants in Round 1, mean scores were calculated and consensus for inclusion and exclusion was determined in Round 2. In Round 3, means were calculated for items that had not reached consensus for inclusion or exclusion in Round 2. Final consensus for inclusion was set at 80% of participants selecting a ‘3’ on a 3-point Likert scale, while exclusion was set at 80% selecting a ‘1’ on the scale. A final list of included and excluded items was created for use in a future consensus meeting. Due to the large volume of included items in the final list, a post-hoc decision was made to create a shorter list of “highly rated” items that reached 90% consensus for inclusion.\n\nAll participants provided consent through an online form preceding the survey of editors and Delphi. In order to stimulate participation and thank participants, an iPad Mini was awarded as a draw prize after the survey of editors and after each round of the Delphi. Participants who wished to be entered into the draw were asked to provide their email address. These email addresses were used for the sole purpose of the draw and removed from the data prior to analysis. All data from this research was pooled for presentation in the results section.\n\n\nResults\n\nA total of 152 participants completed either the survey of editors’ perceptions and assessment of their training needs or the Delphi process (Figure 1). As described in Figure 1, four respondents did not complete the survey of editors but completed one round each of the Delphi and their responses were included in the analysis. We presume these four respondents were invited by other participants as access was not restricted for completing the Delphi.\n\nDemographic data. A total of 148 editors from around the world accessed and completed the survey of their perceptions (Table 1 & Supplementary File S2). Respondents were mainly those replying to the notices for participation posted by the World Association of Medical Editors (WAME) and Cochrane. Nearly 2/3 of respondents were male. Close to 2/3 had more than seven years of experience as an editor. The majority of editors indicated that their journal had provided formal or informal training related to their job and that they had sought both formal and informal training beyond what was provided by their employer. The large majority indicated having formal or informal training in research methods and statistics.\n\n*This question allowed participants to enter multiple responses\n\nEditor Perceptions. The degree to which participants perceive that expertise-related items are important to their job as editor. Of the 18 questions related to participants’ perceptions of the importance of particular expertise-related items to their job as editor, two items reached consensus for inclusion (Table 2). The highest consensus was for ‘expertise in research methods’ (85.4%), followed by ‘expertise in dealing with publication ethics, including conflicts of interest of authors, reviewers, and editors and the editorial board’ (80.4%). None of the items reached consensus for exclusion.\n\n* = Reached 80% consensus of ≥6 on a 7-point Likert scale with endpoints of Not Important/Not Much/Not Well and Very Important/Very Much/Very Well\n\n1Question asked: “Please rate THE IMPORTANCE of the following expertise-related items to the performance of your job as editor”; % indicates the percentage of respondents endorsing ≥6 on a 7-point Likert scale with endpoints of Not Important and Very Important\n\n2Question asked: “Please rate THE IMPORTANCE of the following skills and experience to the performance of your job as editor”; % indicates the percentage of respondents endorsing ≥6 on a 7-point Likert scale with endpoints of Not Important and Very Important\n\n3Question asked: “Please rate HOW MUCH YOU POSSESS the following expertise in your job as editor”; % indicates the percentage of respondents endorsing ≥6 on a 7-point Likert scale with endpoints of Not Much and Very Much\n\n4Question asked: “Please rate HOW WELL YOU PERFORM the following skills or HOW MUCH YOU POSSESS the following experience in your job as editor”; % indicates the percentage of respondents endorsing ≥6 on a 7-point Likert scale with endpoints of Not Well and Very Well\n\nThe degree to which participants perceive that particular skills and experience are important to their job as editor. Of the 20 questions related to perceptions of the importance of particular skills and experience related to their job as editor, four items reached consensus for inclusion (Table 2). The highest consensus was for ‘behaving with integrity/professionalism’ (94.4%), followed by ‘using good judgment in decision-making’ (93.7%), ‘language/writing skills’ (90.1%), and ‘author and peer reviewer correspondence; how to evaluate peer reviews, draft a revision letter and evaluate an author response letter and revision’ (86.6%). No items reached consensus for exclusion.\n\nThe degree to which participants perceive they possess particular expertise-related items related to their job as editor. Of the 18 questions related to participants’ perceptions of how much they possess particular expertise related to their job as editor, no items reached consensus for inclusion (Table 2). The highest rated item was ‘expertise in research methods’ (72.1%), followed by ‘expertise in the publication process (decision-making aspects) for research papers, commentary, and correspondence’ (70.6%), ‘expertise in the subject areas in which your journal publishes’ (69.9%), and ‘expertise in dealing with authorship issues’ (60.7%). No items reached consensus for exclusion.\n\nThe degree to which participants perceive they perform particular skills and possess particular experience related to their job as editor. Of the 20 questions related to participants’ perceptions of how much they thought they performed particular skills and possessed particular experience related to their job as editor, three items reached consensus for inclusion (Table 2). The highest consensus was for ‘behaving with integrity/professionalism’ (90.2%), followed by ‘using good judgment in decision-making’ (87.5%), and ‘language/writing skills’ (81.5%). No items reached consensus for exclusion.\n\nRanked training needs. Training needs from participants’ ranked top ten lists were categorized into 109 unique items (Dataset 1). Of the 114 respondents to this question, the top priority listed was training in statistics; mentioned by 36.8% of respondents, with a median ranking of 2 (IQR=2). The second ranked need was for training in research methods; mentioned by 21.9% of respondents, with a median ranking of 2 (IQR=1.5). The third highest training need was in publication ethics; mentioned by 20.2% of respondents, with a median ranking of 3 (IQR=2). The fourth highest need was in recruiting and dealing with peer reviewers; mentioned by 17.5% of respondents, with a median ranking of 3 (IQR=4). The fifth highest training need was in indexing of journals; mentioned by 15.8% of respondents, with a median ranking of 2 (IQR=1).\n\nWe compiled a list of 202 unique competency-related statements identified in our previous research9 and 12 additional statements identified by participants in the survey of editors in Phase 1 (Supplementary File S3). These 214 items were categorized into seven areas:\n\nJournal publishing (29 competency-related statements)\n\nPublication ethics and research integrity (23 competency-related statements)\n\nJournal editing (46 competency-related statements)\n\nJournal promotion (23 competency-related statements)\n\nDealing with authors (27 competency-related statements)\n\nEditor qualities and characteristics (43 competency-related statements)\n\nDealing with peer reviewers (23 competency-related statements)\n\nRound 1. Eighty-three people participated, all of whom had completed the survey of editors. Of the 214 items listed, 88 items reached consensus for inclusion (≥4 on a 5-point Likert scale). Only one item – ‘act with integrity and accountability’ - was rated as 5 out of 5 by more than 80% of respondents. The items with the highest average score were ‘act with integrity and accountability’ (4.84), ‘identify and address allegations of fraud or plagiarism’ (4.70), ‘act on concerns about plagiarism, data fabrication, or an authorship issue and follow up with authors and then institutions’ (4.65), and ‘request full disclosure of potential conflicts of interest by the authors’ (4.65). No items reached consensus for exclusion (≤2 on a 5-point Likert scale) (see Supplementary File S3 for participants’ comments from all 3 rounds of the Delphi).\n\nRound 2. Eighty-three people participated, 80 of whom had completed the survey of editors and 68 of whom participated in Round 1. Of the 214 items listed, 99 items reached consensus for inclusion (≥4 on a 5-point Likert scale). Sixteen items were added for assessment based on the suggestions made by Round 1 participants, of which 4 reached consensus for inclusion, bringing the total of included items to 103. The items with the highest average score in Round 2 were ‘act with integrity and accountability’ (4.82), ‘identify and address allegations of fraud or plagiarism’ (4.68), ‘demonstrate the ability to assess the quality of papers’ (4.64), ‘Demonstrate accountability to authors and ensure they are treated with fairness, courtesy, and objectivity’ (4.63), and ‘request full disclosure of potential conflicts of interest by the authors’ (4.61). Again, no items achieved consensus for exclusion (≤2 on a 5-point Likert scale).\n\nRound 3. Round 3 included 73 participants, 72 of whom participated in the survey of editors, 58 of whom participated in both previous rounds, and 10 of whom participated in only one of the two previous rounds. The 103 items that reached consensus in Round 2 were not re-rated in Round 3. Additionally, 5 items with >80% consensus but a rating of between 3.95 and 3.99, as well as one item rated 4.0 but with only 78% consensus were inadvertently left out of the Round 3 Delphi and were therefore added (with a note) to the final list of included competency-related statements. This, therefore, left a total of 121 items to be rated in Round 3. None of these items reached consensus in Round 3, leaving a total of 109 included items at the end of Round 3. Similar to Rounds 1 and 2, no items achieved consensus for exclusion in this round.\n\nDue to the large volume of included items after 3 rounds of the Delphi, the post-hoc decision was made (JG, KDC, LS, DM) to further narrow down the list to a more manageable size. This was done by identifying items that achieved 90% consensus for inclusion in Rounds 2 or 3. This produced 23 “highly rated” items, (Table 3), leaving 86 “included” items with 80% consensus (Dataset 2). With no items having reached consensus for exclusion, the Delphi exercise was completed with 121 items that did not reach consensus for inclusion or exclusion.\n\n*The competencies are presented in the order in which they appeared in the Delphi.\n\n\nDiscussion\n\nThe results from the survey of editors revealed some patterns. First, every item on the list of participant perceptions (Table 2) occupied the same positional ranking for both the degree to which participants perceived an item as important and the degree to which they perceived that they possessed the expertise or skill. Also, consensus was higher on every item for the degree to which participants perceived particular skills or expertise as important compared to the degree to which they possessed each of these skills. This would seem to indicate that the expertise and skills that editors thought were most important were also those for which they believed themselves to be most competent. However, in examining the data more closely, we can see that the largest gaps between the perceived importance and the perceived possession of particular expertise and skills occurred among items where consensus for inclusion was achieved. In particular, ‘expertise in research methods’, ‘expertise in dealing with publication ethics…’, ‘language/writing skills’, and ‘author and peer reviewer correspondence…’ all had near or above double –digit differences between perceptions of the degree of importance vs. the degree to which participants believed they possessed this expertise or skill. This finding could point to the possibility that despite having more training in the areas they deem most important, editors still may not feel adequately trained in some of these areas.\n\nWhen comparing the top five items on participants’ ranked list of training needs with editors’ perceptions, their perceptions of competency in these areas (based on similarly-themed items in the respondent’s perceptions of their own knowledge, skills, and abilities) varied from moderate competency (for research methods, publication ethics, and recruiting and dealing with peer reviewers) to low competency (for statistics and indexing).\n\nIn the Delphi, participants were quite consistent in their ratings, reflected by the fact that no new items were scored as a 4 or above by 80% of participants after the first round of ratings. Also, although it appears from our sample that editors believed that nearly all of the knowledge, skills, and abilities identified in the scoping review and environmental scan were at least somewhat important to the role of scientific editor, none of these items were rated 2 or lower by 80% of respondents in any round of the Delphi. This could indicate that editors see their role as encompassing a very large number of important interrelated skills, abilities, and knowledge.\n\nGenerally, there appears to be some agreement between the survey of editors’ perceptions and the Delphi process across many items. When provided with an expanded list of potential competencies in the Delphi (from 38 items in the survey of editors to 230 items in the Delphi), many of the highest rated items from the survey of editors’ perceptions remained among the highest rated in the Delphi. For example, of the six items that achieved consensus in the survey of editors’ perceptions, four were similar to items on the ‘highly rated’ list from the Delphi, while the remaining two (‘writing/language skills’ and ‘using good judgment in decision-making’) were similar to items in the ‘included’ list from the Delphi. Additionally, the top 5 items listed in participants’ top 10 training needs were all included in the final list of competency-related statements arising from the Delphi, with the 2nd, 3rd, and 4th most cited training needs similar to items in the ‘highly rated’ list from the Delphi. This finding may suggest that some of the most critical skills related to the position of scientific editor may also be some of the ones for which editors feel the least trained. However, it’s unclear whether this is due to the central importance of these elements (and the need for thorough, ongoing training), a true lack of training (whether in terms of availability or quality), both of these factors, or some other reason(s).\n\nThere were a number of limitations in this research. The limited number of respondents for both the survey of editors’ perceptions and the modified Delphi, the fact that the study was conducted in English, and the fact that the survey was completed primarily by medical journal editors may limit the generalizability of the findings to the wider pool of scientific editors around the world. Additionally, for the modified Delphi, we chose to only invite respondents from the survey of editors (however we did not restrict participation to only this group). This decision was made since we believed we had used all of the most pertinent communication channels to recruit participants for the survey of editors, therefore (given our biomedical focus) we were unlikely to gain many more respondents by putting out a further call for participation in the modified Delphi. Moreover, although efforts were made to regroup similar items from participants’ top 10 lists of training needs, this subjective process may have failed to regroup some items that could be seen as similar while combining other items that could be judged to be different from one another. A further limitation is in regards to the interpretation of competency-related statements to be rated by participants in the Delphi. While efforts were made to preserve the original wording of competency-related statements in the scoping review, some of the participants’ comments relayed difficulties in understanding some items and misinterpreting others. This may have led to the mis-rating of a few items by some participants. This limitation should be offset at least to some extent by our use of median scores rather than means.\n\n\nConclusion\n\nThis research provides an insight into the perceptions of scientific editors of biomedical journals from around the world regarding the importance of particular expertise and skills in their role as scientific editors and the degree to which they believe they possess these competencies. This information complements the competency-related statements identified in the scoping review, as it enabled those who might benefit from educational efforts to identify the most important competency-related statements from their standpoint and to self-identify their greatest needs. Together with a previous scoping review and environmental scan of the literature, these findings were used to inform a consensus-building process in which a minimum set of core competencies for scientific editors of biomedical journals was determined at a consensus meeting by a wide-ranging group of stakeholders (to be reported in a future paper). The findings from both the survey and the modified Delphi process reported on here are critical for understanding knowledge and training gaps among scientific editors when designing curriculum around these core competencies in the future.\n\n\nData Availability\n\nDataset 1. Ranked list of training needs\n\nThe dataset lists all of the training needs named by participants (regrouped into categories of similar items) in their respective lists of top 10 training needs from the survey of editors.\n\n10.5256/f1000research.12400.d17599816\n\nDataset 2. All data for Delphi\n\nThe dataset is a summary of the data collected over the three rounds of the Delphi process. We considered items with 80% consensus of 4 or higher (out of 5) as \"Included\", and items with 90% consensus of 4.5 or higher as \"Highly Ranked\".\n\n10.5256/f1000research.12400.d17599917",
"appendix": "Competing interests\n\n\n\nDM is supported by a University of Ottawa Research Chair. SB and HM are part of the Cochrane Central Editorial Unit. JD leads the Cochrane Collaboration’s test evaluation activities. PG is a Coordinating Editor for the Cochrane Infectious Diseases Group. MW participated in the creation and dissemination of a survey to WAME members on medical journal editor professionalism in June 2015 and in the development of a series of sessions on medical journal editor professionalism for the WAME International Conference for Medical Journal Editors in New Delhi held in October 2015. Her participation in the study described herein was independent of WAME. JG, SS, LS, KDC, PT, and PB all have no competing interests to declare. VB is Chair of the Committee on Publication Ethics (COPE). COPE had no role in the paper except to distribute the survey.\n\n\nGrant information\n\nThis project is funded by Cochrane, Elsevier, and BioMed Central. The funding was provided to David Moher.\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 all of the respondents for taking the time to complete surveys. We also thank Cochrane, the Committee on Publication Ethics (COPE), the World Association of Medical Editors (WAME), the European Association of Science Editors (EASE), the Council of Science Editors (CSE), the Eastern Mediterranean Association of Medical Editors (EMAME), and PLoS One (A journal of the Public Library of Science) for their involvement in the survey and Delphi process. Finally, we thank Elizabeth Wager and Miranda Cumpston for their involvement in this research and the larger project to develop core competencies for scientific editors.\n\n\nSupplementary material\n\nSupplementary File S1 – Questions for survey of editors’ perceptions.\n\nClick here to access the data.\n\nSupplementary File S2 – Text answers for survey of editors’ perceptions.\n\nClick here to access the data.\n\nSupplementary File S3 – Delphi text responses for all 3 rounds.\n\nClick here to access the data.\n\n\nReferences\n\nWorld Medical Association: World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013; 310(20): 2191–4. PubMed Abstract | Publisher Full Text\n\nWong VS, Callaham ML: Medical journal editors lacked familiarity with scientific publication issues despite training and regular exposure. J Clin Epidemiol. 2012; 65(3): 247–52. PubMed Abstract | Publisher Full Text\n\nGalipeau J, Moher D, Campbell C, et al.: A systematic review highlights a knowledge gap regarding the effectiveness of health-related training programs in journalology. J Clin Epidemiol. 2015; 68(3): 257–65. PubMed Abstract | Publisher Full Text\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\nGarrow J, Butterfield M, Marshall J, et al.: The reported training and experience of editors in chief of specialist clinical medical journals. JAMA. 1998; 280(3): 286–7. PubMed Abstract | Publisher Full Text\n\nWager E, Fiack S, Graf C, et al.: Science journal editors’ views on publication ethics: results of an international survey. J Med Ethics. 2009; 35(6): 348–53. PubMed Abstract | Publisher Full Text\n\nCollier R: No favour, no friends: parsing the qualifications for a journal’s editor-in-chief. CMAJ. 2011; 183(4): 415–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarrett H, Bion JF: An international survey of training in adult intensive care medicine. Intensive Care Med. 2005; 31(4): 553–61. PubMed Abstract | Publisher Full Text\n\nHarrison LM, Davis MV, MacDonald PD, et al.: Development and implementation of a public health workforce training needs assessment survey in North Carolina. Public Health Rep. 2005; 120 Suppl 1: 28–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdgren G: Developing a competence-based core curriculum in biomedical laboratory science: a Delphi study. Med Teach. 2006; 28(5): 409–17. PubMed Abstract | Publisher Full Text\n\nPenciner R, Langhan T, Lee R, et al.: Using a Delphi process to establish consensus on emergency medicine clerkship competencies. Med Teach. 2011; 33(6): e333–9. PubMed Abstract | Publisher Full Text\n\nPenciner R, Woods RA, McEwen J, et al.: Core competencies for emergency medicine clerkships: results of a Canadian consensus initiative. CJEM. 2013; 15(1): 24–33. PubMed Abstract | Publisher Full Text\n\nHsu CC, Sandford BA: The Delphi technique: making sense of consensus. Practical assessment, research & evaluation. 2007; 12(10): 1–8. Reference Source\n\nGreen B, Jones M, Hughes D, et al.: Applying the Delphi technique in a study of GPs’ information requirements. Health Soc Care Community. 1999; 7(3): 198–205. PubMed Abstract | Publisher Full Text\n\nPutman JW, Spiegel AN, Bruininks RH: Future directions in education and inclusion of students with disabilities: A Delphi investigation. Except Children. 1995; 61(6): 553–576. Publisher Full Text\n\nGalipeau J, Cobey KD, Barbour V, et al.: Dataset 1 in: 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. F1000Research. 2017. Data Source\n\nGalipeau J, Cobey KD, Barbour V, et al.: Dataset 2 in: 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. F1000Research. 2017. Data Source"
}
|
[
{
"id": "25637",
"date": "12 Sep 2017",
"name": "Thomas A. Lang",
"expertise": [
"Reviewer Expertise Experience assessing the training needs of medical journal editors",
"years of experience working with such editors and providing some elements of their training"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 surveyed 150 editors for their opinions of the knowledge and skills needed to edit scientific journals. In a modified Delphi process, the editors then rated the importance of dozens of competencies identified in a previous scoping review to create a list of minimum competencies to be addressed in editor-training programs.\n\nThe survey and development processes seem reasonable and straightforward, and the results have high face validity, raise no concerns, and do not appear to be controversial. The authors accomplished their objective, which was to create an evidence-based list of core competencies that can form the basis for training editors of biomedical journals.\n\nThe wording of the competency statements is not always parallel or specific, but the thrust of each is clear enough that reasonable people could develop each further.\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": "25638",
"date": "15 Sep 2017",
"name": "Chris Graf",
"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 no recommendations to the authors that must be addressed. This report is part of a larger study, to identify core competencies for biomedical journal editors. The methods seem appropriate, and are well described. The results are presented clearly. The supplemental materials provide the materials (survey questions) and data. The conclusions are apt.\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/6-1634
|
https://f1000research.com/articles/6-255/v1
|
13 Mar 17
|
{
"type": "Research Article",
"title": "Structural brain abnormalities in 12 persons with aniridia",
"authors": [
"Madison K. Grant",
"Anastasia M. Bobilev",
"Jordan E. Pierce",
"Jon DeWitte",
"James D. Lauderdale",
"Madison K. Grant",
"Anastasia M. Bobilev",
"Jordan E. Pierce",
"Jon DeWitte"
],
"abstract": "Background: Aniridia is a disorder predominately caused by heterozygous loss-of-function mutations of the PAX6 gene, which is a transcriptional regulator necessary for normal eye and brain development. The ocular abnormalities of aniridia have been well characterized, but mounting evidence has implicated brain-related phenotypes as a prominent feature of this disorder as well. Investigations using neuroimaging in aniridia patients have shown reductions in discrete brain structures and changes in global grey and white matter. However, limited sample sizes and substantive heterogeneity of structural phenotypes in the brain remain a challenge. Methods: Here, we examined brain structure in a new population sample in an effort to add to the collective understanding of anatomical abnormalities in aniridia. The current study used 3T magnetic resonance imaging to acquire high-resolution structural data in 12 persons with aniridia and 12 healthy demographically matched comparison subjects. Results: We examined five major structures: the anterior commissure, the posterior commissure, the pineal gland, the corpus callosum, and the optic chiasm. The most consistent reductions were found in the anterior commissure and the pineal gland; however, abnormalities in all of other structures examined were present in at least one individual. Conclusions: Our results indicate that the anatomical abnormalities in aniridia are variable and largely individual-specific. These findings suggest that future studies investigate this heterogeneity further, and that normal population variation should be considered when evaluating structural abnormalities.",
"keywords": [
"MRI",
"PAX6",
"neuroanatomy"
],
"content": "Introduction\n\nAniridia is panocular, congenital, and progressive disorder with an occurrence of approximately 1 in 83,000 live births1,2. Aniridia is best characterized by the lack of or hypoplasia of the iris (for which it is named), in addition to several other ocular abnormalities, which culminate in reduced visual acuity3. Due to the progressive nature of the disease, individuals usually develop multiple ocular abnormalities, such as keratopathy, corneal vascularization and opacification, glaucoma, anterior chamber fibrosis, and cataracts4–7. Although aniridia is most well known for its ocular phenotypes, the condition has a number of other abnormalities, including sensory, neural, cognitive, and auditory processing abnormalities8,9.\n\nThe development of aniridia in humans is linked to heterozygous loss-of-function mutations to the PAX6 gene, which encodes a highly conserved transcription factor critical for normal eye and neural development1. The vast majority of aniridia cases (80%) are associated with mutations in PAX62,10. Functional mutations in this gene can be either sporadic or familial, and causal mutations in aniridia encompass a large number of variants. The majority of these variants are single nucleotide polymorphisms, which lead to a premature termination codon, and are found across the PAX6 locus10,11. PAX6 is expressed in the developing eye, brain, and spinal cord, and is required for aspects of anatomical and functional development of the central nervous system (CNS) and visual system12. Within the CNS, PAX6 is involved in patterning, regionalization, and the formation of neural circuits13–16. Previous studies of patients with aniridia using structural magnetic resonance imaging (MRI) have shown abnormalities in major fiber tracts and subcortical structures of the brain, including the anterior commissure9,17–20, posterior commissure18,20, corpus callosum9,19,20, pineal gland18,20, optic chiasm20, and olfactory bulb17,18. The most consistently reported abnormalities are found in the anterior commissure, pineal gland, and optic chiasm; abnormalities in the posterior commissure, corpus callosum, and olfactory bulb are found in fewer than 35% of patients examined. Additionally, studies have shown conflicting results regarding grey matter volume differences in aniridia, with reports of both increases and decreases in whole brain grey matter21,22. Most recently, it has been shown that there is an accelerated age-related increase in cortical thinning of the inferior parietal lobe and prefrontal/premotor areas in both brain hemispheres in aniridia compared to healthy patients23.\n\nWhile several previous studies have investigated structural brain abnormalities in patients with aniridia, the variance in brain structures affected and extent of anatomical abnormalities is high. The variance observed in the published literature may be interpreted as a result of genetic differences in patient samples, either directly related to disease-causing mutations or modifier effects caused by genomic differences across subjects. The current study sought to investigate gross anatomical correlates of aniridia in a new population sample. Results from this study will serve as a comparison for previous studies, as well as contribute to what is known about the distribution of neuroanatomical phenotypes in the aniridia population as a whole.\n\n\nMethods\n\nA total of 14 individuals with aniridia and 15 healthy comparison individuals participated in the current study. Data from two participants with aniridia were excluded from analyses (due to a significant artifact and missing data). One healthy subject was excluded due to an anatomical abnormality, and two others were excluded because they did not match the demographic profile of an individual in the aniridia group included in the analysis. The remaining 12 individuals with aniridia (7 females; mean age=36 years, SD=15) and 12 age- and gender-matched comparisons (7 females; mean age=35 years, SD=14) were included in the analyses (Table 1). Healthy comparison subjects were recruited through flyers posted in the community. Participants with aniridia were recruited through the Aniridia Foundation International Conference held in 2011 in Athens, Georgia and had been clinically diagnosed with aniridia. Exonic sequencing of the PAX6 gene (11p13) (OMIM: 607108) was conducted at the University of Georgia, as previously described10,24. All mutations, which can be found in Table 1, have been submitted to the Human PAX6 Allelic Variant Database (http://lsdb.hgu.mrc.ac.uk/home.php?select_db=PAX6), as part of a previous genotype identification study10. Three of the participants with aniridia belonged to the same family, and all other participants included in the analyses were unrelated. After written informed consent was obtained and MRI safety screening was conducted, all participants completed an MRI session in which a high-resolution structural scan was obtained. The Institutional Review Board of the University of Georgia approved all activities prior to subject recruitment, data collection, and data analysis (project number: 2011-10862-1; STUDY00003122).\n\nGender, age, mutation, and structural abnormalities of both aniridia subjects and healthy comparisons. Subject ID: Numbers are matched subjects, A refers to aniridia subjects, C refers to healthy comparisons. ND, not determined.\n\nAll data were collected on a 3T GE Signa MRI (General Electric, Milwaukee, WI, USA) at the University of Georgia’s Bio-Imaging Research Center. To obtain a high-resolution structural scan, images were acquired with a T1-weighted 3D FSPGR sequence [echo time (TE)=min full, flip angle=20°; field of view (FOV)=240 mm × 240 mm; matrix size=256 × 256, 150 axial slices; in-slice resolution=0.94 × 0.94 mm; slice thickness=1.2 mm].\n\nMR images were transferred to a DICOM image format and analyzed using two software programs, SPM run on MATLAB and OsiriX. For SPM analysis, DICOM files were converted to nifti format and analyzed using Statistical Parametric Mapping Software (SPM8; Wellcome Trust Centre for Neuroimaging; http://www.fil.ion.ucl.ac.uk/spm/) run on a MATLAB software platform (MATLAB Release 2015b; The Mathworks, Inc., Natick, MA, USA). SPM software was used to compare aniridia subjects to their demographically matched comparison subjects. DICOM files were additionally analyzed using Osirix Lite DICOM viewer (OsiriX v5.6,;Pixmeo SARL, Bernex, Switzerland) and all figures generated using this software. All 24 individual subjects’ data were visually inspected for gross anatomical abnormalities in two independent sessions by the first author (MG) and a radiologist (JDW). The radiologist was completely blinded to patient genotype when examining the MRI scans. This study focused on evaluating five major brain structures: anterior commissure, posterior commissure, pineal gland, corpus callosum, and optic chiasm.\n\n\nResults\n\nWhole brain analysis was used to identify major structural abnormalities in aniridia patients. All neuroanatomical results are reported in Table 1, which includes subject demographics and mutations. We analysed five major brain structures that demonstrate clear anatomical abnormalities in aniridia patients. Our study showed that all 12 aniridia patients had a reduced anterior commissure when compared to their demographically matched healthy comparisons, as shown in Figure 1. The posterior commissure was reduced in 5/12 aniridia patients and normal in 7/12 of aniridia patients (Figure 1). Of the five aniridia patients with reduced posterior commissures, one had a reduced commissure at the midline. The pineal gland was affected in all 12 aniridia patients: absent in two (Figure 1), highly reduced in one, and reduced in nine. The corpus callosum was slightly thinned in 3/12 aniridia patients and normal in 9/12 aniridia patients (Figure 2). The optic chiasm was reduced in 7/12 of patients (Figure 2) and normal in 5/12 patients.\n\nAxial cerebral T1-weighted magnetic resonance images, slice thickness 1.2mm. (A) Subject 12C: Arrow shows normal anterior commissure; arrowhead shows normal posterior commissure; dashed arrow shows normal pineal gland. (B) Subject 12A: Arrow shows reduced anterior commissure; arrowhead shows normal posterior commissure; dashed arrow shows absence of pineal gland.\n\nCoronal cerebral T1-weighted magnetic resonance images, slice thickness 1.2mm. (A) Subject 2C: Arrow shows normal optic chiasm. (B) Subject 2A: Arrow shows reduced optic chiasm. Arrowheads in A and B denote normal corpus callosum.\n\nIn an effort to consider normal structural variation in the healthy population, we also evaluated healthy comparisons for structural brain abnormalities. We saw a reduced anterior commissure in two of the healthy comparisons and a reduced posterior commissure in one healthy comparison. The pineal gland showed the most variance within the healthy comparison group with one subject with no visible pineal gland, three healthy comparisons with slightly reduced to reduced pineal glands, and eight healthy comparisons with normal pineal glands. All healthy comparisons had normal corpus callosums and optic chiasms. A full description of which structures showed abnormalities in both the aniridia and healthy comparison groups are reported in Table 1. These findings provide context for asserting disease-related changes in the aniridia population, in the current study as well as others.\n\n\nDiscussion\n\nThe most commonly reported neuroanatomical abnormality in MRI studies of aniridia patients is the anterior commissure. Previous studies have reported changes in the anterior commissure with some cases described as reductions and others reported as complete absence of the structure9,17–19. Consistent with these reports, our study identified a reduction in the anterior commissure in all 12 aniridia patients, but none of the patients lacked the structure. The posterior commissure has also been evaluated in previous studies: one study reports that it is present in all subjects while the other study presented evidence that one patient had an absent structure while the others had normal posterior commissures18,20. We found no individuals lacking the posterior commissure, and fewer than half of our aniridia patients seemed to have an abnormal structure. Interestingly though, it seems as if one patient exhibited a reduction of the posterior commissure at the midline with thickened bundles adjacent to the midline suggesting that commissure formation was incomplete. PAX6 has a known role in formation of the posterior commissure in rodents, so it is likely that this phenotype in humans is a direct consequence of PAX6 deficiency15. In agreement with previous findings, we also see abnormal or absent pineal glands in our entire patient population. This finding is consistent with sleep regulation deficits in persons with aniridia reported in other studies25. The corpus callosum has also been a structure commonly evaluated in aniridia MRI studies, with many reporting reductions in corpus callosum thickness and severe agenesis9,19,20. However, we found very few patients present with a reduced or abnormal corpus callosum, and propose that the slight reduction we see in three of our patients falls within normal population variation. Unlike most previous studies, we examined the optic chiasm, and found a reduction in the structure in more than half of our patients. This reduction could be a developmental consequence of the disorder or a progressive phenotype associated with reduced levels of PAX6. Anatomical abnormality findings seem to be highly dependent on population sample, and a larger collective sample in the literature will help us get closer to understanding common disease traits and variation.\n\nAs described above, we observed a reduction in the anterior commissure in every patient we evaluated and a reduction of the posterior commissure in five patients, but no individuals completely lacked either structure. Our study utilized a high resolution 3T MRI for data acquisition, while most other studies used a 1.5T MRI. Signal to noise ratio from 3T MRIs are almost double that of 1.5T MRIs, which will lead to an improved image quality and resolution from the 3T magnet26. We propose that the difference in observing a reduced versus absent anterior/posterior commissure may be due to a difference in scan resolution between images captured from a 3T versus 1.5T magnet. We see multiple patients in our group who have a severely reduced anterior commissure, and identifying this abnormality using a 1.5T magnet may be more difficult than when using a 3T. Additionally, the posterior commissure is smaller than the anterior commissure naturally, making it more difficult to distinguish between presence and absence in a scan. Lower scan resolution may not capture small structures such as these commissures, especially if they are reduced in size, leading to a false judgment of their absence.\n\nA recent study has found an age component to cortical thickness in aniridia patients when compared to healthy individuals. The study found that in patients with aniridia there is an accelerated reduction in cortical thickness of the inferior parietal lobe and prefrontal/premotor areas in both brain hemispheres23. Adding to this age component seen in the Yogarajah (2016) study, there are also population differences in brain anatomy, even within healthy groups, between younger subjects and older subjects27. Additionally, as we show in our study, there are anatomical abnormalities even within healthy, unaffected participants. This makes it vitally important for careful selection of comparison subjects, and presents a caveat for interpreting differences we see in this and other clinical populations.\n\nIn addition to abnormal structural findings in patient populations with aniridia, multiple studies have assessed volumetric differences in grey and white matter in the brains of aniridia versus healthy comparisons. Similar to the findings in gross structural differences, much variation exists between reports. Some studies show an increase in grey matter volume in aniridia patients compared to healthy control groups, while others find both increases and decreases depending on brain region21,22. Changes in white matter findings follow the same suit with some reports of reductions in white matter and others finding both reductions and increases21,22. Even more interestingly, structures, such as the anterior commissure, posterior commissure, and pineal gland, show no deviation from healthy in these group-wise comparisons. This suggests that either the abnormalities seen in these structures are not as common among aniridia patients as previously thought, or that the characteristics of anatomical changes observed in aniridia patients have a high degree of variability within the population. Due to the consistency of abnormalities in structures like the anterior commissure in our study, as well as others, we predict the latter explains this discrepancy.\n\n\nConclusions\n\nThe current study investigated anatomical brain abnormalities correlated to aniridia in a new population sample in an effort to serve as a comparison to previous studies. Our aim was to contribute to what is known about the distribution of neuroanatomical phenotypes in the aniridia population as a whole. Although we found similar neuroanatomical abnormalities as previous studies, we find the severity is not as great as previously reported. The anterior commissure and pineal gland seem to be the structures most affected in the aniridia patients we examined, and we do see abnormalities in the posterior commissure, corpus callosum and the optic chiasm, albeit at lower frequency than previously reported. We believe the neuroanatomical abnormalities seen in aniridia populations have a high level of variability, and future studies should be aimed at collecting more patient MRI scans so that the breadth of abnormalities can be assessed.\n\n\nEthical approval and consent\n\nMRI sessions were completed after written informed consent was obtained and MRI safety screening was completed. All activities were approved by the Institutional Review Board of the University of Georgia prior to subject recruitment and data collection. All individuals who participated in this study provided consent for their demographic, mutation information, and images to be published.\n\n\nData availability\n\nDataset 1: Aniridia and healthy comparison MRI data: Structural MRI DICOM files for 12 aniridia and 12 healthy comparison individuals. Files are in DICOM format and labeled according to subject I.D. found in Table 1. These files can be opened using SPM software run in Matlab or OsiriX DICOM viewer (see Methods section). doi, 10.5256/f1000research.11063.d15379928\n\nMutation information that has been presented here is also available through PAX6 Allelic Variant Database (http://lsdb.hgu.mrc.ac.uk/home.php?select_db=PAX6).",
"appendix": "Author contributions\n\n\n\nAMB and JDL conceived this study as part of a large neuroimaging effort in this clinical population. JEP provided technical expertise on MRI imaging and data acquisition, and assisted AMB with experimental design and data collection. MKG analyzed the data first followed by JDW, who analyzed the data second (independently) to determine structural abnormalities in each brain. MKG and AMB prepared the first draft of the manuscript. All authors were involved in the revisions of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by a grant from the Sharon Stewart Aniridia Research Trust to AMB and JDL.\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\nAMB is a Franklin Foundation Neuroimaging Fellow and an ARCS Scholar.\n\nCollection of magnetic resonance imaging data was made possible by the University of Georgia Bioimaging Research Center. The authors wish to thank Ms. Kimberly Mason for her assistance with data collection and subject screening, Ms. Jill Nerby, Director and Founder of Aniridia Foundation International, and the participants in this study for their support of this research.\n\n\nReferences\n\nHanson IM, Seawright A, Hardman K, et al.: PAX6 mutations in aniridia. Hum Mol Genet. 1993; 2(7): 915–920. PubMed Abstract | Publisher Full Text\n\nGrønskov K, Olsen JH, Sand A, et al.: Population-based risk estimates of Wilms tumor in sporadic aniridia. A comprehensive mutation screening procedure of PAX6 identifies 80% of mutations in aniridia. Hum Genet. 2001; 109(1): 11–18. PubMed Abstract | Publisher Full Text\n\nNelson LB, Spaeth GL, Nowinski TS, et al.: Aniridia. A review. Surv Ophthalmol. 1984; 28(6): 621–42. PubMed Abstract | Publisher Full Text\n\nGrant WM, Walton DS: Progressive changes in the angle in congenital aniridia, with development of glaucoma. Am J Ophthalmol. 1974; 78(5): 842–847. PubMed Abstract | Publisher Full Text\n\nMcCulley TJ, Mayer K, Dahr SS, et al.: Aniridia and optic nerve hypoplasia. Eye (Lond). 2005; 19(7): 762–764. PubMed Abstract | Publisher Full Text\n\nNishida K, Kinoshita S, Ohashi Y, et al.: Ocular surface abnormalities in aniridia. Am J Ophthalmol. 1995; 120(3): 368–375. PubMed Abstract | Publisher Full Text\n\nTsai JH, Freeman JM, Chan CC, et al.: A progressive anterior fibrosis syndrome in patients with postsurgical congenital aniridia. Am J Ophthalmol. 2005; 140(6): 1075–1079. PubMed Abstract | Publisher Full Text\n\nThompson PJ, Mitchell TN, Free SL, et al.: Cognitive functioning in humans with mutations of the PAX6 gene. Neurology. 2004; 62(7): 1216–1218. PubMed Abstract | Publisher Full Text\n\nBamiou DE, Musiek FE, Sisodiya SM, et al.: Deficient auditory interhemispheric transfer in patients with PAX6 mutations. Ann Neurol. 2004; 56(4): 503–509. PubMed Abstract | Publisher Full Text\n\nBobilev AM, McDougal ME, Taylor WL, et al.: Assessment of PAX6 alleles in 66 families with aniridia. Clin Genet. 2016; 89(6): 669–77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHingorani M, Williamson KA, Moore AT, et al.: Detailed ophthalmologic evaluation of 43 individuals with PAX6 mutations. Invest Ophthalmol Vis Sci. 2009; 50(6): 2581–90. PubMed Abstract | Publisher Full Text\n\nKim J, Lauderdale JD: Analysis of Pax6 expression using a BAC transgene reveals the presence of a paired-less isoform of Pax6 in the eye and olfactory bulb. Dev Biol. 2006; 292(2): 486–505. PubMed Abstract | Publisher Full Text\n\nManuel M, Price DJ: Role of Pax6 in forebrain regionalization. Brain Res Bull. 2005; 66(4–6): 387–393. PubMed Abstract | Publisher Full Text\n\nSimpson TI, Price DJ: Pax6; a pleiotropic player in development. Bioessays. 2002; 24(11): 1041–1051. PubMed Abstract | Publisher Full Text\n\nMastick GS, Davis NM, Andrew GL, et al.: Pax-6 functions in boundary formation and axon guidance in the embryonic mouse forebrain. Development. 1997; 124(10): 1985–1997. PubMed Abstract\n\nOsumi N: The role of Pax6 in brain patterning. Tohoku J Exp Med. 2001; 193(3): 163–174. PubMed Abstract\n\nSisodiya SM, Free SL, Williamson KA, et al.: PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans. Nat Genet. 2001; 28(3): 214–216. PubMed Abstract | Publisher Full Text\n\nMitchell TN, Free SL, Williamson KA, et al.: Polymicrogyria and absence of pineal gland due to PAX6 mutation. Ann Neurol. 2003; 53(5): 658–663. PubMed Abstract | Publisher Full Text\n\nBamiou DE, Free SL, Sisodiya SM, et al.: Auditory interhemispheric transfer deficits, hearing difficulties, and brain magnetic resonance imaging abnormalities in children with congenital aniridia due to PAX6 mutations. Arch Pediatr Adolesc Med. 2007; 161(5): 463–469. PubMed Abstract | Publisher Full Text\n\nAbouzeid H, Youssef MA, ElShakankiri N, et al.: PAX6 aniridia and interhemispheric brain anomalies. Mol Vis. 2009; 15: 2074–83. PubMed Abstract | Free Full Text\n\nFree SL, Mitchell TN, Williamson KA, et al.: Quantitative MR image analysis in subjects with defects in the PAX6 gene. Neuroimage. 2003; 20(4): 2281–2290. PubMed Abstract | Publisher Full Text\n\nEllison-Wright Z, Heyman I, Frampton I, et al.: Heterozygous PAX6 mutation, adult brain structure and fronto-striato-thalamic function in a human family. Eur J Neurosci. 2004; 19(6): 1505–12. PubMed Abstract | Publisher Full Text\n\nYogarajah M, Matarin M, Vollmar C, et al.: PAX6, brain structure and function in human adults: advanced MRI in aniridia. Ann Clin Transl Neurol. 2016; 3(5): 314–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPierce JE, Krafft CE, Rodrigue AL, et al.: Increased functional connectivity in intrinsic neural networks in individuals with aniridia. Front Hum Neurosci. 2014; 8: 1013. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHanish AE, Butman JA, Thomas F, et al.: Pineal hypoplasia, reduced melatonin and sleep disturbance in patients with PAX6 haploinsufficiency. J Sleep Res. 2016; 25(1): 16–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWood R, Bassett K, Foerster V, et al.: PROS AND CONS OF 1.5 T MRI VERSUS 3.0 T MRI. Reference Source\n\nCoffey CE, Lucke JF, Saxton JA, et al.: Sex differences in brain aging. a quantitative magnetic resonance imaging study. Arch Neurol. 1998; 55(2): 169–179. PubMed Abstract | Publisher Full Text\n\nGrant MK, Bobilev AM, Pierce JE, et al.: Dataset 1 in: Structural brain abnormalities in 12 persons with aniridia. F1000Research. 2017. Data Source"
}
|
[
{
"id": "21598",
"date": "06 Apr 2017",
"name": "Veronica van Heyningen",
"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 report on structural brain changes in 12 aniridia patients examined by structural MRI studies, using 12 selected matching controls for comparison. The title and abstract convey details appropriately.\nI should say at the outset that I have no expertise in MRI methodology and cannot compare the techniques used here with those used elsewhere, including our own papers.\n\nIt is clear, however, that MRI has advanced technically to a significant extent since the first set of papers was published in 2001-3. We need to know how directly comparable the results are. Certainly Sisodiya et al. in 2001 (ref 17) Mitchell et al. and 2003 (ref 18) used voxel-based morphometry and less powerful MRI measurements but showed similar findings with observed reduction or absence of the anterior commissure and olfactory bulb (ref 17) and of the pineal (ref 18). The current study does not mention study of the olfactory bulb. Interestingly olfactory function was also shown to be reduced and very occasionally absent in the first study (ref 17). Reduced interhemispheric transfer is thought to underlie the observed auditory processing problems reported in children with aniridia in ref 19. The current study reported in this paper does not undertake functional smell or auditory tests. Although what seems to be the same cohort, judging by the reported mutations, has been studied by functional fMRI by Pierce et al (ref 24). Surprisingly this overlapping author group (with the current paper) reports an apparently increased functional connectivity in the patients studied in the current paper. The earlier studies by other groups do not look at these functional connectivity changes. It is regrettable that the later studies, including Yogarajah et al (ref 23) did not follow this up.\n\nOtherwise the major findings are remarkably similar for this study and the previous studies to explore 12 or more cases. The controls in ref 17 and 18 are larger numbers (100) and not directly paired controls. The study in Ref 20 where the posterior commissure reduction was observed, only looked at the index case from three families with aniridia and PAX6 mutations. Reference 23 re-examines many of the same cases as Ref 17 and 18, but about a decade later and with more powerful MRI using surface-based morphometry. The reduced cortical measurements are more obvious in these older cases. The observed reduction of the optic chiasm reported here in 7/12 cases is an interesting new finding, first suggested in the one of the three families studied by Abouzeid et al, ref 20.\n\nMinor comments\nIn the Introduction the PAX6 mutations are described as mostly “single nucleotide polymorphisms”. This is incorrect the changes are extremely rare variants. There are no polymorphisms of any note in the extremely highly conserved PAX6 gene.\n\nIt is a pity that the patient numbering is different in earlier fMRI work from this group (ref 24). Indeed it is not made clear that ref 24 studies the same cohort as observed in the current paper.\n\nIt would help to have the protein effects of the mutations in Table 1.\n\nCan the olfactory bulb status be observed with the current cohort? It would be interesting to see that reported.",
"responses": [
{
"c_id": "2973",
"date": "01 Sep 2017",
"name": "Madison Grant",
"role": "Author Response",
"response": "Reviewer: Veronica van Heyningen, Institute of Ophthalmology, University College London, London, UK We thank Dr. van Heyningen for the helpful comments and suggestions, which improved the manuscript. Our responses are listed below. Comments to the Author:Minor comments In the Introduction the PAX6 mutations are described as mostly “single nucleotide polymorphisms”. This is incorrect the changes are extremely rare variants. There are no polymorphisms of any note in the extremely highly conserved PAX6 gene.- The text was modified to read: “The majority of these variants are nonsense mutations, which lead to a premature termination codon, and are found across the PAX6 locus.” It is a pity that the patient numbering is different in earlier fMRI work from this group (ref 24). Indeed it is not made clear that ref 24 studies the same cohort as observed in the current paper.- Numbering of patients has been amended to reflect the same numbering used in the previous paper (ref 24). We have additionally added a supplementary table (Supplementary Table 2) that matches DICOM file names to patient ID from this paper and previous papers using the same patient group. Additionally, we have added a sentence in methods (Subjects) to state that this study and ref 24 used the same patients. It would help to have the protein effects of the mutations in Table 1.- Protein effects have been added to Table 1. Can the olfactory bulb status be observed with the current cohort? It would be interesting to see that reported.- While we agree that the olfactory bulb would be an interesting and important addition, we elected not to pursue this structure because constraints inherent to our data. We have amended methods (MRI data analysis) to discuss explain why we decided not to investigate olfactory bulb in this patient sample."
}
]
},
{
"id": "21372",
"date": "18 Apr 2017",
"name": "Lutz Jäncke",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and extremely important study reporting specific neuroanatomical features in patient group suffering from a rare disease affecting the visual system: aniridia. I have listed my comments and suggestions below.\nIntroduction The introduction is fine and discusses the current literature. However, I would suggest to improve a bit the study question. The study question as it is used in the current version is very broad and does not come to the point (e.g.,\"The current study sought to investigate gross anatomical correlates of aniridia in a new population sample. Results from this study will serve as a comparison for previous studies, as well as contribute to what is known about the distribution of neuroanatomical phenotypes in the aniridia population as a whole.\"). I would suggest to include a few statements what this new study will add to the literature and what the \"problems\" of the older studies are? In addition, it could be helpful for the reader to learn the very new and maybe innovative aspects of this study.\nMethods Subjects: Some additional information about the subjects could be helpful (cognitive ability, handedness, language lateralization). Some of these variables have been shown to be strongly related to the anatomical measures for which the authors are reporting the specific aniridia features.\nStatistics: It is not entirely clear how the authors did their between-group comparisons. Did they compare the patients only on a visual-descriptive basis or did they perform a statistical analysis (which I would suggest). When doing a statistical analysis, one could compare single subjects to the mean of the control group. Thus, it would be possible to test each patient against the mean of the healthy control group. Using this comparison it would also be possible to calculate effect size measures for each subject.\nBy the way, did the use a priorly defined regions of interest?\nDiscussion The discussion is fine.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "2972",
"date": "01 Sep 2017",
"name": "Madison Grant",
"role": "Author Response",
"response": "Dr. Lutz Jäncke, Department of Psychology (Neuropsychology) and INAPIC, University of Zurich, Zurich, Switzerland We thank Dr. Jäncke for the helpful comments and suggestions, which improved the manuscript. Our responses are listed below.Introduction Improve study question Introduction has been revised to improve, clarify, and expand the description of our study question. Suggest: to include a few statements what this new study will add to the literature and what the \"problems\" of the older studies are? In addition, it could be helpful for the reader to learn the very new and maybe innovative aspects of this study. Introduction has been revised to include more information on new aspects for this study. In particular we have emphasized at the end of the “Introduction” section that this study utilized a 3T magnet instead of a 1.5T, allowing for higher resolution when examining small structures such as the anterior and posterior commissure. Additionally, we also clarified, at the end of the “Introduction” section, that our study examined five structures shown to be abnormal in the aniridia population. Previous studies focused on only a subset of structures in each patient rather than all five. By examining all five structures in a new population of aniridia patients, we have added to the collective knowledge on structural brain abnormalities present within the disorder. Methods Subjects: additional information: cognitive ability, handedness, language lateralization We added handedness and predicted protein effect to Table 1. We do not have data on cognitive ability or language lateralization for our participants. Statistics: It is not entirely clear how the authors did their between-group comparisons. Did they compare the patients only on a visual-descriptive basis or did they perform a statistical analysis (which I would suggest). When doing a statistical analysis, one could compare single subjects to the mean of the control group. Thus, it would be possible to test each patient against the mean of the healthy control group. Using this comparison it would also be possible to calculate effect size measures for each subject. For this study, the structural changes in Table 1 were concluded by two independent reviews of the MRI scans using a visual-descriptive basis. The methods have been updated to clarify the visual-descriptive assessment. Volumetric analysis was conducted for the corpus callosum using Freesurfer software and results have been added to the manuscript (see supplementary Table 1 and supplementary Figure 1). Findings from this volumetric analysis confirmed our structural examination of the corpus callosum for all subjects. We did not conduct quantitative volumetric analyses on the anterior and posterior commissures as these are relatively small structures and the plane of view was not consistent within all subjects. This is due to variation in slice location within each brain. Because of this we decided to rely on our visual-descriptive assessment of these two structures for our dataset. While not ideal, a visual-descriptive assessment of the anterior and posterior commissures is consistent with previous reports comparing these structures between PAX6 normal individuals and individuals with PAX6 mutations (e.g. Sisodiya et al. 2001; Abouzeid et al., 2009) and captures relative changes between these two groups. By the way, did the use a priorly defined regions of interest? Yes, our Methods section (MRI data analysis) has been revised to state how we determined regions of interest. We used literature review to define which structures would be examined, and had ourthe radiologist (JD) examined the MRI scans for identification of additional regions of interest. DiscussionThe discussion is fine."
},
{
"c_id": "3039",
"date": "19 Sep 2017",
"name": "Lutz Jäncke",
"role": "Reviewer Response",
"response": "Thank you so much. Well done!"
}
]
}
] | 1
|
https://f1000research.com/articles/6-255
|
https://f1000research.com/articles/6-588/v1
|
27 Apr 17
|
{
"type": "Systematic Review",
"title": "What is open peer review? A systematic review",
"authors": [
"Tony Ross-Hellauer"
],
"abstract": "Background: “Open peer review” (OPR), despite being a major pillar of Open Science, has neither a standardized definition nor an agreed schema of its features and implementations. The literature reflects this, with a myriad of overlapping and often contradictory definitions. While the term is used by some to refer to peer review where the identities of both author and reviewer are disclosed to each other, for others it signifies systems where reviewer reports are published alongside articles. For others it signifies both of these conditions, and for yet others it describes systems where not only “invited experts” are able to comment. For still others, it includes a variety of combinations of these and other novel methods. Methods: Recognising the absence of a consensus view on what open peer review is, this article undertakes a systematic review of definitions of “open peer review” or “open review”, to create a corpus of 122 definitions. These definitions are then systematically analysed to build a coherent typology of the many different innovations in peer review signified by the term, and hence provide the precise technical definition currently lacking. Results: This quantifiable data yields rich information on the range and extent of differing definitions over time and by broad subject area. Quantifying definitions in this way allows us to accurately portray exactly how ambiguously the phrase “open peer review” has been used thus far, for the literature offers a total of 22 distinct configurations of seven traits, effectively meaning that there are 22 different definitions of OPR in the literature. Conclusions: Based on this work, I propose a pragmatic definition of open peer review as an umbrella term for a number of overlapping ways that peer review models can be adapted in line with the ethos of Open Science, including making reviewer and author identities open, publishing review reports and enabling greater participation in the peer review process.",
"keywords": [
"open peer review",
"Open Science",
"scholarly communication",
"research evaluation",
"publishing"
],
"content": "Introduction\n\n“Open review and open peer review are new terms for evolving phenomena. They don’t have precise or technical definitions. No matter how they’re defined, there’s a large area of overlap between them. If there’s ever a difference, some kinds of open review accept evaluative comments from any readers, even anonymous readers, while other kinds try to limit evaluative comments to those from ”peers“ with expertise or credentials in the relevant field. But neither kind of review has a special name, and I think each could fairly be called “open review” or “open peer review”.” - Peter Suber, email correspondence, 20071.\n\nAs with other areas of “open science” (Pontika et al., 2015), “open peer review” (OPR) is a hot topic, with a rapidly growing literature that discusses it. Yet, as has been consistently noted (Ford, 2013; Hames, 2014; Ware, 2011), OPR has neither a standardized definition, nor an agreed schema of its features and implementations. The literature reflects this, with a myriad of overlapping and often contradictory definitions. While the term is used by some to refer to peer review where the identities of both author and reviewer are disclosed to each other, for others it signifies systems where reviewer reports are published alongside articles. For others it signifies both of these conditions, and for yet others it describes systems where not only “invited experts” are able to comment. For still others, it includes a variety of combinations of these and other novel methods. The previous major attempt to resolve these elements systematically to provide a unified definition (Ford, 2013), discussed later, unfortunately ultimately confounds rather than resolves these issues.\n\nIn short, things have not improved much since Suber made his astute observation. This continuing imprecision grows more problematic over time, however. As Mark Ware notes, “it is not always clear in debates over the merits of OPR exactly what is being referred to” (Ware, 2011). Differing flavours of OPR include independent factors (open identities, open reports, open participation, etc.), which have no necessary connection to each other, and very different benefits and drawbacks. Evaluation of the efficacy of these differing variables and hence comparison between differing systems is therefore problematic. Discussions are potentially side-tracked when claims are made for the efficacy of “OPR” in general, despite critique usually being focussed on one element or distinct configuration of OPR. It could even be argued that this inability to define terms is to blame for the fact that, as Nicholas Kriegskorte has pointed out, “we have yet to develop a coherent shared vision for “open evaluation” (OE), and an OE movement comparable to the OA movement” (Kriegeskorte, 2012).\n\nTo resolve this, I undertake a systematic review of the definitions of “open peer review” or “open review”, to create a corpus of more than 120 definitions. These definitions have been systematically analysed to build a coherent typology of the many different innovations in peer review signified by the term, and hence provide the precise technical definition that is currently lacking. This quantifiable data yields rich information on the range and extent of differing definitions over time and by broad subject area. Based on this work, I propose a pragmatic definition of OPR as an umbrella term for a number of overlapping ways that peer review models can be adapted in line with the ethos of Open Science, including making reviewer and author identities open, publishing review reports and enabling greater participation in the peer review process.\n\n\nBackground\n\nPeer review is the formal quality assurance mechanism whereby scholarly manuscripts (e.g. journal articles, books, grant applications and conference papers) are made subject to the scrutiny of others, whose feedback and judgements are then used to improve works and make final decisions regarding selection (for publication, grant allocation or speaking time). This system is perhaps more recent than one might expect, with its main formal elements only in general use since the mid-twentieth century in scientific publishing (Spier, 2002). Researchers agree that peer review per se is necessary, but most find the current model sub-optimal. Ware’s 2008 survey, for example, found that an overwhelming majority (85%) agreed that “peer review greatly helps scientific communication” and that even more (around 90%) said their own last published paper had been improved by peer review. Yet almost two thirds (64%) declared that they were satisfied with the current system of peer review, and less than a third (32%) believed that this system was the best possible (Ware, 2008). A recent follow-up study by the same author reported a slight increase in the desire for improvements in peer review (Ware, 2016)\n\nWidespread beliefs that the current model is sub-optimal can be attributed to the various ways in which traditional peer review has been subject to criticism. Peer review has been variously accused of:\n\nUnreliability and inconsistency: Reliant upon the vagaries of human judgement, the objectivity, reliability, and consistency of peer review are subject to question. Studies show reviewers’ views tend to show very weak levels of agreement (Kravitz et al., 2010; Mahoney, 1977), at levels only slightly better than chance (Herron, 2012; Smith, 2006). Studies suggest decisions on rejection or acceptance are similarly inconsistent. For example, Peters and Ceci’s classic study found that eight out of twelve papers were rejected for methodological flaws when resubmitted to the same journals in which they had already been published (Peters & Ceci, 1982). This inconsistency is mirrored in peer review’s inability to prevent errors and fraud from entering the scientific literature. Reviewers often fail to detect major methodological failings (Schroter et al., 2004), with eminent journals (whose higher rejection rates might suggest more stringent peer review processes) seeming to perform no better than others (Fang et al., 2012). Indeed, Fang and Casadevall found that the frequency of retraction is strongly correlated with the journal impact factor (Fang & Casadevall, 2011). Whatever the cause, recent sharp rises in the number of retracted scientific publications (Steen et al., 2013) testify that peer review sometimes fails in its role as the gatekeeper of science, allowing errors to enter the literature. Peer review’s other role, of filtering the best work into the best journals, also seems to fail. Many articles in top journals remain poorly cited, while many of the most highly-cited articles in their fields are published in lower-tier journals (Jubb, 2016).\n\nDelay and expense: The period from submission to publication at many journals can often exceed one year, with much of this time taken up by peer review. This delay slows down the availability of results for further research and professional exploitation. The work undertaken in this period is also expensive, with the global costs of reviewers’ time estimated at £1.9bn in 2008 (Research Information Network [RIN], 2008), a figure which does not take into account the coordinating costs of publishers, or the time authors spend revising and resubmitting manuscripts (Jubb, 2016). These costs are greatly exacerbated by the current system in which peer review is managed by each journal, such that the same manuscript may be peer reviewed many times over as it is successively rejected and resubmitted until it finds acceptance.\n\nUnaccountability and risks of subversion: The “black-box” nature of traditional peer review gives reviewers, editors and even authors a lot of power to potentially subvert the process. Lack of transparency means that editors can unilaterally reject submissions or shape review outcomes by selecting reviewers based on their known preference for or aversion to certain theories and methods (Travis & Collins, 1991). Reviewers, shielded by anonymity, may act unethically in their own interests by concealing conflicts of interest. Smith, an experienced editor, for example, reports reviewers stealing ideas and passing them off as their own, or intentional blocking or delaying publication of competitors’ ideas through harsh reviews (Smith, 2006). Equally, they may simply favour their friends and target their enemies. Authors, meanwhile, can manipulate the system by writing reviews of their own work via fake or stolen identities (Kaplan, 2015).\n\nSocial and publication biases: Although often idealized as impartial, objective assessors, in reality studies suggest that peer reviewers may be subject to social biases on the grounds of gender (Budden et al., 2008; Lloyd, 1990; Tregenza, 2002), nationality (Daniel, 1993; Ernst & Kienbacher, 1991; Link, 1998), institutional affiliation (Dall’Aglio, 2006; Gillespie et al., 1985; Peters & Ceci, 1982), language (Cronin, 2009; Ross et al., 2006; Tregenza, 2002) and discipline (Travis & Collins, 1991). Other studies suggest so-called “publication bias”, where prejudices against specific categories of works shape what is published. Publication bias can take many forms. First is a preference for complexity over simplicity in methodology (even if inappropriate, c.f. Travis & Collins, 1991) and language (Armstrong, 1997). Next, “confirmatory bias” is theorized to lead to conservatism, biasing reviewers against innovative methods or results contrary to dominant theoretical perspectives (Chubin & Hackett, 1990; Garcia et al., 2016; Mahoney, 1977). Finally, factors like the pursuit of “impact” and “excellence” (Moore et al., 2017) mean that editors and reviewers seem primed to prefer positive results over negative or neutral ones (Bardy, 1998; Dickersin et al., 1992; Fanelli, 2010; Ioannidis, 1998), and to disfavour replication studies (Campanario, 1998; Kerr et al., 1977).\n\nLack of incentives: Traditional peer review provides little in the way of incentives for reviewers, whose work is almost exclusively unpaid and whose anonymous contributions cannot be recognised and hence rewarded (Armstrong, 1997; Ware, 2008).\n\nWastefulness: Reviewer comments often add context or point to areas for future work. Reviewer disagreements can expose areas of tension in a theory or argument. The behind-the-scenes discussions of reviewers and authors can also guide younger researchers in learning review processes. Readers may find such information helpful and yet at present, this potentially valuable additional information is wasted.\n\nIn response to these criticisms, a wide variety of changes to peer review have been suggested (see the extensive overview in Walker & Rocha da Silva, 2015). Amongst these innovations, many have been labelled as “open peer review” at one time or another.\n\nThe diversity of the definitions provided for open peer review can be seen by examining just two examples. The first one is, to my knowledge, the first recorded use of the phrase “open peer review”:\n\n“[A]n open reviewing system would be preferable. It would be more equitable and more efficient. Knowing that they would have to defend their views before their peers should provide referees with the motivation to do a good job. Also, as a side benefit, referees would be recognized for the work they had done (at least for those papers that were published). Open peer review would also improve communication. Referees and authors could discuss difficult issues to find ways to improve a paper, rather than dismissing it. Frequently, the review itself provides useful information. Should not these contributions be shared? Interested readers should have access to the reviews of the published papers.” (Armstrong, 1982)\n\n“[O]pen review makes submissions OA [open access], before or after some prepublication review, and invites community comments. Some open-review journals will use those comments to decide whether to accept the article for formal publication, and others will already have accepted the article and use the community comments to complement or carry forward the quality evaluation started by the journal. ” (Suber, 2012)\n\nWithin just these two examples, there are already a multitude of factors at play, including the removal of anonymity, the publishing of review reports, interaction between participants, crowdsourcing of reviews, and making manuscripts public pre-review, amongst others. But each of these are distinct factors, presenting separate strategies for openness and targeting differing problems. For example, disclosure of identities aims usually at increasing accountability and minimizing bias, c.f. “referees should be more highly motivated to do a competent and fair review if they may have to defend their views to the authors and if they will be identified with the published papers” (Armstrong, 1982). Publication of reports, on the other hand, also tackles problems of incentive (reviewers can get credit for their work) and wastefulness (reports can be consulted by readers). Moreover, these factors need not necessarily be linked, which is to say that they can be employed separately: identities can be disclosed without reports being published, and reports published with reviewer names withheld, for example.\n\nThis diversity has led many authors to acknowledge the essential ambiguity of the term “open peer review” (Hames, 2014; Sandewall, 2012; Ware, 2011). The major attempt thus far to bring coherence to this confusing landscape of competing and overlapping definitions, is Emily Ford’s paper “Defining and Characterizing Open Peer Review: A Review of the Literature” (Ford, 2013). Ford examined thirty-five articles to produce a schema of eight “common characteristics” of OPR: signed review, disclosed review, editor-mediated review, transparent review, crowdsourced review, prepublication review, synchronous review, and post-publication review. Unfortunately, however, Ford’s paper fails to offer a definitive definition of OPR, since despite distinguishing eight “common characteristics” of OPR, Ford nevertheless tries to reduce it to merely one: open identities: “Despite the differing definitions and implementations of open peer review discussed in the literature, its general treatment suggests that the process incorporates disclosure of authors’ and reviewers’ identities at some point during an article’s review and publication” (p. 314). Summing up her argument elsewhere, she says: “my previous definition … broadly understands OPR as any scholarly review mechanism providing disclosure of author and referee identities to one another” (Ford, 2015). But the other elements of her schema do not reduce to this one factor. Many definitions do not include open identities at all. This hence means that although Ford claims to have identified several features of OPR, she in fact is asserting that there is only one defining factor (open identity), which leaves us where we started. Ford’s schema is also problematic elsewhere: it lists “editor-mediated review” and “pre-publication review” as distinguishing characteristics, despite these being common traits of traditional peer review; it includes questionable elements such as the purely “theoretical” “synchronous review”; and some of its characteristics do not seem to be “base elements”, but complexes of other traits – for example, the definition of “transparent review” incorporates other characteristics such as open identities (which Ford terms “signed review”) and open reports (“disclosed review”).\n\n\nMethod: A systematic review of previous definitions\n\nTo resolve this ambiguity, OpenAIRE performed a review of the literature for articles discussing “open review” or “open peer review”, extracting a corpus of 122 definitions of OPR. I first searched Web of Science (WoS) for “TOPIC: (”open review\" OR “open peer review”)”, with no limitation on date of publication, yielding a total of 137 results (searched on 12th July 2016). These records were then each individually examined for relevance and a total of 57 were excluded. 21 results (all BioMed Central publications) had been through an OPR process (which was mentioned in the abstract) but did not themselves touch on the subject of OPR; 12 results used the phrase “open review” to refer to a literature review with a flexible methodology; 12 results were for the review of objects classed “out of scope” (i.e. academic articles, books, conference submissions, data – examples included guidelines for clinical or therapeutic techniques, standardized terminologies, patent applications, and court judgements); 7 results were not in the English language; and 5 results were duplicate entries in WoS. This left a total of 80 relevant articles which mentioned either “open peer review” or “open review”. This set of articles was further enriched with 42 definitions from sources found through searching for the same terms in other academic databases (e.g., Google Scholar, JSTOR, disciplinary databases), Google (for blog articles) and Google Books (for books), as well as following citations in relevant bibliographies and literature reviews. The dataset is available online (Ross-Hellauer, 2017, http://doi.org/10.5281/zenodo.438024).\n\nEach source was then individually examined for its definition of OPR. Where no explicit definition (e.g. “OPR is …”) was given, implicit definitions were gathered from contextual statements. For instance, “reviewers can notify the editors if they want to opt-out of the open review system and stay anonymous” (Janowicz & Hitzler, 2012) is taken to endorse a definition of OPR as incorporating open identities. In a few cases, sources defined OPR in relation to the systems of specific publishers (e.g., F1000Research, BioMed Central and Nature), and so were taken to implicitly endorse those systems as definitive of OPR.\n\n\nResults\n\nThe number of definitions of OPR over time show a clear upward trend, with the most definitions in a single year coming in 2015. The distribution shows that except for some outlying definitions in the early 1980s, the phrase “open peer review” did not really enter academic discussion until the early 1990s. At that time, the phrase seems to have been used largely to refer to non-blinded review (i.e. open identities). We then see a big upswing from the early-mid 2000s onwards, which perhaps correlates with the rise of the rise of the openness agenda (especially open access, but also open data and open science more generally) over that period (Figure 1). Most of the definitions, 77.9% (n=95), come from peer-reviewed journal articles, with the second largest sources being books and blog posts. Other sources include letters to journals, news items, community reports and glossaries (Figure 2). As shown in Figure 3, the majority of definitions (51.6%) were identified to be primarily concerned with peer-review of STEM-subject material, while 10.7% targeted material from Social Sciences and Humanities material. The remainder (37.7%) were interdisciplinary. Meanwhile, regarding the target of the OPR mentioned in these articles (Figure 4), most were referring to peer review of journal articles (80.7%), with 16% not specifying a target (16%), and a small number of articles also referring to review of data, conference papers and grant proposals.\n\nOf the 122 definitions identified, 68% (n=83) were explicitly stated, 37.7% (n=46) implicitly stated, and 5.7% (n=7) contained both explicit and implicit information.\n\nThe extracted definitions were examined and classified against an iteratively constructed taxonomy of OPR traits. Nickerson et al. (2013) advise that the development of a taxonomy should begin by identifying the appropriate meta-characteristic – in this case distinct individual innovations to the traditional peer review system. An iterative approach then followed, in which dimensions given in the literature were applied to the corpus of definitions and gaps/overlaps in the OPR taxonomy identified. Based on this, new traits or distinctions were introduced so that in the end, a schema of seven OPR traits was produced (defined below):\n\nOpen identities\n\nOpen reports\n\nOpen participation\n\nOpen pre-review manuscripts\n\nOpen final-version commenting\n\nOpen interaction\n\nOpen platforms\n\nThe core traits are easily identified, with just three covering more than 99% of all definitions: Open identities combined with open reports cover 116 (95.1%) of all records. Adding open participations leads to a coverage of 121 (99.2%) records overall. As seen in Figure 5, open identities is by far the most prevalent trait, present in 90.1% (n=110) of definitions. Open reports is also present in the majority of definitions (59.0%, n=72), while open participation is part of around a third. Open pre-review manuscripts (23.8%, n=29) and open interaction (20.5%, n=25) are also a fairly prevalent part of definitions. The outliers are open final version commenting (4.9%) and open platforms (1.6%).\n\nThe various ways these traits are configured within definitions can be seen in Figure 6. Quantifying definitions in this way allows us to accurately portray exactly how ambiguously the phrase “open peer review” has been used thus far, for the literature offers a total of 22 distinct configurations of seven traits, effectively meaning that there are 22 different definitions of OPR in the literature.\n\nA “power law” distribution can be observed in the distribution of these traits, with the most popular configuration (open identities) accounting for one third (33.6%, n=41) and the second-most popular configuration (open identities, open reports) accounting for almost a quarter (23.8%, n=29) of all definitions. There then follows a “long-tail” of less-frequently found configurations, with more than half of all configurations being unique to a single definition.\n\n\nDiscussion: The traits of open peer review\n\nI next offer a detailed analysis of each of these traits, detailing the issues they aim to resolve and the evidence to support their effectiveness.\n\nOpen identity peer review, also known as signed peer review (Ford, 2013; Nobarany & Booth, 2015) and “unblinded review” (Monsen & Horn, 2007), is review where authors and reviewers are aware of each other’s identities. Traditional peer review operates as either “single-blind”, where authors do not know reviewers’ identities, or “double-blind”, where both authors and reviewers remain anonymous. Double-blind reviewing is more common in the Arts, Humanities and Social Sciences than it is in STEM (science, technology, engineering and medicine) subjects, but in all areas single-blind review is by far the most common model (Walker & Rocha da Silva, 2015). A main reason for maintaining author anonymity is that it is assumed to tackle possible publication biases against authors with traditionally feminine names, from less prestigious institutions or non-English speaking regions (Budden et al., 2008; Ross et al., 2006). Reviewer anonymity, meanwhile, is presumed to protect reviewers from undue influence, allowing them to give candid feedback without fear of possible reprisals from aggrieved authors. Various studies have failed to show that such measures increase review quality, however (Fisher et al., 1994; Godlee et al., 1998; Justice et al., 1998; McNutt et al., 1990; van Rooyen et al., 1999). As Godlee and her colleagues have said, “Neither blinding reviewers to the authors and origin of the paper nor requiring them to sign their reports had any effect on rate of detection of errors. Such measures are unlikely to improve the quality of peer review reports” (Godlee et al., 1998). Moreover, factors such as close disciplinary communities and internet search capabilities, mean that author anonymity is only partially effective, with reviewers shown to be able to identify authors in between 26 and 46 percent of cases (Fisher et al., 1994; Godlee et al., 1998).\n\nProponents of open identity peer review argue that it will enhance accountability, further enable credit for peer reviewers, and simply make the system fairer: “most importantly, it seems unjust that authors should be “judged” by reviewers hiding behind anonymity” (van Rooyen et al., 1999). Open identity peer review is argued, moreover, to potentially increase review quality, as it is theorised that reviewers will be more highly motivated and invest more care in their reviews if their names are attached to them. Opponents counter this by arguing that signing will lead to poorer reviews, as reviewers temper their true opinions to avoid causing offence. To date, studies have failed to show any great effect in either direction (McNutt et al., 1990; van Rooyen et al., 1999; van Rooyen et al., 2010). However, since these studies derive from only one disciplinary area (medicine), the results cannot taken as representative and hence further research is undoubtedly required.\n\nOpen reports peer review is where review reports (either full reports or summaries) are published alongside the relevant article. The main benefits of this measure is in making currently invisible but potentially useful scholarly information available for re-use. There is increased transparency and accountability that comes with being able to examine normally behind-the-scenes discussions and processes of improvement and assessment, and a potential to further incentivize peer reviewers by making their peer review work a more visible part of their scholarly activities (thus enabling reputational credit).\n\nReviewing is hard work. Research Information Network reported in 2008 that a single peer review takes an average of four hours, at an estimated total annual global cost of around £1.9 billion (Research Information Network, 2008). Once an article is published, however, these reviews usually serve no further purpose than to reside in publisher’s long-term archives. Yet those reviews contain information that remains potentially relevant and useful in the here-and-now. Often, works are accepted despite the lingering reservations of reviewers. Published reports can enable readers to consider these criticisms themselves, and “have a chance to examine and appraise this process of ”creative disagreement\" and form their own opinions” (Peters & Ceci, 1982). Making reviews public in this way also adds another layer of quality assurance, as the reviews are open to the scrutiny of the wider scientific community. Moreover, publishing reports also aims at raising the recognition and reward of the work of peer reviewers. Adding review activities to the reviewer’s professional record is common practice; author identification systems currently also add mechanisms to host such information (e.g. via ORCID) (Hanson et al., 2016). Finally, open reports give young researchers a guide (to tone, length, the formulation of criticisms) to help them as they begin to do peer review themselves.\n\nThe evidence-base against which to judge such arguments is not great enough to enable strong conclusions, however. Van Rooyen and her colleagues found that open reports correlate with higher refusal rates amongst potential reviewers, as well as an increase in time taken to write review but no concomitant effect on review quality (van Rooyen et al., 2010). Nicholson and Alperin’s small survey, however, found generally positive attitudes: “researchers … believe that open review would generally improve reviews, and that peer reviews should count for career advancement” (Nicholson & Alperin, 2016).\n\nOpen participation peer review, also known as “crowdsourced peer review” (Ford, 2013; Ford, 2015), “community/public review” (Walker & Rocha da Silva, 2015) and “public peer review” (Bornmann et al., 2012), allows the wider community to contribute to the review process. Whereas in traditional peer review editors identify and invite specific parties (peers) to review, open participation processes invite interested members of the scholarly community to participate in the review process, either by contributing full, structured reviews or shorter comments. It may be that comments are open to anybody (anonymous or registered), or some credentials might first be required (e.g., Science Open requires an ORCID profile with at least five published articles). Open participation is often used as a complement to a parallel process of solicited peer review. It aims to resolve possible conflicts associated with editorial selection of reviewers (e.g. biases, closed-networks, elitism) and possibly improve the reliability of peer review by increasing the number of reviewers (Bornmann et al., 2012). Reviewers can come from the wider research community, as well as those traditionally under-represented in scientific assessment, including representatives from industry or members of special-interest groups, for example patients in the case of medical journals (Ware, 2011). This has the potential to open the pool of reviewers beyond those identified by editors to include all potentially interested reviewers (including those from outside academia), and hence increase the number of reviewers for each publication (though in practice this is unlikely). Evidence suggests this practice could help increase the accuracy of peer review. For example, Herron (2012) produced a mathematical model of the peer review process which showed that “the accuracy of public reader-reviewers can surpass that of a small group of expert reviewers if the group of public reviewers is of sufficient size”, although only if the numbers of reader-reviewers exceeded 50.\n\nCriticisms of open participation routinely focus on questions about reviewers’ qualifications to comment and the incentives for doing so. As Stevan Harnad has said: “it is not clear whether the self-appointed commentators will be qualified specialists (or how that is to be ascertained). The expert population in any given speciality is a scarce resource, already overharvested by classical peer review, so one wonders who would have the time or inclination to add journeyman commentary services to this load on their own initiative” (Harnad, 2000). Moreover, difficulties in motivating self-selecting commentators to take part and deliver useful critique have been reported. Nature, for example, ran an experiment from June to December 2006 inviting submitting authors to take part in an experiment where open participation would be used as a complement to a parallel process of solicited peer reviews. Nature judged the trial to have been unsuccessful due to the small number of authors wishing to take part (just 5% of submitting authors), the small number of overall comments (almost half of articles received no comments) and the insubstantial nature of most of the comments that were received (Fitzpatrick, 2011). At the open access journal Atmospheric Chemistry and Physics (ACP), which publishes pre-review discussion papers for community comments, only about one in five papers is commented upon (Pöschl, 2012). Bornmann et al. (2012) conducted a comparative content analysis of the ACP’s community comments and formal referee reviews and concluded that the latter – tending to focus more on formal qualities, conclusions and potential impact – better supported the selection and improvement of manuscripts. This all suggests that although open participation might be a worthwhile complement to traditional, invited peer review, it is unlikely to be able to fully replace it.\n\nOpen interaction peer review allows and encourages direct reciprocal discussion between reviewers, and/or between author(s) and reviewers. In traditional peer review, reviewers and authors correspond only with editors. Reviewers have no contact with other reviewers, and authors usually have no opportunity to directly question or respond to reviewers’ comments. Allowing interaction amongst reviewers or between authors and reviewers, or between reviewers themselves, is another way to “open up” the review process, enabling editors and reviewers to work with authors to improve their manuscript. The motivation for doing so, according to (Armstrong, 1982), is to “improve communication. Referees and authors could discuss difficult issues to find ways to improve a paper, rather than dismissing it”.\n\nSome journals enable pre-publication interaction between reviewers as standard (Hames, 2014). The EMBO Journal, for example, enables “cross-peer review,” where referees are “invited to comment on each other’s reports, before the editor makes a decision, ensuring a balanced review process” (EMBO Journal, 2016). At eLife, reviewers and editor engage in an “online consultation session” where they come to a mutual decision before the editor compiles a single peer review summary letter for the author to give them a single, non-contradictory roadmap for revisions (Schekman et al., 2013). The publisher Frontiers has gone a step further, including an interactive collaboration stage that “unites authors, reviewers and the Associate Editor – and if need be the Specialty Chief Editor – in a direct online dialogue, enabling quick iterations and facilitating consensus” (Frontiers, 2016).\n\nPerhaps even more so than other areas studied here, evidence to judge the effectiveness of interactive review is scarce. Based on anecdotal evidence, Walker & Rocha da Silva (2015) advise that “[r]eports from participants are generally but not universally positive”. To the knowledge of the author, the only experimental study that has specifically examined interaction among reviewers or between reviewers and authors is that of Jeffrey Leek and his colleagues, who performed a laboratory study of open and closed peer review based on an online game and found that “improved cooperation does in fact lead to improved reviewing accuracy. These results suggest that in this era of increasing competition for publication and grants, cooperation is vital for accurate evaluation of scientific research” (Leek et al., 2011). Such results are encouraging, but hardly conclusive. Hence, there remains much scope for further research to determine the impact of cooperation on the efficacy and cost of the review process.\n\nOpen pre-review manuscripts are manuscripts that are immediately openly accessible (via the internet) in advance, or in synchrony with, any formal peer review procedures. Subject-specific “preprint servers” like arXiv.org and bioRxiv.org, institutional repositories, catch-all repositories like Zenodo or Figshare and some publisher-hosted repositories (like PeerJ Preprints) allow authors to short-cut the traditional publication process and make their manuscripts immediately available to everyone. This can be used as a complement to a more traditional publication process, with comments invited on preprints and then incorporated into redrafting as the manuscript goes through traditional peer review with a journal. Alternatively, services which overlay peer-review functionalities on repositories can produce functional publication platforms at reduced cost (Boldt, 2011; Perakakis et al., 2010). The mathematics journal Discrete Analysis, for example, is an overlay journal whose primary content is hosted on arXiv (Day, 2015). The recently released Open Peer Review Module for repositories, developed by Open Scholar in association with OpenAIRE, is an open source software plug-in which adds overlay peer review functionalities to repositories using the DSpace software (OpenAIRE, 2016). Another innovative model along these lines is that of ScienceOpen, which ingests articles metadata from preprint servers and contextualizes them by adding altmetrics and other relational information, before offering authors peer review.\n\nIn other cases, manuscripts are submitted to publishers in the usual way but made immediately available online (usually following some rapid preliminary review or “sanity check”) before the start of the peer review process. This approach was pioneered with the 1997 launch of the online journal Electronic Transactions in Artificial Intelligence (ETAI), where a two-stage review process was used. First, manuscripts were made available online for interactive community discussion, before later being subject to standard anonymous peer review. The journal stopped publishing in 2002 (Sandewall, 2012). Atmospheric Chemistry and Physics uses a similar system of multi-stage peer review, with manuscripts being made immediately available as “discussion papers” for community comments and peer review (Pöschl, 2012). Other prominent examples are F1000Research and the Semantic Web Journal.\n\nThe benefits to be gained from open pre-review manuscripts is that researchers can assert their priority in reporting findings – they needn’t wait for the sometimes seemingly endless peer review and publishing process, during which they might fear being scooped. Moreover, getting research out earlier increases its visibility, enables open participation in peer review (where commentary is open to all), and perhaps even, according to (Pöschl, 2012), increases the quality of initial manuscript submissions.\n\nOpen final-version commenting is review or commenting on final “version of record” publications. If the purpose of peer review is to assist in the selection and improvement of manuscripts for publication, then it seems illogical to suggest that peer review can continue once the final version-of-record is made public. Nonetheless, in a literal sense, even the declared fixed version-of-record continues to undergo a process of improvement (occasionally) and selection (perpetually).\n\nThe internet has hugely expanded the range of effective action available for readers to offer their feedback on scholarly works. Where before only formal routes like the letters to the journal or commentary articles offered readers a voice, now a multitude of channels exist. Journals are increasingly offering their own commentary sections. Walker & Rocha da Silva (2015) found that of 53 publishing venues reviewed, 24 provided facilities to enable user-comments on published articles – although these were typically not heavily used. Researchers seem to see the worth of such functionalities, with almost half of respondents to a 2009 survey believing supplementing peer review with some form of post-publication commentary to be beneficial (Mulligan et al., 2013). But users can “publish” their thoughts anywhere on the Web – via academic social networks like Mendeley, ResearchGate and Academia.edu, via Twitter, or on their own blogs. The reputation of a piece of work is continuously evolving as long as it remains the subject of discussion.\n\nImprovements based on feedback happen most obviously in the case of so-called ‘living’ publications, like the Living Reviews group of three disciplinary journals in the fields of relativity, solar physics and computational astrophysics, publishing invited review articles which allow authors to regularly update their articles to incorporate the latest developments in the field. Here, even where the published version is anticipated to be the final version, it remains open to future retraction or correction. Such changes are often fueled by social media, as in the 2010 case of #arseniclife, where social media critique over flaws in the methodology of a paper claiming to show a bacterium capable of growing on arsenic resulted in refutations being published in Science. The Retraction Watch blog is dedicated to publicizing such cases.\n\nA major influence here has been the independent platform Pubpeer which proclaims itself a “post-publication peer review platform”. When its users swarmed to critique a Nature paper on STAP (Stimulus-Triggered Acquisition of Pluripotency) cells, PubPeer argued that its “post-publication peer review easily outperformed even the most careful reviewing in the best journal. The papers’ comment threads on PubPeer have attracted some 40000 viewers. It’s hardly surprising they caught issues that three overworked referees and a couple of editors did not. Science is now able to self-correct instantly. Post-publication peer review is here to stay” (PubPeer, 2014).\n\nOpen platforms peer review is review facilitated by a different organizational entity than the venue of publication. Recent years have seen the emergence of a group of dedicated platforms which aim to augment the traditional publishing ecosystem by de-coupling review functionalities from journals. Services like RUBRIQ and Peerage of Science offer “portable” or “independent” peer review. A similar service, Axios Review, operated from 2013 to 2017. Each platform invites authors to submit manuscripts directly to them, then organises review amongst their own community of reviewers and returns review reports. In the case of RUBRIQ and Peerage of Science, participating journals then have access to these scores and manuscripts and so can contact authors with a publishing offer or to suggest submission. Axios meanwhile, directly forwarded the manuscript, along with reviews and reviewer identities, to the author’s preferred target journal. The models vary in their details – RUBRIQ, for example, pays its reviewers, whereas Axios operated on a community model where reviewers earned discounts on having their own work reviewed – but all aim in their ways to reduce inefficiencies in the publication process, especially the problem of duplication of effort. Whereas in traditional peer review, a manuscript could undergo peer review at several journals, as it is submitted and rejected, then submitted elsewhere, such services need just one set of reviews which can be carried over to multiple journals until a manuscript finds a home (hence “portable” review).\n\nOther decoupled platforms aim at solving different problems. Publons seeks to address the problem of incentive in peer review by turning peer review into measurable research outputs. Publons collects information about peer review from reviewers and publishers to produce reviewer profiles which detail verified peer review contributions that researchers can add to their CVs. Overlay journals like Discrete Mathematics, discussed above, are another example of open platforms. Peter Suber (quoted in Cassella & Calvi, 2010) defines the overlay journal as “An open-access journal that takes submissions from the preprints deposited at an archive (perhaps at the author’s initiative), and subjects them to peer review…. Because an overlay journal doesn’t have its own apparatus for disseminating accepted papers, but uses the pre-existing system of interoperable archives, it is a minimalist journal that only performs peer review.” Finally, there are the many venues through which readers can now comment on already-published works (see also “open final-version commenting” above), including blogs and social networking sites, as well as dedicated platforms such as PubPeer.\n\n\nConclusion: A unified definition of open peer review\n\nWe have seen that the definition of “open peer review” is contested ground. Our aim here has been to provide some clarity as to what is being referred to when this term is used. By analyzing 122 separate definitions from the literature I have identified seven different traits of OPR which all aim to resolve differing peer review problems. Amongst the corpus of definitions there are 22 unique configurations of these traits –so 22 distinct definitions of OPR in the literature. Given this is such a contested concept, in my view the only sensible way forward is to acknowledge the ambiguity of this term, accepting that it is used as an umbrella concept for a diverse array of peer review innovations.\n\nThe theme that unifies these diverse traits is Open Science. Factors like opening identities, reports and participation all bespeak the ethos of Open Science in trying, in their differing and overlapping ways, to bring greater transparency, accountability, inclusivity and flexibility to the restricted traditional model of peer review.\n\nBased upon this analysis I offer the following unified definition:\n\nOPR definition: Open peer review is an umbrella term for a number of overlapping ways that peer review models can be adapted in line with the ethos of Open Science, including making reviewer and author identities open, publishing review reports and enabling greater participation in the peer review process. The full list of traits is:\n\nOpen identities: Authors and reviewers are aware of each other’s identity\n\nOpen reports: Review reports are published alongside the relevant article.\n\nOpen participation: The wider community to able to contribute to the review process.\n\nOpen interaction: Direct reciprocal discussion between author(s) and reviewers, and/or between reviewers, is allowed and encouraged.\n\nOpen pre-review manuscripts: Manuscripts are made immediately available (e.g., via pre-print servers like arXiv) in advance of any formal peer review procedures.\n\nOpen final-version commenting: Review or commenting on final “version of record” publications.\n\nOpen platforms: Review is de-coupled from publishing in that it is facilitated by a different organizational entity than the venue of publication.\n\n\nData availability\n\nDataset including full data files used for analysis in this review: http://doi.org/10.5281/zenodo.438024 (Ross-Hellauer, 2017).\n\n\nNotes\n\n1The provenance of this quote is uncertain, even to Suber himself, who recently advised in a personal correspondence (19.8.2016): “I might have said it in an email (as noted). But I can’t confirm that, since all my emails from before 2009 are on an old computer in a different city. It sounds like something I could have said in 2007. If you want to use it and attribute it to me, please feel free to note my own uncertainty!”",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work is funded by the European Commission H2020 project OpenAIRE2020 (Grant agreement: 643410, Call: H2020-EINFRA-2014-1).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe author thanks Birgit Schmidt (University of Goettingen), Arvid Deppe (University of Kassel), Jon Tennant (Imperial College London, ScienceOpen), Edit Gorogh (University of Goettingen) and Alessia Bardi (Istituto di Scienza e Tecnologie dell'Informazione) for discussion and comments that led to the improvement of this text. Birgit Schmidt created Figure 1.\n\n\nSupplementary material\n\nSupplementary file 1: PRISMA checklist. The checklist was completed with the original copy of the manuscript.\n\nClick here to access the data.\n\nSupplementary file 2: PRISMA flowchart showing the number of records identified, included and excluded.\n\nClick here to access the data.\n\n\nReferences\n\nArmstrong JS: Barriers to Scientific Contributions: The Authors Formula. Behav Brain Sci. Cambridge University Press (CUP). 1982; 5(02): 197–199. Publisher Full Text\n\nArmstrong JS: Peer Review for Journals: Evidence on Quality Control Fairness, and Innovation. Sci Eng Ethics. Springer Nature. 1997; 3(1): 63–84. Publisher Full Text\n\nBardy AH: Bias in reporting clinical trials. Br J Clin Pharmacol. Wiley-Blackwell. 1998; 46(2): 147–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoldt A: Extending ArXiv.Org to Achieve Open Peer Review and Publishing. J Scholarly Publ. University of Toronto Press Inc. (UTPress), 2011; 42(2): 238–42. 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Publisher Full Text\n\nPontika N, Knoth P, Cancellieri M, et al.: Fostering Open Science to Research Using a Taxonomy and an ELearning Portal. In Proceedings of the 15th International Conference on Knowledge Technologies and Data-Driven Business - i-KNOW 15. Association for Computing Machinery (ACM). 2015. Publisher Full Text\n\nPöschl U: Multi-stage open peer review: scientific evaluation integrating the strengths of traditional peer review with the virtues of transparency and self-regulation. Front Comput Neurosci. Frontiers Media SA. 2012; 6: 33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPubPeer: Science Self-Corrects – Instantly. PubPeer: The Online Journal Club. 2014. Reference Source\n\nResearch Information Network: Activities, Costs and Funding Flows in the Scholarly Communications System in the UK: Report Commissioned by the Research Information Network (RIN). 2008. 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Publisher Full Text\n\nvan Rooyen S, Delamothe T, Evans SJ: Effect on Peer Review of Telling Reviewers That Their Signed Reviews Might Be Posted on the Web: Randomised Controlled Trial. BMJ. BMJ. 2010; 341: c5729–c5729. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Rooyen S, Godlee F, Evans S, et al.: Effect on peer review of telling reviewers that their signed reviews might be posted on the web: randomised controlled trial. BMJ. BMJ. 1999; 318(7175): 23–27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWalker R, Rocha da Silva P: Emerging trends in peer review-a survey. Front Neurosci. Frontiers Media SA. 2015; 9: 169. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWare M: Peer Review: Benefits, Perceptions and Alternatives. Publishing Research Consortium 4, 2008. Reference Source\n\nWare M: Peer Review: Recent Experience and Future Directions. New Review of Information Networking. Informa UK Limited. 2011; 16(1): 23–53. Publisher Full Text\n\nWare M: Peer Review Survey 2015. Publishing Research Consortium. 2016. Reference Source"
}
|
[
{
"id": "22299",
"date": "08 May 2017",
"name": "Richard Walker",
"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\nThis is a useful, well-written article that helps to clarify some of the “fuzziness” concerning the concept of “Open Peer Review”.\n\nThe author makes a systematic search of the literature, fully and correctly detailing his methods in the Supplementary Materials. His main conclusion is that “Open peer review is an umbrella term for a number of overlapping ways that peer review models can be adapted in line with the ethos of Open Science…”. The data from the systematic review fully justifies this conclusion. The references are comprehensive and up to date.\n\nOn this basis, I believe the quality of the article is already sufficient to justify publication. I would like, nonetheless to suggest some possibilities for improvement.\n\nWeak conclusions\n\nThe author’s conclusions, while correct, are weak. The rapid growth in references to Open Peer Review in the literature suggests that interest in OPR is growing rapidly. It would be useful to point this out. It would also be useful to point out that 110/122 references in his survey talk about “Open Identities” and 72 talk about Open Reports”. This suggests to me that the core sense of Open Peer Review lies precisely in the use of Open identifies and Open Reports and that other aspects are more peripheral. If this were my article (which it is not) I would make this core/periphery distinction more explicit. The author observes correctly that there is still very little evidence about the effectiveness or otherwise of different forms of Open Peer Review. This is another issue that it would be good to bring out in the conclusions.\n\n“Power distribution” -\n\nThe author claims that the configurations of OPR traits “follow a power-law distribution”. Readers will understand what he means. However a power law is a functional relationship between two quantities – and here I see only one (the number of configurations). Power laws play no further part in the author’s argument. So I suggest it would be better to avoid the term. What the author could say, correctly, is that there are a couple of very common configurations and a lot of rarer ones. This links to the idea of a “core” and “peripheral” concepts of OPR.\n\nReasons for open reports\n\nThe author correctly argues that Open Identities provide an incentive to reviewers to do their work thoroughly. I suggest that the same applies to “Open Reports”. No reviewer wants to expose himself/herself as lazy or blatantly unfair.\n\nDetailed points.\n\nP4: I suggest the author replaces “Unaccountability” with “Lack of accountability” P6: In the methods, there seem to be two literature surveys, the first by OpenAire (never mentioned again in the rest of the article), the second by the author. The author should clarify exactly who did what and how he used the OpenAire search P6: The text at the top of column 2 starts in the middle of a sentence. I think something is missing. P7: In Figure 4, it is not clear what is the metric. Is it the number of Journal Articles/Grant proposals etc. or is it the number of distinct definitions found in journals etc? It would be good to clarify what is meant by “Data,Journal Articles” P7: The author writes that “for the literature offers a total of 22 distinct configurations of seven traits, effectively meaning that there are 22 different definitions of OPR in the literature.\" In reality he found 22 configurations in his, necessarily limited survey. I am certain the literature contains many more. I suggest he corrects his initial statement to make this clear. P7: The definition of “Open identities”, “Open Reports” etc. is given in the discussion. I suggest it would be useful to insert the definitions, earlier, immediately after the introduction of the schema (column 2 p 7) P8: It might be worth mentioning that some publishers (like Frontiers) favor a system of Open Peer Review which publishes reviewers’ names, only when articles are accepted, thereby avoiding the risk of self-censorship by critical reviewers.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": [
{
"c_id": "2992",
"date": "01 Sep 2017",
"name": "Tony Ross-Hellauer",
"role": "Author Response",
"response": "Richard Walker: “This is a useful, well-written article that helps to clarify some of the “fuzziness” concerning the concept of “Open Peer Review”. The author makes a systematic search of the literature, fully and correctly detailing his methods in the Supplementary Materials. His main conclusion is that “Open peer review is an umbrella term for a number of overlapping ways that peer review models can be adapted in line with the ethos of Open Science…”. The data from the systematic review fully justifies this conclusion. The references are comprehensive and up to date. On this basis, I believe the quality of the article is already sufficient to justify publication. I would like, nonetheless to suggest some possibilities for improvement.” Tony Ross-Hellauer: I'd like to personally thank the reviewer for their time and care in undertaking this review, which has helped strengthen the paper, especially with regards to the suggestions for strengthening the conclusion. RW: Weak conclusions - The author’s conclusions, while correct, are weak. The rapid growth in references to Open Peer Review in the literature suggests that interest in OPR is growing rapidly. It would be useful to point this out. It would also be useful to point out that 110/122 references in his survey talk about “Open Identities” and 72 talk about Open Reports”. This suggests to me that the core sense of Open Peer Review lies precisely in the use of Open identifies and Open Reports and that other aspects are more peripheral. If this were my article (which it is not) I would make this core/periphery distinction more explicit. The author observes correctly that there is still very little evidence about the effectiveness or otherwise of different forms of Open Peer Review. This is another issue that it would be good to bring out in the conclusions. TRH: I agree that the conclusion was weak and welcome the suggestions for improvement. I have written an extended conclusion which I believe addresses all these points. RW: ““Power distribution” - The author claims that the configurations of OPR traits “follow a power-law distribution”. Readers will understand what he means. However a power law is a functional relationship between two quantities – and here I see only one (the number of configurations). Power laws play no further part in the author’s argument. So I suggest it would be better to avoid the term. What the author could say, correctly, is that there are a couple of very common configurations and a lot of rarer ones. This links to the idea of a “core” and “peripheral” concepts of OPR.” TRH: Text changed to \"The distribution of traits shows two very popular configurations and a variety of rarer ones ...\" RW: “Reasons for open reports - The author correctly argues that Open Identities provide an incentive to reviewers to do their work thoroughly. I suggest that the same applies to “Open Reports”. No reviewer wants to expose himself/herself as lazy or blatantly unfair.” TRH: Agreed - I have added text to this effect: \"It could also increase review quality, as the thought of their words being made publicly available could motivate reviewers to be more thorough in their review activities. \" RW: “P4: I suggest the author replaces “Unaccountability” with “Lack of accountability”” TRH: Text changed as suggested. RW: “P6: In the methods, there seem to be two literature surveys, the first by OpenAire (never mentioned again in the rest of the article), the second by the author. The author should clarify exactly who did what and how he used the OpenAire search”TRH: There was only one lit review, done by the main author as part of the OpenAIRE project - for clarity, I've changed the reference to OpenAIRE to the first person singular (\"I\") RW: “P6: The text at the top of column 2 starts in the middle of a sentence. I think something is missing.” TRH: I've changed the structure of this sentence to be less confusing: \"Sixty-eight percent (n=83) of the 122 definitions identified were explicitly stated, 37.7% (n=46) implicitly stated, and 5.7% (n=7) contained both explicit and implicit information.\" RW: “P7: In Figure 4, it is not clear what is the metric. Is it the number of Journal Articles/Grant proposals etc. or is it the number of distinct definitions found in journals etc? It would be good to clarify what is meant by “Data,Journal Articles”” TRH: This is explained in the text at the bottom of p5 of v1: \"Meanwhile, regarding the target of the OPR mentioned in these articles (Figure 4), most were referring to peer review of journal articles (80.7%), with 16% not specifying a target (16%), and a small number of articles also referring to review of data, conference papers and grant proposals.\" RW: “P7: The author writes that “for the literature offers a total of 22 distinct configurations of seven traits, effectively meaning that there are 22 different definitions of OPR in the literature.\" In reality he found 22 configurations in his, necessarily limited survey. I am certain the literature contains many more. I suggest he corrects his initial statement to make this clear.” TRH: Added text to sentence to read \"in the literature examined here.\" RW: “P7: The definition of “Open identities”, “Open Reports” etc. is given in the discussion. I suggest it would be useful to insert the definitions, earlier, immediately after the introduction of the schema (column 2 p 7)”TRH: Text added as suggested. RW: “P8: It might be worth mentioning that some publishers (like Frontiers) favor a system of Open Peer Review which publishes reviewers’ names, only when articles are accepted, thereby avoiding the risk of self-censorship by critical reviewers.” TRH: This is a good suggestion."
}
]
},
{
"id": "22301",
"date": "11 May 2017",
"name": "Theodora Bloom",
"expertise": [
"Reviewer Expertise I am a journal editor. I have operated a couple of different variants of open peer review."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper addressing a question that is important to journal editors and publishers as well as the wider ‘open science’ community, namely what is meant by open peer review. I have three significant concerns that need to be addressed, followed by more minor annotations and comments.\n\nMajor issues, in the order they arise in the article\n\nWould this meet most established criteria for a systematic review? Although the author completes a PRISMA checklist, he also notes firstly that he searched Web of Science, then that he added a bunch of other sources, and finally, “This set of articles was further enriched with 42 definitions from sources found through searching for the same terms in other academic databases (e.g., Google Scholar, JSTOR, disciplinary databases), Google (for blog articles) and Google Books (for books), as well as following citations in relevant bibliographies and literature reviews.\" This suggests that it is not really systematic (although the author is to be applauded for providing the data he worked from). There is a lack of clarity about the universe of literature that is being assessed, and of details about how it was assessed. In my view, the article is still a worthwhile undertaking, despite being non-systematic, but the title ought to reflect this.\n\nIn my view the author does not pay enough attention to one important variant of open review, namely real-time review in the open, in which either invited reviewers or ‘the crowd’ comment on an article, with comments being posted as they are ready, rather than at the end of a formal process of peer review and decision-making. Conversely, the inclusion of ‘open platforms’ seems very confusing to me, as they do not have many of the criteria of openness that define the other flavours of open review that the author describes, but instead are about decoupling peer review from publication. Indeed Figure 6 makes plain that as a trait open platforms only occur twice in the 122 definitions considered. I would bet that the ‘real-time’ variant occurs far more often, and ought to be included as one of the key traits within the umbrella definition.\n\nIn a paper that aims to define open peer review, it is unfortunate that the author doesn’t spend longer considering alternative definitions of peer review. Throughout the article he appears to conflate editorial selection (whether a journal accepts a manuscript) with technical review (whether the work is sound and properly reported). Thus when he talks about the “problems with peer review” he is sometimes talking about reviewers not spotting technical problems, sometimes about editors rejecting articles that don’t suit their taste, and sometimes about authors going through cycles of editorial rejection to achieve a high impact publication. Conflating these various things does not provide a sound foundation on which to build a definition of open peer review. This conflation is made worse when, for example, it is implied that the only reason for retraction is error. Thus most of the first three paragraphs of the ‘background’ section, and much of what follows about biases, incentives and wastefulness are muddled, and the references and evidence do not all support the broad claims made about ‘peer review’ (itself an ‘umbrella term’).\n\nMore minor concerns, with page numbers (PDF version) of the corresponding text\nAbstract and later: The author needs to decide throughout the article whether he is singular (as he appears to be) or uses the royal ‘we’.\n\np7 – it is not helpful to list the seven types of openness without definitions. Even if the lengthy discussion of them follows later, I was desperate for some brief definition, in particular for the last two, and was labouring under a misapprehension about the meaning of the fourth, until much later in the article when I discovered what was meant by ‘open pre-review manuscripts’.\n\np8 – proponents of open identity review in medicine would also point out that it makes conflicts of interest much more apparent and subject to scrutiny.\n\np8 – some journals use open reports without open identities – i.e. posting reports with published articles but without identifying the reviewer (e.g. http://embor.embopress.org/about#Transparent_Process). The author writes as if open reports must always have open identities.\n\np10 – I think ‘open pre-review manuscripts’ is the wrong name for what the author is describing. At first I thought this meant the practice of posting the authors’ version of a manuscript alongside the peer review history (as is done by The BMJ, for example). But I think the author means ‘Open posting before formal review’ (which some call preprints). He might like to consider the suggestions by Neylon et al. about this issue of terminology (http://biorxiv.org/content/early/2016/12/09/092817)\n\np10 – I wonder if the author has any evidence that PubPeer has been ‘a major influence’?\n\np11 – I don’t think ‘open platforms’ is the right term either. (Although as noted above, I don’t really think this section belongs in the discussion at all, if it is to remain I would strongly recommend renaming it.) In publishing terms a platform is ‘where’ you publish articles, and the author is here discussing an aspect of how you get to the point of publication, and in particular peer review services (which as far as I can tell de facto meet only rather limited criteria of openness). I think what the author is describing is peer review options decoupled from journals (see Priem and Hemminger, Front Comput Neurosci 2012; 6: 19) and as noted I don’t understand why these have a place in a definition of open peer review.\n\np11 – Conclusion. I don’t believe the author presents a unified definition of open peer review, for all the reasons discussed above, but he does present most of the traits that together come under the umbrella term.\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? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": [
{
"c_id": "2993",
"date": "01 Sep 2017",
"name": "Tony Ross-Hellauer",
"role": "Author Response",
"response": "Theodora Bloom: “This is an interesting paper addressing a question that is important to journal editors and publishers as well as the wider ‘open science’ community, namely what is meant by open peer review. I have three significant concerns that need to be addressed, followed by more minor annotations and comments.” Tony Ross-Hellauer: I'd like to thank the reviewer for their time and care in undertaking this review, which has helped strengthen the paper, especially with regards to the suggestions for strengthening the analysis of the problems with peer review. TB: Would this meet most established criteria for a systematic review? Although the author completes a PRISMA checklist, he also notes firstly that he searched Web of Science, then that he added a bunch of other sources, and finally, “This set of articles was further enriched with 42 definitions from sources found through searching for the same terms in other academic databases (e.g., Google Scholar, JSTOR, disciplinary databases), Google (for blog articles) and Google Books (for books), as well as following citations in relevant bibliographies and literature reviews.\" This suggests that it is not really systematic (although the author is to be applauded for providing the data he worked from). There is a lack of clarity about the universe of literature that is being assessed, and of details about how it was assessed. In my view, the article is still a worthwhile undertaking, despite being non-systematic, but the title ought to reflect this. TRH: I have strengthened the description in the methods section to address this criticism. I do believe this article meets the criteria for PRISMA systematic reviews (the relevant sections from the PRISMA checklist being: \"7. - Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched\"; and \"8. Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated.\" ) TB: “In my view the author does not pay enough attention to one important variant of open review, namely real-time review in the open, in which either invited reviewers or ‘the crowd’ comment on an article, with comments being posted as they are ready, rather than at the end of a formal process of peer review and decision-making. Conversely, the inclusion of ‘open platforms’ seems very confusing to me, as they do not have many of the criteria of openness that define the other flavours of open review that the author describes, but instead are about decoupling peer review from publication. Indeed Figure 6 makes plain that as a trait open platforms only occur twice in the 122 definitions considered. I would bet that the ‘real-time’ variant occurs far more often, and ought to be included as one of the key traits within the umbrella definition.” TRH: I thank the reviewer for pointing out the gap in not mentioning what they term \"real-time review in the open\" - but I would respectfully disagree with the reviewer that this feature constitutes a core trait. In my view it depends upon other traits, including open reports, commenting, participation and especially open pre-review manuscripts. As this feature depends (in my view) most fully upon open pre-review manuscripts (since the manuscript would need to be online to begin the process), I have included mention of this option in the discussion section for that trait with the added text: \"Finally, making manuscripts openly available in advance of review allows comments to be posted as they are received, either from invited reviewers or the wider community, and enabling readers to follow the process of peer-review in real-time.\" TB: “In a paper that aims to define open peer review, it is unfortunate that the author doesn’t spend longer considering alternative definitions of peer review. Throughout the article he appears to conflate editorial selection (whether a journal accepts a manuscript) with technical review (whether the work is sound and properly reported). Thus when he talks about the “problems with peer review” he is sometimes talking about reviewers not spotting technical problems, sometimes about editors rejecting articles that don’t suit their taste, and sometimes about authors going through cycles of editorial rejection to achieve a high impact publication. Conflating these various things does not provide a sound foundation on which to build a definition of open peer review. This conflation is made worse when, for example, it is implied that the only reason for retraction is error. Thus most of the first three paragraphs of the ‘background’ section, and much of what follows about biases, incentives and wastefulness are muddled, and the references and evidence do not all support the broad claims made about ‘peer review’ (itself an ‘umbrella term’).” TRH: Since the aim of this article was to clarify, I am very grateful to the reviewer for pointing out the ways in which this section actually confuses things. It was certainly not my intention to insinuate that OPR is a panacea for all problems with traditional peer review. I have added text to address the reviewer's comments: (a) The description of traditional peer review in the Background section has been revised to clarify the role of peer review in scholarly communication; (b) Two new sections have been added to the discussion which make clearer (1) the particular problems with traditional peer review that each OPR trait aims to address, and (2) how each trait can be related to the broader agenda of Open Science (a new figure is also added).TB: “Abstract and later: The author needs to decide throughout the article whether he is singular (as he appears to be) or uses the royal ‘we’.” TRH: Corrected TB: “p7 – it is not helpful to list the seven types of openness without definitions. Even if the lengthy discussion of them follows later, I was desperate for some brief definition, in particular for the last two, and was labouring under a misapprehension about the meaning of the fourth, until much later in the article when I discovered what was meant by ‘open pre-review manuscripts’. “ TRH: Definitions added. TB: “p8 – proponents of open identity review in medicine would also point out that it makes conflicts of interest much more apparent and subject to scrutiny.” TRH: Added text to address this: \"Finally, a reviewer for this paper advises that “proponents of open identity review in medicine would also point out that it makes conflicts of interest much more apparent and subject to scrutiny” (Bloom, 2017). \" TB: “p8 – some journals use open reports without open identities – i.e. posting reports with published articles but without identifying the reviewer (e.g. http://embor.embopress.org/about#Transparent_Process). The author writes as if open reports must always have open identities.” TRH: Added sentence to clarify: \"Often, although not in all cases (e.g., EMBO reports, http://embor.embopress.org), review names are published alongside the reports. \" TB: “p10 – I think ‘open pre-review manuscripts’ is the wrong name for what the author is describing. At first I thought this meant the practice of posting the authors’ version of a manuscript alongside the peer review history (as is done by The BMJ, for example). But I think the author means ‘Open posting before formal review’ (which some call preprints). He might like to consider the suggestions by Neylon et al. about this issue of terminology (http://biorxiv.org/content/early/2016/12/09/092817)” TRH: I agree the terminology could be misconstrued, but am not sure the reviewer's suggestion is preferable (the phrase \"posting\" could be thought ambiguous) - to avoid ambiguity for future readers, I have added definitions on p7 where I introduce the terms. TB: “p10 – I wonder if the author has any evidence that PubPeer has been ‘a major influence’?” TRH: I’ve weakened the terminology to \"An important platform in this regard has been major influence here has been the independent platform Pubpeer \" TB: “p11 – I don’t think ‘open platforms’ is the right term either. (Although as noted above, I don’t really think this section belongs in the discussion at all, if it is to remain I would strongly recommend renaming it.) In publishing terms a platform is ‘where’ you publish articles, and the author is here discussing an aspect of how you get to the point of publication, and in particular peer review services (which as far as I can tell de facto meet only rather limited criteria of openness). I think what the author is describing is peer review options decoupled from journals (see Priem and Hemminger, Front Comput Neurosci 2012; 6: 19) and as noted I don’t understand why these have a place in a definition of open peer review.” TRH: I respectfully disagree with the reviewer here - the word \"platform\" seems to be used more broadly - e.g., PubPeer considers itself an \"online platform for post-publication peer review\" - however, I agree that the terminology might be confusing and so have changed the wording to \"Open platforms (“decoupled review”)\". The point about open platforms being a fringe trait is valid (I've added text to the open platforms section in the discussion to strengthen the acknowledgement of this) - it was included simply because it was observed to be part of the two definitions cited. TB: “p11 – Conclusion. I don’t believe the author presents a unified definition of open peer review, for all the reasons discussed above, but he does present most of the traits that together come under the umbrella term.” TRH: I have removed the term \"unified\" as this is no doubt contentious"
}
]
},
{
"id": "22575",
"date": "15 May 2017",
"name": "Bahar Mehmani",
"expertise": [
"Reviewer Expertise Peer review innovations"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTony provides an overview of different definitions of Open Peer Review, acknowledging the ambiguity of the term “open peer review” and the probable impact of such ambiguity on evaluation of the efficiency of open peer review. The author has created then seven OPR traits based on WoS data driven taxonomy. In conclusion though he suggests accepting the existing ambiguity is the only way: “given this is such a contested concept, in my view the only sensible way forward is to acknowledge the ambiguity of this term, accepting that it is used as an umbrella concept for a diverse array of peer review innovations”. This doesn’t seem to be solving the issue he raises at in beginning. On the contrary considering all peer review innovations as forms of OPR might worsen the current situation. Initiatives like “registered reports”, “shortening review deadlines” or “reviewer recognition”, which are meant to address result-biased peer review, incentivizing reviewers, and boosting review speed, respectively, are certainly peer review innovations but cannot be directly considered as part of the umbrella term of open peer review. Hence the article deserves a stronger conclusion, including perhaps a suggested guideline of clarifying what type of “open peer review” beforehand of any evaluation/discussion using authors seven classifications.\nAuthor can surely strengthen the conclusion by highlighting the lack of evidence in efficiency claims about some of the 7 traits mentioned in the article.\nI suggest a double checking the term “Post-publication peer review” in WoS search results. Author reports he has used “Open Final version commenting” for this process in making taxonomy reporting only 6 results. However, Post-publication peer review seem to produce a higher number of documents when I searched the term in Scopus results in 55 documents (11 for 2015 and 19 for 2016). This might impact the result reported in Fig. 6 reporting unique configurations of OPR. Although the author reports 22 different definitions there, the figures shows 23 of them.\nIt is also worth mentioning most of OPR initiatives mentioned in the article are not directly addressing all of shortcomings of the current peer review process. Mainly because one should distinguish between editorial process and peer review process. Issues such as delay and expense are universal to single/double/open peer review as they are part of the editorial process.\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? Partly",
"responses": [
{
"c_id": "2991",
"date": "01 Sep 2017",
"name": "Tony Ross-Hellauer",
"role": "Author Response",
"response": "Bahar Mehmani: „Tony provides an overview of different definitions of Open Peer Review, acknowledging the ambiguity of the term “open peer review” and the probable impact of such ambiguity on evaluation of the efficiency of open peer review. The author has created then seven OPR traits based on WoS data driven taxonomy. In conclusion though he suggests accepting the existing ambiguity is the only way: “given this is such a contested concept, in my view the only sensible way forward is to acknowledge the ambiguity of this term, accepting that it is used as an umbrella concept for a diverse array of peer review innovations”. This doesn’t seem to be solving the issue he raises at in beginning. On the contrary considering all peer review innovations as forms of OPR might worsen the current situation. Initiatives like “registered reports”, “shortening review deadlines” or “reviewer recognition”, which are meant to address result-biased peer review, incentivizing reviewers, and boosting review speed, respectively, are certainly peer review innovations but cannot be directly considered as part of the umbrella term of open peer review. Hence the article deserves a stronger conclusion, including perhaps a suggested guideline of clarifying what type of “open peer review” beforehand of any evaluation/discussion using authors seven classifications.” Tony Ross-Hellauer: I thank the reviewer for their time and care in preparing this helpful review report, which has helped to improve the second version. I understand the concern about merely accepting ambiguity to be no way to overcome confusion. I share this view and have now added/altered text to make my intentions clearer - \"Given that OPR is such a contested concept, in my view the only sensible way forward is to acknowledge the ambiguity of this term, accepting that it is used as an umbrella concept for a diverse array of peer review innovations. Although it could be argued that merely accepting the status quo in this way does not help resolve possible confusion regarding usage, I would argue that quantifying the ambiguity of usage and mapping the distinct traits enables future discussion to start from a firmer basis that (1) acknowledges that people often mean different things when they use this term, and (2) clarifies in advance exactly which OPR traits are under discussion.\" BM: “Author can surely strengthen the conclusion by highlighting the lack of evidence in efficiency claims about some of the 7 traits mentioned in the article.” TRH: I have included this point in the expanded conclusion. BM: “I suggest a double checking the term “Post-publication peer review” in WoS search results. Author reports he has used “Open Final version commenting” for this process in making taxonomy reporting only 6 results. However, Post-publication peer review seem to produce a higher number of documents when I searched the term in Scopus results in 55 documents (11 for 2015 and 19 for 2016). This might impact the result reported in Fig. 6 reporting unique configurations of OPR.” TRH: Although there is obvious overlap between the terms \"open peer review\" and \"post-publication peer review\", and so the literature on PPPR is useful to understand these phenomena, this study is only an attempt to categorize uses of the former term - hence, articles on PPPR which did not mention OPR were out of scope. BM: “Although the author reports 22 different definitions there, the figures shows 23 of them.” TRH: The original figure was wrong and has been corrected. The final row of the original figure was a duplicate of the 12th entry (n=1, open reports, participation, manuscripts). I thank the reviewer very much for their attention to detail in spotting this silly error! Hence, it is correct that there are 22 different definitions. BM: “It is also worth mentioning most of OPR initiatives mentioned in the article are not directly addressing all of shortcomings of the current peer review process. Mainly because one should distinguish between editorial process and peer review process. Issues such as delay and expense are universal to single/double/open peer review as they are part of the editorial process.” TRH: I address this point with added text in the background and conclusion sections."
}
]
},
{
"id": "22576",
"date": "22 May 2017",
"name": "Emily Ford",
"expertise": [
"Reviewer Expertise library and information science",
"scholarly communication. scholarly publishing"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 definition of open science needs to be clearly stated in the Introduction in order to strengthen the frame of the whole paper. Is the definition you are using of open science fully accepted and not contested? If so, then great, but if not, then it becomes murkier and you might want to spend time unpacking the tension there. Also in the last sentence of the Intro, what is that ethos of open science?\nIntroduction: Background\nWould it be useful to unpack some counter arguments on the reasons peer review in its current state of blinded does not work? For example, in the delay and expense portion, how does flipping the model to use APCs change the cost at all? And what happens to unfunded research when the model is flipped? Does that create a disparity that only well funded research is readily available? This might create yet another stratification of scholarly publishing and science communication, which one would assume open science is trying to diminish. I realize that you do some of this in the discussion section, but I find there is a gap in the discussion of the economic argument.\nIntroduction: Contested Meaning\nI appreciate your thoughtful criticisms of past works that have been unable to do what you are doing in this article. Being the author of one of them, however, I would like to make some points. Would like to point out that while I understand the lack of a definition in my authored article (Defining & Characterizing Open Peer Review - 2013) is problematic, it was never my intent to fully describe it, but I had to use a scope for my systematic review, and that scope was identity disclosure. Please note that in my concluding remarks on that paper that I recommended a definition be more tightly defined, and it never claimed to define it wholesale. Your research does a good job picking up the task that other papers were unable to accomplish.\nMethodology\nI would like to hear more in your methodology section about the searching for and selection of social sciences and humanities literature, as I think there might be some gaps in your data set based on this approach. You provide your search terms for Web of Science, but not the other databases and search engines. Including this would strengthen your methodology section. To me, the treatment of social sciences and humanities in this study is one of its weaknesses. Please outline the limitations of your research method. The methods section should be strengthened for better understanding of social sciences and humanities approaches, as well as limitations, for the paper to be more scientifically sound.\n\nResults\nI am not a statistician, nor am I a quantitative researcher, so I cannot provide a robust review of your results when it comes to these facets. Figures and tables are helpful to translate findings and ideas presented.\n\nDiscussion:\nThis section is well organized and easy to understand. I appreciate the presentation of criticisms of OPR in this section. Open participation and open interaction sections would be greatly enhanced and do a great service to your consideration of the social sciences and humanities disciplines if you engaged with Fitzpatrick’s work presented in the Mellon White paper as well the Logos article. Additionally, there is an article not included in your data set (was it out of scope?), in Social Epistemology, that may help. It would be good to engage more deeply with the question: Is OPR changing the role and purpose of peer review itself? There seems to be evidence of this by the mentorship offered at eLife, the encouragement of reviewers to engage with one another at Frontiers, and generally by collaborative approaches to review that OPR enables. In my view these approaches make peer review more robust, including more than just vetting, fact checking, and some substantive critical feedback. To this end, you will need to more clearly define in your introduction and throughout the paper the assumed purpose of peer review, which you offer us in the open final-versioning commenting portion of the discussion section. Open platforms section: While I agree that today platforms are an enormous part of our work in communicating science and engaging with our colleagues across the globe. That being said, I would like to point out that the process of peer review could be completely decoupled from a platform. The reason I mention this is that for some individuals and perhaps some disciplines, it might be difficult to get one’s head around the distinction between a peer review process and its technological implementation. To me they are distinct, and it is merely digital technology that assists us in allowing OPR to unfold. It behooves shy away from techno determinism when it comes to the possibilities presented by OPR.\nConclusion\nI think you have a solid finding, but I would like to point out one more quibble. “Open science” is not a term embraced in the social sciences and humanities. Again, since you are couching your definition under the ethos of open science you will need to better describe open science, and make a bridge for social science and humanities disciplines. If an overarching definition of OPR is to be fully accepted by all disciplines, it needs to be inclusive of all of them. This is wherein the tension lies, where the community-based aspect of OPR in the social sciences and humanities (digital humanities?) are much more pronounced in the meaning making of the process. How can you better acknowledge the disciplinary tensions in the paper? Or would you like to scope your findings differently?\nFinal thoughts\nThis paper is well written and organized logically, which make it quite readable and easy to follow. The main weakness of your paper is the lack of nuance addressed between STEM and social sciences and humanities disciplines. Engaging in the the tension between the approaches to and understanding of peer review and OPR in different disciplines will greatly strengthen your paper. Presenting better the limitations of your method and clarifying your method as noted above will help scope the paper to be more scientifically sound. Finally, clearly define and scope Open Science so that your proposed definition is more understandable. This will greatly strengthen not only the paper, but the definition itself.\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? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": [
{
"c_id": "2990",
"date": "01 Sep 2017",
"name": "Tony Ross-Hellauer",
"role": "Author Response",
"response": "Emily Ford: “Introduction: The definition of open science needs to be clearly stated in the Introduction in order to strengthen the frame of the whole paper. Is the definition you are using of open science fully accepted and not contested? If so, then great, but if not, then it becomes murkier and you might want to spend time unpacking the tension there. Also in the last sentence of the Intro, what is that ethos of open science?” Tony Ross-Hellauer: I'd like to thank the reviewer for their very thoughtful and helpful comments. The inclusion of more consideration of the SSH perspective, especially, definitely strengthens the paper. EF: “Introduction: Background - Would it be useful to unpack some counter arguments on the reasons peer review in its current state of blinded does not work? For example, in the delay and expense portion, how does flipping the model to use APCs change the cost at all? And what happens to unfunded research when the model is flipped? Does that create a disparity that only well funded research is readily available? This might create yet another stratification of scholarly publishing and science communication, which one would assume open science is trying to diminish. I realize that you do some of this in the discussion section, but I find there is a gap in the discussion of the economic argument.” TRH: Open Participation relies to an extent on OA (have added a sentence on this), but I’m afraid I don't see a further connection here. Although OPR is of course related to OA journals (in that they have tended to be more likely to experiment with OPR), surely if the same system of (traditional, blinded) peer review is in use, the basic costs (for review) will be the same? I agree that a fully-APC based OA model of publishing has the potential to exclude less well-resourced institutions (especially outside the developed West), but do not follow how this wider argument is connected to OPR. In any case, I believe these considerations fall out of scope of this review (although it would be interesting to follow them up elsewhere).EF: \"Introduction: Contested Meaning - I appreciate your thoughtful criticisms of past works that have been unable to do what you are doing in this article. Being the author of one of them, however, I would like to make some points. - would like to point out that while I understand the lack of a definition in my authored article (Defining & Characterizing Open Peer Review - 2013) is problematic, it was never my intent to fully describe it, but I had to use a scope for my systematic review, and that scope was identity disclosure. Please note that in my concluding remarks on that paper that I recommended a definition be more tightly defined, and it never claimed to define it wholesale. - Your research does a good job picking up the task that other papers were unable to accomplish.\"TRH: Thanks for clarifying this. EF “Methodology - I would like to hear more in your methodology section about the searching for and selection of social sciences and humanities literature, as I think there might be some gaps in your data set based on this approach. You provide your search terms for Web of Science, but not the other databases and search engines. Including this would strengthen your methodology section. To me, the treatment of social sciences and humanities in this study is one of its weaknesses. Please outline the limitations of your research method. The methods section should be strengthened for better understanding of social sciences and humanities approaches, as well as limitations, for the paper to be more scientifically sound.” TRH: I have expanded the methodology section to better specify search terms and databases used, and included a statement regarding limitations of the search strategy. EF: “Results - I am not a statistician, nor am I a quantitative researcher, so I cannot provide a robust review of your results when it comes to these facets. Figures and tables are helpful to translate findings and ideas presented.” TRH: No response required. EF: “Open participation and open interaction sections would be greatly enhanced and do a great service to your consideration of the social sciences and humanities disciplines if you engaged with Fitzpatrick’s work presented in the Mellon White paper as well the Logos article. Additionally, there is an article not included in your data set (was it out of scope?), in Social Epistemology (http://dx.doi.org/10.1080/02691728.2010.498929), that may help. It would be good to engage more deeply with the question: Is OPR changing the role and purpose of peer review itself? There seems to be evidence of this by the mentorship offered at eLife, the encouragement of reviewers to engage with one another at Frontiers, and generally by collaborative approaches to review that OPR enables. In my view these approaches make peer review more robust, including more than just vetting, fact checking, and some substantive critical feedback. To this end, you will need to more clearly define in your introduction and throughout the paper the assumed purpose of peer review, which you offer us in the open final-versioning commenting portion of the discussion section.” TRH: I have added consideration of disciplinary differences in the results and discussion sections. In particular, I have added a new figure to show the breakdown of traits by discipline, and added more consideration of the philosophical reasons to consider open participation and interaction. The question of whether OPR is changing the role of peer review per se is an excellent one, but I feel it is out of scope for this paper (which is already long enough!). The article by Fitzpatrick in Social Epistemology was deemed out of scope as it does not mention OPR (save for one mention in a block of quoted text) - the ideas underlying \"peer-to-peer review\" are no doubt related to the idea of OPR, but the scope here is only those papers which discuss OPR and give an explicit or implicit definition. EF: “Open platforms section: While I agree that today platforms are an enormous part of our work in communicating science and engaging with our colleagues across the globe. That being said, I would like to point out that the process of peer review could be completely decoupled from a platform. The reason I mention this is that for some individuals and perhaps some disciplines, it might be difficult to get one’s head around the distinction between a peer review process and its technological implementation. To me they are distinct, and it is merely digital technology that assists us in allowing OPR to unfold. It behooves shy away from techno determinism when it comes to the possibilities presented by OPR.” TRH: This is an important point - I have added a sentence to the section on final version commenting \"In this sense, peer review can be decoupled not only from the journal, but also from any particular platform.\" EF: “Conclusion - I think you have a solid finding, but I would like to point out one more quibble. “Open science” is not a term embraced in the social sciences and humanities. Again, since you are couching your definition under the ethos of open science you will need to better describe open science, and make a bridge for social science and humanities disciplines. If an overarching definition of OPR is to be fully accepted by all disciplines, it needs to be inclusive of all of them. This is wherein the tension lies, where the community-based aspect of OPR in the social sciences and humanities (digital humanities?) are much more pronounced in the meaning making of the process. How can you better acknowledge the disciplinary tensions in the paper? Or would you like to scope your findings differently?“ TRH: I have added an explicit reference to Open Science, specifically noting that I use the term to include all academic disciplines. I've also added reference to the disciplinary differences found, and included a concluding note that this area needs further research. EF: “Final thoughts - This paper is well written and organized logically, which make it quite readable and easy to follow. The main weakness of your paper is the lack of nuance addressed between STEM and social sciences and humanities disciplines. Engaging in the the tension between the approaches to and understanding of peer review and OPR in different disciplines will greatly strengthen your paper. Presenting better the limitations of your method and clarifying your method as noted above will help scope the paper to be more scientifically sound. Finally, clearly define and scope Open Science so that your proposed definition is more understandable. This will greatly strengthen not only the paper, but the definition itself.” TRH: Restatement of the above points - see answers above."
}
]
}
] | 1
|
https://f1000research.com/articles/6-588
|
https://f1000research.com/articles/5-2667/v1
|
14 Nov 16
|
{
"type": "Case Report",
"title": "Case Report: Whole exome sequencing identifies a novel frameshift insertion c.1325dupT (p.F442fsX2) in the tyrosine kinase domain of BTK gene in a young Indian individual with X-linked agammaglobulinemia",
"authors": [
"Amit Rawat",
"Shamsudheen Karuthedath Vellarikkal",
"Ankit Verma",
"Rijith Jayarajan",
"Anju Gupta",
"Surjit Singh",
"Anita Chopra",
"Rajive Kumar",
"Vinod Scaria",
"Sridhar Sivasubbu",
"Amit Rawat",
"Shamsudheen Karuthedath Vellarikkal",
"Ankit Verma",
"Rijith Jayarajan",
"Anju Gupta",
"Surjit Singh",
"Anita Chopra",
"Rajive Kumar"
],
"abstract": "X-linked agammaglobulinemia (XLA) is an extremely rare inherited primary immunodeficiency characterized by recurrent bacterial infections, decrease in number of mature B cells and low serum immunoglobulins. XLA is caused by mutations in the gene encoding Bruton's tyrosine kinase. We report a case of a young Indian boy suspected to have XLA. Immunophenotyping was performed for the affected child using CD20, CD19 and CD3 antibodies. Whole exome sequencing was performed using trio-based approach. The variants were further analyzed using capillary sequencing in the trio as well as maternal grandmother. Initial immunophenotyping in the affected child showed decreased count of CD19+ B cells. To strengthen the clinical findings and confirm the diagnosis of XLA, we performed whole exome sequencing. Our analysis identified a novel frameshift insertion (c.1325dupT) in the BTK gene, which was further validated by Sanger sequencing. Our approach shows the potential in using whole exome sequencing to pinpoint the molecular lesion, enabling timely diagnosis and genetic counseling, and potentially offering prenatal genetic testing for the family.",
"keywords": [
"X-linked agammaglobulinemia",
"Bruton's tyrosine kinase",
"Whole exome sequencing",
"Flow cytometry",
"B-lymphocytes"
],
"content": "Introduction\n\nPrimary immunodeficiencies are congenital defects in the immune defence mechanisms of the host against invading pathogens. X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disorder (OMIM# 300755) characterized by recurrent infections causing pneumonia, conjunctivitis, gastrointestinal infections, otitis media and sinopulmonary infections1, which may require frequent hospitalization. The disease is extremely rare with an estimated prevalence of 3–6 per 1,000,000 males2 and is inherited in an X-linked recessive manner. The disease arises from genetic defects, due to which the mature B lymphocytes are either low in number or completely absent in the bloodstream while also exhibiting a complete absence of serum immunoglobulins1. The absence of immunoglobulins results in a compromised humoral immune response, which makes the affected individual extremely vulnerable to infections with encapsulated bacteria and enteroviruses. Molecular genetic studies have conclusively mapped the genetic locus of XLA to the gene encoding for the Bruton’s tyrosine kinase (BTK)3. A comprehensive mutation database (BTKbase) lists over 700 unique mutations associated with XLA that affect the activity of BTK protein4.\n\n\nCase report\n\nA five year old boy of north Indian origin, born out of a non-consanguineous marriage (Figure 1A) presented to the hospital with headache, fever and a history of recurrent infections requiring hospitalization. The antenatal and perinatal periods were uneventful. His clinical history revealed that the child had been hospitalized for septicaemia and underwent treatment with intravenous (IV) antibiotics for 2 weeks at one year of age. Later, at 2.5 years, the child developed fever with swelling in right knee joint, that was diagnosed as septic arthritis of the right knee joint. The child was again hospitalized at 5 years of age for pyogenic meningitis. The culture of cerebrospinal fluid was found to be positive for Pseudomonas aeruginosa. On close examination, the tonsils were found to be absent and there was no peripheral lymphadenopathy. The immunoglobulin profile revealed serum level of IgG 50 mg/dl (200–700 mg/dl), IgA 5 mg/dl (40–200 mg/dl) and IgM 9 mg/dl (50–200 mg/dl). The hemogram showed hemoglobin concentration of 9.5 g/dl (11.5–15.5 g/dl), total leukocyte count 14800/cumm (6000–13500/cumm), platelet count 931000/cumm (150000–400000/cumm) and erythrocyte sedimentation rate ESR 18mm/1st hr (0–14 mm/1st hr).\n\nA). Pedigree of the family B). Flow cytometric immunophenotyping of peripheral blood lymphocytes gated on side scatter/CD45 plot. In CD19/CD3 plot, arrow depicts the count of B lymphocytes (CD19+) and T lymphocytes (CD3+). C). Heat map showing mutation spectrum corresponding to BTK protein structure and hotspots are marked with black diamond symbol. BTK domains (PH, TH, SH3, SH2 and Kinase) are marked in respective colors. The exons encoding specific region of domain and amino acids span for each domain is represented in schematic of BTK. A red triangle represents the novel variation p.F442fsX2, which lies in exon 14 and BTK kinase domain. The widely studied p.Y551 is also marked with black arrow. Pairwise alignment between wild type and truncated protein sequence is performed by using EMBOSS online software. D). The chromatogram depicts capillary sequencing results of c.1325dupT (p.F442fsX2) (marked with asterisks).\n\nThe blood flow cytometric analysis of the affected child was performed to evaluate the status and count of mature B cells. The patient (III.1) has only CD3+ lymphocytes as observed on CD19/CD3 dot plot and there was complete absence of CD19+ cells (0.02%) (Figure 1B). This observation was consistent with the diagnosis of XLA5. The patient had no family history of immunodeficiency and no such characteristics were present in any other family members.\n\nThe blood samples were collected and processed for genomic DNA isolation by salting out method6. We performed the whole exome sequencing using trio-based approach (patient, mother and father). In brief, the whole-exome library was prepared using Nextera rapid capture expanded exome kit (Illumina Inc., USA) according to manufacturer’s standard protocol. Sequencing was performed on Illumina Hiseq2500 platform (Illumina Inc., USA) with 130bp paired-end reads. Reads were trimmed using Trimmomatic v0.337 and aligned to reference genome hg19 (GRCh37) by Stampy v1.0.238 along with BWA v0.7.12-r10399. PCR duplicates were marked using Picard tools v1.127. Variations were called using Platypus v0.7.910 and annotated using ANNOVAR11. Analysis revealed a novel frameshift insertion c.1325dupT in exon 14 of the BTK gene. The mutation was found to be homozygous in child and heterozygous in mother. The identified mutation c.1325dupT has not yet been reported in the BTKbase4 and absent in public as well as internal control databases, which confirms the novelty of the variation. The mutation evaluation by SIFT Indel tool (http://sift.bii.a-star.edu.sg/www/SIFT_indels2.html, 12) was predicted to be damaging and cause nonsense mediated decay (confidence score 0.858). Further in silico analysis suggested that the mutation causes Isoleucine at 443 residue in BTK to be replaced by Histidine and introduces a premature stop codon at 444 residue, which lies in the kinase domain of the BTK protein (Figure 1C).\n\nThe variation was further validated by PCR amplification of region encompassing the variation using specific primer sets (Forward primer: 5’-CCCCAAATGCTACTGAGATGGT-3’ and Reverse primer: 3’-CCTATTTCTACCCCAGTAGGGA-5’) with the annealing temperature of 59°C using Brazilian taq polymerase (Invitrogen, USA) according to manufacturer instruction. PCR products were purified using Qiaquick PCR purification kit (QIAGEN, Germany). Capillary sequencing was performed using BigDye-terminator chemistry on 3130xl Genetic Analyzer (Applied Biosystems, USA). Analysis revealed that the mutation was homozygous in child (III.1), heterozygous in mother (II.3) and absent in father (II.2) and maternal grandmother (I.4) (Figure 1D).\n\n\nDiscussion\n\nXLA is a primary immunodeficiency disorder characterized by recurrent infections causing pneumonia, conjunctivitis, gastrointestinal infections, otitis media and sinopulmonary infections1. Whole exome sequencing has been increasingly used to identify mutations in rare genetic diseases mainly due to the speed, cost and amenability as compared to traditional capillary sequencing13. Recent reports have suggested the application of whole exome sequencing for mutation detection in a variety of primary immunodeficiency cases14,15.\n\nIn the present report, we performed whole exome sequencing using a trio-based approach for a child from an Indian family who presented to the clinic with the suspected diagnosis of XLA. The lack of readily available specific gene sequencing assays coupled with absence of a next-generation sequences (NGS) based targeted gene panels for XLA provided the impetus for attempting exome sequencing.\n\nOur exome sequencing analysis revealed a novel frameshift insertion c.1325dupT in exon 14 of the BTK gene. The mutation was found to be homozygous in patient and heterozygous in unaffected mother, which was further validated by capillary sequencing. This confirmed the X-linked inheritance and carrier status of the mother for the mutation. The mutation was found to be absent in unaffected father and maternal grandmother. The identified mutation c.1325dupT was found to be novel and damaging due to truncation of the BTK at 444 residue of kinase domain. The mutation excludes functionally well characterized active site residue Y551 of the protein. Additionally, nonsense mutation at the codon Y425X, E441X, Q459X and Q497X is known to cause loss of kinase activity of BTK, which has been previously demonstrated using in vitro kinase activity assay in Japanese individuals16. Since c.1325dupT (p.F442fsX2) lies in the vicinity of the above mentioned well studied codon positions, the effect of the mutation is expected to be damaging to BTK. Currently the patient is on intravenous immunoglobulin replacement therapy (15 g every 3–4 weekly) and is responding well.\n\nIn summary, our flow cytometry data and exome sequencing analysis are well correlated for confirming the diagnosis of XLA. The outcome from the present study strongly supports the pathogenicity of identified novel mutation in BTK gene.\n\n\nConsent\n\nWritten informed consent was obtained the parents of the child.\n\n\nData availability\n\nThe raw sequencing data are available at NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) with accession number SRR3439009.",
"appendix": "Author contributions\n\n\n\nAR, AG and SS clinically characterized the patient and collected blood samples for the study. SKV, AV and RJ isolated the DNA, prepared the exome enrichment and performed sequencing, analysis and validation. AC, RK performed the immuno-characterization of the family. SSivasubbu and VS designed the study and oversaw all the experiment and validation. AR, AV, SKV, SSivasubbu and VS contributed in writing the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nSS and VS acknowledge funding from the Council of Scientific and Industrial Research (CSIR), India through Grant No. BSC0212.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgement\n\nAuthors also acknowledge Ms. Rowmika Ravi and Vigneshwar Senthivel for proofreading and the GUaRDIAN consortium for support.\n\n\nReferences\n\nOchs HD, Smith CI: X-linked agammaglobulinemia. A clinical and molecular analysis. Medicine (Baltimore). 1996; 75(6): 287–99. PubMed Abstract | Publisher Full Text\n\nConley ME: B cells in patients with X-linked agammaglobulinemia. J Immunol. 1985; 134(5): 3070–4. PubMed Abstract\n\nTsukada S, Saffran DC, Rawlings DJ, et al.: Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell. 1993; 72(2): 279–90. PubMed Abstract | Publisher Full Text\n\nVäliaho J, Smith CI, Vihinen M: BTKbase: the mutation database for X-linked agammaglobulinemia. Hum Mutat. 2006; 27(12): 1209–17. PubMed Abstract | Publisher Full Text\n\nConley ME, Broides A, Hernandez-Trujillo V, et al.: Genetic analysis of patients with defects in early B-cell development. Immunol Rev. 2005; 203(1): 216–34. PubMed Abstract | Publisher Full Text\n\nMiller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988; 16(3): 1215. PubMed Abstract | Publisher Full Text | Free 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\nLunter G, Goodson M: Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 2011; 21(6): 936–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Durbin R: Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010; 26(5): 589–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRimmer A, Phan H, Mathieson I, et al.: Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications. Nat Genet. 2014; 46(8): 912–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang K, Li M, Hakonarson H: ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010; 38(16): e164. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu J, Ng PC: SIFT Indel: predictions for the functional effects of amino acid insertions/deletions in proteins. PLoS One. 2013; 8(10): e77940. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoycott KM, Vanstone MR, Bulman DE, et al.: Rare-disease genetics in the era of next-generation sequencing: discovery to translation. Nat Rev Genet. 2013; 14(10): 681–91. PubMed Abstract | Publisher Full Text\n\nAngulo I, Vadas O, Garcon F, et al.: Phosphoinositide 3-kinase δ gene mutation predisposes to respiratory infection and airway damage. Science. 2013; 342(6160): 866–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLucas CL, Kuehn HS, Zhao F, et al.: Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110δ result in T cell senescence and human immunodeficiency. Nat Immunol. 2014; 15(1): 88–97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHashimoto S, Tsukada S, Matsushita M, et al.: Identification of Bruton’s tyrosine kinase (Btk) gene mutations and characterization of the derived proteins in 35 X-linked agammaglobulinemia families: a nationwide study of Btk deficiency in Japan. Blood. 1996; 88(2): 561–73. PubMed Abstract"
}
|
[
{
"id": "17566",
"date": "20 Jan 2017",
"name": "Mohamed Badawy Hassan Tawfik Abdel-Naser",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report is interesting.\n\nThe authors unnecessarily repeated several sentences and phrases e.g. in the Introduction section the sentence \"X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disorder ....\" has been repeated in the discussion section. Also, in the case report section the phrase \"of the right knee joint\".\n\nThe findings of the whole exome sequencing analysis should be regarded as interesting findings because a causal relationship with X-linked agammaglobulinemia needs additional cases and animal studies.",
"responses": []
},
{
"id": "20086",
"date": "10 Feb 2017",
"name": "Saharuddin Bin Mohamad",
"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\nXLA is a rare disease which gave this case report high value for being indexed. However I have some concerns regarding the report as below:\nSince the patient was suspected with XLA and BTK gene is well known as the cause of the condition. Why Whole Exome Sequencing (WES) was conducted for this case? Why not conduct targeted sequencing for BTK since that would be much cheaper and easier to analyse. Since WES was conducted, I think authors should provide other results from WES analysis such as variant analysis, etc.\n\nThe results in Figure 1D for heterogeneous needs further explanation. It seems like the heterogeneous is not having two peaks at the asterisks position, but having two peaks at 5 positions downstream to the asterisks.\n\nI would like to suggest evidence to be provided for the truncated BTK gene due to the mutation (Reverse transcriptase-PCR).\n\nI would like to suggest the authors to referred to other database with bigger number of samples such as Exome Aggregation Consortium and 1000 Genomes database (not only BTKbase).",
"responses": [
{
"c_id": "2523",
"date": "02 Mar 2017",
"name": "Amit Rawat",
"role": "Author Response",
"response": "Why Whole Exome Sequencing (WES) was conducted for this case? The differential diagnosis in a case of hypogammaglobulinemia with absent B cells would include X-linked agammaglobulinemia and autosomal recessive forms of hypogammaglobulinemia. X-linked agammaglobulinemia is caused by mutations in the BTK gene, but there is wide list of genes implicated in autosomal recessive forms of hypogammaglobulinemia. These include PIK3R1, GRB1, AGM7, IMD36, LRRC8A, KIAA1437, AGM5, BLNK, SLP65, AGM4 IGHM, MU, AGM1, CD79B, IGB, B29, AGM6, CD79A, IGLL1, IGO, IGL5, VPREB2, AGM2 AGMX2, XLA2, IMD6, IMD1, AGMX1 Therefore, a whole exome sequencing was used as diagnostic strategy in this case The variant found in the index case was also checked in the ExAC and not found to be reported there."
},
{
"c_id": "2913",
"date": "31 Aug 2017",
"name": "Vinod Scaria",
"role": "Author Response",
"response": "XLA is a rare disease which gave this case report high value for being indexed. However I have some concerns regarding the report as below:1. Since the patient was suspected with XLA and BTK gene is well known as the cause of the condition. Why Whole Exome Sequencing (WES) was conducted for this case? Why not conduct targeted sequencing for BTK since that would be much cheaper and easier to analyse. Since WES was conducted, I think authors should provide other results from WES analysis such as variant analysis, etc.Clarification 1: Targeted sequencing of BTK is not readily available in the India. In many cases, due to the unavailability of ready services, whole exome sequencing is an attractive alternative due to the speed, cost-effectiveness as well as the coverage of genes. Indeed the targeted sequencing for BTK was performed previously by another organization outside the country, which was reported negative, which prompted us to explore additional genes involved. In addition, number of genes have been previously implicated in agammaglobulinemia including PIK3R1, GRB1, AGM7, IMD36, LRRC8A, KIAA1437, AGM5, BLNK, SLP65, AGM4 IGHM, MU, AGM1, CD79B, IGB, B29, AGM6, CD79A, IGLL1, IGO, IGL5, VPREB2, AGM2, AGMX2, XLA2, IMD6, IMD1, AGMX apart from BTK.The whole exome sequencing generated a total of 67437 variants in the patient, and 2046 and 891 variants for the mother and father respectively. Analysis of the variants are summarized in Table 1.Analysis revealed four variants in genes related to agammaglobulinemia. Out of which three were with an allele frequency quite common in the Indian population. The BTK variant was previously not described in the variant frequency databases. The variants are summarized in Table 2.2. The results in Figure 1D for heterogeneous needs further explanation. It seems like the heterogeneous is not having two peaks at the asterisks position, but having two peaks at 5 positions downstream to the asterisks.Clarification 2: The asterisk position represents the site where ‘A’ base is inserted. The frameshift starts to be noticed after 1 peak due to AAA repeats. Please see Figure.3. I would like to suggest evidence to be provided for the truncated BTK gene due to the mutation (Reverse transcriptase-PCR).Clarification 3: We could not avail the RNA sample for the case for further studies. We have added a sentence in the discussion to highlight this caveat. Ample evidence suggests that the nonsense mutation at the codon Y425X, E441X, Q459X and Q497X is known to cause loss of kinase activity of BTK, which has been previously demonstrated using in vitro kinase activity assay in Japanese individuals (Hashimoto et al. 1996) . Since c.1325dupT (p.F442fsX2) lies in the vicinity of the above mentioned well studied codon positions, the effect of the mutation may be damaging to BTK.Ref: Hashimoto S, Tsukada S, Matsushita M, et al.: Identification of Bruton’s tyrosine kinase (Btk) gene mutations and characterization of the derived proteins in 35 X-linked agammaglobulinemia families: a nationwide study of Btk deficiency in Japan. Blood. 1996;88(2):561–73. 8695804 4. I would like to suggest the authors to referred to other database with bigger number of samples such as Exome Aggregation Consortium and 1000 Genomes database (not only BTKbase).Clarification 4: The suggestions have been incorporated in the revised manuscript. Databases such as ExAC, 1000genome and al-mena (Koshy et al. 2017) are mentioned in the manuscript.Ref: Koshy R, Ranawat A, Scaria V. al mena: a comprehensive resource of human genetic variants integrating genomes and exomes from Arab, Middle Eastern and North African populations. Journal of Human Genetics. 2017 Jun 22."
},
{
"c_id": "2998",
"date": "04 Sep 2017",
"name": "Vinod Scaria",
"role": "Author Response",
"response": "There is bit correction in the total number of variations mentioned in comment box (See Clarification 1). The whole exome sequencing generated a total of 67437 variants in the patient, and 61429 and 31074 variants for the mother and father respectively."
}
]
}
] | 1
|
https://f1000research.com/articles/5-2667
|
https://f1000research.com/articles/6-1602/v1
|
30 Aug 17
|
{
"type": "Research Note",
"title": "First record of the Ligia baudiniana species complex in the American Gulf of Mexico Coastline, as confirmed by morphological and molecular approaches",
"authors": [
"Carlos A. Santamaria",
"Edgar T. Bischoff III",
"Moe Aye",
"Keith W. Phillips",
"Victoria Overmeyer",
"Edgar T. Bischoff III",
"Moe Aye",
"Keith W. Phillips",
"Victoria Overmeyer"
],
"abstract": "Ligia isopods exhibit a constrained morphology that makes identification difficult. In the Greater Caribbean, a convoluted taxonomic history has left the distributional limits of Ligia baudiniana unclear. To date, no confirmed records of this species exist from the American Gulf of Mexico. Herein, we report the presence of L. baudiniana in Sarasota-Manatee Florida, as confirmed by morphological and molecular approaches. This is the first record of this species in the region and a ~300Km extension of its range. Specimens were collected in mangroves, underscoring the importance of protecting these habitats.",
"keywords": [
"Isopoda",
"cryptic species",
"Sarasota",
"Crustacea",
"Ligia exotica"
],
"content": "Introduction\n\nThe isopod genus Ligia includes ~40 nominal species, most of which inhabit a narrow range in the upper rocky intertidal habitats. In the Greater Caribbean Region (i.e. the Caribbean and adjacent regions), a single endemic species is currently considered valid: Ligia baudiniana1,2. The species has been reported from Brazil3, the Caribbean islands4, the Pacific coastlines of Central and South America5–7, Bermuda8, Bahamas4, and in southern Florida9,10 and the Everglades4; however, doubt over historical records have left the distributional limits of L. baudiniana unclear.\n\nL. baudiniana was described from specimens collected in the San Juan de Ulua Fort in Veracruz, Mexico. Milne-Edwards’ original species description11 focuses on characters that are of limited taxonomic importance12, lacks illustrations, and does not provide an account of male reproductive structures now known to be useful in Ligia taxonomy12–14. Indeed, the terse description and source origin of the type material (i.e., artificial substrate) have led to confusion on whether L. baudiniana is a synonym of L. exotica or a valid species15,16, and to records and specimens identified as L. baudiniana to be re-classified as L. exotica (3 and references). This is particularly true for specimens from the American Gulf of Mexico coastlines, as most records appear to have been reclassified as L. exotica. Furthermore, a wide-ranging survey of Ligia in the Gulf of Mexico from Texas to Florida has shown artificial habitats in the region to harbor only L. exotica (unpublished study; Hurtado LA, Mateos M, Wang C, Santamaria CA, Jung J, Khalaji-Pirbalouty V, and Kim W).\n\nThe taxonomic confusion between L. baudiniana and L. exotica is complicated by the presence of a Ligia species endemic to habitats throughout the Greater Caribbean, Gulf of Mexico excluded, that is easily recognized by a unique male gonopod morphology that is readily distinguishable from L. exotica (Figure 1), and that has been attributed to L. baudiniana by Andersson5, Rouse9, Schultz4,8, and Schultz and Johnson10. A recent molecular study demonstrated that Ligia exhibiting this trait form a well-supported monophyletic clade composed of several cryptic and highly divergent lineages endemic to the region14. The combination of these studies suggests that L. baudiniana as currently recognized: (a) is an endemic species to the Greater Caribbean Region; (b) can be identified using both molecular and morphological tools; and (c) appears to have a broad geographic range that includes the Caribbean islands, the Pacific coastlines of Central America to Ecuador, Bermuda, Bahamas, and southern Florida.\n\nThis trait was used to putatively identify specimens collected in this study. The appendix masculine of L. exotica is shown in II-E. Photographs in panel II are reproduced under a Creative Commons license from Santamaria et al. (2014)14.\n\nIn southern Florida, L. baudiniana is reported from the Florida Keys9,10 and the Everglades4, while no confirmed records from the American Gulf of Mexico exist to date. In this study, we use molecular and morphological approaches to identify specimens collected from Sarasota and Manatee counties in Florida as L. baudiniana. Our findings extend the confirmed range of this species ~300-km into the Gulf of Mexico coastline of Florida and represent the first confirmed record of L. baudiniana in the American Gulf of Mexico coastline.\n\n\nMethods\n\nLigia specimens were collected by hand across the Sarasota-Manatee counties in Florida (Table 1, Figure 2) and field preserved in 70% EtOH. No permits were necessary for collections. Specimens were identified to species by inspecting the morphology of the apex of the endopod of the second pleopod of 15–25 male Ligia specimens per site, with individuals putatively identified as L. baudiniana if they exhibited a large process bifurcating close to the apex of the appendix masculina (Figure 1A), as proposed by Schultz4,8 and confirmed by Santamaria et al.14. A subset of specimens was deposited in the Invertebrate Collections of the Biodiversity Research and Teaching Collections (BRTC) at Texas A&M University (http://brtc.tamu.edu/).\n\nNew records are in bold.\n\nLocations are: (SRQ1) End of Tiara Drive; (SRQ2) Quick Point; (SRQ3) Joan M. Durante Community Park; (SRQ4) Leffis Key; (SRQ5) Near Coquina Beach. Detailed locality information can be found in Table 1. The smaller panel presents the distribution of L. baudiniana lineages reported to date throughout the Caribbean and its adjacent region.\n\nMorphological identifications were corroborated using a mitochondrial barcoding approach. We extracted total genomic DNA from pleopods/pereopods for a subset of individuals putatively identified as L. baudiniana using the ZR Quick-gDNA Miniprep Kit. Previously described primers and conditions were used to PCR-amplify and sequence a 658-bp fragment of the Cytochrome Oxidase I gene (COI, primers LCO1490/HCO2198; 17). Positive amplicons were cleaned and sequenced at the University of Arizona Genetics Core (UAGC). Sequences were assembled in Geneious R v8.1.7.\n\nWe combined nucleotide sequences produced in this study with publicly available ones for L. baudiniana and L. exotica (Table 1). We used default settings to align the resulting dataset using the MUSCLE Alignment18 tool in Geneious R v8.1.7. No signs of misaligned regions or pseudo-genes were observed in the resulting alignment. The final alignment was imported into MEGA v7.0.1819, where we estimated a neighbor-joining tree under Kimura’s 2-parameter model (hereafter K2P; 20) and uniform rates. Support for the relationships within the tree were estimated by conducting 1,000 bootstrap replicates. Lastly, we calculated K2P genetic distances between haplotypes produced by this study, L. exotica, and previously reported L. baudiniana clades14.\n\n\nResults\n\nMolecular identifications produced results congruent with morphological identifications. We obtained 12 unique COI haplotypes from a total of 25 individuals putatively identified as L. baudiniana. Haplotypes produced in this study were highly similar to each other (COI K2P 0.00–2.81%, Table 2) and to those reported from localities in the Florida Keys, The Bahamas, northern Cuba, Cozumel, and Bermuda (COI K2P 0.50–6.08%, Table 2). Haplotypes were moderately to highly divergent from L. baudiniana from other localities in the Caribbean (COI K2P 14.44–24.90%, Table 2), and highly divergent from L. exotica (COI K2P 20.32–25.18%). The neighbor-joining analysis produced similar results (Figure 3), nesting all haplotypes produced in this study in a well-supported clade (Bootstrap Support = 100) with the Clade C reported by Santamaria et al.14. All unique haplotypes have been deposited in GenBank (Table 1).\n\nThe top diagonals show minimum and maximum divergences between lineages, with lower diagonals presenting average genetic distances between clades. Within-group divergences are shown in the middle diagonal (in bold) in the order: minimum, maximum, and average divergence.\n\nMolecular identifications of putative L. baudiniana samples from Sarasota were made using K2P distances. All haplotypes for Ligia from Sarasota-Manatee counties (denoted by an *) are placed with previously reported haplotypes from the North Caribbean Clade reported by Santamaria et al.14 in a well-supported clade (values near nodes represent bootstrap support values). Branches are drawn to scale, with colors and labels corresponding with those used by Santamaria et al.14. The COI haplotype obtained from topotypes of L. baudiniana by Santamaria et al.14 is denoted by a †.\n\n\nDiscussion\n\nMorphological and molecular evidence confirm that our sampled individuals represent L. baudiniana. These new records represent the first confirmed presence of this species in the Gulf of Mexico coastlines of the USA and extend the recognized range of the species ~300 km northward from a previous confirmed record from Florida Bay. Positive identifications in this study were made using both morphological and molecular characters. These findings are important as Florida’s rich coastal biodiversity faces serious threats such as sea-level rise, introduction of alien species, urbanization, habitat loss, and species displacements21.\n\nAll L. baudiniana specimens collected in our surveys were found in coastal mangrove forests with no specimens found in >10 surveyed artificial habitats. This suggests that coastal development in the American Gulf of Mexico may have led to the replacement of a native species with an introduced one via the removal of mangrove habitats for the establishment of artificial substrates. Additional work is needed to establish whether L. baudiniana is present in other mangrove habitats along the Gulf of Mexico, thus clarifying the northern limits of this species’ range.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to acknowledge Taylor Greenan, Kathleen Newell, and Sharla C. Rafferty for their help in the field and the laboratory. We would also like to thank the University of South Florida Sarasota-Manatee for funding and access to shared facilities.\n\n\nSupplementary material\n\nSupplementary File 1: Alignment of COI gene sequences for all sequenced individuals and L. baudiniana and L. exotica sequences from GenBank.\n\nClick here to access the data.\n\n\nReferences\n\nLeistikow A, Wägele JW: Checklist of the terrestrial isopods of the new world (Crustacea, Isopoda, Oniscidea). Rev Bras Zool. 1999; 16(1): 1–72. Publisher Full Text\n\nSchmalfuss H: World catalog of terrestrial isopods (Isopoda: Oniscidea). Serie A Nr, Stuttgarter Beiträge zur Naturkunde Series A. 2003; 654: 1–341. Reference Source\n\nVan Name WG: The American land and fresh-water isopod Crustacea. Bulletin of the American Museum of Natural History. 1936; 71: 1–535. Reference Source\n\nSchultz GA: Terrestrial isopod crustaceans mainly from the West Indies and adjacent regions, 1. Tylos. and Ligia. Stud Fauna Curacao Caribb Isl. 1974; 45(77): 162–73. Reference Source\n\nAndersson A: South American terrestrial isopods in the collection of the Swedish State Museum of Natural History. Arkiv för zoologi new series. 1960; 12: 537–70. Reference Source\n\nEspinosa-Perez MD, Hendrickx ME: A comparative analysis of biodiversity and distribution of shallow-water marine isopods (Crustacea: Isopoda) from polar and temperate waters in the East Pacific. Belg J Zool. 2006; 136(2): 219–47. Reference Source\n\nLeistikow A: Terrestrial isopods from Costa Rica and a redescription of Ischioscia variegata (Dollfus, 1893) (Crustacea: Isopoda: Oniscidea). Can J Zool. 1997; 75(9): 1415–64. Publisher Full Text\n\nSchultz GA: Ecology and systematics of terrestrial isopod crustaceans from Bermuda (Oniscoidea). Crustaceana Supplement. 1972; (3): 79–99. Reference Source\n\nRouse WL: Littoral crustacea from southwest Florida. Quarterly Journal of the Florida Academy of Science. 1969; 32: 127–52. Reference Source\n\nSchultz GA, Johnson C: Terrestrial isopod crustaceans from Florida (Oniscoidea). Tylidae, Ligiidae, Halophilosciidae, Philosciidae, and Rhyscotidae. J Crustacean Biol. 1984; 4(1): 154–71. Publisher Full Text\n\nMilne-Edwards H: Histoire naturelle des Crustacés, comprenant l’anatomie, la physiologie et la classification de ces animaux. Paris: Librairie Encyclopédique de Roret; 1840. Publisher Full Text\n\nJackson HG: A revision of the isopod genus Ligia (Fabricius). J Zool. 1922; 92(3): 683–703. Publisher Full Text\n\nKhalaji-Pirbalouty V, Wägele JW: Two new species of Ligia Fabricius, 1798 (Crustacea: Isopoda: Ligiidae) from coasts of the Persian and Aden gulfs. Organisms Diversity & Evolution. 2010; 10(2): 135–45. Publisher Full Text\n\nSantamaria CA, Mateos M, Hurtado LA: Diversification at the narrow sea-land interface in the Caribbean: phylogeography of endemic supralittoral Ligia isopods. Front Ecol Evol. 2014; 2: 42. Publisher Full Text\n\nBudde-Lund G: Crustacea Isopoda Terrestria per Familias et Genera et Species. Descripta: Sumtibus Auctoris; 1885; 1–319. Publisher Full Text\n\nRichardson H: The marine and terrestrial isopods of the bermudas with descriptions of new genera and species. Transactions of the Connecticut Academy of Arts and Sciences. 1902; 11: 277–310. Reference Source\n\nFolmer O, Black M, Hoeh W, et al.: DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994; 3(5): 294–9. PubMed Abstract\n\nEdgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32(5): 1792–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumar S, Stecher G, Tamura K: MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016; 33(7): 1870–4. PubMed Abstract | Publisher Full Text\n\nKimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16(2): 111–20. PubMed Abstract | Publisher Full Text\n\nReece JS, Noss RF, Oetting J, et al.: A vulnerability assessment of 300 species in Florida: threats from sea level rise, land use, and climate change. PLoS One. 2013; 8(11): e80658. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "26865",
"date": "11 Oct 2017",
"name": "Mary K. Wicksten",
"expertise": [
"Reviewer Expertise Marine 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\nVery nice work, well supported with clear diagrams and photographs. Double-check spelling: Introduction line 4: doubt has or doubts have...I wonder if further work will show that the reports from the eastern Pacific constitute a sibling species but this information is not pertinent to acceptance of the current manuscript. It might be worthwhile to point out that mangroves either do not occur or do not support characteristic communities in the northern Gulf of Mexico.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "25492",
"date": "16 Nov 2017",
"name": "Stefano Taiti",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very interesting paper which can provide clear differences between two species of Ligia which have been mixed up in papers published up to the middle of last century.\n\nI agree that the main differential character between L. baudiniana and L. exotica is the shape of male pleopod 2 endopod, even if several other morphological characters differ between the two species (e.g. shape of telson). In the text and Figure 1 the male pleopod 1 endopod is called “appendix masculina” or “gonopod”. I would avoid using these terms, since they are not in use in the taxonomy of terrestrial isopods, also because they might not refer to the same appendage. In Ligia the male modifications are present mainly in the pleopod 2 endopod, but in other genera of Oniscidea they are present on the pleopod 1 endopod or even on both pleopod 1 and 2 endopods.\n\nLigia baudiniana is reported also from the Galapagos.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1602
|
https://f1000research.com/articles/6-1588/v1
|
29 Aug 17
|
{
"type": "Research Article",
"title": "The utility of serum 25-Hydroxyvitamin-D and body mass index in the work-up of patients presenting to a bone health clinic",
"authors": [
"Matthew R. McCarley",
"Kelsey L. Wise",
"Daniel C. Jupiter",
"Ronald W. Lindsey",
"Gordon L. Klein",
"Kelsey L. Wise",
"Daniel C. Jupiter",
"Ronald W. Lindsey",
"Gordon L. Klein"
],
"abstract": "Background: 25-hydroxyvitamin-D (25[OH]D) and Dual-energy x-ray absorptiometry (DEXA) are routinely evaluated in bone health clinics, but existing literature is conflicting with regard to whether these factors predict fragility fractures. We hypothesized that both serum 25(OH)D levels and bone density are lower in patients who have sustained fragility fracture(s) prior to initial presentation compared to those patients who have not. Methods: We reviewed the charts of 102 consecutive patients presenting to a single-center Bone Health Clinic, comprising 11 males and 91 females with a mean age of 68 and range of 50 to 92. Demographic data, serum 25(OH)D levels, fracture history, and DEXA scans were obtained at the initial visit. Results: 64 patients had previously sustained a fragility fracture, and 38 patients had not. 25(OH)D levels were similar in the fracture and non-fracture groups (37.12±17.02 ng/mL versus 38.55±16.42, p=0.676). DEXA T-scores were similar between fracture and non-fracture groups (-2.28±1.33 versus -1.82±1.1, p=0.075). Patients with rheumatoid arthritis (RA) (n=7) had lower 25(OH)D levels upon presentation (mean 22.57±8.46 versus 38.77±16.67, p=0.001). BMI was inversely correlated with 25(OH)D level (Pearson correlation [R] =-0.211, p=0.033). Age was inversely correlated with DEXA T-score (R=-0.269, p-0.009), whereas BMI was positively correlated with DEXA T-score (R=0.259, p=0.013). The other demographic variables and risk factors studied were not significantly associated with either 25(OH)D levels or DEXA T-scores. Within the fracture group, DEXA T-scores were lower for patients who had sustained a hip fracture (n=15) compared to those who had sustained a fragility fracture elsewhere (-3.12±1.02 versus -2.03±1.32, p=0.004), but their 25(OH)D levels did not differ (34.33±25.49 versus 37.98±13.69, p=0.602). Conclusions: In this cohort of patients referred to a Bone Health Clinic, serum 25(OH)D levels and DEXA T-scores did not differ between those patients who had sustained a fragility fracture from those who had not.",
"keywords": [
"fragility fracture",
"vitamin D",
"osteoporosis",
"bone health"
],
"content": "Introduction\n\nFragility fractures are defined by their low-energy nature, occurring from a fall or impact from a standing height or lower. They are the result of an underlying problem in the bone itself—for example, low density or abnormal remodeling—and, therefore, potentially preventable. Low bone mineral density (BMD) has been widely accepted as the target for treatment and prevention of these fractures, given its high prevalence and economic burden. Fifty-five million adults in the U.S. have low bone density, as defined by osteopenia or osteoporosis, sustain 1.5 million fragility fractures annually, and cost an estimated $22 billion annually for osteoporosis-related care1–2. Primary prevention is the ultimate goal; unfortunately, in clinical practice the diagnosis of low BMD is often not made until after fragility fractures have already occurred.\n\nA key explanation for this diagnostic lag is the limitations BMD measurements have in predicting fragility fracture risk via existing methods, particularly dual-energy x-ray absorptiometry (DEXA). Measurements of BMD obtained by this test provide a snapshot of a patient’s bone density at a single time point, but bone metabolism is a dynamic process that is not fully reflected by BMD measurements alone. As many as 50% of patients with fragility fractures do not have osteoporosis as defined by their bone density3–5. Recognizing this limitation, the World Health Organization developed the Fracture Risk Assessment Tool (FRAX) to guide treatment and prevention strategies for fragility fractures. This online resource (https://www.sheffield.ac.uk/FRAX/) calculates 10-year fracture risk by integrating BMD with patient risk factors and is based on clinical data from a global cohort encompassing over 250,000 person-years of observation6–7. However, FRAX does not account for all patient factors that likely have an impact, and among these include fall risk, bone turnover markers, or certain medications. Moreover, FRAX has been shown to underestimate fracture risk in patients with diabetes, for example, as this particular risk factor is excluded from the calculation8.\n\nIn particular, FRAX does not account for serum 25-hydroxyvitamin-D [25(OH)D] levels. Vitamin D plays a key role in bone metabolism, and its supplementation is a mainstay in osteoporosis treatment and prevention, given its key role in bone metabolism9–12. Hypovitaminosis D is defined as a serum level <30 ng/ml and is present in nearly 70% of the U.S. adult population, according to National Health and Nutrition Examination Survey (NHANES) data13. Although the relationships between 25(OH)D levels and BMD, as well as that between BMD and fragility fractures, have been reported14–19, these previous studies focused primarily on hip and vertebral fragility fractures20–28. The relationship between 25(OH)D levels, fragility fractures, clinical risk factors, and BMD in a single cohort of referral patients has not been studied.\n\nRecently, our orthopedic surgery department established a referral service for patients who have either sustained or been identified as being at risk for sustaining fragility fractures. Serum 25(OH)D levels, demographic data, clinical risk factor assessments, and BMD measurements using DEXA are routinely obtained for all patients at their initial office visit. The objective of this study is to evaluate the relationships between the data collected and the prevalence of fragility fractures in this cohort of referral patients. We hypothesized that both BMD measurements and serum 25(OH)D levels are lower in patients with a prior fragility fracture compared to those without.\n\n\nMethods\n\nFollowing approval by our institutional review board, the charts of patients presenting to our Bone Health Clinic were reviewed. Patients age 50 and older without a history of primary metabolic bone disorders (osteomalacia, Paget disease, or primary hyperparathyroidism) or oncologic bone disorders (primary or metastatic disease involving the skeleton) were included. Patients were included regardless of their vitamin D supplementation status prior to presentation. At the initial visit, each patient’s demographic data (age, race, and sex), body mass index (BMI), smoking history, corticosteroid use, and medical comorbidities (in particular, diabetes mellitus [DM] and rheumatoid arthritis [RA]) were recorded. A serum 25(OH)D level (ng/mL) was obtained for all patients. In addition, a DEXA scan to measure areal BMD T-scores at the lumbar spine, femoral neck, total hip, and distal radius (g/cm2) was obtained for those patients who had not had a DEXA within the six months prior to their first visit. The lowest BMD T-score value among the anatomical sites scanned was analyzed, consistent with our clinical practice preferences in guiding treatment decisions. Prior history of fragility fractures, if applicable, was documented in detail and included the mechanism of injury, date of injury, fracture site, and number of fractures sustained. (Dataset 1)\n\nDiscrete data, including patient demographics and risk factors, were described using means (SD) or proportions, as appropriate. Normality of the continuous variables, including 25(OH)D level, BMD measurements, BMI, and age, was assessed using QQ plots and boxplots. All categorical and continuous variables were compared between the fracture and no fracture groups using the Student t-test, chi-squared, or Fisher’s exact test, as appropriate. A subgroup analysis was also performed using these methods among the fracture patients to compare patients with a prior hip fracture to those with a prior fragility fracture elsewhere. Multivariate logistic regression analysis was performed including all covariates with a p-value less than 0.2 in the bivariate analyses, as well as 25(OH)D and BMD, to see whether these markers are independent predictors of referral group (i.e., prior occurrence of fragility fracture). Association of age, race, gender, BMI (and any other variables) with each of the 25-D and BMD measurements were performed with Pearson correlation or t-test as appropriate. A multivariate linear regression was also performed for each of BMD and 25-D level including all covariates with a p-value less than 0.2 in the above bivariate analyses to see whether these covariates are independently associated with 25-D or BMD. The Pearson correlations between BMD and 25(OH)D were computed within the two referral groups separately, as well as within the entire sample. For all analyses, a two-tailed p < 0.05 was considered significant. Bootstrap analysis and permutation tests were used to confirm bivariate results when there were concerns about normality of data.\n\n\nResults\n\nOne hundred and fourteen patients’ charts were identified. Twelve of these patients were excluded: ten lacked either a 25(OH)D level or DEXA scan available for review, and two had a diagnosis of primary hyperparathyroidism. Therefore, 102 patients were included in the study for analysis. 91 of these patients were women, and 11 were men. Sixty-four presented with a prior history of a fragility fracture (55 women, 9 men), while 38 of them presented without a prior history of a fragility fracture (36 women, 2 men). Mean patient age was 68, with a range of 50 to 92. Ninety patients were white, seven were black, and five were of other races. Average patient BMI was 28.71 and ranged from 15.14 to 45.58. The average 25(OH)D level was 37.66 ng/ml and ranged from 11 to 105 ng/ml. There were 12 patients with DM, seven patients with RA, four with a prior history of or current usage of corticosteroids, and 47 with a positive smoking history.\n\nPrior fragility fractures occurred in 64 patients. The fractures recorded included six distal radius fractures, four fractures of the upper extremity other than distal radius, 15 hip fractures, 18 fractures of the lower extremity other than hip, 13 fractures of the thoracic spine, and 23 fractures of the lumbar spine. Fifty-one patients presented with one prior fragility fracture, 11 presented with two prior fragility fractures, and two presented with three. Eleven fragility fractures had occurred within one month prior to initial presentation, 28 occurred between one and six months prior, five occurred between six and 12 months prior, five occurred more than one year prior, and 15 occurred at an unknown prior time.\n\nThe fracture and non-fracture groups did not differ significantly with regard to sex, race, smoking history, steroid use, DM or RA. (Table 1) Additionally, the two groups did not differ significantly with regard to age, BMI, BMD value, or serum 25(OH)D level. (Table 2)\n\nIn the subset of patients with a prior fragility fracture (n=64), those who had sustained a hip fracture (n=15) had significantly lower BMD values compared to those who had sustained a fracture elsewhere (mean -3.12 vs. -2.03, p=0.004). There were no significant differences with regard to serum 25(OH)D level or any of the other variables measured when comparing these two subgroups. (Table 3 and Table 4)\n\nSex, race, smoking history, DM, and steroid use were not significantly associated with measured serum 25(OH)D levels. However, patients with RA had significantly lower serum 25(OH)D levels upon presentation compared to non-RA patients (mean 22.57 vs. 38.77, p=0.001). (Table 5) Age and lowest BMD value were also not significantly associated with measured serum 25(OH)D level. However, BMI was negatively correlated with serum 25(OH)D level (R=-0.211, p=0.033). (Table 6, Figure 1)\n\nA scatterplot of patients’ serum 25(OH)D levels as a function of their body mass index reveals a negative correlation (r=-0.211, p=0.033).\n\nSex, race, smoking history, DM, RA, and steroid use were not significantly associated with the lowest BMD measurement. (Table 7) However, age was negatively correlated with low BMD value (R=-0.269, p=0.009), whereas BMI was positively correlated with low BMD value (R=0.259, p=0.013). (Table 8, Figure 2 and Figure 3)\n\nA scatterplot of patients’ lowest measured DEXA T-score as a function of their body mass index reveals a positive correlation (r=0.259, p=0.013).\n\nA scatterplot of patients’ lowest measured DEXA T-score among as a function of their age reveals a negative correlation (r=-0.228, p=0.021).\n\nA power analysis calculation with the criteria set at 80% power and a p-value of 0.05 indicates a sample size requirement of 1692 patients in each group to detect the difference in serum 25(OH)D levels between fracture and non-fracture patients that was observed in this study.\n\n\nDiscussion\n\nFracture liaison services are increasing in popularity as an adjunct to traditional orthopedic fracture care. These liaison services have been successful in providing more comprehensive medical evaluation and treatment for some fragility fracture patients29–32. Post-fracture osteoporosis care continues to be a treatment gap, since the majority of patients with fragility fractures are likely to “fall through the cracks” after their orthopedic care, without a proper referral system in place33–35. Our Bone Health Clinic aims for primary and secondary prevention of fragility fractures and, thereby, has expanded its indications for referral to also include patients who have yet to fracture prior to their initial visit.\n\nFracture and non-fracture groups did not differ with respect to any of the demographic variables evaluated. This suggests that our non-fracture patients were a suitable control group. Although our control group is valid on the basis of our interest in studying this specific referral population, this data is not generalizable to all adults age 50 and over in the U.S. This differs from previous studies which utilize publicly available data, such as NHANES, to estimate fragility fracture risk26,28.\n\nIn this study, serum 25(OH)D was not associated with fragility fractures. The literature regarding the predictive value of 25(OH)D levels in the setting of fragility fractures is conflicting, and this may be because most existing cohort studies analyze different fragility fracture sites. Swanson et al., Bakhtiyaroya et al., and LeBoff et al. studied fragility fractures of the hip and found lower 25(OH)D levels in patients with hip fractures as compared to controls24,25,27. However, Maier et al. studied vertebral fragility fractures and found no significant difference in 25(OH)D levels of patients admitted with vertebral fragility fractures compared to a control group23. Furthermore, Rozental et al. found no significant difference in 25(OH)D levels in patients who had sustained a fragility fracture of the distal radius as compared to controls36. Our study analyzed all fragility fracture sites, with the exception of our subgroup evaluation of 25(OH)D level in hip-fracture patients compared to non-hip-fracture patients. Our findings agree with prior studies and suggest that the predictive value of serum 25(OH)D levels may depend upon the specific fracture site. In a population study of nearly 5,000 patients, Looker found that 25(OH)D levels were a significant linear predictor of major osteoporotic fracture and a significant quadratic predictor of hip fracture. However, in the same study 25(OH)D levels did not predict fractures beyond 10 years after presentation26. This further supports the notion that fragility fracture sites differ with respect to the predictive value of 25(OH)D, with hip fractures having the strongest association. Further cohort studies with greater numbers are needed to distinguish the utility of 25(OH) among the various fracture sites.\n\nThe literature regarding the predictive value of BMD is also conflicting. Melton et al. found that low BMD measurements predicted fragility fractures at the hip and lumbar spine at long-term follow-up of ten years18. However, Schuit et al. found that less than half of the patients in their cohort with non-vertebral fractures had low BMD5. Furthermore, Marshall et al. performed a meta-analysis of 11 studies evaluating the relative risk for fracture with decreases in BMD and concluded that although BMD can be useful to predict fracture risk on a population level, it cannot predict which individuals will fracture37. Our findings confirm this data, as we found that BMD measurements are not associated with fragility fractures as a whole, except in patients sustaining hip fractures compared to those with fractures elsewhere. One potential explanation for the lack of fracture predictability using BMD in this study is that, for those patients with fragility fractures, the lowest DEXA value measured was not necessarily in the fractured area. Bone strength is derived not only from bone quantity but also bone quality, which consists of structure (micro- and macroarchitecture), turnover, and material properties—all of which are not assessed with DEXA. Our finding that BMD decreases with age is also consistent with existing literature38–40.\n\nIn our study, all patients were considered to have sustained a fragility fracture if the fracture occurred after a fall from standing height or lower. It is possible that any high-energy fractures sustained by these patients previously, and which were excluded in our calculations using this definition, could have actually resulted in fragility fractures had the mechanism of injury been low energy. It has been reported that the exclusion of high-energy fractures underestimates the prevalence of fragility fractures in the community, and as a result BMD measurements have been recommended following all trauma in older adults regardless of the nature of the energy41. This knowledge, along with the findings of a previous study which found a high prevalence of hypovitaminosis D among patients admitted to an orthopedic trauma service42, suggests that older adult patients warrant post-fracture care work-up regardless of the mechanism of injury.\n\nWith regard to the clinical risk factors we studied, patients with RA had lower serum 25(OH)D levels. This confirms existing data which has well established the linkage between RA disease activity and severity, and decreased vitamin D synthesis43,44. However, we cannot conclude whether these patients’ 25(OH)D levels were the result of their disease.\n\nThe effect BMI has on fracture risk is difficult to determine based on our data, as we found a positive association of BMI with BMD and a negative association with 25(OH)D levels. Ekwaru et al. determined that the serum 25(OH)D level significantly drops as BMI increases among individuals with vitamin D supplementation45. We speculate that the concentration gradient of 25(OH)D may shift from serum to fat stores as BMI increases. On the other hand, the increased bone density afforded by being overweight may simply be due to the increased response of bone to mechanical stress, according to Wolff’s Law, as has been shown clinically46. Both of these associations with BMI were weak, so it is unclear how significantly BMI affects each of these markers. We conclude that obesity may confound the overall assessment of fracture risk in these patients.\n\nLimitations of the present study include its retrospective nature as well as a small, homogeneous sample which is underpowered to detect the difference in 25(OH)D levels or DEXA values between the fracture and non-fracture groups. Given the reported high prevalence of hypovitaminosis D in the U.S. population, we were surprised to find that many of the patients studied had normal serum 25(OH)D levels. This is likely the result of supplementation by patients prior to referral. Future studies including a larger number of patients, in particular those who have not received prior vitamin D supplementation or other pharmacologic treatment are needed. While 25(OH)D levels may be of value in predicting fracture risk in treatment-naive patients, in those already on supplementation we found that its utility is largely limited to serving as a reference for dosing.\n\nOur goal with this study to provide preliminary data on a referral population of patients deemed to have poor bone health will prove useful in the establishment of similar programs elsewhere, in light of the increasing trend in healthcare toward quality and outcomes measures. We conclude that the initial assessment of patients presenting to Bone Health Clinic is complex and requires a holistic approach, taking into consideration a multitude of factors.\n\n\nData Availability\n\nDataset 1: Demographic Data, Fracture Status, and Serum 25-Hydroxy-Vitamin D Levels for the Bone Health Clinic Patient Cohort\n\nEach row represents a single patient’s information which is separated into columns based on topic of interest. These include age, sex, race, body mass index (BMI), fracture status on initial presentation, anatomical location of fracture(s) (if applicable), whether a patient with a prior fracture had sustained a hip fracture or multiple fractures, the time from most recent fracture sustained, DEXA T-scores included in separate columns with the lowest of the three listed in a separate column, serum 25(OH)D level, serum parathyroid hormone (PTH) level, rheumatoid arthritis status, smoking status, diabetes mellitus, and corticosteroid use. Y=yes, N=no. 10.5256/f1000research.12484.d175581",
"appendix": "Competing interests\n\n\n\nThe authors have no competing interests.\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 acknowledge Randal Morris who assisted with institutional review board (IRB) submission, coordination of data collection, and manuscript preparation. We also acknowledge Suzanne Simpson who assisted with copyediting of manuscript\n\n\nReferences\n\nWright NC, Looker AC, Saag KG, et al.: The recent prevalence of osteoporosis and low bone mass in the United States based on bone mineral density at the femoral neck or lumbar spine. J Bone Miner Res. 2014; 29(11): 2520–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlume SW, Curtis JR: Medical costs of osteoporosis in the elderly Medicare population. Osteoporos Int. 2011; 22(6): 1835–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStone KL, Steeley DG, Lui LY, et al.: BMD at multiple sites and risk of fracture of multiple types: long-term results from the Study of Osteoporotic Fractures. J Bone Miner Res. 2003; 18(11): 1947–54. PubMed Abstract | Publisher Full Text\n\nWainwright SA, Marshall LM, Ensrud KE, et al.: Hip fracture in women without osteoporosis. J Clin Endocrinol Metab. 2005; 90(5): 2787–93. PubMed Abstract | Publisher Full Text\n\nSchuit SC, van der Klift M, Weel AE, et al.: Fracture incidence and association with bone mineral density in elderly men and women: the Rotterdam Study. Bone. 2004; 34(1): 195–202. PubMed Abstract | Publisher Full Text\n\nSilverman SL, Calderon AD: The utility and limitations of FRAX: a US perspective. Curr Osteoporos Rep. 2010; 8(4): 192–197. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWatts NB, Ettinger B, LeBoff MS: FRAX facts. J Bone Miner Res. 2009; 24(6): 975–9. PubMed Abstract | Publisher Full Text\n\nGiangregorio LM, Leslie WD, Lix LM, et al.: FRAX underestimates fracture risk in patients with diabetes. J Bone Miner Res. 2012; 27(2): 301–308. PubMed Abstract | Publisher Full Text\n\nInstitute of Medicine: Dietary reference intakes for calcium and vitamin D. Washington, DC: National Academies Press. 2010. Reference Source\n\nBischoff-Ferrari HA, Willett WC, Wong JB, et al.: Fracture prevention with vitamin D supplementation: a meta-analysis of randomized controlled trials. JAMA. 2005; 293(18): 2257–64. PubMed Abstract | Publisher Full Text\n\nTang BM, Eslick GD, Nowson C, et al.: Use of calcium or calcium in combination with vitamin D supplementation to prevent fractures and bone loss in people aged 50 years and older: a meta-analysis. Lancet. 2007; 370(9588): 657–66. PubMed Abstract | Publisher Full Text\n\nJackson RD, LaCroix AZ, Gass M, et al.: Calcium plus Vitamin D supplementation and the risk of fractures. N Engl J Med. 2006; 354(7): 669–83. PubMed Abstract | Publisher Full Text\n\nYetley EA: Assessing the vitamin D status of the US population. Am J Clin Nutr. 2008; 88(2): 558S–564S. PubMed Abstract\n\nGernero P, Munoz F, Sornay-Rendu E, et al.: Associations of vitamin D status with bone mineral density, bone turnover, bone loss and fracture risk in healthy postmenopausal women. The OFELY study. Bone. 2007; 40(3): 716–22. PubMed Abstract | Publisher Full Text\n\nKota S, Jammula S, Kota S, et al.: Correlation of vitamin D, bone mineral density and parathyroid hormone levels in adults with low bone density. Indian J Orthop. 2013; 47(4): 402–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCollins D, Jasani C, Fogelman I, et al.: Vitamin D and bone mineral density. Osteoporos Int. 1998; 8(2): 110–4. PubMed Abstract | Publisher Full Text\n\nSiris ES, Miller PD, Barrett-Connor E, et al.: Identification and fracture outcomes of undiagnosed low bone mineral density in postmenopausal women: results from the National Osteoporosis Risk Assessment. JAMA. 2001; 286(22): 2815–22. PubMed Abstract | Publisher Full Text\n\nMelton LJ 3rd, Atkinson EJ, O’Fallon WM, et al.: Long-term fracture prediction by bone mineral assessed at different skeletal sites. J Bone Miner Res. 1993; 8(10): 1227–33. PubMed Abstract | Publisher Full Text\n\nGutiérrez OM, Farwell WR, Kermah D, et al.: Racial differences in the relationship between vitamin D, bone mineral density, and parathyroid hormone in the National Health and Nutrition Examination Survey. Osteoporos Int. 2011; 22(6): 1745–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCauley JA, LaCroix AZ, Wu L, et al.: Serum 25-hydroxyvitamin D concentrations and risk for hip fractures. Ann Intern Med. 2008; 149(4): 242–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPieper CF, Colon-Emeric C, Caminis J, et al.: Distribution and correlates of serum 25-hydroxyvitamin D levels in a sample of patients with hip fracture. Am J Geriatr Pharmacother. 2007; 5(4): 335–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeringer T, Heyburn G, Finch M, et al.: Prevalence of vitamin D inadequacy in Belfast following fragility fracture. Curr Med Res Opin. 2006; 22(1): 101–5. PubMed Abstract | Publisher Full Text\n\nMaier GS, Seeger JB, Horas K, et al.: The prevalence of vitamin D deficiency in patients with vertebral fragility fractures. Bone Joint J. 2015; 97-B(1): 89–93. PubMed Abstract | Publisher Full Text\n\nBakhtiyaroya S, Lesnyak O, Kyznesova N, et al.: Vitamin D status among patients with hip fracture and elderly control subjects in Yekaterinburg, Russia. Osteoporos Int. 2006; 17(3): 441–6. PubMed Abstract | Publisher Full Text\n\nLeBoff MS, Kohlmeier L, Hurwitz S, et al.: Occult vitamin D deficiency in postmenopausal US women with acute hip fracture. JAMA. 1999; 281(16): 1505–11. PubMed Abstract | Publisher Full Text\n\nLooker AC: Serum 25-Hydroxyvitamin D and risk of major osteoporotic fractures in older U.S. adults. J Bone Miner Res. 2013; 28(5): 997–1006. PubMed Abstract | Publisher Full Text\n\nSwanson CM, Srikanth P, Lee CG, et al.: Associations of 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D With Bone Mineral Density, Bone Mineral Density Change, and Incident Nonvertebral Fracture. J Bone Miner Res. 2015; 30(8): 1403–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLooker AC, Mussolino ME: Serum 25-hydroxyvitamin D and hip fracture risk in older U.S. white adults. J Bone Miner Res. 2007; 23(1): 143–50. PubMed Abstract | Publisher Full Text\n\nMiller AN, Lake AF, Emory CL: Establishing a fracture liaison service: an orthopaedic approach J Bone Joint Surg Am. 2015; 97(8): 675–81. PubMed Abstract | Publisher Full Text\n\nMcLellan AR, Gallacher SJ, Fraser M, et al.: The fracture liaison service: success of a program for the evaluation and management of patients with osteoporotic fracture. Osteoporos Int. 2003; 14(12): 1028–34. PubMed Abstract | Publisher Full Text\n\nCooper MS, Palmer AJ, Seibel MJ: Cost-effectiveness of the Concord Minimal Trauma Fracture Liaison Service, a prospective, controlled fracture prevention study. Osteoporos Int. 2012; 23(1): 97–107. PubMed Abstract | Publisher Full Text\n\nHuntjens KM, van Geel TA, van den Bergh JP, et al.: Fracture liaison service: impact on subsequent nonvertebral fracture incidence and mortality. J Bone Joint Surg Am. 2014; 96(4): e29. PubMed Abstract | Publisher Full Text\n\nCharalambous CP, Kumar S, Tryfonides M, et al.: Management of osteoporosis in an orthopaedic department: audit improves practice. Int J Clin Pract. 2002; 56(8): 620–1. PubMed Abstract\n\nHawker G, Ridout R, Ricupero M, et al.: The impact of a simple fracture clinic intervention in improving the diagnosis and treatment of osteoporosis in fragility fracture patients. Osteoporos Int. 2003; 14(2): 171–8. PubMed Abstract | Publisher Full Text\n\nSimonelli C, Killeen K, Mehle S, et al.: Barriers to osteoporosis identification and treatment among primary care physicians and orthopedic surgeons. Mayo Clin Proc. 2002; 77(4): 334–8. PubMed Abstract\n\nRozental TD, Herder LM, Walley KC, et al.: 25-Hydroxyvitamin-D and Bone Turnover Marker Levels in Patients with Distal Radial Fracture. J Bone Joint Surg Am. 2015; 97(20): 1685–93. PubMed Abstract | Publisher Full Text\n\nMarshall D, Johnell O, Wedel H: Meta-analysis of how well measures of bone mineral density predict occurrence of osteoporotic fractures. BMJ. 1996; 312(7041): 1254–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRiggs BL, Wahner HW, Dunn WL, et al.: Differential changes in bone mineral density of the appendicular and axial skeleton with aging: relationship to spinal osteoporosis. J Clin Invest. 1981; 67(2): 328–35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMajumdar S, Genant HK, Grampp S, et al.: Correlation of trabecular bone structure with age, bone mineral density, and osteoporotic status: in vivo studies in the distal radius using high resolution magnetic resonance imaging. J Bone Miner Res. 1997; 12(1): 111–8. PubMed Abstract | Publisher Full Text\n\nSteiger P, Cummings SR, Black DM, et al.: Age-related decrements in bone mineral density in women over 65. J Bone Miner Res. 1992; 7(6): 625–32. PubMed Abstract | Publisher Full Text\n\nSanders KM, Pasco JA, Ugoni AM, et al.: The exclusion of high trauma fractures may underestimate the prevalence of bone fragility fractures in the community: the Geelong osteoporosis study. J Bone Miner Res. 1998; 13(8): 1337–42. PubMed Abstract | Publisher Full Text\n\nZellner BS, Dawson JR, Reichel LM, et al.: Prospective nutritional analysis of a diverse trauma population demonstrates substantial hypovitaminosis D. J Orthop Trauma. 2014; 28(9): e210–5. PubMed Abstract | Publisher Full Text\n\nKostoglou-Athanassiou I, Athanassiou P, Lyraki A, et al.: Vitamin D and rheumatoid arthritis. Ther Adv Endocrinol Metab. 2012; 3(6): 181–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKerr GS, Sabahi I, Richards JS, et al.: Prevalence of vitamin D insufficiency/deficiency in rheumatoid arthritis and associations with disease severity and activity. J Rheumatol. 2011; 38(1): 53–9. PubMed Abstract | Publisher Full Text\n\nEkwaru JP, Zwicker JD, Holick MF, et al.: The importance of body weight for the dose response relationship of oral vitamin D supplementation and serum 25-hydroxyvitamin D in healthy volunteers. PLoS One. 2014; 9(11): e111265. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFelson DT, Zhang Y, Hannan MT, et al.: Effects of weight and body mass index on bone mineral density in men and women: the framingham study. J Bone Miner Res. 1993; 8(5): 567–73. PubMed Abstract | Publisher Full Text\n\nMcCarley MR, Wise KL, Jupiter DC, et al.: Dataset 1 in: The utility of serum 25-Hydroxyvitamin-D and body mass index in the work-up of patients presenting to a bone health clinic. F1000Research. 2017. Data Source"
}
|
[
{
"id": "27195",
"date": "26 Oct 2017",
"name": "Maria Lucia Fleiuss de Farias",
"expertise": [
"Reviewer Expertise Endocrinology and metabolic bone diseases"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nVitamin D deficiency is indeed endemic, taking 30ng/mL as the lower limit of normality for serum 25(OH)D. And this seems correct, at least concerning bone health in postmenopausal women and elderly people. In this population D-deficiency has been associated with secondary hyperparathyroidism, increased bone turnover and loss, as well as propensity to falls, and all these factors contribute to increased fracture risk. The results presented here suggest that 25(OH)D and bone mineral density are not associated with fractures. However, some points must be considered.\nThe authors need to clarify if there was an active search for non-clinical vertebral fractures using image methods (X-rays, VFA), as they correspond to more than half of all vertebral fractures in elderly population. If not, at least some of the patients could have been erroneously considered “non-fractured” which would interfere with future comparisons.\n\nMost previous documented fractures occurred several months before this study, and patients on adequate vitamin D supplementation were included. This would interfere with the diagnosis of vitamin D status at the time of fracture, as recognised in text “We were surprised to find that many of the patients studied had normal serum 25(OH)D levels. This is likely the result of supplementation by patients prior to referral.....” and also interfere with the valuation of absolute values at the study, as mentioned “While 25(OH)D levels may be of value in predicting fracture risk in treatment-naive patients, in those already on supplementation we found that its utility is largely limited to serving as a reference for dosing”. Thus the suggestion that D-deficiency is not associated with osteoporotic fractures because mean vitamin D levels were similar between patients with and without fractures should be reconsidered.\n\nA comment on previous use of antiosteoporosis drugs at the time fractures ocurred should be included, as this could interfere with BMD values at the study and the conclusions.\n\nPlease comment on the reasons why variables whose correlation / association was not significant (p=0.2) in the univariate analysis were considered in the multivariate.\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? No\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "26624",
"date": "06 Nov 2017",
"name": "Nicole C. Wright",
"expertise": [
"Reviewer Expertise Osteoporosis 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 authors evaluated the association between 25(OH)D, BMI, BMD, and fractures in their fracture liason like service in their orthopedic clinic. The relationship between vitamin D, BMD, and fractures has been widely studied in the field, so this study is not unique; however, utilizing low BMD and/or low vitamin D as a reference tool for FLS and FLS like program is a unique idea, so evaluating how it is associated with fractures is an interesting research topic. However, the presentation of the data was not the best.\nFirst, in the discussion section, there were a few articles that would could have been referenced. 1) the racial differences in vitamin D biology with respect to bone health. The researchers did not properly present the racial background of their study, but this could be relevant to other researchers. 2) they did not cite the meta-analysis about BMI, BMD, and fractures. After adjusting to BMD, there is a relationship between BMI and fracture differs.\n\nAlthough the methods and analyses were sound and well laid out, the authors did not mention what statistical program was used for the analyses.\n\nThe interpretation of the data was very confusing at times, particularly with respect to the tables. The formatting of the tables was not good. Traditionally Table 1 is a description of the population. The authors sort of put descriptive data in multiple tables, with different outcomes and/or exposures in the same columns. It was very confusing to read.\nPlease also see edits made to the article PDF here.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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/6-1588
|
https://f1000research.com/articles/6-1583/v1
|
29 Aug 17
|
{
"type": "Opinion Article",
"title": "Intertraffic of Endothelin 1 and Thrombospondin 1 between lungs and myocard via pulmonary circulation can alter cardiac loads, tissue integrity and atrial blood coagulability",
"authors": [
"Sven Kurbel"
],
"abstract": "Similar expression patterns of mRNA profiles for Endothelin 1 (ET-1) and Thrombospondin 1 (TSP1) from GeneAtlas U133A, gcrma, suggest that these two mediators are dominantly synthesized in the myocard and lungs. This paper proposes that intertraffic of these two mediators between myocard and lungs via pulmonary and coronary vessels optimizes cardiopulmonary functions and maintains their tissue integrity. A controlled delivery of endocrine mediators to the left and to the right heart is done by the coronary sinus (CS) that drains venous blood to the right atrium, and Thebesian veins (TVs) that open in all four heart cavities. Myocard and pulmonary capillaries are connected as a bidirectional portal system. Mediators from lungs can directly influence myocardial cells after entering the coronary circulation, while mediators in the myocardial venous blood that drain into the right heart will initially affect lungs. Strain induced myocardial ET-1 secretion into the right heart and pulmonary circulation increases the right heart afterload by constricting pulmonary vasculature. The same action reduces the left heart preload. Pulmonary ET-1 secretion can protect lungs from overperfusion by increasing the left heart afterload through constriction of peripheral arterioles. Chronic tissue overexposure to ET-1 leads to pulmonary and myocardial fibrosis. TSP1 availability is important in tissues under mechanical stress, since TSP1 is an adhesive glycoprotein involved in cell-to-cell and cell-to-matrix interactions. Pulmonary and myocardial TSP1 secretion that enter the fibrillating left atrium can mimic actions of the platelet-derived TSP1 in promoting the thrombus formation.",
"keywords": [
"mRNA tissue expression",
"Endothelin-1",
"Thrombospondin 1",
"myocard",
"lungs",
"pulmonary fibrosis",
"heart-lung interaction",
"atrial fibrillation"
],
"content": "Introduction\n\nUnexpectedly similar expression patterns of mRNA profiles for Endothelin 1 (ET-1) (http://biogps.org/#goto=genereport&id=1906) and Thrombospondin 1 (TSP1) (http://biogps.org/#goto=genereport&id=7057) in GeneAtlas U133A (at http://biogps.org)1, suggest that these two mediators are dominantly synthesized in myocard and lungs.\n\nET-1 is known as one of the strongest vasoconstrictors, while TSP1 is one of the key inhibitors of angiogenesis that is also important in the maintenance of tissue integrity and remodeling. A direct link between these two mediators and cardiopulmonary circulation seems to exist, since it has been reported that loss-of-function thrombospondin-1 mutations were found in familial pulmonary hypertension2, a clinical condition treatable with bosentan, an ET-1 receptor blocker3. An attractive possibility is that TSP1 and ET-1 might act as a pair of opposing mediators in the regulation of resistance in pulmonary vessels.\n\nThis paper proposes that endocrine intertraffic of these two mediators between myocard and lungs via pulmonary and coronary vessels might be important in optimizing cardiopulmonary function and maintaining tissue integrity of both organs.\n\nAn important issue is whether mediators synthesized in the myocard can be selectively diverted between the pulmonary circuit and the systemic circuit. Here the proposed idea is that a controlled delivery to the left and to the right heart is done by the peculiar myocardial venous circulation that includes coronary sinus (CS) that drains venous blood to the right atrium, and Thebesian veins (TVs) that open in all four heart cavities4,5, particularly numerous at the ventricle apex and at papillary muscles base6.\n\nIt was observed that the blood flow through TVs depends on the heart muscle stretching. In inactive heart muscle perfused through coronaries under pressure, more fluid can escape through TVs than through CS7, suggesting that fluid drainage via Thebesian veins in comparison to other veins might be more important in situations when heart chambers are stretched, due to the volume load.\n\nIf we look at the cardiopulmonary circulation in more detail (Figure 1), the pulmonary capillaries are in the serial position to the myocardial capillaries, meaning that mediators from lungs can directly influence myocardial cells after entering the coronary circulation. Myocardial veins to the right heart (mainly CS and also the right heart TVs) place myocardial capillaries in the serial position, in front of the pulmonary capillaries. Any mediator in the myocardial venous blood drained in the right heart will initially affect lungs before being diluted in the systemic circulation.\n\nThis unique setting can be described as a double portal system in which myocard and pulmonary capillaries are bidirectionally connected via short endocrine loops. Other, better-known examples of portal circulations in our body are unidirectional, since the tissue of the secondary capillaries (adenohypophysis or liver) has no way of endocrine action directed selectively on the primary organ (hypothalamus in the former and stomach and intestines in the latter). Instead of that mediators from secondary capillaries return to the heart and enter pulmonary circulation.\n\nThe pulmonary capillaries are in the serial position to the myocard, so mediators from lungs can directly influence myocardial cells, after entering the coronary circulation. Myocardial venous blood that drain into the right heart (mainly via coronary sinus (CS) and also via the right heart Thebesian veins (TVs)) initially affects lungs before being diluted in the systemic circulation.\n\nIt should be noted that although this paper is focused on ET-1 and TSP1, due to their high synthesis rates in lungs and myocard, various other substances can act through these short, portal-type loops that connect cardiopulmonary organs.\n\nBoth lungs and myocard are rich in various receptors, so local perfusion clearance is expected for many mediators in the blood that passes through lungs and myocard.\n\nAs shown in Table 1, any surplus of myocardial mediators can leave the heart muscle through the left heart Thebesian veins and enter the systemic circulation, or leak to the right heart through CS and the right heart TVs. In the latter case, the right heart veins act as a shortcut from myocard to pulmonary circulation.\n\nOn the other hand, substances synthesized in lungs enter the left heart and aorta. Some of these can directly affect the myocard via coronary arteries, and their surplus can return to the lungs via CS and the right heart TVs.\n\nAn important consequence of the described setting is that for any disorder caused by direct cardiopulmonary endocrine loops it can be expected to remain undetectable by measuring the peripheral vein blood levels of involved mediators. This means that new diagnostic methods are probably required to recognize patients with problems caused by an alteration in cardiopulmonary control loops.\n\nFrom the U133A data, in the probeset 218995 with Endothelin-1 (EDN1) data (http://biogps.org/#goto=genereport&id=1906), among 176 measurements of different tissue samples, only four values stand out. Two values are human cardiac myocytes (Clonetics Cat # CC-2582) with mRNA expression values of 502.7 and 118.1, and the other two are lung samples with values of 343.5 and 307.5. The mean EDN1 value for all reported tissue samples is only 13.28, clearly suggesting that cardiac myocytes and lungs synthesize much more ET-1 than all other tested tissues and cells.\n\nA separate issue is the identification of target tissues for the circulating ET-1. Data of endothelin A receptor (gene EDNRA) (http://biogps.org/#goto=genereport&id=1909) were extracted from the probeset 216235. High EDNRA mRNA levels were reported in uterus, cardiac myocytes, heart, prostate, lungs, liver, appendix and skeletal muscle. In the second probeset 204463, uterus, prostate and lungs have the highest EDNRA values, while in the probeset 204464, fetal lungs and uterus standout as tissues with the highest EDNRA mRNA values.\n\nA plausible interpretation is that lungs and cardiac myocytes produce so much ET-1 that the surplus of ET-1 molecules leaks into the circulation. Other tissues are able to synthesize ET-1 in modest quantities, probably acting as a paracrine mediator8. This interpretation is in concordance with papers that report cardiac secretion of ET-1 in animal models9,10.\n\nTable 1 is based on the expectation that the myocardial ET-1 synthesis increases due to stretching and increased work of the cardiac muscle, during heavy exercise as well as in pathologic conditions that dilate heart chambers. Exposure to humoral and paracrine mediators also influences the ET-1 synthesis. So, although atrial TVs can remain open during almost the whole heart cycle, with the exception of the short atrial systole (visible as the P-R interval on ECG), the ventricular TVs probably drain more blood during late diastole, when ventricles are stretched to maximal length and under low pressure. During ventricular systole TVs openings are reduced in size, the intraventricular pressure is higher than in the heart veins, particularly in the Left ventricle, so most of the myocardial venous blood probably leaves through the coronary sinus.\n\nWhen considering the mRNA distributions for ET-1 and its receptor A together, here the proposed interpretation is that an elaborate regulatory system mediated by ET-1 selectively tunes resistance in the pulmonary and in the systemic circulation to meet ever-changing demands during rest and exercise (Table 1):\n\n• Strain induced secretion of cardiac ET-1 via coronary sinus and the right heart TVs into the pulmonary circulation increases the right heart afterload by constricting pulmonary vasculature. The same action reduces the left heart preload and thus reduces myocard strain on the left side.\n\n• Pulmonary secreted ET-1 can protect lungs from overperfusion by increasing the left heart afterload through constriction of peripheral arterioles. This will also reduce the venous return to the right heart (the preload of the right heart) and thus reduce flow in the pulmonary circuit.\n\n• Some of the pulmonary ET-1 molecules inevitably enter coronary circulation. In the long run, chronic myocard exposure to ET-1 secreted from lungs may lead to the heart muscle hypertrophy.\n\n• It is similar for the myocardial ET-1 secreted in the left heart via Thebesian veins. This secretion also increases the left heart afterload by constricting peripheral arteries and the consequence is the reduction of the right heart preload due to the reduced venous return.\n\nThese simple changes in resistance of pulmonary and systemic arteries help the myocard to adapt to many different settings of physical work, particularly to quickly alleviate extremely low afterload and thus reduce the increased venous return (the right heart preload) during intense muscular work.\n\nIt is known that blood levels of thrombospondin 1 can be altered in patients with ischemic disease11,12, malignancy, particularly in pancreatic cancer patients13 or metabolic problems14. Based on many TSP1 roles, some synthesis is expected to be almost ubiquitous, but only few cells/tissues require so much thrombospondin 1 that the local surplus normally leaks into circulation.\n\nThere are two important aspects of the reported TSP1 mRNA values (http://biogps.org/#goto=genereport&id=7057). The first is that almost all tested cells/tissues contained this mRNA, and the second is that few cells/organs have shown high levels. Among the main sources of TSP1 synthesis are Cardiac Myocytes, Bronchial Epithelial Cells, Smooth Muscle Cells and Uterus. All these cells/tissues are unique by their special ability to actively or passively adapt to changes in the cellular shape and surface area. Cardiac myocytes and smooth muscle cells form tissue layers that contract by reducing their area in a manner of bidimensional shrinkage:\n\n• During smooth muscle contractions in arterial vessels, digestive organs or uterus, axially undefined smooth muscle cells are not constrained. They change their shape by bidimensional shrinking of the covered area.\n\n• During myocardial contractions, many small interconnected cells form a meshwork. Although individual myocard cells can contract only along their axial skeleton, the myocard layers bidimensionally shrink, due to the linear shortening of cellular units that form meshwork layers.\n\n• In lung tissue, there is no active contraction during inspiration and expiration, since the physical work of breathing is done by skeletal muscles that change the thoracic volume. Lungs expand passively according to the intrathoracic pressure and available space. These passive changes of alveolar volume force alveolar cells to adapt in their shape and covering area.\n\nIn all these three examples, changes in the cell shape are repetitive. This means that the tissue structure integrity strongly depends on interconnections between cells. This is a point where plentiful TSP1 availability becomes important, since TSP1 is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions, binds to fibrinogen, fibronectin, laminin, type V collagen and integrins15. This means that beside other factors (glucose, insulin14,16), the local synthesis of TSP1 is probably related to the average mechanical stress, in tubular structures governed by the Law of Laplace.\n\nBased on the mentioned link of ET-1 and TSP1 to pulmonary fibrosis, it can be assumed that other health issues can be linked to the imbalance in pulmonary or myocardial synthesis of these two mediators.\n\nTable 2. shows possible damage of supraphysiologic myocardial and pulmonary ET-1 secretion. Two auto-endocrine loops that start and end in the same tissue are proposed. The first possibility is that the presence of endothelin A receptors on cardiac myocytes can explain the ET-1 mediated heart hypertrophy. Beside pulmonary ET-1, the left heart Thebesian veins can increase deliverance of ET-1 to the coronary circulation and lead to heart hypertrophy17 and to the diastolic dysfunction18.\n\nSupraphysiological ET-1 exposure can damage organs and tissues (LV - left ventricle; RV - right ventricle).\n\nThe second autocrine loop is possible in patients with a significant left-to-right cardiac shunt. Surplus of pulmonary ET-1, due to increased pulmonary perfusion can leak through the shunt opening from the left heart to the right side. Once on the right side, the excess ET-1 leaves the right ventricle and through pulmonary arteries reach lungs. This recycling of pulmonary ET-1 via left-to-right shunt can lead to pulmonary hypertension and fibrosis. Both conditions are components of the Eisenmenger's syndrome, often treated by bosentan, a blocker of endothelin receptors19.\n\nSome patients with long-lasting atrial fibrillation never suffer from peripheral embolism, despite not being on anticoagulant therapy, while others experience peripheral artery embolisms despite regular therapy20. Only rare patients develop thrombi in the right atrium, although both atria are fibrillating, with similar magnitude reductions in ejection velocities20. These observations point to the unsolved question why is the left atrium so much more prone to the thrombus formation during fibrillation.\n\nIt is here assumed that the local rate of TSP1 synthesis probably depends on the local myocardial and pulmonary pathology, some patients produce a surplus of myocardial TSP1 due to cardiac dilatation, myocytes stretching or metabolic conditions, while diverse pulmonary problems can augment leaking of pulmonary TSP1 into circulation.\n\nAs shown in Table 3, the right atrium receives the systemic venous blood, normally poor in TSP1. On the other hand, the coronary venous blood via CS and TVs, normally contain some myocardial TSP1, but endocrine factors or local condition within the heart muscle can increase the TSP1 content. The situation is similar in the right ventricle.\n\nThe left atrium is exposed to two separate thrombospondin 1 sources, from lungs via pulmonary veins and from myocard via left atrial Thebesian veins. It can be presumed that in many pulmonary or cardiac patients, these two organs often secrete a surplus of TSP1, thus making TSP1 blood levels increased in the left heart, particularly in the left atrium. In short, this means that the left atrium and ventricle are only chambers that receive both pulmonary and myocardial TSP1.\n\nA thin and TSP1-rich juxtamural layer of blood surrounding Thebesian veins can temporary form during the end-diastolic atrial and ventricular phases. In a normal heart this should not increase the risk of thrombus formation, since all secreted TSP1 will be quickly diluted by the inevitable systole that mixes all blood during ejection.\n\nHere the proposed sequence of events starts with the onset of atrial fibrillation:\n\n• Since there are no systoles to mix the atrial content, TSP1 is not adequately diluted and washed away from atrial cavities:\n\n○ In the right atrium, systemic venous blood is poor in TSP1 and the buildup of cardiac TSP1 via CS and TVs depends on myocard stretching and endocrine factors. In many patients, the right atrial TSP1 levels probably remain well below the increased risk of thrombus formation.\n\n○ In the left atrium, pulmonary disorders increase TSP1 levels in blood that enters from pulmonary veins. Dilated atrial walls continuously leak venous blood that contains myocardial TSP1. These two sources build up the juxtamural TSP1 concentration in the vicinity of the TVs openings, thus stratifying the juxtamural layer of blood in peripheral parts of the left atrium with TSP1.\n\n• Blood carried TSP1 molecules that enter the fibrillating left atrium mimic actions of the platelet-derived TSP1 in promoting the thrombus formation. Increased local availability of TSP1 protects endothelium-bound and subendothelial von Willebrand factor from degradation by plasma ADAMTS13, as it has been reported for soluble or local platelet-released TSP1 in a shear field21. This TSP1 action, combined with stagnant blood flow in peripheral parts of the left atrium, secures platelet tethering, thrombus adherence and growth.\n\nSince levels of ET-1 and TSP1 in the peripheral vein is not expected to be adequately related to the pulmonary and coronary levels of these two mediators, here proposed interpretations can be tested by taking blood samples from other vascular segments.\n\nOne possibility is to measure ET-1 values in samples of blood taken during systole and diastole from the right atrium (near the coronary sinus) and from aorta (below the aortal valve):\n\n• If ET-1 levels in the right atrial blood are lower than in aorta, or show inverse oscillations during the heart cycle, this would prove that Thebesian veins of the left heart side act as an important direct shortcut for ET-1 from the myocard to systemic arteries.\n\nOn the other hand, it might be important to measure the TSP1 level in simultaneously taken samples of blood from a systemic artery and from a central vein:\n\n• Higher TSP1 levels in the arterial blood are expected if pulmonary and myocardial TSP1 into circulation is increased due to disorders of these two organs, making the patient prone to peripheral artery embolisms in the case of atrial fibrillation.\n\n• In the opposite case with higher TSP1 levels in central veins, the overall TSP1 leakage from peripheral tissues back to the heart is increased, possibly due to metabolic disorders or tumors and this setting increases the risk of thrombus formation in peripheral veins. This can be related to the Trousseau's syndrome, associated with the pancreatic, gastric and lung cancer.",
"appendix": "Competing interests\n\n\n\nThe author declares not to have any competing interests regarding the content of this article.\n\n\nGrant information\n\nThis theoretical paper was financed through grant 21921923822426 from the Croatian Ministry of Science, Education and Sport.\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\nSu AI, Wiltshire T, Batalov S, et al.: A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci U S A. 2004; 101(16): 6062–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaloney JP, Stearman RS, Bull TM, et al.: Loss-of-function thrombospondin-1 mutations in familial pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol. 2012; 302(6): L541–54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChannick RN, Simonneau G, Sitbon O, et al.: Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo-controlled study. Lancet. 2001; 358(9288): 1119–23. PubMed Abstract | Publisher Full Text\n\nKurbel S, Marić S, Gros M: Do Thebesian veins and arterioluminal vessels protect against myocardial edema occurrence? Med Hypotheses. 2009; 73(1): 38–9. PubMed Abstract | Publisher Full Text\n\nGregg DE: Effect Of Coronary Perfusion Pressure Or Coronary Flow On Oxygen Usage Of The Myocardium. Circ Res. 1963; 13: 497–500. PubMed Abstract | Publisher Full Text\n\nAnsari A: Anatomy and clinical significance of ventricular Thebesian veins. Clin Anat. 2001; 14(2): 102–10. PubMed Abstract | Publisher Full Text\n\nWearn JT: The Role Of The Thebesian Vessels In The Circulation Of The Heart. J Exp Med. 1928; 47(2): 293–315. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKurbel S, Kurbel B, Kovacić D, et al.: Endothelin-secreting tumors and the idea of the pseudoectopic hormone secretion in tumors. Med Hypotheses. 1999; 52(4): 329–33. PubMed Abstract | Publisher Full Text\n\nIwanaga Y, Kihara Y, Hasegawa K, et al.: Cardiac endothelin-1 plays a critical role in the functional deterioration of left ventricles during the transition from compensatory hypertrophy to congestive heart failure in salt-sensitive hypertensive rats. Circulation. 1998; 98(19): 2065–73. PubMed Abstract | Publisher Full Text\n\nSakai S, Miyauchi T, Sakurai T, et al.: Endogenous endothelin-1 participates in the maintenance of cardiac function in rats with congestive heart failure. Marked increase in endothelin-1 production in the failing heart. Circulation. 1996; 93(6): 1214–22. PubMed Abstract | Publisher Full Text\n\nKrishna SM, Golledge J: The role of thrombospondin-1 in cardiovascular health and pathology. Int J Cardiol. 2013; 168(2): 692–706. PubMed Abstract | Publisher Full Text\n\nKaiser R, Grotemeyer K, Kälsch T, et al.: Decreased TSP1 following percutaneous coronary intervention is associated with major adverse cardiac events in ST-elevation myocardial infarction. Clin Hemorheol Microcirc. 2013; 54(1): 59–73. PubMed Abstract | Publisher Full Text\n\nJenkinson C, Elliott VL, Evans A, et al.: Decreased Serum Thrombospondin-1 Levels in Pancreatic Cancer Patients Up to 24 Months Prior to Clinical Diagnosis: Association with Diabetes Mellitus. Clin Cancer Res. 2016; 22(7): 1734–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMatsuo Y, Tanaka M, Yamakage H, et al.: Thrombospondin 1 as a novel biological marker of obesity and metabolic syndrome. Metabolism. 2015; 64(11): 1490–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBonnefoy A, Hantgan R, Legrand C, et al.: A model of platelet aggregation involving multiple interactions of thrombospondin-1, fibrinogen, and GPIIbIIIa receptor. J Biol Chem. 2001; 276(8): 5605–12. PubMed Abstract | Publisher Full Text\n\nMaile LA, Allen LB, Hanzaker CF, et al.: Glucose regulation of thrombospondin and its role in the modulation of smooth muscle cell proliferation. Exp Diabetes Res. 2010; 2010: pii: 617052. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCheng CP, Ukai T, Onishi K, et al.: The role of ANG II and endothelin-1 in exercise-induced diastolic dysfunction in heart failure. Am J Physiol Heart Circ Physiol. 2001; 280(4): H1853–60. PubMed Abstract\n\nBurlew BS, Weber KT: Cardiac fibrosis as a cause of diastolic dysfunction. Herz. 2002; 27(2): 92–8. PubMed Abstract | Publisher Full Text\n\nGalie N, Beghetti M, Gatzoulis MA, et al.: Bosentan therapy in patients with Eisenmenger syndrome: a multicenter, double-blind, randomized, placebo-controlled study. Circulation. 2006; 114(1): 48–54. PubMed Abstract | Publisher Full Text\n\nSubramaniam B, Riley MF, Panzica PJ, et al.: Transesophageal echocardiographic assessment of right atrial appendage anatomy and function: comparison with the left atrial appendage and implications for local thrombus formation. J Am Soc Echocardiogr. 2006; 19(4): 429–33. PubMed Abstract | Publisher Full Text\n\nJurk K, Clemetson KJ, de Groot PG, et al.: Thrombospondin-1 mediates platelet adhesion at high shear via glycoprotein Ib (GPIb): an alternative/backup mechanism to von Willebrand factor. FASEB J. 2003; 17(11): 1490–2. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "25407",
"date": "12 Sep 2017",
"name": "David C. Randall",
"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:\n\nThis is a well-written, provocative ‘Opinion Article’ that uses, in part, published observations to propose a novel hypothesis concerning the roles of Endothelin 1 (ET‐1) and Thrombospondin 1 (TSP1) in circulatory regulation. A few logical inferences upon which the supposed relationships are predicated are not immediately clear to this reviewer, but this should be easily rectified. The author’s focus upon the regulatory role of substance delivery via Thebesian veins and via flow through the coronary sinus (in their role as a “bidirectional portal system”) is notable since, to my knowledge, these structures have received almost no attention in this context.\nI confess to relative ignorance of the actions of ET‐1, but the pulmonary vasculature (or pulmonary smooth muscle) responds differently from the systemic in some circumstances. I asked two of my respiratory/pulmonary colleagues what the action of ET‐1 was in the pulmonary circulation, and neither knew. Please clarify ET‐1 effects on pulmonary (i.e., vs. systemic) at some appropriate point in this ms (i.e., affirm that ET‐1 vasoconstricts the pulmonary circulation).\nThe author uses the word ‘myocard’ rather than ‘myocardium’; the latter is common English usage in this reviewer’s experience, and his use of ‘myocard’ instead was a bit distracting. The author might want to consider changing his usage to ‘myocardium’ if (and only if) he considers it likely that the majority of his readers will be more familiar with ‘myocardium’.\n\nSPECIFIC COMMENTS\nAbstract: The first of the uncertain logical links occurs in the first sentence of the abstract: it is unclear to me why ‘similar expression profiles’ necessarily implies that the two mediators are “dominantly synthesized in the myocard and lungs.” It is acceptable to me to leave this statement as is and clarify the logical link in the full text, but this is foundational to the author’s argument and should be sufficiently expanded to enhance clarity where most appropriate.\n\nIntroduction: The opening sentence again ‘suggests’ dominant synthesis in myocard and lungs. I suggest that amplification here of this logical sequence would be the best and easiest place to make this clearer. If I’m missing something simple, I apologize! The first sentence of this next (2nd) paragraph probably needs a reference.\n\np. 3, right column, para. 2 :It was observed…: Please reference this observation.\n\nTable 1: This is a formidable table whose ‘message’ is difficult to ferret out; simplify if practical. (Note the error in middle column, row 1: <10 L/min.)\n\nIn the sentence that introduces Table 1 the word ‘surplus’ (of myocardial mediators) is used; I’m not convinced that this word is appropriate: even if only modest amounts of mediator are secreted, must not some percentage pass through the TV and CS? (Likewise on p. 5, left column, para. 3.) P 4 (of 8), right column, 3rd paragraph: The antecedent of ‘it’ in the first sentence (…endocrine loops it can be expected…) is not clear to me. I assume ‘it’ is ET‐1 or TSP1, but, as written, ‘disorder’ would seem to be the antecedent.\n\np. 5, left column, para. beginning “Table 1 is based…”: The ‘expectation’ that stretching of the myocardium (or dilation and thereby patency of the TV in the LV) increases synthesis is important to the author’s thesis; please reference or give some validation for this ‘expectation’.\n\nNext para: The intended meaning of the opening sentence (When considering…) is not clear.\n\np. 5, top of right column: I admit to being a bit ‘picky’, but the use of ‘it’ (It is similar…) seems weak to me. Also, would ‘into’ not be better than ‘in’ here (…ET‐1 secreted into the left heart…)\n\np. 5, last para: I do not understand what you mean by ‘repetitive’ in this opening sentence. Cyclic? (If so, I don’t think this would be correct.) My confusion is increased by the logical connection between this (repetitive changes) and the next sentence (tissue structure integrity and cellular interconnection dependent upon repetitive.) Please clarify.\n\np. 6, “Disorders possibly…” paragraph: Would ‘produced by’ be better than ‘of’ so sentence might read “Table 2. Shows possible damage produced by supraphysiologic…”?\n\np. 6, left column, final line: …the excess ET‐1 … reachES (not reach) lungs. In this second autocrine loop are you referring to a loop made via the TV which would exist in all people, but become functionally important in only certain patients (e.g., Eisenmenger’s syndrome)?\n\n(I found the proposed application of your concepts to thrombus formation in the LA to be particularly interesting, though at points somewhat harder to follow and integrate with Table 3.)\n\np. 6, last para. introducing Table 3: Do you mean “As shown in the middle row, right column of Table 3…”? In other words, it would be helpful to the reader to ‘walk him through’ Table 3 a bit more in the text.\n\np. 7, Part V: Consider replacing the word ‘prove’ with the word ‘test’. In the opening sentence of this section “Since levels of ET‐1 and TSP1 in the peripheral vein ARE not expected…”\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
},
{
"id": "26137",
"date": "20 Oct 2017",
"name": "Mingyi Yao",
"expertise": [
"Reviewer Expertise Cardiovascular pharmacology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 proposed a possible connection between endothelin-1 (ET-1) and thrombospondin 1 (TSP1) based on their mRNA profiles and random clinical observations. Whether the mRNA profile predicts protein expression of ET-1 and TSP1 is still waiting for verification. The role of ET-1 in pathology of cardio-pulmonary diseases such as pulmonary arterial hypertension (PAH) has been well recognized and thus therapy targeting ET-1/ETA receptor developed. Involvement of TSP1 in clinical PAH received attention till recent years. Isenberg JS and his group revealed that expressions of TSP1, ET-1 and ETA receptors as well are increased in patients with PAH1,2. Abnormally high level of tissue and circulatory ET-1 is consistently found in all WHO classification of PAH3-5, suggesting that upregulation of ET-1 is independent of type I repeat domains of TSP1. TSP1 modulates ET-1 and ETA receptor expression via association with cognate receptor CD47 through its C-terminus. This explains the reason why nonselective ET receptor antagonist is effective in managing PAH. It seems that that ET-1 and ETA receptor are downstream of TSP1 but not vice versa, based on currently available evidence. Anatomical knowledge of the myocardial venous circulation helps to interpret persistent deteriorating effect of elevated TSP1 on the cardio-pulmonary system, in addition to its long half-life of approximately 9 hours. The presence of high level of TSP1 in the heart not only exposes the high risk of thrombus, also contribute to the left ventricle heart failure6.\nBased on references listed above, connection between TSP1 and ET-1 under pathologic condition is found exist. The article is suggested to convert to a mini review incorporating these findings along with the mRNA data.\nSpecific comments:\nIntroduction: Link between loss-of-function TSP1 mutations found in familial PAH and Bosentan therapy for PAH is not convincing to support the hypothesized possibility of opposing effect of TSP1 and ET-1. TSP1 can interact with up to 83 ligands/receptors via various domains7. Mutation of repeat type I domains of TSP1 only affects its interaction with TGFβ which is not involved in regulation of ET-1 or ETA receptor expression.\n\nInformation provided by Figure 1 is useful to explain circulation of ET-1 and TSP1 in the cardio-pulmonary circulation; however, it is hard to follow.\n\nDoes “surplus” mean excess?\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": []
}
] | 1
|
https://f1000research.com/articles/6-1583
|
https://f1000research.com/articles/6-1559/v1
|
24 Aug 17
|
{
"type": "Research Article",
"title": "Exploring Vietnamese co-authorship patterns in social sciences with basic network measures of 2008-2017 Scopus data",
"authors": [
"Tung Manh Ho",
"Ha Viet Nguyen",
"Thu-Trang Vuong",
"Quang-Minh Dam",
"Hiep-Hung Pham",
"Quan-Hoang Vuong",
"Tung Manh Ho",
"Ha Viet Nguyen",
"Thu-Trang Vuong",
"Quang-Minh Dam",
"Hiep-Hung Pham"
],
"abstract": "Background: Collaboration is a common occurrence among Vietnamese scientists; however, insights into Vietnamese scientific collaborations have been scarce. On the other hand, the application of social network analysis in studying science collaboration has gained much attention all over the world. The technique could be employed to explore Vietnam’s scientific community. Methods: This paper employs network theory to explore characteristics of a network of 412 Vietnamese social scientists whose papers can be found indexed in the Scopus database. Two basic network measures, density and clustering coefficient, were taken, and the entire network was studied in comparison with two of its largest components. Results: The networks connections are very sparse, with a density of only 0.47%, while the clustering coefficient is very high (58.64%). This suggests an inefficient dissemination of information, knowledge, and expertise in the network. Secondly, the disparity in levels of connection among individuals indicates that the network would easily fall apart if a few highly-connected nodes are removed. Finally, the two largest components of the network were found to differ from the entire networks in terms of measures and were both led by the most productive and well-connected researchers. Conclusions: High clustering and low density seems to be tied to inefficient dissemination of expertise among Vietnamese social scientists, and consequently low scientific output. Also low in robustness, the network shows the potential of an intellectual elite composed of well-connected, productive, and socially significant individuals.",
"keywords": [
"Social network analysis",
"science collaboration",
"network characteristics",
"network visualization",
"research output."
],
"content": "1. Introduction\n\nIn early 2017, the Vietnamese public was once again disappointed to find out there was no Vietnamese universities in the Times Higher Education’s ranking of the top 300 universities in Asia. There was no shortage of experts’ attempts to explain this disappointing situation; many pointed to the fact that Vietnamese universities have not put enough focus on research. Being aware of the demand for improving research capacity, the Ministry of Education and Training has recently issued a number of policies and proposals addressing the issue head-on. Figuring among the many efforts is the issuance of circular No. 08/2017/TT-BGDĐT (issued on 14th April, 2017) mandating doctoral students must have papers published in Scopus and Web of Science-indexed journals, the doctoral dissertation instructors must also have international publications. There has also been a proposal to mandate that candidates for the titles of Professor and Associate Professor must have international publications. Although these changes and proposals were met with both excitement and dread by the public, it is noteworthy that those who criticize the new regulations do not argue against the changes. Rather, their main concern is “when” or the timeline to adopt these policies: whether these changes are too abrupt.\n\nIn other words, people on both sides of the arguments express their desire to improve research capacity in Vietnam. The question remains is “how”: How to increase the quantity and quality of scientific publications in Vietnamese social sciences? The answer seems to be related to the spread of information and expertise in the scientific community, which may call for quantitative methods. However, the field of quantitative research on scientific activities and research policy in Vietnam is still nascent. Even though there have been several studies on the status of scientific publications in Vietnam, none has been carried out with a sole focus on social sciences – a field often criticized for having low productivity1,2. In addition, the technique of social network analysis is yet to be applied in the case of Vietnam, despite its potentials in explaining and predicting scientific performance. A study on the nature of scientific co-authorship among Vietnamese social scientists using network statistical analysis would yield valuable insights for policy-makers and educators in Vietnam.\n\nOver the years, the application of network statistical analysis on science collaboration has become pervasive; it has gleaned many insights into the dynamics of scientific activities as well as the properties of scholars’ networks. By exploring a number of databases from different fields such as biomedical research, physics and computer science, Newman showed that scientific collaboration networks seem to form “small worlds”, in which any randomly chosen pair of scientists would be separated only through a few intermediate collaborators. Another interesting aspect is that there are different degrees of clustering of scientists in different fields, suggesting the differences in social organizations3. In a 2004 study of sociology collaboration networks by exploring of 30 years’ worth of data in the field, from 1963 to 1999, Moody discovered that participation in the network depends on the research major, and scholars who are more inclined to quantitative work are more likely to collaborate than those in non-quantitative work4. In 2008, on the relationship between structural and socio-academic communities of co-authorship networks, Rodriguez and Pepe applied different community detection algorithms into the network of scholars in the field of wireless communication and sensors networks. They found out that even in interdisciplinary fields and multi-institutional research groups, co-authorship is heavily influenced by departments and institutional affiliations. In 2010, a study of network analysis on co-authorship and citation networks using topic-modelling path-finding algorithms showed that productive authors tend to cite and directly collaborate with colleagues sharing the same research interests5.\n\nNot only the application of network statistics is useful in characterizing the nature of scientist networks, it also provides a powerful tool to study and predict scientific performance such as productivity or research impact. A study on the effects of co-authorship on the performance of scholars using regression model and social network analysis showed that researchers who have a strong connection to only one co-author among a group of connected co-authors perform better than those who have many connections to the same group. The study also suggests it is possible to use professional social network of researchers to predict future performance6. In 2013, a group of Taiwanese researchers examined co-authorship networks and research impact through social capital perspective. There are six indicators of social capital in the study: degree centrality, closeness centrality, betweenness centrality, prolific co-author count, team exploration, and publishing tenure. The team found that betweenness centrality is the most influential factor affecting citations of publications7. Using data from library and information science in China, a Chinese research team constructed a network of co-authors, then compared an author’s centrality values with his/her citations. They found a high correlation between these two elements8.\n\nMeanwhile, in Vietnam, network statistics analysis has never been employed to study scientific activities. However, there have been a few attempts to study quantifiable aspects of scientific activities among Vietnamese scholars. Previous studies showed that Vietnam has a low scientific production rate in South East Asia, only equivalent to 13.33% of Singapore and 29% of Thailand in the period of 1991–20107,9. The total scientific output in Vietnam increased about 16 papers per year during the 1996–2001 period and increased by 20% from 2002 to 2010. It is worth noticing that the share of international collaboration was about 77% of the total publications, of which Japan was the largest collaborating country, followed by the United States, France, South Korea, and United Kingdom10,11. Furthermore, most of the key authors of these international projects did not come from Vietnam but from other countries (Manh 2015)10. Mathematics was the only field where domestic output proportion was larger than the international. The largest segment was of biology and agriculture, in which 80–90% of published works involved inter-country collaborations. As for social sciences in Vietnam, a study on a sample of 412 Vietnamese scholars who have international publications in Scopus during the period of 2008–2017 has revealed that more than 90% of social scientists have published at least one co-written article (indexed in Scopus), and they worked in collaborations 13 times on average12.\n\nIn short, faced with the current public desire to improve scientific output in social sciences in Vietnam, there is a shortage of in-depth quantitative analysis on the situation of information diffusion and of scientific output in the network of Vietnam scientists. Given the high frequency of co-authoring among social scientists in Vietnam, a network statistical analysis on collaboration among Vietnamese social scientists as the vector of connection would prove to be valuable. It would be interesting to see how network analysis – a technique first developed for studying networks in the natural world – yield valuable insights into the dissemination of knowledge and information of scientific nature among scholars in Vietnam.\n\nThis study aims to describe the basic properties of a co-authorship network in a sample of 412 Vietnamese social scientists who have published in Scopus-indexed journals and have online profiles, in the period of 2008–2017.\n\nFirst, through analyzing the vertex degree distribution in the network, the study will discuss the concept of robustness of the network, which means how well-connected the network could remain if certain nodes and edges are removed. Then through the number of cliques and components, the study will describe the basic structure of the network. Furthermore, using metrics such as density and clustering coefficient, the status of the communication and exchange of scientific knowledge and expertise in the network will be analyzed.\n\nSecond, the study does not only provide numerical understanding of the network but also shows various ways in which it can be graphically represented. In doing so, the study will discuss the usefulness of several techniques of network graphical representation that can be applied to facilitate one’s understanding of the network.\n\nFinally, the study will extract two of the largest components - one of the largest groups of connected scientists, then explore its characteristics. By comparing this component with the network of 412 Vietnamese social scientists, the study will provide deeper analysis on the concepts visited above.\n\n\n2. Results\n\nUsing R, the dataset employed in this paper counts 412 vertices in the Nodes list and 401 edges in the Edges list. Each vertex or node can be different in terms of degree. The average vertex degree is 1.95 with standard deviation 2.26. This means on average, one Vietnamese social scientist co-authors with about two other Vietnamese authors. Figure 1 visualizes the distribution of vertex degrees and shows the disparity between the least and most well-connected authors. (Figure 1 can be plotted using the command in Supplementary File 1 “Rcommands_fig1.doc”.)\n\nThis is a histogram of vertex degree distribution of the full 412-node network. The degree of each node is measured as the number of co-authored papers, or connections, of each individual in the dataset.\n\nAn overwhelming majority of researchers - about 280 out of 412 - possesses degree from 0 to 2; only about 50 researchers have a vertex degree of 3-4, and the number of authors with higher degree decreases dramatically from degree 4 upwards. In other words, most researchers in Vietnam have less than two connections – less than two co-authored papers – and only very few has more than four. Clearly, rather than being composed of mostly people with the same level of connections, the network consists of a few very well-connected people, while the rest does not have many connections at all. It can be inferred that it would be possible to break the network into multiple components if we just removed those few well-connected nodes (people of degree higher than 5) or their links. In network analysis literature, how well-connected a network remains when some vertices and edges are taken out is referred to as robustness13. Thus, in this study, the degree distribution reveals that the network of Vietnamese social scientists is not robust. This effect can be seen more visibly when we explore the characteristics of one of the biggest components of this network.\n\nTo explore the structure and cohesiveness of the network, it is useful to look at censuses of cliques, components, graph density, and transitivity (Commands for calculation of the network metrics can be found in Supplementary File 2 “Rcommands_metrics.doc”).\n\nBy generating a census of cliques of all sizes, we can get a general sense of the structure of the network:\n\nAs shown in Table 1, in this network, there are 412 nodes (clique of size 1), 401 edges (clique of size 2), 281 triangles (cliques of size 3), 201 cliques of size 4, and so on. The largest clique is size 9, of which there is only one.\n\nA clique is a subset of vertices that are fully cohesive, meaning that all vertices are connected by one link. This table lists all clique sizes that exist within the dataset and the number of cliques in each size category.\n\nA graph is considered to be connected if every node could be reached by any other node (i.e. if for any two nodes, there is a walk between the two). Looking at Table 2, we can see that the network of Vietnamese social scientists is not connected; there are 125 components of size 1. About 30% of the scientists in this study are isolated nodes in the network, possibly because they either work alone or work exclusively with foreigners. Alternatively, the five biggest components (size 11, 15, 16, 27 and 43) together takes up another 30%, while the rest consists of all middle-sized components (size 2–9).\n\nA component is a subgraph in which every vertex can be reached from every other, no matter how many links constitute the path. This table lists all component sizes that exist within the dataset and the number of components in each size category\n\nBy calculating the density and transitivity of the graph, it can be seen that the network is very sparse. The density of the graph is 0.0047, indicating only about 0.47% of potential edges are realized in this network. On the other hand, when three vertices are connected at all, there is a better than a 50/50 chance they will form a triangle (clique of size 3): The global clustering coefficient of the collaboration graph is 0.5862, indicating that nearly 59% of connected triples have formed triangles. Given that there is a clear relationship between the speed of the spread of information and clustering coefficient; the higher the clustering coefficient, the slower the information spread14, it is reasonable to assume when two scientists co-author in a scientific paper, there is a great deal of knowledge and expertise to be communicated and exchanged. Hence, the low density and high clustering coefficient of the network suggests that the dissemination of knowledge and expertise among 412 Vietnamese social scientists in this study is not happening as smoothly as possible.\n\nVisual representations of the network is done through figure plotting in R. Commands for data set-up required for figure plotting can be found in Supplementary file 3 “Rcommands_graph.doc”.\n\nThere are several ways to visually represent the network. Here, the study aims to strike a balance between creating a graph both visually attractive and useful in facilitating the statistical understanding of the previous histogram and analysis.\n\nFigure 2 was conceived as a primary representation of the network, highlighting vertex degree, density, transitivity, and robustness using various visual cues. Among many attributes of the nodes that have been collected (region, age, title, etc.), biological gender has been chosen as the basis because of its relatively simple binary nature. In this study, blue color represents male and red represents female. Such simplicity is hoped to make the graph more aesthetically appealing. Meanwhile, the size of each vertex is determined by the number of edges incident on each node – in other words, by the vertex degree. Hence, the higher the number of edges incident upon a vertex, the bigger the vertex is. This is to make visible the gap between the well-connected scientists and the more isolated ones, one of the most striking features of the network as shown in section 2.1. For layouts, among all those available in R(v3.1.1)’s igraph packages, layout Fruchterman-Reingold is chosen because it makes the structure of the network nicely perceptible: 30% of nodes fall into five largest components, 40% are middle-size components, and the 125 left are isolated nodes (recall the statistics on components in section 2.1). Commands for plotting this figure can be found in the Supplementary File 4 (“Rcommands_fig2.doc”).\n\nThis figure is a visualization of the full 412-node network in Fruchterman-Reingold layout. Nodes are color-coded based on author gender (blue for male, red for female). Node sizes are based on node degrees. Edges are represented by a line connecting concerned nodes.\n\nSeeking more insights on the network, a community detection algorithm was run on the data, which resulted in Figure 3, a second visualization that complemented Figure 2. (This can be performed using the commands provided in Supplementary File 5 “Rcommands_fig3.doc”.) Looking at the biggest components in Figure 3, one can see a new pattern emerges: though the big components are fully connected, they do not seem to be one big close group; rather, they seem to consist of a few smaller communities of very closely connected scientists, and these communities are linked together by one or two vertices acting as weak links. The algorithm does indeed break the two big groups into smaller communities with one or two vertices that connect these communities.\n\nThis figure is a visualization of the full 412-node network in Fruchterman-Reingold layout with community detection. Potential communities are partitioned using colored regions with boundaries. Colors are mostly to facilitate visual perception and irrelevant to the understanding of the data.\n\nIn the next section, the two largest components, component size 43 and component size 27, will be studied more in-depth.\n\nRecall that component is a technical term in network theory that refers to a maximally connected subgraph, in which any two vertices can be reached from another via a path consisting of any number of edges and nodes. Thus, any graph can be constituted by many different components. In this study, the network of 412 Vietnamese social scientists is the sum total of 179 components of various size, ranging from 1 to 43; the two largest components have 43 and 27 nodes each. One can treat such components as independent networks in and of themselves. In this section, the characteristics of these two largest components will be explored and compared with the whole network. From this point on, the components will be called Comp43 and Comp27, and the original network will be dubbed Net412. As one might expect, as we zoom in, there will be differences in the properties of the components in question and that of the network as a whole. Table 3 summarizes and compares the basic metrics of Comp43, Comp27 and Net412.\n\nVertex degree is the number of edges incident upon a vertex. Density is the frequency of realized edges (connections) relative to potential edges (connections). Transitivity (or clustering coefficient) is the relative frequency with which connected triples of vertices form triangles. Net412 is the full 412-node network consisting of the entire dataset. Comp43 and Comp27 are the 43-node and 27-node components, respectively, which are subsets in which every vertex can be reached by every other.\n\nIn all network metrics, Comp27 scores the highest. Specifically, in terms of density of connections, Net412 is the sparsest, 0.47%. The density of Comp43 (7.20%) is 14-fold that of Net 412, and the same characteristic in Comp27 (22.51%) is 44-fold compared to that of the whole network. Regarding average vertex degree, Comp27 is the highest followed by Comp43 then Net412. Concerning global clustering coefficient (or transitivity), Comp27 towers over Net412 by 11 percentage points (70% versus 59%), while the latter is in turn over 2 times higher than Comp43 (70% versus 32%).\n\nHigh clustering and low density suggest a certain level of inefficiency in the spread of knowledge and expertise (as explained in section 1.1 on the characteristics of the network of 412 Vietnamese social scientists); either could be the cause of the other. Thus, from the network metrics, one would expect Comp27’s dissemination of scientific knowledge and expertise to be less efficient than Comp43. In fact, even though the density of connection in Comp27 is about 3 times that of Comp43, its effects would be limited because of the higher clustering. One can then ask how to verify that high clustering cancels the good effects of even high density. Supposing that better dissemination of scientific knowledge and expertise can be observed in a better scientific output, we could look at the mean value of total publications of scientists in each network for insights on the aforementioned question. Indeed, as Table 3 shows, Comp43 performs better than Comp27 in terms of scientific output – almost 3 times higher, 5.53 versus 2.00.\n\nThe difference in scientific output between Comp43 and Comp27 can be viewed in Figure 4 below. Commands for plotting Figure 4 (left and right) can be found in Supplementary File 6 and Supplementary File 7 (“Rcommands_fig4left.doc” and “Rcommands_fig4right.doc” respectively.)\n\nThis figure is a visualization of the full 43-node and 27-node components in Fruchterman-Reingold layout. Nodes are color-coded based on author gender (blue for male, red for female). Node sizes are based on node degrees. Edges are represented by a line connecting concerned nodes.\n\nBesides revealing the differences in scientific output of the two networks, Figure 4 also reveals that nodes in both networks seems to revolve around one or two important nodes with higher level of scientific output. In Comp43, it is node s004 and in Comp27, it is node s067 and s219 (the visible blue and red dots on the left side of Figure 4). It is interesting that these three nodes have highest numbers of edges incident upon them in their respective networks; s004 has a degree of 11, highest in Comp43; s067 has a degree of 13 and s319 has a degree of 16, also highest in Comp27. If these important vertices are to be removed, the networks would break apart into several smaller components. This feature was referred to in section 5.1 through the concept of robustness, and it should be noted that Net412 is not robust. The situation is the same for Comp43 and Comp27. In Figure 5, the histogram distributing the degrees of nodes in these networks shows a clear disparity in vertex degree.\n\nCommands for plotting Figure 5 (left and right) in R can be found in Supplementary File 8 and Supplementary File 9 (“Rcommands_fig5left.doc” and “Rcommands_fig5_right.doc” respectively).\n\nThese are histograms of vertex degree distribution of the 43-node and 27-node components. The degree of each node is measured as the number of co-authored papers, or connections, of each individual in the dataset.\n\n\n3. Discussion\n\nAfter performing social network analyses on a sample of 412 social scientists in Vietnam, whose information has been gathered primarily from their Scopus profiles.\n\nFirst, the study has shown that the network has a low level of connection with only 0.47% of all potential edges realized, and high in clustering with 59% chance a connected triple would close into a triangle. These two characteristics together suggest a reality that the communication and exchange of knowledge and expertise among the Vietnamese social scientists are not very efficient. In addition, the degree distribution reveals that it would be difficult for the network to stay well-connected when a few highly-connected nodes and their edges are removed; or, in network theory’s terminology, the network is not very robust.\n\nSecond, in this study, network visualization is shown to be useful not only in facilitating quantitative understanding but also in discovering new insights into the structures of the network. By applying appropriate techniques of graph plotting, the disparity of the level of connections and the structure of the network can be easily visualized. Using the community detection algorithm, an interesting fact about these biggest groups is unraveled: they mostly comprised of smaller and tightly connected communities with one or two vertices connecting these altogether.\n\nThird, close investigations show that the two largest components in the network have different characteristics from the 412-node-graph. Both smaller networks have more connections than the big one, but in terms of clustering, the 43-node-graph has a much higher level of clustering. Despite these differences, all the three networks resemble in low level of robustness and high disparity in terms of degree distribution, which means when the most connected people are removed from the networks, these latter would immediately be decomposed into several smaller groups. Most strikingly, the two smaller networks seem to be led by the most productive researchers in them, who also have the most connections.\n\nGiven the mostly high transitivity of all three networks, it could be remarked that the original 412-node network could be considered more or less a sum of smaller communities centered around well-connected nodes. On a more ego-centric and contextual note, there seems to be a relationship between the social status (their position in an institution, for example) of an individual in the network and his or her importance to the network (whether he/she has the most connections or being central to many connections in some ways) as well as his or her scientific output, as suggested by the examples of node s004, s067 and s219. These individuals are few and far between in a network of high disparity in vertex degree, and present a stark contrast with their peers in terms of both connections and productivity. They have the potentials to form a group of intellectual elites.\n\nFinally, there is still much to be learned from both the dataset of 412 social scientists and the network that can be constructed from the raw data. For example, though the study has hinted at the difference in scientific output of two networks (comparison of Comp43 and Comp27 in section 2.3), it is worth considering a more systematic examination of the relationship between a network’s properties and the scientific output of the vertices it contains. Thus, finding out whether a correlation among these variables exists does merit further investigation. Another promising area of research is the exploration of diversity in scientific co-authorship. In this study, node color is coded by gender (section 2.2), but other attributes such as age, region, work, titles, etc. can also be added to the analysis as well.\n\nThis paper cannot claim to have exhausted the toolkits that social network analysis could provide. There are still many other aspects of the network worthy of further investigation. How would the network turn out if other dimensions such as weights or durability of the relational data are added to the analysis? How useful are certain aspects of the network in predicting scientific performance? How would this network evolve over time? Not only intellectually stimulating, these important questions are of tremendous practical value for policy-makers and educators, particularly when their decision-making concerns education policies and research organizations. Further investigation in this area of research and on this topic is thus necessary.\n\n\n4. Materials and methods\n\nThe data for this study was derived from a dataset on the productivity of Vietnamese scientists in the field of social sciences collected by Vuong & Associates. The investigation, which took place within two months from March to April 2017, was conducted under the license V&A/03/2017, issued on 15 March, 2017.\n\nFirst, we constructed a file that contains data on all the attributes of each author, called a “Nodes list” (Dataset 1: \"20170725_net412_ NODES.csv\"). The data collection process was monitored regularly to ensure its reliability, including the following steps: first, the research team used sources such as personal and institutional websites of authors, websites of journals where their works were published, Google Scholar, and Scopus database to collect data. Then, to check the accuracy of the information, we compare various online sources where each author’s information can be found; for example, Google scholar versus Scopus, personal websites versus institutional websites. After this process, the research team obtained a complete dataset of 412 scholars’ information, consisting of: (i) age, sex, region; (ii) affiliations; (iii) fields of study; (iv) the number of publications in Scopus, (v) the number of research years since the Master graduation; (vi) the number of researchers they collaborated with; (vii) whether or not they have the title of “Professor/Assoc. Professor”. All of this essentially constitutes the node.\n\nBased on this information, we then construct our relational data, which is called an “Edges list” (Dataset 2: \"20170729_net412_LINKS.csv\"). We consider two authors as exhibiting a co-authorship tie when they appear together in a scientific publication. Each time the same two authors appear together in a paper, it is counted toward the “weight” of the tie. The example of an edges list can be seen in the following figure. The data was then processed and analyzed using statistical software R (v3.3.1). Figure 6 shows an example of how relational data is handled in the study. To illustrate, in the first row of the table on the left side, a published paper being co-authored by scientists ID s004, s076 and s079 is recorded into the database first. Then on the right side, co-authorship relations among these three scholars are recorded; and the weight is the count of how many times each pair co-authors.\n\nIn these figures, a fraction of the construction of Edges lists is shown. The table on the right shows how we record 4 published articles in which 5 Vietnamese scientists coded as s004, s005, s076, s079, s080 take part as co-authors. The table on the right shows every pair that have collaborated at least once among these 5 scientists, as well as the number of collaborations of each pair, which are considered the “weight” of the relation.\n\nThe data for Comp43 and Comp27 were manually extracted from the full dataset. Nodes lists (Dataset 3 “20170719_comp43_NODES.csv” and Dataset 5 \"20170726_comp27_NODES.csv\") and Links lists (Dataset 4 “20170719_comp43_LINKS.csv” and Dataset 6 \"20170729_comp27_LINKS.csv\") for Comp43 and Comp27 respectively were constructed by picking relevant edges and nodes from the original lists.\n\nThe method employed in this study was statistical analysis of network data. There were several reasons why we choose this method. First, the prevalence of co-authorship in research efforts among Vietnamese scientists as shown in the literature review naturally prompts us to ponder on how the co-authors cooperate and the kinds of interactions that exist among them. Second, as we find out that social network analysis has been applied widely all over the world in the study of scientific collaborations, we expect a match between our interest in characterizing collaboration among Vietnamese social scientists and the technical tools this approach provide. Finally, the help of statistical software allows us to create graphic representation of the network, which supplements all the rigorous numerical analysis with a more intuitive way of understanding interactions among actors in the network.\n\nIn this study, we will only focus on a descriptive analysis of our network data. The study is strictly limited to the interactions among Vietnamese scholars only. There are two caveats with regards to the method and the scope of the analysis. First, as the collaborations with foreign scholars are not accounted for in this study, certain interesting features of the networks can be lost. For example, a foreign scholar could cooperate with two Vietnamese scholars, but these Vietnamese scholars might not publish together. Thus, a link is missing. The cumulative effects of this kind of missing links can make the network appear much less connected than it actually is. Second, network analysis is first developed to solve problems in areas such as mathematics, chemistry, electrical circuits, operational research, and computer science before being applied by sociologists in mid-20th Century to study social network, hence, we can expect there are inherent limits to the explanatory power of the technique.\n\nIn order to understand the visualization of a network, it is important to familiarize oneself with the terminologies of statistical network analysis. Here, we provide an explanation of terms that are relevant for the scope and purpose of this paper. More technical explanations of the terms in this paper can be found in Statistical analysis of network data with R15, and Social Network Analysis: A Handbook, Second edition16.\n\nA graph G= (V, E) is a mathematical structure consisting of a set V of vertices (or nodes) and a set E of edges (or links); elements of E are links between a pair of distinct vertices belongs to set V. When two nodes are connected to each other by an edge, they are said to be adjacent. In this study, a vertex represents a Vietnamese social scientist, which means the total number of vertices is 412. An edge represents a relationship between two distinct Vietnamese social scientists. A concept that connects edge and vertex is degree; a degree of a vertex is the counts of the number of edges incident upon that vertex. For instance, if there are three edges incident upon a vertex, the degree of that vertex is three.\n\nNotice that depending on the attribute of the relationships between two vertices, an edge might or might not have a direction, thus there might be a need to specify the ordering of the pair of vertices in each edge in set E. A directed graph is a graph where each edge in E has an ordering to its vertices; an undirected graph is a graph where an edge needs not to be defined by the ordering in the vertices. In this study, since the relationship among co-authors is considered to be neutral, the graph that shows their relational ties will be undirected.\n\nTo understand the structure of a network, two fundamental concepts are clique and component. A clique is a subset of vertices that are fully cohesive, in that, all vertices within this subset are connected by edges. For example, a node is a clique of size one, an edge is a clique of size two, a triangle is a clique of size three, and so on. A component is a subgraph, in which, every vertex can be reached from every other. It is easy to see the different between a clique and a component. In a clique, every two nodes must be connected by an edge or in other words, they must be adjacent; while in a component, every two nodes might or might not be connected by an edge, but they must be somehow connected through a path consisting of a number of other edges and nodes.\n\nRegarding the structure of a network, it is natural to wonder about the level of cohesion of the network: How frequent do the edges appear? How likely do three connected nodes close into a clique size 3? These questions can be answered using the concept of density and global clustering coefficient, also known as transitivity. The density of a graph is the frequency of realized edges relative to potential edges. It can be calculated using the following formula:\n\ndensity = 2l/[n(n-1)]\n\nin which l is the numbers of links (or edges), and n is the number of nodes (or vertices). The clustering coefficient (or transitivity) measures the relative frequency with which connected triples of vertices form triangles:\n\nclT(G) = 3τΔ(G)/τ3(G)\n\nin which τΔ(G) is the number of triangles in the graph G; and τ3(G) the number of subgraphs consist of three vertices connected by two edges, i.e. connected triples.\n\nArmed with understanding of relevant technical concepts, we are able to explore the characteristics of the network of 412 Vietnamese social scientists.\n\n\n5. Conclusions\n\nWith the purpose of understanding the structure and characteristics of the network of 412 Vietnamese social scientists, the study has applied the technique of social network analysis to give a sense of the structure of the network, the level of connection as well as the level of clustering in the network. In the last parts of this paper, we zoomed into the two largest components of the network and compare their relevant characteristics together with the network of the entire sample (in line with the spirit of 17).\n\nRemarks corresponding to each characteristic along with insights into the robustness of the network and the spread of scientific knowledge and expertise in the network have been extracted and discussed. The high clustering of the entire network of 412 Vietnamese social scientists and low density shared by both the original network and its two component networks, seem to be closely related to inefficient dissemination of academic expertise. Both of these in turn lead to modest scientific output, which is at the heart of the perpetual discussions on research capacity in Vietnam. Furthermore, the network, low in robustness, is only held together by a few well-connected scholars, who seem to also hold significant social positions. This suggests the existence of certain intellectual elites who could perhaps propel Vietnamese scientific output.\n\n\nData Availability\n\nDataset 1: \"20170725_net412_NODES.csv\" This dataset contains all 412 individuals in the study and their attributes. Each individual is considered a node (vertex) in the network. 10.5256/f1000research.12404.d17492918\n\nDataset 2: \"20170729_net412_LINKS.csv\" This dataset lists the number of co-written articles between all 412 authors of the network, where relevant. Each collaboration is counted as a link (edge) in the network. 10.5256/f1000research.12404.d17493019\n\nDataset 3: “20170719_comp43_NODES.csv” This dataset contains 43 individuals in the 43-node component and their attributes. Each individual is considered a node (vertex) in the component. 10.5256/f1000research.12404.d17493120\n\nDataset 4: “20170719_comp43_LINKS.csv” This dataset lists the number of co-written articles between the 43 authors of the 43-node component, where relevant. Each collaboration is counted as a link (edge) in the component. 10.5256/f1000research.12404.d17493221\n\nDataset 5: \"20170726_comp27_NODES.csv\" This dataset contains 27 individuals in the 27-node component and their attributes. Each individual is considered a node (vertex) in the component. 10.5256/f1000research.12404.d17493322\n\nDataset 6: \"20170729_comp27_LINKS.csv\" This dataset lists the number of co-written articles between the 27 authors of the 27-node component, where relevant. Each collaboration is counted as a link (edge) in the component. 10.5256/f1000research.12404.d17493423",
"appendix": "Author contributions\n\n\n\nQ.H.V., T.M.H. conceived and designed the study; all contributors (T.M.H., H.V.N., T.T.V., Q.M.D., H.H.P., Q.H.V.) contributed equally for the remaining tasks of the research and manuscript preparation, i.e. data set preparation, analysis, interpreting results, and writing and checking the paper.\n\n\nCompeting interests\n\n\n\nThe authors declare no conflict of interest.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe would like to thank Vuong & Associates for their research initiative The Network of Vietnamese Social Scientists (NVSS), which enabled the research process and provided the raw data for the study. We particularly thank Dam Thu Ha and Nghiem Phu Kien Cuong for their excellent research assistance.\n\n\nSupplementary Materials\n\nSupplementary File 1: Commands for plotting Figure 1: “Rcommands_fig1.doc” This file contains the R command used to plot the histogram of vertex degree distribution of the full 412-node network shown in Figure 1.\n\nClick here to access the data.\n\nSupplementary File 2: Commands for computing network metrics: ”Rcommands_metrics.doc” This file contains the commands that compute network metrics.\n\nClick here to access the data.\n\nSupplementary File 3: Commands for graph plotting: “Rcommands_graph.doc” This file contains the commands that set up the data for graph plotting in R.\n\nClick here to access the data.\n\nSupplementary File 4: Commands for plotting Figure 2: “Rcommands_fig2.doc” This file contains the R commands used to plot the visualization of the full 412-node network in Fruchterman-Reingold layout shown in Figure 2.\n\nClick here to access the data.\n\nSupplementary File 5: Commands for plotting Figure 3: “Rcommands_fig3.doc” This file contains the R commands used to run a community detection algorithm on the full 412-network and visually present in on the base of Figure 3.\n\nClick here to access the data.\n\nSupplementary File 6: Commands for plotting Figure 4 – left: “Rcommands_fig4left.doc” This file contains the R commands used to plot the visualization of the 43-node component shown in Figure 4 (left).\n\nClick here to access the data.\n\nSupplementary File 7: Commands for plotting Figure 4 – right: “Rcommands_fig4right.doc” This file contains the R commands used to plot the visualization of the 27-node component shown in Figure 4 (right).\n\nClick here to access the data.\n\nSupplementary File 8: Commands for plotting Figure 5 – left: “Rcommands_fig5left.doc” This file contains the R commands used to plot the histogram of vertex degree distribution of the 43-node component shown in Figure 5 (left). These commands should be used in continuation with the data set-up from “Rcommands_fig4left.doc”.\n\nClick here to access the data.\n\nSupplementary File 9: Commands for plotting Figure 5 – right: “Rcommands_fig5right.doc” This file contains the R commands used to plot the histogram of vertex degree distribution of the 27-node component shown in Figure 5 (right). These commands should be used in continuation with the data set-up from “Rcommands_fig4right.doc”.\n\nClick here to access the data.\n\n\nReferences\n\nNguyen VT: Nang suat khoa hoc Viet Nam qua cong bo quoc te 2001 – 2015. Vietnam Journal of Science and Technology. 2016; 2006(10): 49–54. Reference Source\n\nHoang VQ, Dung TT, Napier NK, et al.: Business education in the emerging economy of Vietnam: Twenty years of expectations, illusions and lessons. In: Innovation in Business Education in Emerging Markets. Alon I, Jones V, McIntyre J, Eds; Palgrave Macmillan: NY, New York. 2013; 96–109. Publisher Full Text\n\nNewman ME: Scientific collaboration networks. I. Network construction and fundamental results. Phys Rev E Stat Nonlin Soft Matter Phys. 2001; 64(1 Pt 2): 016131. PubMed Abstract | Publisher Full Text\n\nMoody J: The structure of a social science collaboration network: Disciplinary cohesion from 1963 to 1999. Am Sociol Rev. 2004; 69(2): 213–238. Publisher Full Text\n\nDing Y: Scientific collaboration and endorsement: Network analysis of coauthorship and citation networks. J Informetr. 2011; 5(1): 187–203. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbbasi A, Altmann J, Hossain L: Identifying the effects of co-authorship networks on the performance of scholars: A correlation and regression analysis of performance measures and social network analysis measures. J Informetr. 2011; 5(4): 594–607. Publisher Full Text\n\nYi Y, Qi W, Wu D: Are CIVETS the next BRICs? A comparative analysis from scientometrics perspective. Scientometrics. 2013; 94(2): 615–628. Publisher Full Text\n\nYan E, Ding Y, Zhu Q: Mapping library and information science in China: A coauthorship network analysis. Scientometrics. 2010; 83(1): 115–131. Publisher Full Text\n\nNguyen TV, Pham LT: Scientific output and its relationship to knowledge economy: an analysis of ASEAN countries. Scientometrics. 2011; 89(1): 107–117. Publisher Full Text\n\nManh HD: Scientific publications in Vietnam as seen from Scopus during 1996–2013. Scientometrics. 2015; 105(1): 83–95. Publisher Full Text\n\nNguyen TV, Ho-Le TP, Le UV: International collaboration in scientific research in Vietnam: an analysis of patterns and impact. Scientometrics. 2017; 110(2): 1035–1051. Publisher Full Text\n\nVuong QH, Ho MT, Vuong TT, et al.: Gender, age, research experience, leading role, and academic productivity of Vietnamese researchers in the social sciences and humanities: exploring a 2008–2017 Scopus dataset. Eur Sci Ed. 2017; 43(3): 51–55. Reference Source\n\nVan Steen M: Graph theory and complex networks. An introduction. 2010, ISBN: 9081540610. Reference Source\n\nWu X, Liu Z: How community structure influences epidemic spread in social networks. Physica A: Statistical Mechanics and its Applications. 2008; 387(2): 623–630. Publisher Full Text\n\nKolaczyk ED, Csárdi G: Statistical analysis of network data with R. Springer: NY, New York, 2014; 65. Publisher Full Text\n\nScott J: Social network analysis: A Handbook. Second edition. Sage: California, 2000, ISBN: 0761963383. Reference Source\n\nVuong QH, Napier NK: Academic research: the difficulty of being simple and beautiful. Eur Sci Ed. 2017; 43(2): 32–33. Publisher Full Text\n\nHo TM, Nguyen HV, Vuong TT, et al.: Dataset 1 in: Exploring Vietnamese co-authorship patterns in social sciences with basic network measures of 2008–2017 Scopus data. F1000Research. 2017a. Data Source\n\nHo TM, Nguyen HV, Vuong TT, et al.: Dataset 2 in: Exploring Vietnamese co-authorship patterns in social sciences with basic network measures of 2008–2017 Scopus data. F1000Research. 2017b. Data Source\n\nHo TM, Nguyen HV, Vuong TT, et al.: Dataset 3 in: Exploring Vietnamese co-authorship patterns in social sciences with basic network measures of 2008–2017 Scopus data. F1000Research. 2017c. Data Source\n\nHo TM, Nguyen HV, Vuong TT, et al.: Dataset 4 in: Exploring Vietnamese co-authorship patterns in social sciences with basic network measures of 2008–2017 Scopus data. F1000Research. 2017d. Data Source\n\nHo TM, Nguyen HV, Vuong TT, et al.: Dataset 5 in: Exploring Vietnamese co-authorship patterns in social sciences with basic network measures of 2008–2017 Scopus data. F1000Research. 2017e. Data Source\n\nHo TM, Nguyen HV, Vuong TT, et al.: Dataset 6 in: Exploring Vietnamese co-authorship patterns in social sciences with basic network measures of 2008–2017 Scopus data. F1000Research. 2017f. Data Source"
}
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[
{
"id": "25520",
"date": "05 Sep 2017",
"name": "Tuyen Quang Tran",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAs a member of the evaluation council on economic studies for Vietnam’s National Foundation for Science and Technology Development (NAFOSTED), I am particularly interested in this research as this is one of the key issues we have been facing in recent years. Its merits can also be seen immediately. Some of my comments follow.\n\nThe authors have shown their heroic act in collecting and preparing the unique data sets at individual levels, which has never been done before.\n\nThe insights are clear and useful, with potentially practical implications for policy makers like us. I like the fact that they focus on basic measures where and when complexities do not necessarily bring more useful understanding.\n\nAll data sets are presented, accompanied by R codes, which have made the replication and reproduction of the results both easy and transparent. Having gone through the paper several times, I am now pleased to approve this work.\n\nA further comment: for more valuable analyses in the future regarding this theme of research, I would suggest the expanding of the current data sets to include Scopus citation data.\n\nI would like to suggest that some more limitation regarding the sources of data and methods. For example, ISI web of science covers less journals than Scopus, or panel data should be better for future research.\n\nI wish the authors every success in their future research attempt.\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": []
},
{
"id": "25518",
"date": "07 Sep 2017",
"name": "Donaldine E . Samson",
"expertise": [
"Reviewer Expertise Online teaching and 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\nGiven my knowledge about Vietnam's education system, publishing experiences, I have found the paper's results compelling and cogent. This represents one of the first attempts in Vietnam to study its social sciences research efforts from the community and collaboration perspectives.\n\nThe authors intentionally employ network data analysis to explain what cross-section data could hardly tell. Scientific soundness of this research is based on earlier work on social networks and basic network metrics. The authors use a precise and unbiased definition of connectedness based on Newman’s study1. The network visualization tools are not only original in this thread of research, they are especially useful in facilitating understanding of the statistical analysis. The paper is well-structured, striking a balance between data and discussions. The results are clear, easy to understand, and presented logically. Despite the focus on Vietnam social science researchers, the approach can also be productively applied to other researcher populations.\n\nThis study of co-authorship patterns can be used to help researchers and research directors understand the structure of research collaboration in social sciences in Vietnam and thus develop procedures, platforms and incentives to increase the robustness of networks and reduce the risk of networks disintegrating with the departure of a highly-connected individual researcher.\n\nThe authors identify the potential emergence of an “intellectual elite.” More study on the effect of this group and ways to increase the benefits and reduce the risks would be interesting as it is not clear whether this intellectual elite is detrimental or beneficial to the maturation of a robust social science research community in Vietnam.\n\nI sincerely hope that the authors will continue this direction of research in social network analysis and look forward to their new results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "25359",
"date": "29 Sep 2017",
"name": "Ly Thi Tran",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting and important paper addresses the nature and structure of the network of Vietnamese social scientists who have published in Scopus-indexed journals in the period of 2008–2017. This is a critical issue to Vietnam given the government’s recent emphasis on enhancing research capacity and scientific outcomes. The topic of networking and co-authorship is very timely given Ministry of Education and Training’s new policies requiring doctoral candidates to publish in Scopus and Web of Science-indexed journals, as a compulsory component of their PhD.\nOverall, the paper is nicely written. The literature review provides an effective overview of the status of network statistics analysis and makes reference to the broader international context as well as the Asia region. The aims of the research are clearly articulated. There is a good balance between the presentation and discussion of the data. Even though I do not have statistical expertise and am unable to comment on the detailed analysis, generally I found the key findings of the research logically presented and easy to follow.\nA key strength of the paper lies in the way the authors use social network analysis to interpret and explain Vietnamese social scientists’ research performance. Based on this analysis framework, interesting insights into the structure of the network, the level of connection as well as the level of clustering in the network have been discussed.\nI have five comments below for the authors to consider in enhancing this research project or expanding this research area:\nMore discussion of the specific implications of this research for improving research capacity and doctoral education would be very useful in the current context of Vietnam. This information would be welcome by policy makers, leaders of universities and research institutes as well as research team, individual researchers and doctoral candidates. Given the scope of this paper, I hope a follow-up article may focus more on this aspect.\n\nA critical issue that should be further explored is what facilitates or inhibits productive and sustainable networks among the researchers rather than just an one-off or brief collaboration or co-authorship?\n\nThe authors mainly focus on using social network analysis to explain research performance but less on predicting research performance and impact. Perhaps a follow-up study can address this issue and may include interviews/survey with the scientists to provide more nuanced understandings about the topic. An issue of great concern is what characteristics and structure of networks are likely to lead to not only research productivity but also research impact.\n\nA further issue is whether the network facilitated at the institutional/organisational level or at the individual researcher level is more sustaining and productive? In this regard, what might be the incentives or support mechanism from the institution and government (for example, targeted funding for network/partnership development or grant/funding scheme in which capacity for research collaboration or network is one of the assessment criteria) needed?\n\nOne of the findings of this study is that “the network shows the potential of an intellectual elite composed of well-connected, productive, and socially significant individuals.” What is the implication of this finding for policy makers and education leaders to facilitate an equitable and inclusive networking and collaboration structure that supports rather than marginalises less established or ‘non-elite’ Vietnamese researchers including early career researchers, researchers from regional universities and researchers who were not exposed to overseas education.\n\nIn sum, I have enjoyed reading this paper and I congratulate the authors for this valuable work! Thank you for the opportunity to read and comment on this paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "3075",
"date": "02 Oct 2017",
"name": "Quan-Hoang Vuong",
"role": "Author Response",
"response": "Dear Professor Ly Tran: We greatly appreciate your comments and assessments of our joint work, one of the first attempts in using network data analysis for a rather complex set of relationships among Vietnamese social researchers. Please rest assured that your encouragement will give us stronger motivation in consummating our future studies. Instead of being complacent with your positive evaluation, on behalf of other co-authors, I will directly address and answer each and every point raised in your review report regarding this first version of the study. On further discussion of the specific implications: We agree with your point, and also believe country-specific addressing will certainly help, especially in the Vietnamese context, and right when the government has been trying to revamp the quality of doctoral studies and to improve the candidature. Apparently, we have fallen short of this expectation as the study is currently focusing on technical aspects of the data and relationships among the listed social researchers. We will be very happy to take this point seriously and address it in the next paper, where we intend to touch on one of the very difficult issues: measuring the so-called \"social sustainability\" of every group of well-connected researchers in the social sciences. This task will be immense as we will have to develop a new set of metrics that can satisfy a number of critical criteria. But we will surely get there. On the sustainable network: This is very much line with our objective as mentioned in the preceding answer. But it is worth discussing a bit more here. We have seen and anticipated your questions from day 1, about 9 months ago, when the first line of data was entered into the raw data set. And the literature has been voluminous regarding this issue. The problem with the extant literature is the nature of the data. Not longitudinal, not cross-section, not panel data can satisfy this, and our current inquiry into the network data has naturally been here for a reason. That says we will need updated picture in the near future to see how partitioned groups (of connected researchers) will grow, in which direction, and what shape they look like and become different, etc. This multi-dimensional view might help answer your concern to some extent (which we are still trying to gauge). On performance and impact: This is true, both your current evaluation and suggestion on future follow-up studies. In fact, before this paper we have published another paper using cross-section data for the same period, addressing part of your question. It can be found at DOI:10.20316/ESE.2017.43.006, in the publication European Science Editing by the European Association of Science Editors. In this paper, we also discussed such issue as gender and age (research experience), and the like. Nonetheless, as you pointed out in your review, we feel the need of going further down the route and will certainly take your advice seriously, perhaps during the next data round for 2008-2018. One of the key issues with impact research for social sciences is the data set for citations as well as for the use of scientific results as key input for policymaking. While citations have become kind of \"standards\", policy inputs could be quite vague. Still, with citations the organizing and collecting of correct data are making less confident. We do have a solution of using advanced algorithms and further enhancing the information system for the job, but these will never be an easy task, and we expect they will take months before completion and ready for testing. So we will have to work even harder on this, and the actualization will not be seen in a short horizon. By answering your specific comments and questions, we just want to convey a key message: this research study is not a one-off attempt, but serves to be one milestone along our research journey, a truly multi-dimensional one, and the data process will be expanding over time. So far we have not even been able to predict how far and how influential the project, named as \"The Network of Vietnamese Social Scientists\", will be as we - the implementing researchers - continue to be surprised by the number of possibilities it may have brought about. The interesting thing is: the project itself will give us a very real opportunity of having new research collaborators for coming pursuits. We once again thank you very much for your kind, polite and articulate opinions, from which we can both learn, and on which we reflect our actual performance and anticipation. On behalf of the authors Quan-Hoang Vuong Western University Hanoi (Vietnam) Université Libre de Bruxelles (Belgium)"
}
]
}
] | 1
|
https://f1000research.com/articles/6-1559
|
https://f1000research.com/articles/6-1558/v1
|
24 Aug 17
|
{
"type": "Method Article",
"title": "recount workflow: Accessing over 70,000 human RNA-seq samples with Bioconductor",
"authors": [
"Leonardo Collado-Torres",
"Abhinav Nellore",
"Andrew E. Jaffe",
"Abhinav Nellore",
"Andrew E. Jaffe"
],
"abstract": "The recount2 resource is composed of over 70,000 uniformly processed human RNA-seq samples spanning TCGA and SRA, including GTEx. The processed data can be accessed via the recount2 website and the recount Bioconductor package. This workflow explains in detail how to use the recount package and how to integrate it with other Bioconductor packages for several analyses that can be carried out with the recount2 resource. In particular, we describe how the coverage count matrices were computed in recount2 as well as different ways of obtaining public metadata, which can facilitate downstream analyses. Step-by-step directions show how to do a gene-level differential expression analysis, visualize base-level genome coverage data, and perform an analyses at multiple feature levels. This workflow thus provides further information to understand the data in recount2 and a compendium of R code to use the data.",
"keywords": [
"RNA-seq",
"visualization",
"differential expression",
"human",
"Bioconductor",
"genomics",
"bioinformatics",
"GTEx",
"TCGA",
"SRA"
],
"content": "Introduction\n\nRNA sequencing (RNA-seq) is now the most widely used high-throughput assay for measuring gene expression. In a typical RNA-seq experiment, several million reads are sequenced per sample. The reads are often aligned to the reference genome using a splice-aware aligner to identify where reads originated. Resulting alignment files are then used to compute count matrices for several analyses such as identifying differentially expressed genes. The Bioconductor project1 has many contributed packages that specialize in analyzing this type of data and previous workflows have explained how to use them2–4. Initial steps are typically focused on generating the count matrices. Some pre-computed matrices have been made available via the ReCount project5 or Bioconductor Experiment data packages such as the airway dataset6. The pre-computed count matrices in ReCount have been useful to RNA-seq methods developers and to researchers seeking to avoid the computationally intensive process of creating these matrices. In the years since ReCount was published, hundreds of new RNA-seq projects have been carried out, and researchers have shared the data publicly.\n\nWe recently uniformly processed over 70,000 publicly available human RNA-seq samples, and made the data available via the recount2 resource at jhubiostatistics.shinyapps.io/recount/ 7. Samples in recount2 are grouped by project (over 2,000) originating from the Sequence Read Archive, the Genotype-Tissue Expression study (GTEx) and the Cancer Genome Atlas (TCGA). The processed data can be accessed via the recount Bioconductor package available at bioconductor.org/packages/recount. Together, recount2 and the recount Bioconductor package should be considered a successor to ReCount.\n\nDue to space constraints, the recount2 publication7 did not cover how to use the recount package and other useful information for carrying out analyses with recount2 data. We describe how the count matrices in recount2 were generated. We also review the R code necessary for using the recount2 data, whose details are important because some of this code involves multiple Bioconductor packages and changing default options. We further show: a) how to augment metadata that comes with datasets with metadata learned from natural language processing of associated papers as well as expression data b) how to perform differential expression analyses, and c) how to visualize the base-pair data available from recount2.\n\n\nAnalysis of RNA-seq data available at recount2\n\nThe recount2 resource provides expression data summarized at different feature levels to enable novel cross-study analyses. Generally when investigators use the term expression, they think about gene expression. But more information can be extracted from RNA-seq data. Once RNA-seq reads have been aligned to the reference genome it is possible to determine the number of aligned reads overlapping each base-pair resulting in the genome base-pair coverage curve as shown in Figure 1. In the example shown in Figure 1, most of the reads overlap known exons from a gene. Those reads can be used to compute a count matrix at the exon or gene feature levels. Some reads span exon-exon junctions (jx) and while most match the annotation, some do not (jx 3 and 4). An exon-exon junction count matrix can be used to identify differentially expressed junctions, which can show which isoforms are differentially expressed given sufficient coverage. For example, junctions 2 and 5 are unique to isoform 2, while junction 6 is unique to isoform 1. The genome base-pair coverage data can be used with derfinder8 to identify expressed regions; some of these could be unannotated exons, which together with the exon-exon junction data could help establish new isoforms.\n\nReads (pink boxes) aligned to the reference genome can be used to compute a base-pair coverage curve and identify exon-exon junctions (split reads). Gene and exon count matrices are generated using annotation information providing the gene (green boxes) and exon (blue boxes) coordinates together with the base-level coverage curve. The reads spanning exon-exon junctions (jx) are used to compute a third count matrix that might include unannotated junctions (jx 3 and 4). Without using annotation information, expressed regions (orange box) can be determined from the base-level coverage curve to then construct data-driven count matrices.\n\nrecount2 provides gene, exon, and exon-exon junction count matrices both in text format and RangedSummarizedExperiment objects (rse)9 as shown in Figure 2. These rse objects provide information about the expression features (for example gene IDs) and the samples. In this workflow we will explain how to add metadata to the rse objects in recount2 in order to ask biological questions. recount2 also provides coverage data in the form of bigWig files. All four features can be accessed with the recount Bioconductor package7. recount also allows sending queries to snaptron10 to search for specific exon-exon junctions.\n\nOnce the rse object has been downloaded and loaded into R, the feature information is accessed with rowRanges(rse) (blue box), the counts with assays(rse)$counts (pink box) and the sample metadata with colData(rse) (green box). The sample metadata can be expanded using add_predictions(rse) (orange box) or with custom code (brown box) matching by a unique sample identifier such as the SRA Run ID. The rse object is inside the purple box and matching data is highlighted in each box.\n\nIn this workflow we will use several Bioconductor packages. To reproduce the entirety of this workflow, install the packages using the following code after installing R 3.4.x from CRAN in order to use Bioconductor version 3.5 or newer.\n\n\n\nOnce they are installed, load all the packages with the following code.\n\n\n\nThe most accessible features are the gene, exon and exon-exon junction count matrices. This section explains them in greater detail. Figure 3 shows 16 RNA-seq reads, each 3 base-pairs long, and a reference genome.\n\n16 RNA-seq un-aligned RNA-seq reads 3 base-pairs long are shown (pink boxes) alongside a reference genome that is 16 base-pairs long (white box).\n\nReads in the recount2 resource were aligned with the splice-aware Rail-RNA aligner11. Figure 4 shows the reads aligned to the reference genome. Some of the reads are split as they span an exon-exon junction. Two of the reads were soft clipped meaning that just a portion of the reads aligned (top left in purple).\n\nSpice-aware RNA-seq aligners such as Rail-RNA are able to find the coordinates to which the reads map, even if they span exon-exon junctions (connected boxes). Rail-RNA soft clips some reads (purple boxes with rough edges) such that a portion of these reads align to the reference genome.\n\nIn order to compute the gene and exon count matrices we first have to process the annotation, which for recount2 is Gencode v25 (CHR regions) with hg38 coordinates. Although recount can generate count matrices for other annotations using hg38 coordinates. Figure 5 shows two isoforms for a gene composed of 3 different exons.\n\nA single gene with two isoforms composed by three distinct exons (blue boxes) is illustrated. Exons 1 and 3 share the first five base-pairs while exon 2 is common to both isoforms.\n\nThe coverage curve is at base-pair resolution so if we are interested in gene counts we have to be careful not to double count base-pairs 1 through 5 that are shared by exons 1 and 3 (Figure 5). Using the function disjoin() from GenomicRanges12 we identified the distinct exonic sequences (disjoint exons). The following code defines the exon coordinates that match Figure 5 and the resulting disjoint exons for our example gene. The resulting disjoint exons are shown in Figure 6.\n\nWindows of distinct exonic sequence for the example gene. Disjoint exons 1 and 2 form exon 1.\n\n\n\nNow that we have disjoint exons, we can compute the base-pair coverage for each of them as shown in Figure 7. That is, for each base-pair that corresponds to exonic sequence, we compute the number of reads overlapping that given base-pair. For example, the first base-pair is covered by 3 different reads and it does not matter whether the reads themselves were soft clipped. Not all reads or bases of a read contribute information to this step, as some do not overlap known exonic sequence (light pink in Figure 7).\n\nAt each exonic base-pair we compute the number of reads overlapping that given base-pair. The first base (orange arrow) has 3 reads overlapping that base-pair. Base-pair 11 has a coverage of 3 but does not overlap known exonic sequence, so that information is not used for the gene and exon count matrices (grey arrow). If a read partially overlaps exonic sequence, only the portion that overlaps is used in the computation (see right most read).\n\nWith base-pair coverage for the exonic sequences computed, the coverage count for each distinct exon is simply the sum of the base-pair coverage for each base in a given distinct exon. For example, the coverage count for disjoint exon 2 is 2 + 2 + 3 = 7 as shown in Figure 8. The gene coverage count is then ∑in coveragei where n is the number of exonic base-pairs for the gene and is equal to the sum of the coverage counts for its disjoint exons as shown in Figure 8.\n\nThe coverage counts for each disjoint exon are the sum of the base-pair coverage. The gene coverage count is the sum of the disjoint exons coverage counts.\n\nFor the exons, recount2 provides the disjoint exons coverage count matrix. It is possible to reconstruct the exon coverage count matrix by summing the coverage count for the disjoint exons that compose each exon. For example, the coverage count for exon 1 would be the sum of the coverage counts for disjoint exons 1 and 2, that is 19 + 7 = 26. Some methods might assume that double counting of the shared base-pairs was performed while others assume or recommend the opposite.\n\nThe coverage counts described previously are the ones actually included in the rse objects in recount2 instead of typical read count matrices. This is an important difference to keep in mind as most methods were developed for read count matrices. Part of the sample metadata available from recount2 includes the read length and number of mapped reads. Given a target library size (40 million reads by default), the coverage counts in recount2 can be scaled to read counts for a given library size as shown in Equation (1). Note that the resulting scaled read counts are not necessarily integers so it might be necessary to round them if a differential expression (DE) method assumes integer data.\n\n\n\nFrom Figure 4 we know that Rail-RNA soft clipped some reads, so a more precise measure than the denominator of Equation (1) is the area under coverage (AUC) which is the sum of the coverage for all base-pairs of the genome, regardless of the annotation as shown in Figure 9. Without soft clipping reads, the AUC would be equal to the number of reads mapped multiplied by the read length. So for our example gene, the scaled counts for a library size of 20 reads would be 3645∗20=16 and in general calculated with Equation (2). The following code shows how to compute the AUC given a set of aligned reads and reproduce a portion of Figure 9.\n\nThe area under coverage is the sum of the base-pair coverage for all positions in the genome regardless of the annotation. It is the area under the base-level coverage curve shown as the light blue area under the pink curve.\n\n\n\n\n\n\n\nThe recount function scale_counts() computes the scaled read counts for a target library size of 40 million reads and we highly recommend using it before doing other analyses. The following code shows how to use scale_counts() and that the resulting read counts per sample can be lower than the target size (40 million). This happens when not all mapped reads overlap known exonic base-pairs of the genome. In our example, the gene has a scaled count of 16 reads for a library size of 20 reads, meaning that 4 reads did not overlap exonic sequences.\n\n\n\nData in recount2 can be used for annotation-agnostic analyses and enriching the known annotation. Just like exon and gene coverage count matrices, recount2 provides exon-exon junction count matrices. These matrices can be used to identify new isoforms (Figure 10) or identify differentially expressed isoforms. For example, exon-exon junctions 2, 5 and 6 in Figure 1 are only present in one annotated isoform. Snaptron10 allows programatic and high-level queries of the exon-exon junction information and its graphical user interface is specially useful for visualizing this data. Inside R, the recount function snaptron_query() can be used for searching specific exon-exon junctions in recount2.\n\nReads spanning exon-exon junctions are highlighted and compared against the annotation. Three of them match the annotated junctions, but one (blue and orange read) spans an unannotated exon-exon junction with the left end matching the annotation and the right end hinting at a possible new isoform for this gene (blue and orange isoform).\n\nThe base-pair coverage data from recount2 can be used together with derfinder8 to identify expressed regions of the genome, providing another annotation-agnostic analysis of the expression data. Using the function expressed_regions() we can identify regions of expression based on a given data set in recount2. These regions might overlap known exons but can also provide information about intron retention events (Figure 11), improve detection of exon boundaries (Figure 12), and help identify new exons (Figure 1) or expressed sequences in intergenic regions. Using coverage_matrix() we can compute a coverage matrix based on the expressed regions or another set of genomic intervals. The resulting matrix can then be used for a DE analysis, just like the exon, gene and exon-exon junction matrices.\n\nSome reads might align with known intronic segments of the genome and provide information for exploring intron retention events (pink read). Some might support an intron retention event or a new isoform when coupled with exon-exon junction data (orange read).\n\nReads that go beyond the known exon boundaries can inform us of whether the annotated boundaries are correct or if there was a run-off transcription event.\n\nHaving reviewed how the coverage counts in recount2 were produced, we can now do a DE analysis. We will use data from 72 individuals spanning the human lifespan, split into 6 age groups with SRA accession SRP04563813. The function download_study() requires a SRA accession which can be found using abstract_search(). download_study() can then be used to download the gene coverage count data as well as other expression features. The files are saved in a directory named after the SRA accession, in this case SRP045638.\n\n\n\n\n\nThe coverage count matrices are provided as RangedSummarizedExperiment objects (rse)9. These objects store information at the feature level, the samples and the actual count matrix as shown in Figure 1 of Love et al., 20163. Figure 2 shows the actual rse objects provided by recount2 and how to access the different portions of the data. Using a unique sample ID such as the SRA Run ID it is possible to expand the sample metadata. This can be done using the predicted phenotype provided by add_predictions()14, pulling information from GEO via find_geo() and geo_characteristics(), or with custom code.\n\nUsing the colData() function we can access sample metadata. More information on these metadata is provided in the Supplementary material of the recount2 paper7, and we provide a brief review here. The rse objects for SRA data sets include 21 columns with mostly technical information. The GTEx and TCGA rse objects include additional metadata as available from the raw sources. In particular, we compiled metadata for GTEx using the v6 phenotype information available at gtexportal.org, and we put together a large table of TCGA case and sample information by combining information accumulated across Seven Bridges’ Cancer Genomics Cloud and TCGAbiolinks15.\n\n\n\nTechnical variables Several of these technical variables include the number of reads as reported by SRA, the actual number of reads Rail-RNA was able to download (which might be lower in some cases), the number of reads mapped by Rail-RNA, whether the sample is paired-end or not, the coverage AUC and the average read length (times 2 for paired-end samples). Note that the sample with SRA Run ID SRR2071341 has about 240.8 million reads as reported by SRA, while it has 120.4 million spots reported in https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR2071341; that is because it is a paired-end sample (2 reads per spot). These details are important for those interested in writing alternative scaling functions to scale_counts().\n\n\n\nBiological information Other metadata variables included provide more biological information, such as the SHARQ beta tissue and cell type predictions, which are based on processing the abstract of papers. This information is available for some of the SRA projects.\n\n\n\nFor some data sets we were able to find the GEO accession IDs, which we then used to create the title and characteristics variables. If present, the characteristics information can be used to create additional metadata variables by parsing the CharacterList in which it is stored. Since the input is free text, sometimes more than one type of wording is used to describe the same information, meaning that we might have to process that information in order to build a more convenient variable, such as a factor vector.\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nAs shown in Figure 2, we can expand the biological metadata information by adding predictions based on RNA-seq data14. The predictions include information about sex, sample source (cell line vs tissue), tissue and the sequencing strategy used. To add the predictions, simply use the function add_predictions() to expand the colData() slot.\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nAdding more information Ultimately, more sample metadata information could be available elsewhere, which can be useful for analyses. This information might be provided in the paper describing the data, the SRA Run Selector or other sources. As shown in Figure 2, it is possible to append information to the colData() slot as long as there is a unique sample identifier such as the SRA Run ID.\n\nFor our example use case, project SRP045638 has a few extra biologically relevant variables via the SRA Run selector https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=SRP045638. We can download that information into text file named SraRunTable.txt by default, then load it into R, sort it appropriately and then append it to the colData() slot. Below we do so for the SRP045638 project.\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nSince we have the predicted sex as well as the reported sex via the SRA Run Selector, we can check whether they match.\n\n\n\n\n\nNow that we have all the metadata available we can perform a DE analysis. The original study for project SRP04563813 looked at differences between 6 age groups: prenatal, infant, child, teen, adult and late life. The following code creates these six age groups.\n\n\n\nMost of the DE signal from the original study was between the prenatal and postnatal samples. To simplify the analysis, we will focus on this comparison.\n\n\n\nAs we saw earlier in Figure 9, it is important to scale the coverage counts to read counts. To highlight the fact that we scaled the counts, we will use a new object name and delete the previous one. However, in practice we would simply overwrite the rse object with the output of scale_counts(rse).\n\n\n\nHaving scaled the counts, we then filter out genes that are lowly expressed and extract the count matrix.\n\n\n\nNow that we have scaled the counts, there are multiple DE packages we could use, as described elsewhere2,3. Since we have 12 samples per group, which is a moderate number, we will use limma-voom16 due to its speed. The model we use tests for DE between prenatal and postnatal samples adjusting for sex and RIN, which is a measure of quality of the input sample. We check the data with multi-dimensional scaling plots (Figure 13 and Figure 14) as well as the mean-variance plot (Figure 15). In a real use case we might have to explore the results with different models and perform sensitivity analyses.\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nHaving run the DE analysis, we can explore some of the top results either with an MA plot (Figure 16) and a volcano plot Figure (17). Both reveal very strong and widespread DE signal.\n\nTesting for prenatal and postnatal DE adjusting for sex and RIN.\n\nTesting for prenatal and postnatal DE adjusting for sex and RIN.\n\n\n\nNow that we have the DE results, we can use some of the tools with the biocView ReportWriting to create a report. One of them is regionReport17, which can create reports from DESeq218 and edgeR19 results. It can also handle limma-voom16 results by making them look like DESeq2 results. To do so, we need to extract the relevant information from the limma-voom objects using topTable() and build DESeqDataSet and DESeqResults objects as shown below. A similar conversion is needed to use ideal20, which is another package in the ReportWriting biocView category.\n\n\n\nHaving converted our limma-voom results to DESeq2 results, we can now create the report, which should open automatically in a browser.\n\n\n\nIf the report doesn’t open automatically, we can open it with browseURL(). A pre-computed version is available as Supplementary File 1.\n\n\n\nUsing clusterProfiler21 we can then perform several enrichment analyses using the Ensembl gene IDs. Here we show how to perform an enrichment analysis using the biological process ontology (Figure 18).\n\n\n\nSeveral other analyses can be performed with the resulting list of differentially expressed genes as described previously2,3, although that is beyond the scope of this workflow.\n\nAs described in Figure 1, recount2 provides data for expression features beyond genes. In this section we perform a DE analysis using exon data as well as the base-pair resolution information.\n\nThe exon and exon-exon junction coverage count matrices are similar to the gene level one and can also be downloaded with download_study(). However, these coverage count matrices are much larger than the gene one. Aggressive filtering of lowly expressed exons or exon-exon junctions can reduce the matrix dimensions if this impacts the performance of the DE software used.\n\nBelow we repeat the gene level analysis for the disjoint exon data. We first download the exon data, add the expanded metadata we constructed for the gene analysis, explore the data (Figure 19), and then perform the DE analysis using limma-voom.\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nJust like at the gene level, we see many exons differentially expressed between prenatal and postnatal samples (Figure 20). As a first step to integrate the results from the two features, we can compare the list of genes that are differentially expressed versus the genes that have at least one exon differentially expressed.\n\nTesting for prenatal and postnatal DE adjusting for sex and RIN.\n\n\n\nNot all differentially expressed genes have differentially expressed exons. Moreover, genes with at least one differentially expressed exon are not necessarily differentially expressed (Figure 21). This is in line with what was described in Figure 2B of Soneson et al., 201522.\n\nThis was just a quick example of how we can perform DE analyses at the gene and exon feature levels. We envision that more involved pipelines could be developed that leverage both feature levels, such as in Jaffe et al., 201723. For instance, we could focus on the differentially expressed genes with at least one differentially expressed exon, and compare the direction of the DE signal versus the gene level signal as shown in Figure 22.\n\n\n\nThe fold change for most exons shown in Figure 22 agrees with the gene level fold change. However, some of them have opposite directions and could be interesting to study further.\n\nrecount2 provides bigWig coverage files (unscaled) for all samples, as well as a mean bigWig coverage file per project where each sample was scaled to 40 million 100 base-pair reads. The mean bigWig files are exactly what is needed to start an expressed regions analysis with derfinder8. recount provides two related functions: expressed_regions() which is used to define a set of regions based on the mean bigWig file for a given project, and coverage_matrix() which based on a set of regions builds a count coverage matrix in a RangedSummarizedExperiment object just like the ones that are provided for genes and exons. Both functions ultimately use import.bw() from rtracklayer24 which currently is not supported on Windows machines. While this presents a portability disadvantage, on the other side it allows reading portions of bigWig files from the web without having to fully download them. download_study() with type = \"mean\" or type = \"samples\" can be used to download the bigWig files, which we recommend doing when working with them extensively.\n\nFor illustrative purposes, we will use the data from chromosome 21 for the SRP045638 project. First, we obtain the expressed regions using a relatively high mean cutoff of 5. We then filter the regions to keep only the ones longer than 100 base-pairs to shorten the time needed for running coverage_matrix().\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nNow that we have a set of regions to work with, we proceed to build a RangedSummarizedExperiment object with the coverage counts, add the expanded metadata we built for the gene level, and scale the counts. Note that coverage_matrix() scales the base-pair coverage counts by default, which we turn off in order to use use scale_counts().\n\n\n\n\n\n\n\nNow that we have a scaled count matrix for the expressed regions, we can proceed with the DE analysis just like we did at the gene and exon feature levels (Figure 23, Figure 24, Figure 25, and Figure 26).\n\nTesting for prenatal and postnatal DE adjusting for sex and RIN.\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nHaving identified the differentially expressed regions (DERs), we can sort all regions by their adjusted p-value.\n\n\n\n\n\n\n\n\n\nSince the DERs do not necessarily match the annotation, it is important to visualize them. The code for visualizing DERs can easily be adapted to visualize other regions. Although, the width and number of the regions will influence the computing resources needed to make the plots.\n\nBecause the unscaled bigWig files are available in recount2, several visualization packages can be used such as epivizr25, wiggleplotr26 and derfinderPlot8. With all of them it is important to remember to scale the data except when visualizing the mean bigWig file for a given project.\n\nFirst, we need to get the list of URLs for the bigWig files. We can either manually construct them or search them inside the recount_url table.\n\n\n\nWe visualize the DERs using derfinderPlot, similar to what was done in the original publication13. However, we first add a little padding to the regions: 100 base-pairs on each side.\n\n\n\nNext, we obtain the base-pair coverage data for each DER and scale the data to a library size of 40 million 100 base-pair reads, using the coverage AUC information we have in the metadata.\n\n\n\nThe function plotRegionCoverage() requires several pieces of annotation information for the plots that use a TxDb object. For recount2 we used Gencode v25 hg38’s annotation, which means that we need to process it manually instead of using a pre-computed TxDb package.\n\nTo create a TxDb object for Gencode v25, first we need to import the data. Since we are working only with chromosome 21 for this example, we can subset it. Next we need to add the relevant chromosome information. Some of the annotation functions we use can handle Entrez or Ensembl IDs, but not Gencode IDs. So we will make sure that we are working with Ensembl IDs before finally creating the Gencode v25 TxDb object.\n\n\n\n\n\n\n\n\n\nNow that we have a TxDb object for Gencode v25 on hg38 coordinates, we can use bumphunter’s27 annotation functions for annotating the original 10 regions we were working with. Since we are using Ensembl instead of Entrez gene IDs, we need to pass this information to annotateTranscripts(). Otherwise, the function will fail to retrieve the gene symbols.\n\n\n\n\n\n\n\nThe final piece we need to run plotRegionCoverage() is information about which base-pairs are exonic, intronic, etc. This is done via the annotateRegions() function in derfinder, which itself requires prior processing of the TxDb information by makeGenomicState().\n\n\n\n\n\n\n\n\n\nWe can finally use plotRegionCoverage() to visualize the top 10 regions coloring by whether they are prenatal or postnatal samples. Known exons are shown in dark blue, introns in light blue.\n\n\n\nIn these plots we can see that some DERs match known exons (Figure 28, Figure 34, Figure 36), some are longer than known exons (Figure 27, Figure 33, Figure 35), and others are exon fragments (Figure 29–Figure 32) which could be due to the cutoff used. Note that Figure 33 could be shorter than a known exon due to a coverage dip.\n\n\nSummary\n\nIn this workflow we described in detail the available data in recount2, how the coverage count matrices were computed, the metadata included in recount2 and how to get new phenotypic information from other sources. We showed how to perform a DE analysis at the gene and exon levels as well as use an annotation-agnostic approach. Finally, we explained how to visualize the base-pair information for a given set of regions. This workflow constitutes a strong basis to leverage the recount2 data for human RNA-seq analyses.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nLCT and AEJ were supported by the National Institutes of Health (grant R21 MH109956-01).\n\nThe author confirms that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe would like to acknowledge the members of Andrew Jaffe (Lieber Institute for Brain Development, Johns Hopkins Medical Campus) and Alexis Battle (Department of Computer Science, Whiting School of Engineering at Johns Hopkins University) labs for feedback on the explanatory figures.\n\n\nSupplementary material\n\nSupplementary File 1: A pre-computed version of the differential expression report.\n\nClick here to access the data.\n\nSupplementary File 2: Session information. This workflow was created using BiocWorkflowTools28.\n\nThe session information is available in this file. The most recent version of this workflow is available via Bioconductor at http://bioconductor.org/help/workflows/.\n\nClick here to access the data.\n\n\nReferences\n\nHuber W, Carey VJ, Gentleman R, et al.: Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. 2015; 12(2): 115–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaw CW, Alhamdoosh M, Su S, et al.: RNA-seq analysis is easy as 1-2-3 with limma, Glimma and edgeR [version 2; referees: 3 approved]. F1000Res. 2016; 5: 1408. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLove MI, Anders S, Kim V, et al.: RNA-Seq workflow: gene-level exploratory analysis and differential expression [version 1; referees: 2 approved]. F1000Res. 2016; 4: 1070. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen Y, Lun AT, Smyth GK: From reads to genes to pathways: differential expression analysis of RNA-Seq experiments using Rsubread and the edgeR quasi-likelihood pipeline [version 2; referees: 5 approved]. F1000Res. 2016; 5: 1438. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrazee AC, Langmead B, Leek JT: ReCount: a multi-experiment resource of analysis-ready RNA-seq gene count datasets. BMC bioinformatics. 2011; 12: 449. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHimes BE, Jiang X, Wagner P, et al.: RNA-Seq transcriptome profiling identifies CRISPLD2 as a glucocorticoid responsive gene that modulates cytokine function in airway smooth muscle cells. PLoS One. 2014; 9(6): e99625. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCollado-Torres L, Nellore A, Kammers K, et al.: Reproducible RNA-seq analysis using recount2. Nat Biotechnol. 2017; 35(4): 319–21. PubMed Abstract | Publisher Full Text\n\nCollado-Torres L, Nellore A, Frazee AC, et al.: Flexible expressed region analysis for RNA-seq with derfinder. Nucleic Acids Res. 2017; 45(2): e9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorgan M, Obenchain V, Hester J, et al.: SummarizedExperiment: SummarizedExperiment container. 2017. Reference Source\n\nWilks C, Gaddipati P, Nellore A, et al.: Snaptron: querying and visualizing splicing across tens of thousands of RNA-seq samples. bioRxiv. 2017. Publisher Full Text\n\nNellore A, Collado-Torres L, Jaffe AE, et al.: Rail-RNA: scalable analysis of RNA-seq splicing and coverage. Bioinformatics. 2016; pii: btw575. PubMed Abstract | Publisher Full Text\n\nLawrence M, Huber W, Pages 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\nJaffe AE, Shin J, Collado-Torres L, et al.: Developmental regulation of human cortex transcription and its clinical relevance at single base resolution. Nat Neurosci. 2015; 18(1): 154–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEllis SE, Collado-Torres L, Leek J: Improving the value of public RNA-seq expression data by phenotype prediction. bioRxiv. 2017. Publisher Full Text\n\nColaprico A, Silva TC, Olsen C, et al.: TCGAbiolinks: an R/Bioconductor package for integrative analysis of tcga data. Nucleic Acids Res. 2016; 44(8): e71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaw CW, Chen Y, Shi W, et al.: voom: Precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 2014; 15(2): R29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCollado-Torres L, Jaffe AE, Leek JT: regionReport: Interactive reports for region-level and feature-level genomic analyses [version 2; referees: 2 approved, 1 approved with reservations]. F1000Res. 2016; 4: 105. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with Deseq2. Genome Biol. 2014; 15(12): 550. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26(1): 139–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarini F: Ideal: Interactive differential expression analysis. 2017. Reference Source\n\nYu G, Wang LG, Han Y, et al.: clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012; 16(5): 284–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSoneson C, Love MI, Robinson MD: Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences [version 2; referees: 2 approved]. F1000Res. 2015; 4: 1521. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJaffe AE, Straub R, Shin JH, et al.: Developmental and genetic regulation of the human cortex transcriptome in schizophrenia. bioRxiv. 2017. Publisher Full Text\n\nLawrence M, Gentleman R, Carey V: rtracklayer: an R package for interfacing with genome browsers. Bioinformatics. 2009; 25(14): 1841–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBravo HC, Chelaru F, Smith L, et al.: Epivizr: R interface to epiviz web app. 2017. Reference Source\n\nAlasoo K: Wiggleplotr: Make read coverage plots from bigwig files. 2017. Reference Source.\n\nJaffe AE, Murakami P, Lee H, et al.: Bump hunting to identify differentially methylated regions in epigenetic epidemiology studies. Int J Epidemiol. 2012; 41(1): 200–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith M, Ole´s A: BiocWorkflowTools: Tools to aid the development of bioconductor workflow packages. 2017. Reference Source"
}
|
[
{
"id": "25456",
"date": "12 Sep 2017",
"name": "Davide Risso",
"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 workflow that describes how to analyze the datasets available through the Recount2 project with Bioconductor. Since many of the state-of-the-art methods for the analysis of RNA-seq data are implemented in R and available through the Bioconductor project, this contribution is an important resource for researchers interested in reanalyzing the impressive amount of data that the authors have processed in the Recount2 project.\nI have a few comments that hopefully will help improve the workflow.\n1. I was a bit confused by the rationale of the scaled coverage counts. And especially on the need for a target library size and the use of scaled counts. Wouldn't it be simpler to divide the coverage by read length (without rescaling)? Wouldn't that result in the actual reads mapped to each region (exon, gene, ...)? I understand that for the derfinder analysis, some rescaling is needed for normalization purposes, but for more \"classic\" analysis (such as gene- or exon-level differential expression) where the counts are normalized later in the workflow, wouldn't starting from 'coverage/readlength' be a more sensible choice?\n2. Sex prediction. This is a really interesting part of the analysis, even though it's not the focus of this workflow. It would be interesting to get the authors' opinion of how to best use this feature on real analyses. For instance, are the 8 misclassified samples likely to be false positives from the classifiers or are they mislabeled samples? What is the recommendation of the authors in such cases? Should these samples be discarded or is there any diagnostics that can be run to make sure that the quality of these samples is not compromised?\n3. I think that the authors should give more details on the design matrix. For instance, why did they decide to include RIN and sex? Why is it important to include these variables in the model? More generally, the workflow lacks details on the limma pipeline. I understand that this is not the focus of the authors' work, but it may be confusing for beginners that don't have a direct experience with limma or voom. The authors could for instance refer the reader to the limma workflow1 for details.\n4. Similarly, there is a lack of details on the GO enrichment analysis. Since there are many types of gene-set enrichment analysis, a paragraph could be added with more details and perhaps some references to explain what enrichment analysis is and what types of hypotheses are tested.\n5. One important advantage of exon-level differential expression is that it can be used to infer alternative splicing. This can be done with the functions 'diffSplice()' and 'topSplice()' in limma or with the DEXSeq package. It would be nice to showcase these functions or at least to mention that they exist.\n6. Is the annotation in Recount2 stable? Or is it constantly updated when a new version of Gencode is released? If the former, it might make sense to package the 'gencode_v25_hg38_txdb' object that the authors create in the workflow so that each user does not have to create it from scratch every time.\nMinor comments:\n- The authors use throughout the paper 'assays(rse)$counts' to access the counts of the 'rse' object. Although this is correct, a clearer and more concise way is 'assay(rse)' (or 'assay(rse, \"counts\")' if the authors want to explicitly state the name of the assay). - Section \"Coverage counts provided by recount2\". The authors say \"Although recount can generate count matrices for other annota- tions using hg38 coordinates. \" But they never say how this can be done. It would be good to add a paragraph on how to do that (which I presume involves creating an alternative txdb object).\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": "25458",
"date": "25 Sep 2017",
"name": "Alejandro Reyes",
"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 by Collado-Torres et al. provides a workflow to analyze public RNA-seq data using ‘recount2’. Recount2 is a resource that provides whole-genome coverage tracks for more than 70,000 RNA-seq experiments. The accompanying R/Bioconductor package ‘recount’ gives programmatic access to download read counts per gene and to estimate read counts for genomic regions of interest. In an RNA-seq pipeline, processing raw data into the formats available through recount2 involves the most time-consuming steps. Thus, recount2 will save many researchers a lot of time.\n\nThe workflow describes how to programmatically access data from recount2 and describes different analyses that can be done using these data. However, I think the authors needs improve and clarify some aspects of the workflow, which I summarize below.\n\nMajor comments:\n\nThe authors use the formula in equation 1 to scale read counts. While I agree that the read counts will be approximately equal to the sum of the coverage divided by the read length, it was not clear why the additional rescaling is needed. I recommend that the authors include a more extensive justification. Also, if the experiments were paired-end, wouldn’t this formula be counting reads instead of sequenced RNA fragments (i.e. double counting)? The section “Enriching annotation” describes several functions and analyses but does not provide any code or examples. Currently, since it is incomplete, it is more distracting than informative. I suggest that the authors either expand this section and add code or drop it. I don’t understand the biological question behind a differential expression analysis at the exon level. Could the authors clarify what the biological question is? If the aim is to find differential exon usage, wouldn’t it be better to use either DEXSeq, DRIMseq, or similar packages that are specifically designed for this analysis?\n\nMinor comments:\n\nThe first three sentences of the introduction need references. The sentence “generally, when investigators use the term expression, they refer to gene expression” is not entirely true. For example, developmental or cell biologists often interpret “expression” as protein expression. For full reproducibility, it would be useful to download the data within R using the SRAdb package instead of downloading it manually. The code that creates the age groups is too complicated (4 embedded ‘ifelse’ statements). I have submitted a pull request with a simplified version of it (https://github.com/LieberInstitute/recountWorkflow/pull/1). Figures 13 and 14 could be merged into a single plot, using shapes and colors to distinguish the different annotations. The same holds for figures 23 and 24.\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? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "25459",
"date": "27 Sep 2017",
"name": "Nick Schurch",
"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 workflow for working with the 70,000 processed RNA-seq datasets that form the recount2 project, using R, and seek to expand on the details presented in the original recount2 publication by describing how coverage count matrices were computed in recount2.\n\nI'm always slightly confused about the point of these workflow papers - this kind of example workflow information seems better suited to the R package vignettes, and for this reason I sometimes find them awkward to review. In addition, a considerably similar set of example workflow information (albeit somewhat less well described) has already been published in the supplementary information for the original recount2 publication from the same authors (doi:10.1038/nbt.3838, specifically see Supp. Text & Figures, and Supp. Notes 3 & 4)1. Indeed, the supplementary info there goes further than this workflow in describing how to compare results from recount2 across several studies (Supp. Note 5). Personally I found the workflow example here somewhat convoluted and difficult to follow in places but I am sure the community will find it useful in helping to use the recount2 resource and perhaps the nature of such an example workflow presented on real data for a package such as this.\n\nHappily, however, the authors also present substantially new and more detailed information in a few key areas. In particular the description of how the recount2 read coverage matrices are computed is useful and interesting and the example showing how to supplement the project metadata with additional information is useful.\n\nSpecific Comments:\n\n1. I don't really like the use of pseudo-maths equations for Eq1 & 2 - I'd like to see the words replaced with algebraic variables with meanings explained in the text.\n\n2. The scaled read counts are not the same as the actual mapping read counts that are typically required by downstream DE tools (which then typically apply their own appropriate normalization to these numbers). I'd like to see recount2 provide the actual mapping read count for features in addition to the scaled read counts. That said (and if I’m understanding things correctly) the manuscript here is a description of what the format of the recount2 data is and that has already been published - so I'm not expecting this to be changed.\n\n3. I really don't understand the sentence: \"Not all differentially expressed genes have differentially expressed exons.\" Surely this is the definition of a DE gene?? I absolutely agree that \"Moreover, genes with at least one differentially expressed exon are not necessarily differentially expressed\" - differential transcript usage is a prime example of where this can happen - but if a gene is DE I'm pretty sure that it must have a DE exon.\n\n4. I don't see the need for figs 28-36 - a single example of the plot type should be sufficient I think for an example workflow.\n\n5. It would be nice if recount2 could also provide information at the transcript level. Have the authors considered augmenting recount2 with salmon quantifications for all the data? (big job and more of a 'feature request' really).\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? 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/6-1558
|
https://f1000research.com/articles/6-1557/v1
|
24 Aug 17
|
{
"type": "Opinion Article",
"title": "The ELIXIR-EXCELERATE Train-the-Trainer pilot programme: empower researchers to deliver high-quality training",
"authors": [
"Sarah L Morgan",
"Patricia M Palagi",
"Pedro L Fernandes",
"Eija Koperlainen",
"Jure Dimec",
"Diana Marek",
"Lee Larcombe",
"Gabriella Rustici",
"Teresa K Attwood",
"Allegra Via",
"Patricia M Palagi",
"Pedro L Fernandes",
"Eija Koperlainen",
"Jure Dimec",
"Diana Marek",
"Lee Larcombe",
"Gabriella Rustici",
"Teresa K Attwood"
],
"abstract": "One of the main goals of the ELIXIR-EXCELERATE project from the European Union’s Horizon 2020 programme is to support a pan-European training programme to increase bioinformatics capacity and competency across ELIXIR Nodes. To this end, a Train-the-Trainer (TtT) programme has been developed by the TtT subtask of EXCELERATE’s Training Platform, to try to expose bioinformatics instructors to aspects of pedagogy and evidence-based learning principles, to help them better design, develop and deliver high-quality training in future. As a first step towards such a programme, an ELIXIR-EXCELERATE TtT (EE-TtT) pilot was developed, drawing on existing ‘instructor training’ models, using input both from experienced instructors and from experts in bioinformatics, the cognitive sciences and educational psychology. This manuscript describes the process of defining the pilot programme, illustrates its goals, structure and contents, and discusses its outcomes. From Jan 2016 to Jan 2017, we carried out seven pilot EE-TtT courses (training more than sixty new instructors), collaboratively drafted the training materials, and started establishing a network of trainers and instructors within the ELIXIR community. The EE-TtT pilot represents an essential step towards the development of a sustainable and scalable ELIXIR TtT programme. Indeed, the lessons learned from the pilot, the experience gained, the materials developed, and the analysis of the feedback collected throughout the seven pilot courses have both positioned us to consolidate the programme in the coming years, and contributed to the development of an enthusiastic and expanding ELIXIR community of instructors and trainers.",
"keywords": [
"training",
"Train-the-Trainer",
"TtT",
"instructor training",
"educational psychology",
"science of learning",
"EXCELERATE",
"ELIXIR"
],
"content": "Introduction\n\nELIXIR (https://www.elixir-europe.org) is a distributed research infrastructure with a mission to manage, provide access to and safeguard the increasing volumes of data being generated by European life scientists. It coordinates and sustains bioinformatics resources across its 21 member states, and helps researchers to more easily find, analyse, share and exchange biological data. As thousands of European scientists make use of ELIXIR’s databases, tools, services and data, providing the necessary bioinformatics training to help them do so most effectively is a key priority, and one of ELIXIR’s main missions (van Gelder et al., 2016).\n\nBioinformatics seldom forms a core part of formal life-science degree programmes in Europe; therefore, PhD students, postdocs and even PIs seek out focused ‘point of need’ training to gain the skills they require to fruitfully expedite their research. Across Europe, the availability of bioinformatics training opportunities varies greatly from one country to another, and the number of available courses is not sufficient to meet demand, most notably in subjects such as the analysis of next-generation sequencing data. This high demand is not yet waning, and more courses will need to be brought online as the life sciences extend into new areas. Expanding the provision of training in Europe requires the development not only of new courses and materials, but also of new instructors able to deliver high-quality courses. However, the ability to develop new trainers through the provision of ‘instructor training’ is not yet available in all countries.\n\nA continual challenge faced by course providers is finding appropriate individuals to deliver training. Bioinformatics is a dynamic, highly practical and evolving field, and is generally best explained by practitioners in the field. But subject specialists often have little or no formal ‘instructor training’, and hence may be unaware either of learning principles and their application to teaching, of how to tailor sessions to specific audiences, of how to assess whether learning is occurring or has occurred, or of how, ultimately, to design, develop and deliver effective training sessions and materials.\n\nThis situation argues for the development of a practical programme to familiarise subject specialists with good training practices (Via et al., 2013; Best Practices in Training, DEPOCEI Report, 2013, http://www.depocei.org/wp-content/uploads/2013/06/DEPOCEI_Final-report_TRAINING-BEST-PRACTICES.pdf), introducing pedagogical/andragogical theory and methods for course design, delivery and assessment. The idea would not be to try to transform would-be instructors into educationalists, but rather, to help them become responsive, reflective instructors able to provide high-quality training to a variety of audiences, and potentially also able to help others develop their own training skills. Many researcher-instructors (researchers whose career and/or personal interests involve teaching/training) are isolated in their own work settings. Having access to a network or community of practice is therefore important, not just for the support it can provide, but also for enabling their continued development, by sharing ideas and teaching practices, and exploiting training opportunities that arise.\n\nIn 2015, ELIXIR received funding for the EXCELERATE project https://www.elixir-europe.org/about-us/how-funded/eu-projects/excelerate) from the European Union’s Horizon 2020 research and innovation programme. One of ELIXIR-EXCELERATE’s main goals is to help ELIXIR support a pan-European training programme to increase bioinformatics capacity and competency.\n\nAgainst this background, the ELIXIR-EXCELERATE Train-The-Trainer (EE-TtT) programme was developed with the following goals in mind:\n\n1. To assemble research-based training materials using reliable, published sources, and use them to compile an EE-TtT package/framework, to be refined/enriched/reviewed during the programme;\n\n2. To run a pilot of at least six EE-TtT courses in the first twelve months, using the initial EE-TtT package;\n\n3. Deliver a technical workshop to learn from experts about the use of clouds and Virtual Machines (VMs) in training, and develop guidelines for integration into the EE-TtT framework accordingly; and\n\n4. Train further instructor trainers in order to better consolidate the ELIXIR training community, and be able to scale up to several ELIXIR Nodes.\n\nThis paper describes the EE-TtT pilot, which was designed and delivered between January 2016 and January 2017. It outlines the focus of the pilot, the content, structure and features of the TtT sessions developed in the first year, and the training materials created so far.\n\n\nThe EE-TtT pilot overview and goals\n\nThe pilot work involved exploring TtT approaches that were already in use within ELIXIR Nodes and related initiatives, and defining a basic framework and curriculum to deliver appropriate training sessions, fit for the needs of the ELIXIR community of bioinformatics instructors (van Gelder et al., 2016). A ‘kick-off’ workshop (see Supplementary Material 1) was held at EMBL-EBI (http://www.ebi.ac.uk), which brought together representatives from ELIXIR member states with external experts in educational psychology and TtT design and delivery from the Wellcome Trust, NIH-BD2K and Software Carpentry (SWC, https://software-carpentry.org) and Data Carpentry (DC, http://www.datacarpentry.org) Foundations. The objectives of the workshop were to:\n\n• Explore the scope of current activities in the areas of impact assessment and TtT;\n\n• List the methods currently being applied to undertake these activities;\n\n• Identify the major requirements/outputs of each activity;\n\n• Produce a basic framework or set of guidelines for initial development of the TtT programme/pilot; and\n\n• Identify an initial group of candidate TtT trainers.\n\nWorkshop participants experienced with bioinformatics training and/or TtT courses or tutoring were invited to elaborate on and present their experiences, and provide five key features a TtT programme should have. The commonalities from the features they provided can be grouped as follows:\n\n1. How TtT sessions should be delivered: they should be interactive and promote sharing of experiences; encourage reflection and development; provide mentoring of new instructors; promote performance videoing and critiquing; provide reference materials for delivering TtT courses; and provide examples of courses to demonstrate possible developments.\n\n2. What TtT sessions should span: they should span pedagogical skills (ability to listen to learners, observe and analyse), and review main evidence-based research results on how learning works; lesson and material planning and preparation with outcomes/aims/learning objectives; effective teaching techniques - practical tips for applying them effectively; evaluation and assessment/feedback by mentor/trainer/learner (including how to handle negative feedback) (Anderson & Krathwohl, 2001; Bloom & Krathwohl, 1956); identifying skills trainees currently have and what new skills they need to develop and becoming a reflective practitioner.\n\n3. Who should be trained in TtT sessions: the target audience should be current or future committed bioinformatics instructors, in small groups of 20 or fewer participants.\n\n4. What are the challenges of TtT programs: the challenges faced are of sustainability, scalability, building community of practice networks, valuing trainer efforts; continued training opportunities.\n\nA second part of the workshop was dedicated to breakout sessions, inviting all participants to discuss:\n\n• Specific aims of TtT provision across ELIXIR-EXCELERATE;\n\n• Framing an ELIXIR-EXCELERATE TtT course;\n\n• Sources of material for delivery; and\n\n• Who will deliver – how to develop a cohort of TtT trainers.\n\nAt the end of the workshop, an outline of the first year of the pilot programme was defined, and individuals to deliver it were identified (intending to train further individuals during the pilot).\n\nIn this paper, consistent with the 'instructor' and 'trainer' definitions adopted by the SWC/DC communities, we use the term 'new instructor' to indicate a TtT course completer ('instructor', here, referring to an individual who teaches bioinformatics) and 'trainers' to refer to those individuals who were, or became, qualified to teach EE-TtT courses.\n\nIn April 2016, the initial pool of instructor trainers met at the University of Cambridge, UK, to start collecting materials, and to discuss the content and structure of the pilot courses. Two models were particularly influential in this process: the TtT programme at EMBL-EBI (Watson-Haigh et al., 2013) and the SWC/DC Instructor training (Teal et al., 2015; Wilson, 2016). The former inspired the four core topics that were selected for the EE-TtT pilot courses (see Table 1 and the following section). The initial content of each core topic was based on the EMBL-EBI TtT and Carpentries Instructor training materials (http://swcarpentry.github.io/instructor-training/). The EMBL-EBI TtT is the longest running programme (since 2012) and is focused on providing practical guidance for developing and delivering engaging training in any aspect of bioinformatics, building a network of support for new trainers to exchange ideas and encouraging them to reflect on their practice and development. The teaching philosophy of the SWC/DC communities is based on key research findings about how people learn, and how best to teach them. It was also decided to adopt a number of ‘challenges’ (i.e., practical exercises); from the Carpentries training we use an exercise in which participants, in groups of three, video each other teaching, then watch the videos and give and act upon feedback. This is combined with exercises from EMBL-EBI training on creating appropriate aims and outcomes, which are peer-reviewed within the group, along with sessions focused on the practicalities of running bioinformatics training. EE-TtT materials are being further developed on the basis of additional exploration of educational psychology principles and theories (see, for example, Ambrose et al., 2010; Brown et al., 2014; Dunlosky et al., 2013; Green, 2015; Lang, 2016)\n\nThe EE-TtT pilot courses, as well as the training materials, are structured in four main sessions. The four sessions allow for flexibility in the mode of delivery and personalisation from the trainers.\n\nThe pilot was delivered in seven EE-TtT courses - each using a slightly different format and focus - and one workshop on ‘Using clouds and virtual machines in bioinformatic training’. Teaching materials were progressively accumulated in a freely accessible GitHub repository (https://github.com/TrainTheTrainer/EXCELERATE-TtT).\n\nThe TtT pilot and training materials are described in more detail below.\n\n\nEE-TtT pilot delivery and outcomes\n\nAs mentioned earlier, the point of the TtT programme is not to produce a new group of educationalists; rather, it is to offer guidance, ideas and tips for training development and delivery based on research-driven educational principles, ultimately aiming to develop a skilled network of instructors able to deliver engaging content in an interactive manner.\n\nDuring discussions at the kick-off workshop in Cambridge (above), the four main elements listed in Table 1 were identified as the basis for the content and delivery of a course that would meet the original TtT aims.\n\nThese four high-level elements were then expanded to create the basis for the content and final structure of the course. The four sessions are built in a flexible / modular manner, so they can be easily delivered according to the needs of the audience. Depending on the type of audience, trainers may decide to emphasise some concepts and activities, while attenuating the relevance of others or even omitting them.\n\n1. Principles of learning. This session includes: terms commonly used in the science of learning (albeit often far from standardised); ‘learning objectives’ and ‘learning outcomes’, their differences and uses; how learning works; how memory works; Bloom’s six categories of cognitive skills (knowledge, comprehension, application, analysis, synthesis and evaluation) (Anderson & Krathwohl, 2001; Bloom & Krathwohl, 1956), how these can generate awareness in teachers, be used to understand the process of learning, and ultimately be applied to develop and deliver a lesson, session or course; most relevant learning theories; learner motivation and demotivation; novices versus experts in learning; the concept of cognitive load; learning methods/activities; active learning and learning-by-doing principles. The seven research-based principles of learning by Ambrose et al., 2010, and their implications for teaching practice, are finally listed.\n\n2. Training techniques. This session proposes a number of training techniques that have been observed to effectively enhance learners’ engagement and facilitate their learning. Good and bad training practices are explored, and what makes a good teacher/trainer is discussed. In this context, the skills matrix for trainers developed by the TtT taskforce of the Global Organisation for Bioinformatics Learning, Education and Training (GOBLET http://mygoblet.org, Attwood et al., 2015), which provides an overview of the major skills required to be a good trainer, is also introduced. Strategies to motivate learners and promote their engagement, and behaviours that demotivate them are also described, as well as attitudes and activities that can be used to integrate active learning/learning-by-doing strategies in a course. How to create a comfortable and engaging environment for learning is explained; methods for bioinformatics training are also considered.\n\n3. Designing sessions. This includes defining the needs of an audience; writing appropriate and realistic learning outcomes using Bloom’s taxonomy; practical methods for lesson design, development and delivery, including the use of learning objectives/outcomes and concept maps; and how to create a concept map and develop a lesson plan. From lesson to session and course design; where to find inspiration. Other related topics include: developing, sharing, archiving and making training materials reusable; training material repositories and resources; training rooms for bioinformatics; reproducibility of training environments; preparatory steps for training delivery.\n\n4. Assessment and feedback. This covers formative versus summative assessment; formative assessment in class to gauge trainee engagement/learning; monitoring training impact in real-time; how to use questionnaires to promote peer instruction and content delivery; how to design diagnostic questionnaires to assess learners’ prior knowledge and mental models (multiple choice questions with distractors), and adapt the training accordingly; using learners’ feedback both to assess training quality and instructor performance and as a tool for course development; using feedback as a reflective practitioner; how to gain useful feedback post-course; short- and long-term feedback.\n\nThroughout the course, a balance is struck between theoretical and practical elements, providing an interactive experience where all participants (trainers and instructors) can readily share their thoughts and ideas. All individuals who join the course have some experience of training or teaching - whether it be training / teaching they have delivered, or training / teaching that they have received; this gives everyone a basis for discussion of what training is and is not, or should and should not be. This exchange of thoughts, ideas and experiences makes for a more engaging course, and seeds early network building, and the formation of links that will hopefully endure.\n\nAs mentioned previously, the nature of the course, structured in a modular fashion, with an emphasis on interaction and sharing, allows for flexibility in the mode of delivery. This also allows for personalisation from the trainers based on their experience, background and familiarity with learning principles, and the needs, priorities and peculiarities of specific audiences and courses.\n\nDuring the initial kick-off workshop, two potential models of delivery were identified: stand-alone courses, and courses delivered alongside a training course on a specific subject. These models were implemented in the pilot courses in a variety of different ways (Table 2), depending on the training opportunities available and the type of learners. The modes of delivery are briefly described below:\n\nEE-TtT pilot courses were delivered using two main models: 1) standalone courses lasting one or two days; 2) courses including at least one additional session in which learners sit in on a live training event as observers and/or helpers. Afterwards, trainers and trainees sit together to discuss what they saw in the live classroom and what impact that may have on their own practice.\n\nOne-day course: stand-alone one-day session, focused on providing the theoretical elements listed above, but nevertheless applying an interactive approach (i.e., ensuring that there is still adequate time for discussion/sharing ideas, etc.).\n\nTwo-day course: the first day focuses on theoretical elements, similar to the one-day course, but day two provides trainees with an opportunity to put into practice some of the elements introduced and discussed in day one. This includes delivering a short ‘training session’ on a topic of their choice and gaining real-time feedback; editing and re-presenting this session to the wider group; working in small groups to expand upon one of their chosen topics to deliver a whole session, including the writing of aims and objectives; drafting requirements to run a course, including computational needs; defining the feedback required both for their practice development and session revision.\n\nCourse alongside training course: essentially the same as the other modes, but running alongside a training course on a specific subject. The idea is that observing experienced trainers at work, putting elements of pedagogical theory into practice in a ‘live’ setting, and then having the chance to discuss their observations with other trainers is a valuable learning experience. Whenever possible, TtT participants are therefore offered the chance to sit in on a training session on a specific bioinformatics/computational subject (Linux shell, Python, RNA-Seq data analysis, etc.) as a supplement to the four core TtT sessions, and time is allocated to discuss the experience.\n\nThe structure, content and presentation of the materials was systematically developed as the pilot progressed, and will continue to be honed and reviewed as the project continues.\n\nMaterials include items for running the sessions (session outlines, slides, activities, etc.), and a set of supporting items, which provide further reading, greater detail, and reinforcement of concepts (references, articles, videos, etc.).\n\nStructure. The structure of the materials for running the sessions follows the four essential elements listed in Table 1.\n\nContent. The content is the result of ideas and materials collected from educational psychology research literature (see, for example, Ambrose et al., 2010; Brown et al., 2014; Green, 2015) and from SWC Instructor Training and EMBL-EBI TtT, and also reports a number of successful strategies drawn from trainers’ experiences. Elements of theory are interleaved with activities and practical exercises.\n\nPresentation. Materials have been made publicly available through a Github repository (https://github.com/TrainTheTrainer/EXCELERATE-TtT) and Supplementary file 2, Supplementary file 3. Core materials are currently written in Markdown, and used as visual support in some of the EE-TtT courses. Feedback received at the end of EE-TtT courses in which we displayed content directly from the GitHub website, suggested we should use less distracting and crowded visual aids. We are therefore working to re-organise them into smaller chunks, using a Markdown converter to produce a more interactive format for presentation during courses. Moreover, and importantly, we are working in close collaboration with GOBLET to restructure the materials based on Findable, Accessible, Interoperable and Reusable (FAIR) standards (Wilkinson et al., 2016). A set of slides is also available in the GitHub repository and used as visual support in some courses.\n\nA more technical workshop on using clouds and VMs in bioinformatics training was held at CSC- IT Center for Science (FI) in May 2016, in order to bring everybody abreast with the full potential of these technologies (https://csc.fi/web/training/-/cloud-vm-bioinformatics). This EE-TtT workshop gathered more than 30 bioinformatics trainers and infrastructure specialists from 13 countries to share their experiences and knowledge on using clouds, VM images and Docker in training.\n\nBioinformatics analyses typically involve a large amount of software and reference data, making the installation process time-consuming. This problem is aggravated in course settings where every participant needs to have an identical installation, sufficient hardware to run it, and, ideally, access to an identical set-up after the course. VMs and the use of cloud computing can provide solutions to these training challenges. VMs can be run on a laptop, server or cloud (with appropriate virtualisation software), and provide all trainees with an identical software environment in which to work. Additionally, to continue their learning, trainees have the potential to use the VM once a course has ended.\n\nAn important element of this workshop was therefore to encourage new instructors to explore and implement the use of VMs and cloud-based systems in their training, and also to provide them with the skills to develop and manage these technologies. The dialogue between bioinformatics instructors and infrastructure providers also allowed the latter to gain valuable knowledge on the special needs that bioinformatics training poses.\n\nAll the workshop materials are available in the GitHub (https://github.com/ekorpela/cloud-vm-workshop/), and the lectures were recorded and made available in YouTube (https://www.youtube.com/playlist?list=PLD5XtevzF3yHDQZkvO_1kIYd7ZPlmY8j4). Guidelines on how to use clouds, VM images and Docker in training, and how to access computing infrastructures providing these technologies, will be released alongside EE-TtT materials.\n\n\nDiscussion and future directions\n\nThis paper describes the Train-the-Trainer pilot project that was designed, developed and carried out in the context of ELIXIR-EXCELERATE. The EE-TtT pilot represents an essential step towards a sustainable and scalable ELIXIR TtT programme, and the development of a network of trainers and instructors within the ELIXIR community. We ran seven courses, trained more than sixty new instructors and started training three new TtT trainers. We also developed - and have already used - a first draft of supporting materials, and have started structuring them according to the FAIR principles. Most importantly, we have gained further insight into both the potential and challenges of delivering such a programme in the ELIXIR context.\n\nParticipant feedback collected at the end of each course has given strong, positive indications of the perceived utility of the programme, but the longer-term impact of the training on the individuals who received it needs to be reviewed. To this end, we will shortly invite all participants to complete a long-term feedback survey, providing us with a more concrete view of the impact this training has had on their own practice. Expectation sessions run during a number of the courses suggested that participants did gain a number of ‘take away’ lessons, and we need to determine whether these were actually put into practice and how this affected the training they delivered.\n\nAnother important issue is how to increase the number of TtT trainers who are able to deliver courses independently. The approach taken to date has been to develop interested trainers by having them initially attend a TtT course as a learner, then attend a second course as an assistant. However, the few new candidate TtT trainers who have undertaken this process, despite long-standing experience in bioinformatics training / teaching, felt that, after attending just one or two EE-TtT courses, they were neither knowledgeable nor experienced enough in the pedagogical elements of the course to teach it themselves.\n\nGiven that most trainers have had no formal training in education theory, it will be essential to get this balance right, if the programme is to scale. We therefore need to learn from this as the course matures, and capitalise on existing experience. This could include identifying opportunities to work with experienced instructors: for example, becoming a SWC/DC instructor trainer is a quite demanding and compelling process, requiring learners to meet once a week for eight weeks to engage in discussions around teaching pedagogy. New instructors then shadow a teaching demonstration and part of an online instructor training event and attend regular meetings of the trainer community. Their training is completed after delivering two instructor-training workshops (ideally, one in person and one online). To remain an active trainer, a new trainer commits to teach at least two instructor training events per year, among the other things (see https://github.com/carpentries/policies/blob/master/trainer-agreement.md). Furthermore, the availability of very detailed and consistent training materials is an extremely helpful support, especially for new instructors; and the presence of an experienced community of trainers, who meet at least twice a month, is key to sharing experiences and hence developing new trainers’ self-confidence.\n\nAnother example from which valuable lessons may be drawn is a recent course run by the EMBL-EBI Node as part of an ongoing collaboration with Australian trainers, in which a group that had previously been participants in a TtT course, with significant training experience, were remotely coached to deliver a very successful TtT session. Their critical feedback will be instrumental in understanding the strengths and weaknesses of the current programme, and the opportunities that may exist to evolve and improve the course, ultimately to create more, and more confident, trainers.\n\nAs part of the ongoing, rigorous process of assessment, we put the outcomes of the EE-TtT pilot programme under a microscope, one year after it started. This was done using a Degrees of Freedom Analysis (Tractenberg, 2017) of the features of the EE-TtT pilot courses, in terms of their utility, feasibility, sustainability of learning, and scalability, with the aim of bringing out strengths and weaknesses of the programme. The results of this detailed analysis are presented in a separate paper (Via et al., 2017).\n\nThanks to the enthusiastic work of a group of individuals involved in the EE-TtT activities, we designed, developed and reviewed a new TtT pilot programme tailored to the training needs of the ELIXIR community. We are now ready to move forward and consolidate the EE-TtT programme for the remainder of the EXCELERATE period and beyond.",
"appendix": "Author contributions\n\n\n\nEK, LL, SLM, PMP and AV co-wrote the EE-TtT subtask proposal. SLM and AV co-led the EE-TtT subtask. PF, SLM, AV were the key people in organising and delivery of EE-TtT courses and in the writing of the materials. PF, JD, DM, GR, PMP and AV were also the local host of one or more EE-TtT courses. JD, LL, DM, PMP and GR were also training assistant in EE-TtT courses. GR & LL were also involved in the initial material preparation. EK was local host and organiser of the workshop on VMs & Cloud in Training. TKA is involved in the restructuring of the materials based on FAIR standards. SLM, PMP and AV co-wrote the manuscript, with all authors critically reviewing it.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by ELIXIR, the research infrastructure for life-science data. ELIXIR received funding from the European Union’s Horizon 2020 research and innovation programme (ELIXIR- EXCELERATE, grant agreement no 676559).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary file 1 - EXCELERATE Train-the-trainer and Training Impact workshop report – This is a document reporting the objectives, agenda, participants, outcomes, and future directions of the EE-TtT ‘kick-off’ workshop described in the manuscript.\n\nClick here to access the data.\n\n\nReferences\n\nAmbrose SA, Bridges MW, Dipietro M, et al.: How Learning Works: Seven Research-Based Principles for Smart Teaching. 2010; ISBN: 978-0-470-48410-4. Reference Source\n\nAnderson LW, Krathwohl DR: A taxonomy for learning, teaching and assessing: A revision of Bloom’s Taxonomy of educational objectives: Complete edition. New York: Longman. 2001. Reference Source\n\nAttwood TK, Bongcam-Rudloff E, Brazas ME, et al.: GOBLET: the Global Organisation for Bioinformatics Learning, Education and Training. PLoS Comput Biol. 2015; 11(4): e1004143. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBloom BS, Krathwohl DR: Taxonomy of educational objectives: the classification of educational goals, by a committee of college and university examiners. Handbook 1: Cognitive domain. New York: Longman. 1956. Reference Source\n\nBrown PC, Roediger HL, McDaniel MA: Make it stick. Harvard University Press, 2014; ISBN 978-0674729018. Reference Source\n\nDunlosky J, Rawon K, Marsh E, et al.: What Works, What Doesn’t. The Scientific American Mind (mind.scientificamerican.com). 2013. Reference Source\n\nGreen E: Building a Better Teacher: How Teaching Works. W W Norton & Co Inc. 2015; ISBN 978-0-393-35108-8. Reference Source\n\nLang JM: Small Teaching: Everyday Lessons from the Science of Learning. ISBN: 978-1-118-94449-3. 2016. Reference Source\n\nTeal TK, Cranston KA, Lapp H, et al.: Data Carpentry: Workshops to Increase Data Literacy for Researchers. International Journal of Digital Curation. 2015; 10(1): 135–143. Publisher Full Text\n\nTractenberg RE: Degrees of Freedom Analysis in educational research and decision-making: Leveraging qualitative and survey data to promote excellence in bioinformatics training and education. Brief Bioinform. In press, 2017.\n\nvan Gelder C, Morgan S, Via A, et al.: Report on the training needs identified across the ELIXIR community. Zenodo. 2016. Publisher Full Text\n\nVia A, Attwood TK, Fernandes PL, et al.: A new pan-European Train-the-Trainer Programme for bioinformatics: Pilot results on feasibility, utility and sustainability of learning. Brief Bioinform. Under review, 2017.\n\nVia A, Blicher T, Bongcam-Rudloff E, et al.: Best practices in bioinformatics training for life scientists. Brief Bioinform. 2013; 14(5): 528–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWatson-Haigh NS, Shang CA, Haimel M, et al.: Next-generation sequencing: a challenge to meet the increasing demand for training workshops in Australia. Brief Bioinform. 2013; 14(5): 563–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilson G: Software Carpentry: lessons learned [version 2; referees: 3 approved]. F1000Res. 2016; 3: 62. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "25365",
"date": "12 Sep 2017",
"name": "Jason J. Williams",
"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 an important contribution to the global effort of improving bioinformatics training. Nearly everywhere in the life sciences, institutions, laboratories, and individuals are seeking out bioinformatics training and expertise to advance their research. There are lots of solutions, but none have emerged as a clear standard.\nThe authors informatively describe the ELIXIR-EXCELERATE train-the-trainers (TtT) pilot as an effort that anyone interested in bioinformatics training will want to pay attention to. The merit of the the ELIXIR TtT approach to not to train on domain science/expertise, but to educate instructors on pedagogical best practices - borrowing from Software/Data Carpentry instructor training and previous EMBL-EBI training efforts.\nThe audience trainers will reach is a common one (the biggest? most critical?); researchers who are seeking 'point of need' training. As bioinformatics becomes more skilled at putting tools in researchers hands (Docker, HPC, easy(er)-to-use software), we must also be concerned about users (who may not have bioinformatics/statistical expertise) and their ability to understand the proper use and functionalities of these tools. Importantly therefore, TtT aims to get these domain bioinformatician-trainers to be aware of educational practice - with the goal of helping them better design learning materials and evaluate if they are successfully engaging their audience in learning.\nOverall - the article is very well written. I don't want to change the focus of the report, but I do offer a few comments that might be improvements or prompt the authors to elaborate:\nOn the \"commonalities for the features\" provided (my page 4 - PDF); Item 1 speaks about \"examples of courses to demonstrate possible developments.\" I was not sure at all what was meant. Should TtT training provide examples of courses where TtT skills are implemented? Something else?\n\n(my page 4) you introduce the SWC/DC community trainer definitions. Someone with no knowledge of SWC/DC might not know what this means or why it's important. In the next paragraph you introduce SWC/DC comprehensively, so maybe integrate this with the paragraph below?\n\n(my page 5) When you introduce the modes of delivery, I was slightly confused. You said two modes were proposed, but are there three in practice: standalone one-day, stand-alone two-day, and a mixed mode with the one/two-day course held next to a science workshop? Minor, but was confused here.\n\nIn the Discussion and future directions section (my page 7) you mention the challenges of delivering your program in ELIXIR context? Might you elaborate on one/some of the special challenges of ELIXIR? This would be especially relevant if you could compare to other programs that might have similar challenges in some ways (H3ABioNet Africa?).\n\nIn the same section as item my 4, you mention SWC/DC instructor training as 8-weeks, they now have a two-day in-person/online version.\n\nIf I could ask a \"follow up\" question of this paper, it would be to see some type of summary/profile of the learners who attended the TtT workshops? How many of them had teaching experience? Gender ratios? Just whatever metrics you could provide. I would be curious about how many bioinformaticians that might not see themselves as trainers might see themselves in this cohort.\n\nThanks for this work.\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": "25363",
"date": "12 Sep 2017",
"name": "Kim T. Gurwitz",
"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, clear, and concise article describing the conceptualization, development, and implementation of the Elixir Excelerate train-the-trainer pilot programme (2016-2017). The article highlights best practices for training bioinformatics trainers in how to deliver bioinformatics training to others. The article itself, together with the programme’s freely available materials (links provided within the article), comprise an excellent resource for those interested in learning about, and/or running, a train-the-trainer programme themselves.\nI have 5 additional comments, for consideration:\nParagraph 2 and 3 of the Introduction outline the current state of bioinformatics training in Europe. While these statements might be widely accepted in Europe, it is suggested that citations are added, where possible, for a non-European audience.\n\nIn paragraph 2 of the Introduction, the authors state that ‘Bioinformatics seldom forms a core part of formal life-science degree programmes in Europe’. Perhaps slightly outside the scope of this article, however, it would be interesting to spend one or two more sentences explaining the interplay between formal degree programmes and ‘point of need’ training courses in Europe. In doing so, one might contextualize some of the challenges that bioinformatics trainers encounter as well as the level, and type, of training that they would be expected to deliver against a backdrop of increasing demand for bioinformatics training. Further, it would be interesting to hear the authors’ opinion on why bioinformatics has not been formally incorporated into formal life-science degree programmes in Europe, especially given its increasing importance in the sciences.\n\nPage 4 para. 9 – there should be two separate hyperlinks to ‘EMBL-EBI TtT’ and ‘Carpentry Instructors’, respectively. Currently, it is formatted as one long hyperlink with no clear distinction. Further, the full url is only provided for the Carpentries website and not for the EMBL-EBI website.\n\nPage 8 para. 6 (‘presentation’) – mentions supplementary files 2 and 3, but links to these are not included at the end of the article?\n\nFrom the text it is apparent that the ‘infrastructure for training set up’ workshop was run as a separate event to the train-the-trainer workshops. Could one argue that infrastructure requirements are integral to teaching bioinformatics as they speak to the learning environment? Perhaps one might consider incorporating an element of this into the core train-the-trainer curriculum, if not already included?\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/6-1557
|
https://f1000research.com/articles/5-2435/v1
|
04 Oct 16
|
{
"type": "Research Note",
"title": "First visual record of rare purple dogwhelks (Nucella lapillus) on the Atlantic coast of Nova Scotia, Canada",
"authors": [
"Sonja M. Ehlers",
"Julius A. Ellrich",
"Sonja M. Ehlers"
],
"abstract": "The dogwhelk Nucella lapillus is a rocky intertidal gastropod of the North Atlantic coast. Individual shell color varies. Common colors range between white and brown, with darker dogwhelks being more affected by heat stress than lighter-colored conspecifics. Other reported shell colors are black, mauve, pink, yellow, and orange from European coasts, red and grey from the Bay of Fundy coast of New Brunswick and Nova Scotia (Canada), and purple, black, gray, yellow, and orange from the coasts of Maine and Massachusetts (USA), with purple being considered as a rare color. On the Atlantic coast of Nova Scotia, dogwhelks are active from April until November, but information on dogwhelk shell color is missing for this coast. On 16 June 2016, we found two purple dogwhelks in the mid-to-high intertidal zone of a moderately wave-exposed rocky shore near Duncans Cove, on the Atlantic coast of Nova Scotia while collecting dogwhelks (n= 1000) for manipulative field experiments. All other dogwhelks collected on that day were of common white and brown colors. During earlier dogwhelk collections in Atlantic Nova Scotia (between 2011-2013) and field surveys in Duncans Cove (between 2014-2016), we did not find any purple dogwhelks, indicating the rareness of this color in that region. Interestingly, the purple dogwhelks were detected on a relatively cool day (12.3 ± 0.4 °C, mean ± se, n= 96 temperature measurements) compared to the intertidal temperatures of all other survey days (≥ 18.2 ± 0.5 °C), suggesting that purple dogwhelks may find it less thermally stressful to venture out of crevices and macroalgal cover under relatively cool temperatures. Our observations provide the first visual record of rare purple dogwhelks on the Atlantic coast of Nova Scotia, Canada.",
"keywords": [
"dogwhelk",
"Nucella lapillus",
"color",
"snail",
"rocky intertidal",
"rareness",
"temperature"
],
"content": "Introduction\n\nThe dogwhelk Nucella lapillus (L. 1758) is a common predatory gastropod in the rocky intertidal of the North Atlantic (Crothers, 1985; Etter, 2007). Individuals vary in shell color. White and brown are common colors (Berry & Crothers, 1974; Crothers, 1983; Crothers, 1985; Etter, 1988), with darker dogwhelks being more affected by physiological stress under high temperatures than lighter-color conspecifics caused by higher energy intake from sunlight (Etter, 1988). Other shell colors reported are black, mauve, pink, yellow, and orange on European coasts (Berry & Crothers, 1974; Moore, 1936), red and grey from the Bay of Fundy coast of New Brunswick and Nova Scotia (Canada) (Colton, 1922, Crothers, 1983), and black, purple, gray, yellow, and orange from the coasts of Maine (Colton, 1922; Crothers, 1983) and Massachusetts (USA) (Etter, 1988). Purple is considered to be a rare color in dogwhelks (Colton, 1922; Etter, 1988). On the Atlantic coast of Nova Scotia, dogwhelks are active from April until November (Hughes, 1972; Hunt & Scheibling, 1998), but information on dogwhelk shell colors is missing for this coast.\n\n\nMethods\n\nOn 16 June 2016, we collected 1000 dogwhelks along 300 m of coastline in the mid-to-high intertidal of a moderately wave-exposed rocky shore near Duncans Cove (44°29’41.22”N, 63° 31’26.66”W), Halifax on the Atlantic coast of Nova Scotia. The dogwhelks were collected for manipulative field experiments to examine nonconsumptive effects (NCEs) of these predators on their prey. Equal dogwhelk quantities were collected by one of us (JAE) for related research projects on dogwhelk NCEs (e.g. Ellrich et al., 2015; Ellrich et al., 2016) in several locations, with similar levels of intertidal elevation and wave-exposure, along the Atlantic coast of Nova Scotia: in Glasgow Head (45°19’12.61”N, 60° 17’34.15”W) in May and June 2011, in Deming Island (45°12’44.31”N, 61° 10’25.99”W) in May 2012, and in Deming Island, Halfway Cove (45°20’58.98”N, 61° 21’46.58”W), and Half Island Cove (45°21’19.77”N, 61° 11’23.73”W) in May and June 2013.\n\nDuring field surveys for another research project near our dogwhelk collection site in Duncans Cove, dogwhelk colors were observed regularly (on 12 August 2014, 1 September 2015, and 21 August 2016). To observe dogwhelk colors, 30 quadrats (25 cm × 25 cm) along a ~150 m transect parallel to the coastline were sampled at random on each survey date. Throughout the entire survey period, intertidal temperature was measured continuously every 30 minutes by two submersible loggers (HOBO Pendant Logger, Onset Computer Corp., Pocasset, MA, USA). Using temperature data from those loggers, we calculated the average intertidal temperature for the all the dates when dogwhelks were collected or observed in Duncans Cove.\n\n\nResults & discussion\n\nIn our collection of dogwhelks near Duncans Cove on 16 June 2016 (n= 1000 dogwhelks), we found two dogwhelks of purple shell color. Our results provide the first visual record of purple dogwhelks on the Atlantic coast of Nova Scotia (Figure 1). The other dogwhelks collected on that day were of common white and brown shell colors. We did not find any other purple dogwhelk during any of our five collections of equal dogwhelk quantities along the Atlantic Coast of Nova Scotia (n= 5000 dogwhelks of brown and white shell color in total) or three field surveys near Duncans Cove (n= 82 dogwhelks of brown and white shell color in total) indicating that purple dogwhelks are rare in that region.\n\nPicture taken near Duncans Cove (44°29’41.22”N, 63° 31’26.66”W), Halifax on the Atlantic coast of Nova Scotia, Canada on 16 June 2016 (picture credit: Julius A. Ellrich).\n\nComparing the average intertidal temperatures of the dogwhelk collection day (12.3 ± 0.4°C, mean ± se, n = 96 temperature measurements, 16 June 2016) and the three dogwhelk observation days (19.5 ± 0.7°C, 12 August 2014; 18.2 ± 0.5°C, 1 September 2015; 22.3 ± 0.5°C, 21 August 2016) in Duncans Cove revealed that the purple dogwhelks were found on a relatively cool day. This suggests that purple dogwhelks may find it less thermally stressful to venture out of crevices and macroalgal cover under relatively cool temperatures. Darker dogwhelks show stronger physiological responses to heat, such as faster desiccation, than lighter-color conspecifics (Etter, 1988). Future experiments could, thus, examine if dogwhelk behavioural responses to temperature are related to shell color, which may contribute to the rareness in the observed purple dogwhelks.\n\n\nData availability\n\nF1000Research: Dataset 1. Intertidal temperatures 12-Aug-2014, 10.5256/f1000research.9707.d137308 (Ehlers & Ellrich, 2016a)\n\nF1000Research: Dataset 2. Intertidal temperatures 01-Sep-2015, 10.5256/f1000research.9707.d137309 (Ehlers & Ellrich, 2016b)\n\nF1000Research: Dataset 3. Intertidal temperatures 12-Jun-2016, 10.5256/f1000research.9707.d137310 (Ehlers & Ellrich, 2016c)\n\nF1000Research: Dataset 4. Intertidal temperatures 21-Aug-2016, 10.5256/f1000research.9707.d137311 (Ehlers & Ellrich, 2016d)",
"appendix": "Author contributions\n\n\n\nSME and JAE conducted the field work. JAE wrote the manuscript and SME provided critical comments to produce the final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe field surveys were funded by a Discovery Grant (#311624) awarded to Ricardo A. Scrosati by the Natural Sciences and Engineering Research Council of Canada (NSERC).\n\nThe funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBerry RJ, Crothers JH: Visible variation in the dog-whelk, Nucella lapillus. J Zool (Lond). 1974; 174(1): 123–148. Publisher Full Text\n\nColton HS: Variation in the Dog Whelk, Thais (Purpura Auct.) lapillus. Ecology. 1922; 3(2): 146–157. Publisher Full Text\n\nCrothers JH: Some observations on shell-shape variation in North American populations of Nucella lapillus (L.). Biol J Linn Soc Lond. 1983; 19(3): 237–274. Publisher Full Text\n\nCrothers JH: Dog-Whelks: an introduction to the biology of Nucella lapillus (L.). Field Stud. 1985; 6: 299–360. Reference Source\n\nEhlers SM, Ellrich JA: Dataset 1 in: First visual record of rare purple dogwhelks (Nucella lapillus) on the Atlantic coast of Nova Scotia, Canada. F1000Research. 2016a. Data Source\n\nEhlers SM, Ellrich JA: Dataset 2 in: First visual record of rare purple dogwhelks (Nucella lapillus) on the Atlantic coast of Nova Scotia, Canada. F1000Research. 2016b. Data Source\n\nEhlers SM, Ellrich JA: Dataset 3 in: First visual record of rare purple dogwhelks (Nucella lapillus) on the Atlantic coast of Nova Scotia, Canada. F1000Research. 2016c. Data Source\n\nEhlers SM, Ellrich JA: Dataset 4 in: First visual record of rare purple dogwhelks (Nucella lapillus) on the Atlantic coast of Nova Scotia, Canada. F1000Research. 2016d. Data Source\n\nEllrich JA, Scrosati RA, Molis M: Predator nonconsumptive effects on prey recruitment weaken with recruit density. Ecology. 2015; 96(3): 611–616. PubMed Abstract | Publisher Full Text\n\nEllrich JA, Scrosati RA, Romoth K, et al.: Adult prey neutralizes predator nonconsumptive limitation of prey recruitment. PLoS One. 2016; 11(4): e0154572. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEtter RJ: Physiological stress and color polymorphism in the intertidal snail Nucella lapillus. Evolution. 1988; 42(4): 660–680. Publisher Full Text\n\nEtter R: Snails. In Denny, M. W., and S. D. Gaines. Encyclopedia of Tidepools & Rocky Shores. University of California Press, Berkeley, California, USA. 2007; 530–537. Reference Source\n\nHughes RN: Annual production of two Nova Scotian populations of Nucella lapillus (L.). Oecologia. 1972; 8(4): 356–370. Publisher Full Text\n\nHunt HL, Scheibling RE: Effects of whelk (Nucella lapillus (L.)) predation on mussel (Mytilus trossulus (Gould), M. edulis (L.)) assemblages in tidepools and on emergent rock on a wave-exposed rocky shore in Nova Scotia, Canada. J Exp Mar Bio Ecol. 1998; 226(1): 87–113. Publisher Full Text\n\nMoore HB: The biology of Purpura lapillus. I. Shell variation in relation to environment. J Mar Biol Assoc U K. 1936; 21(1): 61–89. Publisher Full Text"
}
|
[
{
"id": "16981",
"date": "14 Oct 2016",
"name": "Simon C. Courtenay",
"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 note reports, for the first time, the presence of a rare colour morph (purple) of the dogwhelk (Nucella lapillis) near Halifax, Nova Scotia, Canada. This is an interesting observation which will be strengthened by two minor revisions to the presentation.\nFirst, it would be helpful in the Abstract and Methods to indicate that dogwhelks were (presumably) sampled during low tide when they were exposed to the air rather than in water.\nSecondly, temperature records are provided which would benefit from clarification. Judging from the range of temperatures, the temperature loggers were exposed to water during some of the tidal cycle and to air the rest of the time (i.e. they were placed in the intertidal). It would be helpful to know at what point in this temperature record the whelks were sampled. It might be that there are better measures of temperature to report than the daily average (and why do you report the sample size for temperatures recorded on one of your sample days (n=96) but not the other days? Should we assume the same n?). Also, the whelks were sampled on June 16, 2016 but the nearest temperature record is June 12, 2016. Yet the abstract talks about the temperature on the day of whelk sampling. Did I miss something?\nFinally, it would be useful to report whether you archived these unusual specimens, and perhaps some of the more usual colour morphs, in case a future researcher wants to look at them for possible morphological differences from the more common colour morphs. You note that there are physiological differences. With that said, I am delighted that there is a place that basic biological observations like these can still be reported. Well done F1000Research!",
"responses": [
{
"c_id": "2951",
"date": "23 Aug 2017",
"name": "Julius Ellrich",
"role": "Author Response",
"response": "This note reports, for the first time, the presence of a rare colour morph (purple) of the dogwhelk (Nucella lapillus) near Halifax, Nova Scotia, Canada. This is an interesting observation which will be strengthened by two minor revisions to the presentation. We appreciate the positive feedback on our report and express our gratitude in the Acknowledgements. We address each comment below. --- First, it would be helpful in the Abstract and Methods to indicate that dogwhelks were (presumably) sampled during low tide when they were exposed to the air rather than in water. We added the information that dogwhelk collections and observations were done during low tide in the Abstract and Methods of the revised manuscript. --- Secondly, temperature records are provided which would benefit from clarification. Judging from the range of temperatures, the temperature loggers were exposed to water during some of the tidal cycle and to air the rest of the time (i.e. they were placed in the intertidal). It would be helpful to know at what point in this temperature record the whelks were sampled. It might be that there are better measures of temperature to report than the daily average (and why do you report the sample size for temperatures recorded on one of your sample days (n=96) but not the other days? Should we assume the same n?). Also, the whelks were sampled on June 16, 2016 but the nearest temperature record is June 12, 2016. Yet the abstract talks about the temperature on the day of whelk sampling. Did I miss something? As noted correctly by the reviewer, the temperature loggers recorded seawater temperature (while submerged) and air temperature (while emerged). The dogwhelks were collected/sampled within two hours around each low tide. The sample size of temperature recordings was identical for all days (n= 96 temperature recordings). The temperature recordings reported for the dogwhelk collection day were actually recorded by our temperature loggers on that day (i.e. 16 June 2016). Unfortunately, we labeled them wrong (i.e. 12 June 2016) when preparing the supplementary material for the manuscript. We apologize for the confusion. However, for the revised manuscript, we chose to replace the temperature information with other environmental information (i.e. wave-exposure and presence of dense mussel patches and seaweed canopies in Duncans Cove), as this information explains our findings of the purple colored dogwhelks more conclusively. --- Finally, it would be useful to report whether you archived these unusual specimens, and perhaps some of the more usual colour morphs, in case a future researcher wants to look at them for possible morphological differences from the more common colour morphs. You note that there are physiological differences. With that said, I am delighted that there is a place that basic biological observations like these can still be reported. Well done F1000Research! Having collected thousands of dogwhelks in Nova Scotia over the years for manipulative experiments, we were very surprised when we discovered the two purple dogwhelks. We, thus, considered them as rare, and decided not to collect them. Instead, we took the picture (Fig. 1) to document our findings. However, we agree that archiving a collection of dogwhelk color morphs including purple individuals could be useful for future comparative studies. As we will continue our research on nonconsumptive predator effects using dogwhelks, we will take this opportunity to deposit a collection of dogwhelk color morphs in a zoological collection. ---"
}
]
},
{
"id": "16798",
"date": "01 Nov 2016",
"name": "Jeff C. Clements",
"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 by Ehlers & Ellrich qualitatively documents the first record of the Nucella lapillus purple morph on the east coast of Canada. The authors also relate the observations of this colour morph to temperature, suggesting that cooler temperatures may allow this colour morph to become more active outside of rock crevasses.\n\nI very much like this article. From my perspective, there are not enough outlets for basic biological observations such as this, which can serve as the basis for novel and important hypotheses. It is comforting to see F1000Research promoting observational records! I recommend that this observation be indexed once minor revisions are made to the article, which I highlight below:\nThe authors state that temperatures on the date of observation (16 June 2016) were cool relative to 3 other sampling dates. However, temperature data is not presented for 16 June (but is presented for 12 June). I would suspect that the temperature data for 12 June is not an accurate representation of those on 16 June. This can be rectified by including HOBO logger data for 16 June (if the authors have it) or by utilizing historical sea surface temperatures from online databases (e.g. DFO, NOAA, GoSL, etc.).\n\nAside from mentioning that some colour morphs are more sensitive to temperatures than others, the authors do not provide much in the way of context for why this species exhibits such tremendous variation in shell colour. What is the ecological and evolutionary benefit (or hindrance) of having such variation in shell colour? A brief mention of this in the introduction and/or discussion would be useful.\n\nAlthough temperature means and their errors are reported in the abstract, I’m not a fan of downloading 4 separate Excel files of temperature data. I would suggest including a single graph of 24 h temperature recordings (or a bar chart of temperature means +- SE) for each sampling date. This will help the reader visualize the differences in temperature between the date when purple morphs were observed and the dates they were not.\n\nI think the authors can add some more discussion points to the Results and Discussion section. Some specific suggestions are highlighted below:\n\nSome mention of the origin of purple morphs in eastern Canada would be nice. Do you think purple morphs have existed in eastern Canada for some time and were simply not observed until now? Or do you think this might be a consequence of ocean warming, with purple morphs moving to more northerly latitudes to avoid high temperatures? This is especially relevant given that the authors suggest that purple colour morphs do not fare well in higher temperatures.\n\nSome additional suggestions for future research are also warranted. For example, targeted sampling over a given time period in which temperatures vary might give a more quantitative understanding of N. lapillus colour morphs on the shores of Nova Scotia. Such a project would not only provide a quantitative description of N. lapillus colour morphs in eastern Canada (which is currently lacking), but could also inform on spatial and temporal overlaps in N. lapillus colour morphs and lend field evidence for behavioural responses to temperature (which would complement the authors’ suggestion of lab experiments well). Genetic testing could also provide evidence for the origin of these animals, advocating for or against a potential northward displacement in response to warming temperatures.",
"responses": [
{
"c_id": "2950",
"date": "23 Aug 2017",
"name": "Julius Ellrich",
"role": "Author Response",
"response": "The article by Ehlers & Ellrich qualitatively documents the first record of the Nucella lapillus purple morph on the east coast of Canada. The authors also relate the observations of this colour morph to temperature, suggesting that cooler temperatures may allow this color morph to become more active outside of rock crevasses. I very much like this article. From my perspective, there are not enough outlets for basic biological observations such as this, which can serve as the basis for novel and important hypotheses. It is comforting to see F1000Research promoting observational records!I recommend that this observation be published once minor revisions are made to the article, which I highlight below:We are pleased about the thoughtful assessment of our article and express our gratefulness in the Acknowledgements of our revised article. Below, we address the comments.---1. The authors state that temperatures on the date of observation (16 June 2016) were cool relative to 3 other sampling dates. However, temperature data is not presented for 16 June (but is presented for 12 June). I would suspect that the temperature data for 12 June is not an accurate representation of those on 16 June. This can be rectified by including HOBO logger data for 16 June (if the authors have it) or by utilizing historical sea surface temperatures from online databases (e.g. DFO, NOAA, GoSL, etc.). The temperature data recorded by our HOBO loggers were actually from the dogwhelk collection day (i.e. 16 June 2016), but not from the 12 June 2016. When compiling the temperature data for the supplementary material of the manuscript, we accidentally labeled these data wrong. We are apologizing for the confusion. However, we excluded the temperature information from our revised manuscript in favor of information (i.e. wave-exposure, presence of mussel patches and seaweed canopies) that explains the finding of the two purple colored dogwhelks more conclusively.---Aside from mentioning that some colour morphs are more sensitive to temperatures than others, the authors do not provide much in the way of context for why this species exhibits such tremendous variation in shell colour. What is the ecological and evolutionary benefit (or hindrance) of having such variation in shell color? A brief mention of this in the introduction and/or discussion would be useful. We included a brief statement on the consequences of shell color variation in the Introduction of the revised manuscript.---3. Although temperature means and their errors are reported in the abstract, I’m not a fan of downloading 4 separate Excel files of temperature data. I would suggest including a single graph of 24 h temperature recordings (or a bar chart of temperature means +- SE) for each sampling date. This will help the reader visualize the differences in temperature between the date when purple morphs were observed and the dates they were not.In the revised manuscript, we replaced the temperature information by other environmental information as outlined above.---4. I think the authors can add some more discussion points to the Results and Discussion section. Some specific suggestions are highlighted below:i. Some mention of the origin of purple morphs in eastern Canada would be nice. Do you think purple morphs have existed in eastern Canada for some time and were simply not observed until now? Or do you think this might be a consequence of ocean warming, with purple morphs moving to more northerly latitudes to avoid high temperatures? This is especially relevant given that the authors suggest that purple color morphs do not fare well in higher temperatures.We think that purple colored dogwhelks have simply not been reported for the Atlantic coast of Nova Scotia yet, because they are relatively rare compared to the large quantities of the brown and white colored individuals commonly found on this coast. Purple colored individuals having moved to more northerly latitudes in response to ocean warming appears unlikely, because dogwhelks have a restricted activity range (which lies within tens of meters), and lack pelagic larval dispersal (Crothers 1985).To add another discussion point to Results & Discussion in the revised manuscript, we included a paragraph that discusses the environmental influences (i.e. wave-exposure, presence of mussel patches and seaweed canopies) that may favor the occurrence of the rare purple colored dogwhelks on the coast.Crothers, J. H. 1985. Dog-Whelks: an introduction to the biology of Nucella lapillus (L.). Field Studies. 6: 299-360.---ii. Some additional suggestions for future research are also warranted. For example, targeted sampling over a given time period in which temperatures vary might give a more quantitative understanding of N. lapillus colour morphs on the shores of Nova Scotia. Such a project would not only provide a quantitative description of n. lapillus colour morphs in eastern Canada (which is currently lacking), but could also inform on spatial and temporal overlaps in N. lapillus colour morphs and lend field evidence for behavioural responses to temperature (which would complement the authors’ suggestion of lab experiments well). Genetic testing could also provide evidence for the origin of these animals, advocating for or against a potential northward displacement in response to warming temperatures. We suggested future research examining dogwhelk activity patterns in relation to shell color and temperature in the Results & Discussion of the revised manuscript. As discussed above, we deem that northward movement of dogwhelks in response to warming temperatures is unlikely.---"
}
]
},
{
"id": "16799",
"date": "01 Dec 2016",
"name": "Mathieu Cusson",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report on a sighting of purple dogwhelks in Nova Scotia. During a sampling of over 1000 dogwhelks, they observed two purple-shelled individuals.\nEhlers and Ellrich indicate that there may be a link between intertidal (air/water?) temperatures and the observation of the purple dogwhelks. A purple colouring is less thermally stressful in colder settings and due to the lower temperatures, the whelks may venture out from crevices and macroalgal cover.\nI work in the St. Lawrence Estuary where we have found dogwhelks in the intertidal zone although at lower quantities than those reported for Nova Scotia. Water and air temperatures are commonly under 12°C in this portion of the estuary, yet my team and I have never observed purple individuals - the typical colours for dogwhelks in this sector being white–beige to dark grey.\nThe findings of Ehlers and Ellrich are of interest and it is worthwhile to pursue the subject with additional observations or experimental manipulations to understand the factors that affect shell colour variability. However, their linking of the purple colour to a single, cold day (lower water temperatures in the intertidal zone) as reported in this manuscript is rather anecdotic. Rather, the authors should list potential factors (both physiological and environmental) that could potentially affect shell coloration instead of linking this observation to a single day and the possible behaviour of two rare individuals. The potential link to behaviour - lower temperatures favouring an active movement of purple-shelled individuals out from shelter - is not convincing. Mauve coloration has been seen along European coasts. As this colour is very similar to purple, in which environmental conditions were these latter observations made? Similarly, if purple has also been reported from Maine, how would these observations from Maine confirm a link to colder temperatures? I suspect that the purple colour may simply be a rare phenotypic trait that may not be at all related to environmental factors (purple shells may be the albino individuals in dogwhelk populations?).\nNevertheless, I do salute the authors for this valuable natural history observation that must be recorded. Thank you as well to F1000 for allowing the publication of such observations.\n\nOther comments: Datasets: I do not understand why the data were separated into four distinct data sets, one for each date. I suggest a graph presenting all average temperatures coupled with the occurrence of white/brown and purple coloration. In the references, the authors list separate temperature data sets (one for each day). These data sources should be combined (not be separate) so as to avoid artificially increasing the number of publications.\nThe observation date of June 16, the day on which the cool temperatures (12.3°C, n = 96) were recorded, is not presented in the companion data sets. Rather, I would prefer including a graph showing the recorded temperature for each relevant date.",
"responses": [
{
"c_id": "2949",
"date": "23 Aug 2017",
"name": "Julius Ellrich",
"role": "Author Response",
"response": "The authors report on a sighting of purple dogwhelks in Nova Scotia. During a sampling of over 1000 dogwhelks, they observed two purple-shelled individuals. Ehlers and Ellrich indicate that there may be a link between intertidal (air/water?) temperatures and the observation of the purple dogwhelks. A purple colouring is less thermally stressful in colder settings and due to the lower temperatures, the whelks may venture out from crevices and macroalgal cover. I work in the St. Lawrence Estuary where we have found dogwhelks in the intertidal zone although at lower quantities than those reported for Nova Scotia. Water and air temperatures are commonly under 12°C in this portion of the estuary, yet my team and I have never observed purple individuals - the typical colours for dogwhelks in this sector being white–beige to dark grey. The findings of Ehlers and Ellrich are of interest and it is worthwhile to pursue the subject with additional observations or experimental manipulations to understand the factors that affect shell colour variability. However, their linking of the purple colour to a single, cold day (lower water temperatures in the intertidal zone) as reported in this manuscript is rather anecdotic. Rather, the authors should list potential factors (both physiological and environmental) that could potentially affect shell coloration instead of linking this observation to a single day and the possible behaviour of two rare individuals. The potential link to behaviour - lower temperatures favouring an active movement of purple-shelled individuals out from shelter - is not convincing. Mauve coloration has been seen along European coasts. As this colour is very similar to purple, in which environmental conditions were these latter observations made? Similarly, if purple has also been reported from Maine, how would these observations from Maine confirm a link to colder temperatures? I suspect that the purple colour may simply be a rare phenotypic trait that may not be at all related to environmental factors (purple shells may be the albino individuals in dogwhelk populations?). Nevertheless, I do salute the authors for this valuable natural history observation that must be recorded. Thank you as well to F1000 for allowing the publication of such observations. We appreciate the overall positive feedback on our manuscript and express our gratitude in the Acknowledgements. In the revised manuscript, we included the information that shell coloration in the dogwhelk Nucella lapillus is presumably inheritable. We agree that purple colored dogwhelks are rare. In addition, we discuss that their occurrence is likely favoured by the habitat characteristics (i.e. wave splash, presence of dense blue mussel beds and seaweed canopies) of the moderately wave-exposed coastline in Duncans Cove, Nova Scotia, which aligns closely with previous findings of rare purple colored dogwhelks along wave-exposed coasts in Massachusetts, USA (Etter 1988). Also, these habitat characteristics likely enhance the probability of finding rare purple colored dogwhelks. Etter, R. 1988. Physiological stress and color polymorphism in the intertidal snail Nucella lapillus. Evolution. 4(2): 660-680. --- Other comments: Datasets: I do not understand why the data were separated into four distinct data sets, one for each date. I suggest a graph presenting all average temperatures coupled with the occurrence of white/brown and purple coloration. In the references, the authors list separate temperature data sets (one for each day). These data sources should be combined (not be separate) so as to avoid artificially increasing the number of publications. In the revised manuscript, we removed the information on temperature in favor of information on wave-exposure as well as mussel bed and seaweed canopy presence. We did so, because we think that this information explains our findings of the two purple colored dogwhelks more conclusively. --- The observation date of June 16, the day on which the cool temperatures (12.3°C, n = 96) were recorded, is not presented in the companion data sets. Rather, I would prefer including a graph showing the recorded temperature for each relevant date. The correct temperature data for 16 June 2016 was actually included, but unfortunately labeled wrong (i.e. 12 June 2016) in the companion data set. We apologize for the confusion. For the reasons discussed above, we removed the information on temperature from the revised manuscript. ---"
}
]
}
] | 1
|
https://f1000research.com/articles/5-2435
|
https://f1000research.com/articles/6-1545/v1
|
22 Aug 17
|
{
"type": "Research Note",
"title": "Growth performance and feed utilization of African catfish Clarias gariepinus fed a commercial diet and reared in the biofloc system enhanced with probiotic",
"authors": [
"Iskandar Putra",
"Rusliadi Rusliadi",
"Muhammad Fauzi",
"Usman M. Tang",
"Zainal A. Muchlisin",
"Iskandar Putra",
"Rusliadi Rusliadi",
"Usman M. Tang",
"Zainal A. Muchlisin"
],
"abstract": "Background The objective of the present study was to evaluate the growth performance and feed utilization of African catfish Clarias gariepinus fed a commercial diet and reared in the biofloc system enhanced with probiotic. Methods The treatment was the frequency of probiotic application into the cultured system, namely, 5-day interval, 10-day interval, and 15-day interval for 60 days of experiment. Biofloc culture was grown in an experiment tank (vol. 2000 L) by mixing the probiotic (Bacillus sp.) 10 mL and molasses 200 mL per liter of water. The fish was stocked into the biofloc system 7 days after cultured at stocking density of 1000 fish tank-1. The fish was fed a commercial diet that contains 38% crude protein, twice a day at satiation. The application of probiotic was reperformed after 5 days, 10 days, and 15 days after stocking. Results The study showed that the growth performance, survival, and feed utilization of African catfish were higher in the treatment at 5-day intervals over 60 days. The ANOVA test showed that the application frequency of probiotic into biofloc system of cultured media had the significant effect on the growth performance, survival rate, and feed utilization of African catfish. Conclusion The best growth performance and feed utilization were found at the application of probiotic into biofloc system at 5-day intervals over 60 days.",
"keywords": [
"Biofloc",
"Probiotic Frequency",
"Survival Rate"
],
"content": "Introduction\n\nFeed is one of the important agro-inputs in aquaculture production system that contributes to approximately 40–60% of production cost1,2 and it has direct effect on the growth rate of the fish3–6. The aquaculture activity is commonly produced waste, for example, feed remains and feces which changes into ammonia and nitrite once the oxygen level is low. In the closed culture system the concentrations of ammonia (NH3) and nitrite (NO2) are increasing rapidly and would be toxic to organisms7,8.\n\nAccording to Asaduzzaman et al.9 and De Schryver et al.10 the intensive application of commercial feed in the aquaculture causes environmental pollution and increases the possibility of the disease outbreak. Therefore, the water quality management is crucial in the aquaculture system. The objective of water quality management is to provide the comfortable environment and meet the optimum requirements for cultured organisms11. According to Gunadi and Hafsaridewi12 the microbial activities can be used to improve water quality and reduce the burden of contamination by fish farming waste. Therefore, the heterotrophic bacteria have promising potency to be applied in the utilization of waste ammonia in the fish culture. Beside, these bacteria are formed as a floc (clumps) in the cultures media; hence it can be used as an alternative feed source for cultured fish13. Biofloc has abilities to suppress the toxic compounds such as ammonia and harmful bacteria (pathogenic) so that the cultured organisms grow well14. Application of biofloc in the cultures system has been reported by several researchers, for example, in the culture of channel catfish14,15, in the South American catfish Rhamdia quelen16, in Nile tilapia Oreochromis niloticus17,18, Farfantepenaeus brasiliensis19, and in the cultured system of the shrimps Litopenaeus vannamei and Penaeus monodon11,20. However, application of biofloc on African catfish Clarias gariepinus cultures has never been reported previously.\n\nAfrican catfish is the popular species for aquaculture business in Southeast Asian countries21. This species has several advantages, for example, resistance to diseases and handling stress and high growth rate22, thus accounting for its commercial importance worldwide23. Nowadays, the fish farmer fed a commercial diet for African catfish. The protein requirement for African catfish ranges from 25% to 40%, lipid 9.5 to 10%, carbohydrates 15 to 30%, vitamins 0.25 to 0.40%, and minerals 1.0%1, with energy level of 2000 cal/g to 3000 cal/g24. In addition, the application of probiotic into African catfish diet has been reported by several researchers, for example, Al-Dohail et al.25, Ige26, and Dennis and Uchenna27. However, application of probiotic combing with biofloc has never been reported previously. Hence, the aim of the study was to evaluate the growth performance and feed utilization of African catfish fed experimental diet reared in the biofloc cultured system and enhanced with probiotic.\n\n\nMethods\n\nThe research was conducted from June 2016 to August 2016 at Aquaculture Technology Laboratory, Faculty of Fishery and Marine Sciences, Riau University, Indonesia. The experiments were carried out within the ethical guidelines provided by the research institution and national or international regulations.\n\nThe completely random design (CRD) method was used in this study. The tested treatment was the frequency of probiotics application (bacteria inoculation), namely, at 5-day interval (treatment A), 10-day interval (treatment B), and 15-day interval (treatment C). The treatment was conducted at three replications. The experimental fish was maintained in the canvas tank (vol. 2000 L) at stocking density of 1000 fishes and reared for 60 days.\n\nThe biofloc was cultured in the nine canvas tanks with a volume of 2000 L. Each tank was filled with water up to a water level of 100 cm or equivalent to 2000 L. Biofloc culture was done by mixing the probiotic (Bacillus sp.) 10 mL and molasses 200 mL L-1 of water and then mixed into the cultures fish tanks and aerated continuously for 7 days to grow the floc.\n\nThe catfish larvae were stocked at the density of 1000 fish tank-1 with average weight 1.12±0.05 g and average total length 4.42±0.09 cm. The application of 10 mL inoculants bacteria with density of Bacillus sp. about 5×1010 colony forming units (CFU) was performed according to respective treatment, that is, 5-day, 10-day, and 15-day intervals. The experimental catfish feed was a commercial diet with 38% crude protein, crude lipid 5%, and crude fiber 6%, mineral mix 13%, and 13% moisture contents. The fish were fed twice a day at satiation. The weight gain of fish was measured every 12 -days for 60 days.\n\nThe weight gain was calculated as follows: W = Wt – Wo, where W is weight gain (g), Wt is the weight of the fish at the end of experiment (g), and Wo is the weight of fish at the start of experiment (g). The daily growth rate, survival rates, and feed utilization were calculated based on Muchlisin et al.28,29 The main water quality parameters such as dissolved oxygen (DO), pH, and temperature were measured using a digital water checker (YSI-550 A, ASTM, Alla, France) at 6-day intervals, while total ammonia nitrogen (TAN) was measured every 6 days using spectrophotometric method30.\n\nThe data were subjective to one-way analysis of variant (ANOVA) test to determine the effect of treatment on the tested parameters and followed by Newman-Keuls multiple range test with a confidence level of 95%, while the water quality of the data was analyzed descriptively.\n\n\nResults\n\nThe ANOVA test showed that the treatment had a significant effect on the weight gain (WG), specific growth rate and survival rate (SGR), feed efficiency (FE), and feed conversion ratio (FCR) (P<0.05). The study showed that the highest weight gain and specific growth rate were recorded at treatment A; these values were different significantly from other treatments. A similar trend was also found in the survival rate (SR) where the highest survival rate was recorded in treatment A, but this value was not different significantly from treatment C (Table 1). The highest feed efficiency and lower feed conversion ratio were also found in fish with application of probiotic into biofloc system at 5-day intervals (treatment A). However, these values were not different significantly from treatment C (probiotic application at 15-day intervals). In addition, the water temperature ranges from 29.50°C to 29.62°C, dissolved oxygen rages from 3.64 mg L-1 to 3.88 mg L-1, and pH ranges from 6.93 to 7.02. In addition, the ammonia (NH3) content ranged from 0.292 mg L-1 to 0.411 mg L-1 and nitrite content ranged from 0.08 mg L-1 to 0.09 mg L-1. Therefore, there were no significant differences regarding water quality among the treatments; however, the quality in treatment A was slightly better compared to two other treatments (Table 2). The data showing the total length, body weight and total feed consumed by fish at every experiment can be found in Dataset 1.\n\nMean of values in the same row followed by a different superscript that are significantly different (p<0.05).\n\n\nDiscussion\n\nThe study showed that the growth performance, survival rate, and feed utilization of African catfish were the highest in the application of probiotic into the biofloc system at 5-day intervals. This was presumably due to the fact that the applications of probiotics every 5-days can maintain the density of bacteria at suitable forms and effectively decompose organic materials well. This is indicated by lower ammonium (NH3) content in treatment A. According to Widanarni31 the application of biofloc into culture system can improve water quality and reduce the burden of contamination of fish culture waste in the surrounding waters. In addition Irianto32 stated that Bacillus sp. can improve the quality of the cultured media by decomposing organic materials, suppress the growth of pathogenic, and balance the microbial and had a positive effect on fish health and growth.\n\nBesides maintaining the water quality, biofloc is also playing an important role as alternative natural feed for cultured fish. This is because the biofloc contains crude protein that reached 48–53%33,34 and therefore the Feed Conversion Ratio (FCR) in treatment A was 0.90 (below 1.00) and the feed efficiency was higher than 100%. This is because of beside fed on the commercial diet the fish was also fed on floc that contain planktons. This value is better than fish fed on commercial diet without application of biofluc33,35,36. According to Azim34 the nutritional quality of biofloc was appropriate at least for herbivorous and omnivorous fish species. In this case, the African catfish is categorized as omnivorous feeding habits35,37.\n\nIt is clear that biofloc contributed to the growth and production of cultured organism as shown in this study. The basic principle of this technology is using the heterotrophic bacteria to manage the C: N ratio in the water media33,38,39. However, biofloc not only contains the bacteria, but also is composed of other microorganisms including microalgae and zooplanktons as food for farmed fish or shrimps33. According to Crab et al.40 biofloc can be consumed and digested well by the shrimp and therefore possibly substitute for artificial commercial feed. Hence, application of biofloc into cultured system can increase feed efficiency up to 13%39. For example, for feed efficiency of African catfish fed a commercial diet without biofloc was 89.83 %33; it was increased up to 110.86% when the biofloc was applied as shown in this study.\n\nIn addition, according to Avnimelech38, the addition of molasses as a source of carbon in aquaculture system can improve the C/N ratio waters and will further reduce inorganic nitrogen in the waters through increased growth of heterotrophic bacteria, where the heterotrophic bacteria will form a floc which can be fed by fish as feed source. Furthermore the C: N ratio of >10: 1 in the fish farming system is the optimum ratio to enhance the biofloc production and minimize the ammonia regeneration 39.\n\n\nConclusion\n\nThe application of probiotic bacteria with different frequencies in the biofloc system had the significant effect on the growth performance, survival rate, and feed utilization of African catfish (Clarias gariepinus). The higher growth performance and best feed utilization were recorded in the application of probiotic into biofloc system at 5-day intervals.\n\n\nData availability\n\nDataset 1: The total length, body weight and total feed consumed by fish at every experiment. 10.5256/f1000research.12438.d17498041",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by the Ministry of Research, Technology and Higher Education (Ristekdikti) of the Republic of Indonesia through the competitive grants scheme (Contract number: 430/UN.19.5.1.3/LT/2016).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors thank the Ministry of Research, Technology and Higher Education of the Republic of Indonesia for supporting this study through the competitive grants scheme 2016 Contract number: 430/UN.19.5.1.3/LT/2016. The appreciation goes to all of our students who helped the authors during experiments in the laboratory.\n\n\nReferences\n\nSahwan MF: The feed fish and shrimp. Penebar Swadaya. Jakarta. 1999.\n\nFadri S, Muchlisin Z, Sugito S: Growth performance, survival rate and feed utilization of Nile tilapia, Oreochromis niloticus fed experimental diet contains jaloh leafs, Salix tetrasperma Roxb at different levels of EM-4 probiotic. Jurnal Ilmiah Mahasiswa Kelautan dan Perikanan Unsyiah. 2016; 1(2): 210–221.\n\nMuchlisin ZA, Hashim R, Chong AS: Preliminary study on the cryopreservation of tropical bagrid catfish (Mystus nemurus) spermatozoa; The effect of extender and cryoprotectant on the motility after short-term storage. Theriogenology. 2004; 62(1–2): 25–34. PubMed Abstract | Publisher Full Text\n\nMuhammadar AA, Mazlan AG, Samat A, et al.: Growth, survival and feed conversion of juvenile tiger grouper Epinephelus fuscoguttatus in different salinity regimes. AACL Bioflux. 2014; 7(4): 241–247. Reference Source\n\nKarina S, Akbar M, Supriatna A, et al.: Replacement of soybean meal with Moringa oleifera leaf meal in the formulated diets of tilapia (Oreochromis niloticus) fingerlings. AACL Bioflux. 2015; 8(5): 790–795. Reference Source\n\nPutra DF, Fanni M, Muchlisin ZA, et al.: Growth performance and survival rate of climbing perch (Anabas testudineus) fed Daphnia sp. enriched with manure, coconut dregs flour and soybean meal. AACL Bioflux. 2016; 9(5): 944–948. Reference Source\n\nSidik AS, Sarwono, Agustina: The effect of stocking density on nitrification rate in a closed recirculating culture system. Jurnal Akuakultur Indonesia. 2002; 1(2): 47–51. Publisher Full Text\n\nSakala ME, Musuka CG: The effect of ammonia on growth and survival rate of tilapia rendalli in quail manured tanks. Int J Aquac. 2014; 22(4): 1–6. Publisher Full Text\n\nAsaduzzaman M, Wahab MA, Verdegem MC, et al.: C/N ratio control and substrate addition for periphyton development jointly enhance freshwater prawn Macrobrachium rosenbergii production in ponds. Aquaculture. 2008; 280(1–4): 117–123. Publisher Full Text\n\nDe Schryver P, Crab R, Defoirdt T, et al.: The basics of bio-flocs technology: The added value for aquaculture. Aquaculture. 2008; 277(3–4): 125–137. Publisher Full Text\n\nNurhatijah N, Muchlisin ZA, Sarong MA, et al.: Application of biofloc to maintain the water quality in culture system of the tiger prawn (Penaeus monodon). AACL Bioflux. 2016; 9(4): 923–928. Reference Source\n\nGunadi B, Hafsaridewi R: Waste utilization aquaculture of catfish (clarias gariepenus) intensive maintenance system with heterotrophic for tilapia. Final Report of Research Activity in 2007. Research and Technology for Freshwater Aquaculture Stattion. Sukamandi. 2007.\n\nCrab R, Avnimelech Y, Defoirdt T, et al.: Nitrogen removal techniques in aquaculture for a sustainable production. Aquaculture. 2007; 270(1–4): 1–14. Publisher Full Text\n\nSchrader KK, Green BW, Perschbacher PW: Development of phytoplankton communities and common off-flavors in a biofloc technology system used for the culture of channel catfish (Ictalurus punctatus). Aquacult Eng. 2011; 45(3): 118–126. Publisher Full Text\n\nGreen BW, Schrader KK, Perschbacher PW: Effect of stocking biomass on solids, phytoplankton communities, common off-flavors, and production parameters in a channel catfish biofloc technology production system. Aquac Res. 2014; 45(9): 1442–1458. Publisher Full Text\n\nPoli MA, Schveitzer R, De Oliveira Nuñer AP: The use of biofloc technology in a South American catfish (Rhamdia quelen) hatchery: Effect of suspended solids in the performance of larvae. Aquac Eng. 2015; 66: 17–21. Publisher Full Text\n\nEkasari J, Zairin M, Putri DU, et al.: Biofloc-based reproductive performance of Nile tilapia Oreochromis niloticus L. broodstock. Aquac Res. 2015; 46(2): 509–512. Publisher Full Text\n\nEmerenciano M, Ballester EL, Cavalli RO, et al.: Biofloc technology application as a food source in a limited water exchange nursery system for pink shrimp Farfantepenaeus brasiliensis (Latreille, 1817). Aquac Res. 2012; 43(3): 447–457. Publisher Full Text\n\nSouza DM, Suita SM, Romano LA, et al.: Use of molasses as a carbon source during the nursery rearing of Farfantepenaeus brasiliensis (Latreille, 1817) in a biofloc technology system. Aquac Res. 2014; 45(2): 270–277. Publisher Full Text\n\nFurtado PS, Gaona CA, Poersch LH, et al.: Application of different doses of calcium hydroxide in the farming shrimp Litopenaeus vannamei with the biofloc technology (BFT). Aquacult Int. 2014; 22(3): 1009–1023. Publisher Full Text\n\nMuchlisin ZA, Nadiya N, Nadiah WN, et al.: Preliminary study on the natural extenders for artificial breeding of African catfish Clarias gariepinus (Burchell, 1822). AACL Bioflux. 2010; 3(2): 119–124. Reference Source\n\nEl Naggar GO, John G, Rezk MA, et al.: Effect of varying density and water level on the spawning response of African catfish Clarias gariepinus: implications for seed production. Aquaculture. 2006; 261(3): 904–907. Publisher Full Text\n\nMuchlisin ZA, Nadiah WN, Nadiya N, et al.: Exploration of natural cryoprotectants for cryopreservation of African catfish, Clarias gariepinus, Burchell 1822 (Pisces: Clariidae) spermatozoa. Czech J Anim Sci. 2015; 60(1): 10–15. Publisher Full Text\n\nSuhendra N: Grouth performance of working catfish Clarias batracus fed an experimental diet with varying levels of protein and energy. Buletin Penelitian Perikanan Darat. 1988; 7(2): 16–23.\n\nAl-Dohail MA, Hashim R, Aliyu-Paiko M: Effects of the probiotic, Lactobacillus acidophilus, on the growth performance, haematology parameters and immunoglobulin concentration in African catfish (Clarias gariepinus, Burchell 1822) fingerling. Aquac Res. 2009; 40(14): 1642–1652. Publisher Full Text\n\nIge BA: Probiotics use in intensive fish farming. Afr J Mic Res. 2013; 7(22): 2701–2711. Publisher Full Text\n\nDennis EU, Uchenna OJ: Use of probiotics as first feed of larval African catfish Clarias gariepinus (Burchell 1822). Annu Res Rev Biol. 2016; 9(2): 1–9. Publisher Full Text\n\nMuchlisin ZA, Arisa AA, Muhammadar AA, et al.: Growth performance and feed utilization of keureling (Tor tambra) fingerlings fed a formulated diet with different doses of vitamin E (alpha-tocopherol). Arch Pol Fish. 2016; 23: 47–52. Publisher Full Text\n\nMuchlisin ZA, Afrido F, Murda T, et al.: The effectiveness of experimental diet with varying levels of papain on the growth performance, survival rate and feed utilization of keureling fish (Tor tambra). Biosaintifika. 2016; 8(2): 172–177. Publisher Full Text\n\nEaton AD, Clesceri LS, Rice EW, et al.: Standard methods for the examination of water and wastewater. 21st edition. American Public Health Association, American Water Works Association, Water Environment Federation. Washington, DC. 2005. Reference Source\n\nWidanarni, Ekasari J, Maryam S: Evaluation of biofloc technology application on water quality and production performance of red tilapia Oreochromis sp. cultured at different stocking densities. Hayati. 2012; 19(2): 73–80. Publisher Full Text\n\nIrianto A: Probiotic for aquaculture. Gadjah Mada University Press, Yogyakarta. 2003.\n\nHastuti S, Subandiyono S: Production performance African catfish (Clarias gariepinus, Burch) is maintained biofloc technology. Fisheries SANTEK Journal. 2014; 10(1): 37–42. Reference Source\n\nAzim ME, Little D, North B: Growth and welfare of Nile tilapia (Oreochromis niloticus) cultured indoor tank using biofloc technology (BFT). Procedings of Aquaculture Conference 2007, 26 February -3 March 2007. San Antonio, Texas, USA. 2007.\n\nMarimuthu K, Ang CC, Muralikrishnan S, et al.: Effect of different feeding frequency on the growth and survival of African catfish (Clarias Gariepinus) fingerlings. Adv Environ Biol. 2010; 4(2): 187–193. Reference Source\n\nJimoh WA, Fagbenro OA, Adeparusi EO: Response of African catfish, Clarias gariepinus (Burchell 1822), fingerlings fed diets containing differently timed wet-heat-treated sesame (Sesamum indicum) seedmeal. Agric Sci. 2014; 5: 1159–1171. Publisher Full Text\n\nRad F, Kurt GI, Bozaoủlu AS: Effects of spatially localized and dispersed patterns of feed distribution on the growth, size dispersion and feed conversion ratio of the African Catfish (Clarias gariepinus). Turk J Vet Anim Sci. 2003; 28: 851–856. Reference Source\n\nAvnimelech Y: Carbon/nitrogen ratio as a control element in aquaculture systems. Aquaculture. 1999; 176(3–4): 227–235. Publisher Full Text\n\nHargreaves JA: Photosynthetic suspended-growth systems in aquaculture. Aquaculture Engineering. 2006; 34(3): 344–363. Publisher Full Text\n\nCrab R, Chielens B, Wille M, et al.: The effect of different carbon sources on the nutritional value of bioflocs, a feed for Macrobrachium rosenbergii postlarvae. Aquac Res. 2010; 41(4): 559–567. Publisher Full Text\n\nPutra I, Rusliadi R, Fauzi M, et al.: Dataset 1 in: Growth performance and feed utilization of African catfish Clarias gariepinus fed a commercial diet reared in the biofloc system enhanced with probiotic. F1000Research. 2017. Data Source"
}
|
[
{
"id": "25287",
"date": "25 Aug 2017",
"name": "Hafrijal Syandri",
"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 comment for abstract: the fish was fed a commercial diet that contain 38% crude protein, twice a day at satiation, could you please show the time?\nA comment for the methodology: 1. Please state the type of commercial feed used? floating or drowned feed? 2. The weight gain of fish measured every 12 day for 60 days, please show the data weight gain every 12-days in bar diagram or line graph? 3. Please explain the version (including city) of software used in statistical 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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "25289",
"date": "05 Sep 2017",
"name": "Rudy Agung Nugroho",
"expertise": [
"Reviewer Expertise Animal Physiology",
"Fish nutrition",
"Fish Immunology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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. Title is suitable and clearly defined the research that has been done.\n2. Abstract is well written and attract the reader. Please add information regarding the CFU of the bacillus (ex: 10 mL inoculants bacteria with density of Bacillus sp. about 5×1010 colony forming units (CFU) ....).\n3. Introduction: the introduction is well constructed and supported with current references.\n4. Methods: a) please be specific on the ethical guidelines that author's performed in this research. Which international/national ethical guidelines. b) Please explain why the author use 5 days interval in this research. Is there any previous/preliminary research?\n5. Results: Survival rate of B2 groups was only 65% (Raw data), any explanation?\n6. Discussion: Good Discussion.\n7. Conclusion: Well constructed summary.\n8. Reference: please revise reference #1 : (Sahwan MF: The feed fish and shrimp. Penebar Swadaya. Jakarta. 1999. ), with original title and give translation. otherwise it cannot be traced.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/6-1545
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https://f1000research.com/articles/6-682/v1
|
16 May 17
|
{
"type": "Method Article",
"title": "Evaluation of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in pure mineral hydrocarbon-based cosmetics and cosmetic raw materials using 1H NMR spectroscopy",
"authors": [
"Dirk W. Lachenmeier",
"Gerd Mildau",
"Anke Rullmann",
"Gerhard Marx",
"Stephan G. Walch",
"Andrea Hartwig",
"Thomas Kuballa",
"Gerd Mildau",
"Anke Rullmann",
"Gerhard Marx",
"Stephan G. Walch",
"Andrea Hartwig",
"Thomas Kuballa"
],
"abstract": "Mineral hydrocarbons consist of two fractions, mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). MOAH is a potential public health hazard because it may include carcinogenic polycyclic compounds. In the present study, 400 MHz nuclear magnetic resonance (NMR) spectroscopy was introduced, in the context of official controls, to measure MOSH and MOAH in raw materials or pure mineral hydrocarbon final products (cosmetics and medicinal products). Quantitative determination (qNMR) has been established using the ERETIC methodology (electronic reference to access in vivo concentrations) based on the PULCON principle (pulse length based concentration determination). Various mineral hydrocarbons (e.g., white oils, paraffins or petroleum jelly) were dissolved in deuterated chloroform. The ERETIC factor was established using a quantification reference sample containing ethylbenzene and tetrachloronitrobenzene. The following spectral regions were integrated: MOSH δ 3.0 – 0.2 ppm and MOAH δ 9.2 - 6.5, excluding solvent signals. Validation showed a sufficient precision of the method with a coefficient of variation <6% and a limit of detection <0.1 g/100 g. The applicability of the method was proven by analysing 27 authentic samples with MOSH and MOAH contents in the range of 90-109 g/100 g and 0.02-1.10 g/100 g, respectively. It is important to distinguish this new NMR-approach from the hyphenated liquid chromatography-gas chromatography methodology previously used to characterize MOSH/MOAH amounts in cosmetic products. For mineral hydrocarbon raw materials or pure mineral hydrocarbon-based cosmetic products, NMR delivers higher specificity without any sample preparation besides dilution. Our sample survey shows that previous methods may have overestimated the MOAH amount in mineral oil products and opens new paths to characterize this fraction. Therefore, the developed method can be applied for routine monitoring of consumer products aiming to minimize public health risks.",
"keywords": [
"mineral oil",
"petrolatum",
"cosmetics",
"hydrocarbons",
"polycyclic aromatic hydrocarbons",
"benzo[a]pyrene",
"magnetic resonance spectroscopy"
],
"content": "Introduction\n\nMineral oil hydrocarbons were suggested as important contaminants of the human body, with possible routes of contamination including air inhalation, food intake, and dermal absorption. A correlation was found between the use of cosmetic products, such as creams or lipsticks, and mineral oil saturated hydrocarbons (MOSH) in human fat tissue and in milk samples collected from women1. The mineral oil aromatic hydrocarbons (MOAH) fraction is under scrutiny because of the genotoxic carcinogenicity of some of its constituents, namely some polycyclic aromatic hydrocarbons (PAH)2. For monitoring of cosmetic products and medicinal products aiming for health risk assessment, a scarcity of analytical methods was noted. This may be explained because the analysis of mineral oil constituents is extremely difficult, and because of their complexity it has been generally unfeasible to resolve the hydrocarbon mixtures into individual components for quantification3. The current method of choice for analysis of hydrocarbons is the application of an online coupled liquid chromatography-gas chromatography with flame ionization detection (LC-GC-FID), which leads to two fractions quantified as sum parameters, MOSH and MOAH4. It is crucial to stress that the acronyms MOSH and MOAH have to be carefully interpreted depending on the actually applied method, e.g. online or offline hyphenated LC-GC-FID or nuclear magnetic resonance (NMR), as described in this paper.\n\nAccording to the principle of LC-GC-FID, MOSH and MOAH fractions are separated from interfering material by LC and are subsumed according to their retention on the gas chromatographic column. But apart from retention behaviour, the assay includes no information about the chemical properties of the compounds. The resulting chromatograms show regions of overlapped peaks for the MOSH and MOAH fractions (so called humps). Chromatographic integration windows for these humps are empirically identified using n-alkanes as markers. The FID has virtually the same response per unit mass for all saturated hydrocarbons and it is only marginally higher for aromatic hydrocarbons, so that the quantification using cyclohexyl cyclohexane (for MOSH) and 1- and 2-methyl naphthalene (for MOAH) as internal standards is possible5,6.\n\nEspecially for the compounds included in the MOAH region, there is no information about the properties of the detected compounds within the defined retention time windows. Specifically no information about the chain length of substituents or how many polycyclic aromatic rings are included, which may have considerable influence on the toxicity of the compounds7. The critical compounds in the class of polycyclic aromatic hydrocarbons (e.g., the European Food Safety Authority (EFSA) PAH4 group, i.e. benz[a]anthracene, benzo[a]pyrene, chrysene and benzo[b]fluoranthene7) are therefore not specifically determined. It is generally difficult to determine single substances in mixtures of multiple compounds such as mineral oils, which may contain several 1000 of substances. On the other hand, it may also not be appropriate to focus the analysis on only a few single compounds, which may overlook some toxicologically important compounds. For the reason of practicability, it was decided to provide such a total view of MOSH/MOAH sum parameters in mineral oil analysis by hyphenated LC-GC-FID.\n\nDue to the problems in using hyphenated LC-GC-FID techniques, which includes requirements for complex equipment, special training of operators and rather long analysis times, the aim of this study was to evaluate another technique, namely NMR spectroscopy for the purpose of MOSH/MOAH analysis. NMR has so far been only applied to determine the relative proportion of aromatic protons in hydrocarbon resins8 or different fractions in cracked gasoline9, but not for quantification of MOSH/MOAH. In contrast to LC-GC-FID, the analysis of aromatic compounds using NMR may be more precise and selective because the physicochemical properties of the chemical structure of the compound is the underlying criterion for the chemical shifts observed in NMR. Hence the NMR evaluation can be regarded as being much more specific because a chemical property (such as an aromatic ring) is evaluated and not just the retention behaviour of compounds.\n\n\nMethods\n\nRaw materials intended to be used as ingredients for cosmetics as well as medicinal products were analysed in the capacity of the CVUA Karlsruhe as Official Medicines and Cosmetics Control Laboratory (OMCL/OCCL) for the German Federal State of Baden-Württemberg.\n\nThe samples were either provided by the local administrative authorities for routine surveillance or were directly obtained using internet-based mail order. The internet-based sampling also included some technical-quality mineral oil products not intended for use in cosmetics, medicines or foods as comparison samples (listed as ‘Technical products’). An overview of the analysed samples is given in Table 1.\n\n(n.d. not detectable).\n\naProducts from different brands and manufacturers were analysed if the same product is listed several times (e.g. for bag balm and vaseline).\n\nThe samples were prepared for measurement by dissolving ~50 mg of sample in 1.5 mL of CDCl3 containing 0.1% tetramethylsilane (TMS) (purity 99%). After membrane filtration using syringe filters with polyester membrane (Chromafil Xtra PET-20/25 0.20 µm, Macherey Nagel, Düren Germany), 600 µl of the solution were poured into an NMR tube for direct measurement.\n\nAll 1H NMR measurements were performed using a Bruker Ascend 400 spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a 5-mm SEI probe PA BBI 400S1 with Z-gradient coils and a Bruker automatic sample changer (Sample Xpress, Bruker Biospin). All spectra were acquired at 300.0K.\n\nNMR spectra were acquired using the Bruker standard zg30 pulse sequence with 32 scans and 2 prior dummy scans (DS). The acquisition parameters were based on a previously described method10.\n\nAll spectra were recorded in the baseopt mode (generates a smooth baseline at zero without offset). The acquisition parameters were kept constant for reference and sample spectra for PULCON measurements according to Monakhova et al.11. For each sample during spectra acquisition, the 90° pulse width (P1 in Bruker terminology) was set at 8 ms, the sweep width (SW) was 20.5504, and the size of the real spectrum (SI) was 131072.\n\nThe data were acquired automatically under the control of ICON-NMR 5.0.6 (Bruker Biospin), requiring 25 min per sample including temperating. All NMR spectra were phased, baseline-corrected and manually integrated using Topspin 3.2 (Bruker Biospin).\n\nThe application of PULCON (PULse length based CONcentration determination) quantification was previously described in detail11. In short, the ERETIC (Electronic REference To access In vivo Concentrations) factor was calculated for each measurement series based on a so-called quantref sample measured as the first sample in the series. The quantref sample was prepared by dissolving 50 mg tetrachloronitrobenzene and 50 mg ethylbenzene in 10 mL of CDCl3 containing 0.1% TMS. The ERETIC factor was determined using the following equation:\n\n\n\nwhere I is the absolute integral, MW is the molecular weight (260.89 for tetrachloronitrobenzene or 106.17 for ethylbenzene), N is the numbers of protons generating the selected signal, and C is the concentration. The following signals were integrated: tetrachloronitrobenzene: δ 7.83-7.64 ppm (N=1, s); ethylbenzene δ 7.23-7.11 ppm (N=3, m), δ 2.75-2.54 ppm (N=2, q), δ 1.35-1.13 ppm (N=3, t). The average ERETIC factor of all four signals was used for further calculations.\n\nThe analyte concentrations in the samples were then calculated using the following equation:\n\n\n\nwhere I is the absolute integral, MW is the molecular weight (128.17 for naphthalene, 138.25 for decalin, 92.09 for glycerol, 228.29 for benz[a]anthracene, 252.32 for benzo[a]pyrene, 228.29 for chrysene, and 252.32 for benzo[b]fluoranthene), ERETIC is the eretic factor (see eq. 1) and N is the numbers of protons generating the selected signal.\n\nThe integral ranges were chosen as follows: MOSH region calculated as decalin equivalents: δ 3.0-0.2 ppm (N=18) except the regions of residual H2O around δ 1.53 ppm; MOAH region calculated as naphthalene equivalents (N=8): δ 9.20–7.55, δ 7.50–7.30, δ 7.22–7.00, and δ 6.97–6.50 (the three regions cut out are due to the residual undeuterated solvent signals of CHCl3 and its 13C satellites); and region of other compounds calculated as glycerol (N=5): δ 6.50-3.00. The integral regions are also shown in Figure 1. For targeted quantification of the EFSA PAH4 group, the following integration regions were used: benzo[a]pyrene δ 9.06–8.98 (N=2), chrysene δ 8.76–8.70 (N=2), benz[a]anthracene δ 9.20–9.12 (N=1) and benzo[b]fluoranthene δ 7.45–7.38 (N=2).\n\nMOAH, mineral oil aromatic hydrocarbons; MOSH, mineral oil saturated hydrocarbons; TMS, tetramethylsilane.\n\nTo check the quality of the value of the ERETIC factor in terms of precise initial balance, sample preparation and NMR experiment, the concentration in a control solution (cyclohexane, 2500 mg l-1, CDCl3, singlet at δ 1.53–1.36 ppm, N = 12) was measured at the end of each measurement series. The recovery had to be 100 ± 5% to perform further calculations for the samples. The limit of detection (LOD) was manually estimated based on a small, but still integratable, signal in the region of PAH412.\n\n\nResults\n\nAn overview of a typical NMR spectrum of a mineral oil-based cosmetic product (final cosmetic product based on pure petroleum jelly) is shown in Figure 1. Both MOSH and MOAH regions show considerable signals, which are however very overlapped due to the multi-mixture character of mineral oils. The MOSH fraction is much more abundant than the MOAH fraction, which is obvious by the much larger signal range between δ 3 and 0.2 ppm. In the MOSH region (magnification in Figure 2), the specific signals for alkane-type CH2 and CH3 groups are separable. In the MOAH region (magnification in Figure 3), such a structural assignment appears not possible as the aromatic protons in the region of δ 7.2 to 6.8 show a much more overlapped behaviour. In the middle of the spectrum, around 4 ppm (Figure 1), a region remains that includes chemical structures not characteristic to either MOSH or MOAH definitions. The region was therefore separately quantified (Table 1, column “other compounds”), and may provide evidence about the magnitude of admixture of non-mineral oil ingredients in the cosmetic formulations.\n\nIn addition to the quality control measure of a control sample in each series, which showed in all cases a coefficient of variation below 5%, a validation by spiking of standard substances into a paraffin oil sample has been conducted (Table 2). The validation results show acceptable recoveries typically between 97 and 102%, but only at the edges of the working range, a higher imprecision was observed (84–111%). The average coefficient of variation (CV) of the validation measurements was 6.1%. Additionally, a reference solution of chrysene (0.55 g/100 g) was measured 6 times with a CV of 2.9% and an average recovery of 99.7%. Finally, the CV of the control sample determined over 10 measurement days was 2.8% and the average recovery was 99.5%. The LOD of the method for PAH4 is in the range of 0.01-0.4 g/100 g (depending on sample weight and spectral background).\n\nTable 1 shows the quantitative results of 27 samples. Most of the samples were vaseline or petroleum jelly type products, which are offered either as cosmetic raw materials, cosmetic products or medicinal products according to EU-Pharmacopeia quality or in a technical grade quality. MOSH and MOAH were quantifiable in all samples. The highest MOAH amounts of 1.10 g/100g and 1.04 g/100 g were found in a vaseline raw material for cosmetics and a technical grade quality vaseline, respectively. The lowest amount of 0.02 g/100 g was detected in a product placed on the market as a final cosmetic product. Liquid mineral oil products were found to have lower MOAH amounts (typically less than 0.1 g/100 g) than solid products.\n\nThe MOSH amount in all samples iterated around 100% as expected in almost pure mineral oil products. The amount of other compounds besides MOSH and MOAH was also generally very low (<1%), with some exceptions of compounded products, such as lip balm or bag balm, which contain other ingredients besides mineral oils. In none of the products were any of the PAH4 group compounds detectable.\n\n\nDiscussion\n\nThe advantages of NMR are the simple sample preparation, which only consists of diluting 50 mg of sample in solvent followed by membrane filtration, and the short measurement time. The measurement time per sample is about 25 min including temperating. NMR is focussed on the actually contained aromatic amounts and hence the results appear to be suitable for toxicological assessment.\n\nThe method validation results were similar to other NMR methods based on PULCON quantification11,13–15 and were judged as acceptable for the application of the method for official cosmetics control purposes16. The LOD of the method was also in agreement with the LOD of <0.1% reported for protons of olefins (δ 4.5-6.7 ppm)8.\n\nAn advantage of NMR is that a full automation (including all steps of spectra processing, integration and calculation) is possible due to the very simple approach of PULCON quantification17. This in combination with the very rapid measurement of NMR allows a very high sample throughput in routine analysis. In the current method development and validation work, the spectra were evaluated only semi-automatically (meaning integration by manual operation of each spectrum). We therefore expect that improved method validation data might be achievable when fully automated spectral processing and integration will be implemented in the next step of routine application of the method.\n\nIn comparison to LC-GC-FID, for which the sum of both fractions (MOSH+MOAH) seldom leads to 100%, it is plausible that this is possible with NMR. During LC-GC-FID only a certain part of the sample is considered by the cutting of certain fractions from the first column to the second column. Additionally, certain integration regions are selected during LC-GC-FID so that not all eluting compounds are included. This corroborates findings by Lommatzsch et al.8, who reported that differences between NMR and LC-GC-FID occur because hydrocarbons of higher molecular weight are not determined by the latter method (leading to lower MOSH amounts in LC-GC-FID than in NMR) and because the saturated part of the molecule is included in the quantification as well (leading to higher MOAH amounts in LC-GC-FID than in NMR). NMR can also simultaneously quantify the range of miscellaneous compounds not belonging to the MOSH/MOAH fractions.\n\nA further advantage of NMR quantification is the use of a specific compound for quantification. Naphthalene-equivalents were chosen as a marker to determine the MOAH fraction, as this is the approach of the European Pharmacopeia method for quantification of total polycyclic aromatic hydrocarbons using UV spectrophotometry18. As a saturated counterpart of naphthalene, we have chosen decalin-equivalents for the MOSH fraction. Decalin is also expected to correspond to the average of aliphatic compounds in mineral oils. However, like in all other techniques applying an index based on single compounds, and due to the variations in composition of mineral hydrocarbons (white oils, petrolatum, microcrystalline waxes, ozokerites, ceresines and paraffines), it can be explained that some samples had MOSH results of over 100%. While NMR clearly gives better selectivity for aromatic structures, similar limitations as in LC-GC-FID have to be accepted, meaning that a complete spectral region is quantified and defined as MOSH or MOAH. This information initially also does not include information about the amount of polycyclic rings. As we have clearly shown that the most relevant signals of PAH compounds are part of the aromatic region (Figure 4), the analysis at least provides information about the maximal possible amount of PAH, which would be much lower in reality.\n\nAs discussed before, the results of our approach and our sample collection show deviations to previous LC-GC-FID results, especially in the toxicologically relevant aromatic region, which were much higher than the NMR results. There is a general lack of literature about MOSH/MOAH contents in cosmetic raw materials. However, compared to the few studies available, our MOAH results are much lower than some data reported by the German Federal Institute for Risk Assessment (BfR)19 with about 1.7–5.0% MOAH in commercial cosmetic products based on petroleum jelly. The results of this study are more consistent with data from Niederer20 that reported a MOAH range of 0.05–4.5% (average 1.2%) in 38 paraffin oils contained in lip-gloss products, but our average is still lower at <1%. Earlier studies with olive oils presenting an offline SPE-GC method assumed that 70–80% of mineral oil is MOSH21. Our results from market samples show that this is almost 100%. The discrepancies to these studies may be interpreted not only by methodological differences, but also by the different sample collectives. We are fully aware that mineral oil contaminants in food from food packaging or other sources have to be strictly distinguished from highly refined mineral oils used as raw materials for cosmetic or medicinal products. Nevertheless, the described discrepancy in terms of the MOSH and MOAH amounts points out the controversy mentioned in the introduction about the definition of MOSH/MOAH and suggests that the results must be carefully interpreted depending on the sample source (contaminant, matrix) and method.\n\nDue to the higher specificity and selectivity of NMR, it must be assumed that the LC-GC approach could overestimate the MOAH fraction by possible co-elution of compounds that have similar retention behaviour, but do not contain aromatic ring systems. We therefore conclude that – at least for the product groups of cosmetics and medicinal products, which are based on more or less pure hydrocarbons (petroleum jelly) - LC-GC results should be complemented by the NMR-approach to perform a suitable risk assessment.\n\nA limitation of using NMR for compounded cosmetics is that the MOAH region is not completely specific for mineral oil aromatic compounds, but may include some other aromatic ingredients as well as certain non-aromatic compounds, such as formic acid and its ester. Therefore, the method is not directly suitable without modifications for compounded cosmetic products (e.g. lipsticks, skin care products), which may contain aromatic ingredients, such as UV filters, preservatives, perfuming or colouring agents. These substances may also contain signals in the aromatic region and therefore lead to an overestimation of MOAH. For these reasons, the current work was mostly focused on pure mineral oil products and raw materials. Future research will include sample preparation steps to separate the MOAH fraction from these other ingredients. For the current study, all these interfering compounds were not expected in most of the researched products, therefore we believe that our NMR method gives reliable results. If such interfering compounds are to be expected in cosmetic products, some form of sample preparation (e.g. clean-up using solid-phase extraction) has to be conducted, and the current results need to be interpreted as tentative and potential overestimations.\n\nWe observed another limitation during preliminary trials with longer chain compounds (wax-like samples), which were not completely soluble in CDCl3 so that under-quantification can be expected for MOSH. This effect has probably no influence on the more toxicologically relevant MOAH compounds, which should be well soluble in CDCl322.\n\nA further limitation is the sensitivity of NMR. This seems to be sufficient for the detection of MOSH and MOAH, but the LOD may be too high for a trace analysis of specific PAH in compounded cosmetic products with small mineral oil amount. Benzo[a]pyrene is limited in mineral hydrocarbon raw materials to 0.005% (w/w) (Annex II number 620 ff. of regulation (EC) No 1223/2009; http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:02009R1223-20170303). However, it is also difficult with any other technique to conduct targeted quantification of single compounds in the complex mixture of mineral oil.\n\n\nConclusions\n\nThe presented method is fit-for-purpose to obtain monitoring data of MOSH/MOAH in raw materials or pure mineral hydrocarbon-based cosmetic products, aimed at market surveillance and public health evaluation. However, risk assessment of MOSH/MOAH in cosmetic products will be a huge challenge in the light of the sum parameter character of these compound groups. The NMR-approach appears to provide an important piece of the puzzle. It is extremely suitable for rapid screening of mineral oil raw materials in terms of MOSH/MOAH. The BfR concluded, based on LC-GC-FID analyses, that MOAH amounts in mineral oil in the percentage range (>1%) are technically avoidable, with the potential to be further reduced to trace amounts19. To substantiate this conclusion on the basis of a statistically representative data set, it is important to firstly develop reliable and reproducible methods, according to article 12 of the EU cosmetics regulation EC/1223/09, and secondly conduct market monitoring.\n\nSuch a market surveillance study of the available mineral hydrocarbons for cosmetics is therefore important to obtain representative data. Mineral hydrocarbon raw materials for cosmetic and medicinal products have to be analysed to get a complete view about statistically founded orientation values for MOAH. Additionally, a specific chromatographic method for the determination of PAH4 appears to be necessary to characterize toxic polycyclic aromatic compounds. Further toxicity studies are necessary, as well as epidemiological studies that need to confirm that MOSH concentrations may accumulate in human fat tissue, with cosmetics being a potentially relevant source of the contamination1,2,23,24.\n\n\nData availability\n\nDataset 1: NMR raw data are provided as JCAMP-DX files in a zipped file. Type of archive file: JCAMP DIFF/DUP with included data types FID+RSPEC+ISPEC; JCAMP version 6.0. The software Topspin 3.2 (Bruker Biospin) was used for data export. The data include the raw and processed spectra of five measurement series, including the 27 samples, as well as quantref and control samples and spectra of standard substances measured for comparison. doi, 10.5256/f1000research.11534.d16120925",
"appendix": "Author contributions\n\n\n\nDWL and TK conceived the study, planned and supervised the measurements and data evaluation. DWL prepared the first draft of the manuscript. GMi, AK, GMa, SGW and AH contributed to the preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors are grateful to Jürgen Geisser for excellent technical assistance.\n\n\nReferences\n\nConcin N, Hofstetter G, Plattner B, et al.: Evidence for cosmetics as a source of mineral oil contamination in women. J Womens Health (Larchmt). 2011; 20(11): 1713–9. PubMed Abstract | Publisher Full Text\n\nBarp L, Kornauth C, Wuerger T, et al.: Mineral oil in human tissues, Part I: concentrations and molecular mass distributions. Food Chem Toxicol. 2014; 72: 312–21. PubMed Abstract | Publisher Full Text\n\nEFSA Panel on Contaminants in the Food Chain (CONTAM): Scientific Opinion on Mineral Oil Hydrocarbons in Food. EFSA J. 2012; 10(6): 2704. Publisher Full Text\n\nBiedermann M, Grob K: On-line coupled high performance liquid chromatography-gas chromatography for the analysis of contamination by mineral oil. Part 1: method of analysis. J Chromatogr A. 2012; 1255: 56–75. PubMed Abstract | Publisher Full Text\n\nDIN EN 16995: Foodstuffs - Vegetable oils and foodstuff on basis of vegetable oils - Determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) with on-line HPLC-GC-FID analysis; German and English version prEN 16995:2016. Berlin, Germany: Beuth Verlag; 2017. Reference Source\n\nTarnow P, Hutzler C, Grabiger S, et al.: Estrogenic Activity of Mineral Oil Aromatic Hydrocarbons Used in Printing Inks. PLoS One. 2016; 11(1): e0147239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEuropean Food Safety Authority (EFSA): Polycyclic Aromatic Hydrocarbons in Food - Scientific Opinion of the Panel on Contaminants in the Food Chain. EFSA J. 2008; 6: 724. Publisher Full Text\n\nLommatzsch M, Biedermann M, Grob K, et al.: Analysis of saturated and aromatic hydrocarbons migrating from a polyolefin-based hot-melt adhesive into food. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2016; 33(3): 473–88. PubMed Abstract | Publisher Full Text\n\nSarpal AS, Kapur GS, Mukherjee S, et al.: PONA analyses of cracked gasoline by 1H NMR spectroscopy. Part II. Fuel. 2001; 80(4): 521–8. Publisher Full Text\n\nKobler H, Monakhova YB, Kuballa T, et al.: Nuclear magnetic resonance spectroscopy and chemometrics to identify pine nuts that cause taste disturbance. J Agric Food Chem. 2011; 59(13): 6877–81. PubMed Abstract | Publisher Full Text\n\nMonakhova YB, Kohl-Himmelseher M, Kuballa T, et al.: Determination of the purity of pharmaceutical reference materials by 1H NMR using the standardless PULCON methodology. J Pharm Biomed Anal. 2014; 100: 381–6. PubMed Abstract | Publisher Full Text\n\nLachenmeier DW, Schönberger T, Ehni S, et al.: A discussion about the potentials and pitfalls of quantitative nuclear magnetic resonance (qNMR) spectroscopy in food science and beyond. Proceedings of the XIII International Conference on the Applications of Magnetic Resonance in Food Science. 2016; 16: 77–85. Publisher Full Text\n\nDreier L, Wider G: Concentration measurements by PULCON using X-filtered or 2D NMR spectra. Magn Reson Chem. 2006; 44(S1): S206–S212. PubMed Abstract | Publisher Full Text\n\nMonakhova YB, Lachenmeier DW, Kuballa T, et al.: Standardless multicomponent qNMR analysis of compounds with overlapped resonances based on the combination of ICA and PULCON. Magn Reson Chem. 2015; 53(10): 821–8. PubMed Abstract | Publisher Full Text\n\nWatanabe R, Sugai C, Yamazaki T, et al.: Quantitative Nuclear Magnetic Resonance Spectroscopy Based on PULCON Methodology: Application to Quantification of Invaluable Marine Toxin, Okadaic Acid. Toxins (Basel). 2016; 8(10): pii: E294. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchönberger T, Monakhova YB, Lachenmeier DW, et al.: Guide to NMR Method Development and Validation - Part I: Identification and Quantification. Eurolab Technical Report No. 01/2014. EUROLAB General Secretariat; 2014. Reference Source\n\nAckermann SM, Dolsophon K, Monakhova YB, et al.: Automated Multicomponent Analysis of Soft Drinks Using 1D 1H and 2D 1H-1H J-resolved NMR Spectroscopy. Food Anal Methods. 2017; 10(3): 827–36. Publisher Full Text\n\nEuropean Pharmacopoeia: Vaselinum album. Ph Eur. 2014; 8: 5201–2.\n\nBfR: Mineralöle in Kosmetika: Gesundheitliche Risiken sind nach derzeitigem Kenntnisstand bei einer Aufnahme über die Haut nicht zu erwarten. Stellungnahme Nr. 014/2015 des BfR vom 26. Mai 2015 [in German]. Berlin, Germany: Bundesinstitut für Risikobewertung (BfR); 2015. Reference Source\n\nNiederer M: Lippenpflegeprodukte/Mineralparaffine (MOSH/MOAH) und allergene Duftstoffe [in German]. Basel, Switzerland: Kantonales Laboratorium. Gesundheitsdepartement des Kantons Basel-Stadt; 2016. Reference Source\n\nMoret S, Barp L, Grob K, et al.: Optimised off-line SPE-GC-FID method for the determination of mineral oil saturated hydrocarbons (MOSH) in vegetable oils. Food Chem. 2011; 129(4): 1898–903. Publisher Full Text\n\nAbdel-Shafy HI, Mansour MSM: A review on polycyclic aromatic hydrocarbons: Source, environmental impact, effect on human health and remediation. Egypt J Petroleum. 2016; 25(1): 107–23. Publisher Full Text\n\nBiedermann M, Barp L, Kornauth C, et al.: Mineral oil in human tissues, part II: characterization of the accumulated hydrocarbons by comprehensive two-dimensional gas chromatography. Sci Total Environ. 2015; 506–507: 644–55. PubMed Abstract | Publisher Full Text\n\nConcin N, Hofstetter G, Plattner B, et al.: Mineral oil paraffins in human body fat and milk. Food Chem Toxicol. 2008; 46(2): 544–52. PubMed Abstract | Publisher Full Text\n\nLachenmeier DW, Mildau G, Krause A, et al.: Dataset 1 in: Evaluation of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in pure mineral hydrocarbon-based cosmetics and cosmetic raw materials using 1H NMR spectroscopy. F1000Research. 2017. Data Source"
}
|
[
{
"id": "22775",
"date": "01 Jun 2017",
"name": "Markus Niederer",
"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 present a new application of NMR to measure MOSH and MOAH in raw materials or in final products (e.g. cosmetics). They present a carefully performed validation of the method and compare the NMR-results with those of established LC-GC methods. Especially, the findings of a possible overestimation of the MOAH fraction or underestimation of the MOSH-fraction by LC-GC seem to be very important in terms of risk assessment. Further, advantages and limitations of NMR are presented very detailed and in an objective manner.\n\nIndexing of the article is recommended because of novelty and technical quality. This paper will be of interest to many scientists in this field of research, especially for toxicological evaluations and for market survey purposes of cosmetic and medicinal products.\nIn my opinion, this manuscript is clearly presented and well organised. It gives adequate references to related work and the abstract provides a quantitative summary.\n\nRemarks:\nThe discussion section could be supplemented with the possibilities of LC-GCxGC-MS in order to characterise components of MOAH.\n\nDiscussion, Page 9, last section: “Due to the higher specificity and selectivity of NMR, it must be assumed… We therefore conclude that …” Please move these statements from the discussion to the conclusion section.\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": "2958",
"date": "22 Aug 2017",
"name": "Dirk W. Lachenmeier",
"role": "Author Response",
"response": "We would like to thank the reviewer for his time and constructive feedback. As requested the discussion was supplemented with mentioning the possibilities of LC-GCxGC-MS. The section was moved from the discussion to the conclusion as requested."
}
]
},
{
"id": "23439",
"date": "13 Jun 2017",
"name": "Lanfranco S. Conte",
"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 submitted manuscript deals with evaluation of MOSH&MAOH that is nowadays an hot topic mainly for foods; a new approach for MOSH and MOAH determination in pure mineral oil based cosmetics and cosmetic raw materials using 1H NMR spectroscopy is described.\nThe NMR methods seem to be standard ones and well established, and already implemented in many instruments even if for different purposes.\nThe technique used seems promising even though, as stated by the author it suffers of approximation: MOAH data are expressed as naphthalene-equivalents, while as known MOAH include a complex mixture of alkylated aromatics, mainly (mono- di- and tri-aromaticis). Maybe a better characterization of these different classes of aromatics could help in reaching better results.\nAs known, PAHs are not typically of petrogenic origin, but mainly originate from combustion processes, so strictly speaking they should not be considered as MOAH (even though they can be present in trace amount in MOH mixture). This should be clarified in the introduction.\nThe author compare results obtained with NMR with those obtained with on-line LC-GC by other authors, but a direct comparison on the same samples is lacking. A comparison with GCxGC data could be also very interesting. Furthermore, it seems worthwhile to check if the sensitiveness of the NMR method meets the requirement usually request for this kind of contaminants.\n\nReference 21 is not cited in a pertinent way. The paper cited deals with optimization of a rapid SPE-GC-FID method for MOSH determination in vegetable oil (not suitable for MOAH determination) and it doesn’t report that 70-80% of mineral oil in olive oil is MOSH, letting to intend that the remaining 20-30% is MOAH (fortunately this is not true). Concerning MOSH content the author added…”Our results from the market samples show that this is almost 100%”. It is not clear if they refer to cosmetics or olive oil. In the latter case they should report the reference or show the data. The paper needs some revision before being reconsidered 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? 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? Yes",
"responses": [
{
"c_id": "2809",
"date": "19 Jun 2017",
"name": "Dirk W. Lachenmeier",
"role": "Author Response",
"response": "Thank you for reviewing our paper. To facilitate our revision, could you please clarify the following point:The paper in Ref. 21 reports: \"The method only determines the MOSH, which is usuallyaround 70–80% of the mineral oil (depending on the source and the raffination).\" (p. 1902, right column, 2nd paragraph of conclusion). We therefore believe that we have not misrepresented the reference and also had no intention to imply indirectly that the remaining 20-30% are MOAH (we will clarify this, of course, during our revision of the paper).However, we also believe that the 70-80% MOSH does not refer to olive oils but to pure mineral oils in general, and this was the reason that we included this statement, which is one of the few quantitative data in the literature available for comparison. Unfortunately, Ref. 21 does not report any reference for the claim of 70-80% MOSH.Could you please clarify your intended meaning in Ref. 21 if this range refers to mineral oils in olive oils or to mineral oils in general, before we try to improve the text of our paper."
},
{
"c_id": "2957",
"date": "22 Aug 2017",
"name": "Dirk W. Lachenmeier",
"role": "Author Response",
"response": "We thank the reviewers for their constructive comments regarding our study. As requested, it was clarified in the introduction that PAHs are not typical constituents of MOAH but may occur as contaminants in this fraction. Regarding comparison with chromatographic methods, see answer above in response #1 to Richard Stadler. Additionally, we want to stress that the sensitivity of NMR is absolutely sufficient for quantification of the MOSH and MOAH fractions (all samples were positive). The sensitivity may only be insufficient for specific quantification of single PAHs, which now has been clarified in the discussion. While we believe that we had correctly cited Ref. 21 (see also comment above) and no clarification from the reviewers was received in the meantime, we have changed the sentence in the discussion to hopefully make this point much clearer, including a specific statement that the remaining 20-30% are not MOAH."
}
]
},
{
"id": "23855",
"date": "28 Jun 2017",
"name": "Richard H. Stadler",
"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 method using NMR to determine MOSH and MOAH in cosmetics and cosmetic raw materials. Overall this work is important, as it provides a complementary approach to screening methods such as GC-FID. I have only a few comments that the authors are invited to comment on:\nClear deviations of NMR versus LC-GC-FID results are described in the paper, for example MOAH amounts are lower by the NMR method. However, no direct comparison on the same sample is reported, which would strengthen their argument.\n\nThe need for a confirmatory approach in foods and food raw materials, albeit using GC-MS, has recently been reported by Spack et al in Food Additives & Contaminants, 2017 (http://dx.doi.org/10.1080/19440049.2017.1306655). This reference should be included on page 10, after the paragraph “…. LC-GC results should be complemented by the NMR-approach (or alternative confirmatory technique such as GC-MS) to perform a suitable risk assessment,……..”.\n\nI am not certain if NMR is the appropriate methodology to determine PAH4, as it will not achieve the required sensitivity / specificity, so less focus should be placed on these compounds. I would suggest that the authors rather identify specific aromatic hydrocarbon markers to possibly identify the origin of the “contamination”, which would be a next step in the research approach.\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": "2956",
"date": "22 Aug 2017",
"name": "Dirk W. Lachenmeier",
"role": "Author Response",
"response": "We thank the referee for his insight and providing suggestions to improve our paper. Regarding direct comparisons between LC-GC-FID and NMR, the referee is correct that direct comparisons of same samples would be preferable. Actually, we have recently purchased the necessary LC-GC-FID equipment, but are still establishing and validating the procedure, which was more challenging than expected. At the current stage, we can therefore not provide the data comparison for all our samples. However three samples, which we had obtained from another laboratory with LC-GC-FID capabilities, confirmed our assumptions. The LC-GC-FID MOAH was 0.8-6.9% while the NMR MOAH was 0.3-1.0%. The LC-GC-FID MOSH was 53-61% while the NMR MOSH was 101-102%. These limited results strengthen our assumption that LC-GC-FID may overestimate MOAH and underestimate MOSH. However, much more samples are needed for a robust conclusion. Thank you for the suggestions. The text suggestion and reference were added as requested. The referee is right that NMR is not appropriate to determine PAH4 due to the required sensitivity (e.g. the DIN EN 16143 procedure using double LC cleaning and GC/MS analysis has a working range of 0.1-15 mg/kg for petroleum products, which cannot be reached by NMR). Nevertheless, NMR would offer the selectivity to distinguish the PAH4 if they should occur at levels above about 0.01%. A sentence was added to the discussion that NMR is not offering sensitivity for targeted quantification of PAH."
}
]
}
] | 1
|
https://f1000research.com/articles/6-682
|
https://f1000research.com/articles/6-1541/v1
|
22 Aug 17
|
{
"type": "Review",
"title": "Protein nano-cages: Novel carriers for optimized targeted remedy",
"authors": [
"Negar Etehad Roudi",
"Neda Saraygord-Afshari",
"Maryam Hemmaty",
"Negar Etehad Roudi",
"Maryam Hemmaty"
],
"abstract": "Since 1980, when the idea of drug-delivery was proposed, various drug-carriers have been developed, including DNA, proteins, liposomes and several other polymer cages, consisting of many well established natural and synthetic nano-particles. All these drug-carriers can self-assemble in the body and can be manipulated for safer delivery into target tissues. By definition, nano-scale drug delivery systems encompass any structure (either cage or particle) in the form of solid colloids, which range in size from 10 nm to 100 nm. Today, optimization of these nano drug-vehicles is a topic in many research centers. Researchers are trying to improve the carrier’s solubility and their loading capacity and also wish to increase the half-life of drug delivery cargos in target tissues. Efforts in recent years have led to the introduction of novel protein nano-cages composed of multiple protein subunits, which self-assemble within a superfine and precise format. Science their introduction these promising structure have shown many unique characteristics, including low toxicity, bio-system compatibility, minor immunogenicity, high solubility, and a relatively easy production in large scale. Herein, we review and discuss the recently developed protein nano-carriers that are used as drug cargos for targeted delivery and/or diagnostic tools.",
"keywords": [
"protein nano-cage",
"targeted therapy",
"drug delivery",
"drug carrier",
"nanotechnology"
],
"content": "Introduction\n\nDrug delivery, which refers to the technologies and approaches used for safely delivering medications into their site of action and enhancing their therapeutic effects, has gained increasing attention over the past decades. The concept of targeted drug delivery is based on the directed delivery of a certain type of drug to a certain type of cell and/or organ. During the past two decades, new approaches have been developed to control and, additionally, to increase drug delivery efficiency, leading to increased treatment competence. For example, increasing the specificity of targeted therapy, controlling the rate for drug release in the target organ and controlling the duration of drug treatment in the tissue1. The concept of controlled drug delivery has been improved gradually, over the last six decades. It began in 1980, by introducing the first sustained release formulation2. The first age of drug carrier development (1980) was focused on the development of oral and transdermal sustained release systems. These efforts have been led to the more sophisticated deciphering of drug release kinetics and the mechanisms involved. Attention during the second milestone (1980–2010) was paid to the development of controlled release systems. Introducing self-regulated drug delivery methods, long-term depot formulations and nanotechnology-based delivery cargos can be mentioned as some of the achievements of this period. Overall, different carriers for drug delivery, in micro and nano-scales have been developed and the number of drug delivery products has been increased dramatically.\n\nNano-scale drug delivery systems, which exhibit a greater therapeutic potential, encompass any nano-particle and/or nano-cage in the form of solid colloids that range in size from 10 to 100 nm1. Different types of materials have been designed for the application in nano-scales, including metal nano-particles (gold, silver), synthetic biodegradable polymers, and natural polymers (DNA, lipids, polysaccharides)3.\n\nGold nano-particles (AuNPs) are widely used as medical cargos, which provide a non-toxic vehicle for drug delivery. In these systems, AuNPs are generally designed so that the golden core meets the stability of the nano-structure, while a coating layer gives the functional properties of the surface, such as charge, hydrophobicity and specificity. An additional alluring property of AuNPs is their ability to interact with thiol functional groups, providing an effective feature for controlled release in the target tissue4.\n\nDNA molecules can be assembled into several predesigned nano-shapes, via an organized hybridization of the sequence-complementary domains5. These folded DNA nano-structures show a great spatial order, which can address their pronounced potential to serve as an applied and effective platform in the fields of bio-nanotechnology, as well as medicine. Of course, low toxicity and biodegradability are the characteristic features of biopolymer-based nano-particles, which leads to a considerable attention to these structures6.\n\nProtein nano-cages are a newly emerging and promising nano-biopolymers. These structures are composed of multiple protein subunits that are self-assembled within a superfine and precise format, while showing an intricate hollow symmetrical cage-like structure at the nano-scale (1/1000 of the average width reported for a strand human hair)4. Considering the characteristic features of these new structures, herein we present and discuss these structures in detail. Different types of protein nano-cages (viral and non-viral) and the methodology of their production will be introduced, and finally the pros and cons of these new nano-cages will be discussed.\n\n\nNon-viral protein nano-cages\n\nProteins that are used in the non-viral protein nano-structures must be capable of showing self-assembly into capsule-like shapes. These proteins naturally exist in nature. This section discusses the four most widely-used proteins: ferritin, small heat shock proteins, β-lactoglobulin and albumin.\n\nThis protein belongs to a family of iron storage proteins and consists of 24 subunits that are assembled to form a hollow sphere, globular protein4. In vertebrates, ferritin subunits are composed of both heavy (H) and a light (L) chains, which are spontaneously assembled together and surround an aqueous cavity within the internal and external diameters of about 8 and 12 nm, respectively. In 1937, ferritin was isolated from horse spleen, but its structural features remained unidentified until 1991, when its crystal structure was resolved and determined7. The Douglas research group was the first to report the characteristics of this protein family. They showed that any changes in the ferritin external surface would not lead to a significant change on its inner surface, which is another fascinating applied feature of this protein8.\n\nProteins of the ferritin family are thermodynamically stable and can tolerate high temperature (up to 85°C) and pH (8.5–9) stress. Hence, they can remain unchanged for a long time even in rigorous ionic situations. In addition, biodegradability and a non-immunogenic manner can be mentioned as the most important characteristics of these proteins, which can be applied widely in medical theranostics. Therefore, this protein family can be broadly used as mineral scaffolds and contrast-agents in MRI, and recently they have been applied for the construction of nano-protein cargoes8.\n\nThe ability of ferritin for binding to a variety of tumor markers makes it a key determinant for a variety of medical applications, especially tumor diagnosis. The H chain of ferritin (Hfn) consists of an arginylglycylaspartic acid (RGD) domain at its N-terminus, by the sequence of -Cys, Phe, Cys, Asp, Gly, Arg, Cys, Asp, Cys-. The RGD domain, also displayed by “C4” symbol (denoting the 4 cysteine residue present in the domain), is the main core of a ferritin protein and participates in its attachment to other structures. Additionally, the RGD domain shows some genetic variations in different species and can be adopted for different applications9. It has been shown that Hfn-c4-RGD (Hfn integrated with RGD or C4 domain) has the ability to target THP-1, a human monocyte cell line that is derived from acute monocyte leukemia10. There are also some other reports that show that the mutant type of Hfn may have a higher performance than its wild type10.\n\nAnother well-known target for the RGD domain of Hfn is epidermal growth factor receptors (EGFR). EGFR is a famous biomarker that shows a high level of expression in some cancer cells. T cell-replacing factor receptor, another highly expressed receptor in cancer cells, is the other target for the RGD domain of ferritin nano-cages8.\n\nOverall, the ability of ferritin nano-particles to co-bind to tumor biomarkers and anti-tumor agents gives them a notable therapeutic property. As an example, the co-binding of the RGD domain of Hfn to both doxorubicin (Dox) and transferrin receptor 1 facilitates the entrance of the drug to tumor cells. However, this targeted and facilitated drug delivery will lead to reduced drug dosage, as well as unwanted multidrug-resistant effect which occurs as the result of reduced peripheral exposure and decreased systemic toxicity via a more effective bio-distribution10,11.\n\nHeat shock proteins (HSP) are a family of proteins that are widely expressed in living cells. They respond to stressful situations, such as heat shock, after which they were first described. The key mission of the HSP family is to maintain the correct 3D structure of proteins that are faced with undesirable denaturing conditions. Different types of HSPs exist in nature, which are named according to their molecular weight - small HSPs (sHSPs) are the category that is placed at the lower end of this classification. One of the most well-established types of sHSPs are those that are derived from Methanococcus jannaschii) MjHSP. MjHSP can form a protein nano-cage by self-assembling of 24 identical subunits into an octahedral structure12. This structure, which is characterized by 16.5 kDa weight and 12 and 6.5 nm size in the outer and inner dimensions, respectively, resembles a typical ferritin nano-cage; but it has a number of large 3 nm pores on its surface, which makes the transport of small molecules across the cage more feasible13,14. In addition, the structure has the benefit of dramatic thermodynamic stability, similarly to ferritin nano-structures15.\n\nMjHSP was first introduced in 2003 by Flenniken et al.16 as a multifunctional nano-scale platform. They demonstrated that this protein nano-cage has variable internal and external surfaces with a variety of reactive amine groups on their structures. The authors also engineered special selectivity for these structures by adding functional thiol groups via polymerase chain reaction-mediated site-directed mutagenesis to enhance the functional reactivity of these nano-structures. Consequently, these genetically modified structures would be able to attach to peptides, various ligand molecules and antibodies, and show versatile therapeutic applications16.\n\nIn addition, the Flenniken research group used a modified thiol group of sHSP to form a hydrogen bond with the maleimide group of Dox, which led to a successful encapsulation and efficient delivery of this chemotherapy agent. Since 24 thiol groups are located in the internal surface of sHSP, it is possible to connect 24 Dox molecules to a single sHSP nano-cage, which can then be gradually released within 24 to 25 hours of incubation in an acidic pH of about 4.5 to 5.5. It has been shown that 50% of these Dox molecules can be released after the first 1.5 hours at pH=5. Effective detachment of Dox from sHSP in lysosomes has been also reported under the same conditions16.\n\nβ-Lactoglobulin (BLG) is a well-known protein that is present in many mammal species. It is the major constituent of the whey obtained from sheep and cow’s milk. It is a relatively small globular protein, and its regular structure comprises of an 8-stranded antiparallel β-sheet, 3-turn structure and an α-helix on the outer surface. Different studies have reported different ligand binding sites on this cone-like protein structure, and have proposed that a variety of hydrophobic ligands can bind successfully to BLG at pH values between 6 and 8.117. For example, retinol and fatty acids can bind to BLG’s calyx binding site. This property of BLG, besides its stability in the acidic pH, makes the structure a noteworthy choice for oral administration of drugs18. Like many other nano-structures, many studies have been conducted to improve the efficacy of nano-BLG particles to use in medical applications. For instance, solvent accessibility of ligands that bind to BLG is the main drawback of this protein as a drug carrier - this problem has been partially fixed by different approaches, such as applying a second coat, such as pectin, to protect the ligand during the delivery process19. Assembly of BLG into nano-structures has been also improved to enhance the capability of these drug delivery systems20,21 .\n\nAlbumins belong to a family of highly water-soluble, globular proteins. Ovalbumin, the main protein of egg white, bovine serum albumin, the albumin derived from cow’s serum, and human serum albumin, the most abundant protein of human serum, can be mentioned as the three major types of this protein family22. The albumin family has been widely used as drug carriers, since they are easily available, have a low cost, have a feasible purification procedure, are stable in a large pH range (4–9), show unusual ligand-binding properties, due to their highly charge nature, and most importantly represent a preferential uptake in inflamed tissue and, surprisingly, tumors. These properties, besides their nontoxic and biodegradable nature, makes them a favorable choice for drug delivery. Assembly of albumins into well-defined nano-structures gives them additional therapeutic advantages, including high tolerance, lack of undesirable interactions with serum, high capacity for drug loading23.\n\n\nVirus-like nano-particles\n\nVirus-like particles (VLPs), are the same as viruses’ capsid or protein coat, with no genetic material. These particles can induce an immune response without the risk of viral infections. Therefore, they can be applied for vaccine development. They can also be used for chemical attachment and genetic fusion. Currently, a variety of VLPs and virus like nano-particles (VLNPs) have been developed and applied.\n\nIn addition to immunogenic properties, VLPs have fascinating characteristics that make them an appropriate choice for nano-medicine, for example high variability in VLP size and shape. VLPs can be formed by association of hemo or hetero viral proteins (VPs), which are all self-assembled together and organized in a highly symmetrical, mono-disperse and stable nano or micro scale structures. Generally speaking, VLNPs can be assembled into icosahedral or helical shapes. Furthermore, although these particles are inherently well suited for nano-medicine, it is also possible to take additional advantage of them by manipulating the content of their functional groups via genetic engineering and changing their constituent of lysine (-NH) or cysteine (-SH) groups. This will be applied where covalent functionalization of VLNPs is the method of choice instead of being a carrier alone. Below, we introduce some of the most relevant VLNPs used as nano-drug carriers.\n\nCowpea chlorotic mottle virus (CCMV). The use of CCMV nanoparticles as drugs and chemical carriers, besides the manipulation of their functional groups to enhance their capabilities, was first made by Douglas et al. in 2002. These authors also tried to mimic the functionality of ferritin, as a well-established iron reservoir in living organisms8. Today, the structural features of these viral nano-particles have been well defined. We know that CCMV’s protective coat consists of 180 identical protein subunits, each composed of 190 amino acids. These identical subunits are self-assembled together to form an icosahedral protein cage, which represents a T=3 symmetry. This complex structure forms in a way so that the amine terminals of the protein chains locate in the interior side of the cavity, making it a favorable option to carry negatively charged components24. This nano-particle, which is 18 nm wide in its interior cavity and 28 nm wide as a whole (twice the diameter of ferritin), shows a very dynamic structure. For example, reassembly, disassembly and swelling of this structure due to pH alteration is a feature of its dynamicity. This is an important point because researchers can utilize the reversible swelling property of CCMV nano-cages, hence the process can lead to the formation of 60 openings of 2 nm diameters, suitable for an appropriate pH-dependent load and release of the medical cargo25.\n\nCCMVs can be easily obtained from the infected leaves of the virus corresponding host, Vigna unguiculata. It can also be obtained by the means of genetic engineering and the use of E. coli or yeast expression systems. When genetic engineering is the method of choice, it would be possible to make some pseudo particles with different functionality. For example, by truncating the N-terminus of the capsid’s subunits, the number of the subunits, present in the resulting particles, will be changed, along with the order of symmetry and shape, rather than icosahedral structures26.\n\nCowpea mosaic virus (CPMV). CPMV is another plant virus with an icosahedral structure. CPMV particles show interesting properties for drug delivery systems. For example, the recovery yield of these particles from their infected host, Vigna unguiculata, is relatively high and cost-effective. The amino acid composition and arrangement of these nano-structures is another outstanding feature. CPMV’s capsid is composed of 60 protein subunits, mainly of two types, small 24 kDa and large 41 kDa units27. These are self-assembled in a 28 nm structure, in which lysine and cysteine residues are located on the outer surface and make them a suitable carrier for many chemicals and drugs. They can also be used to improve the functionality of the structure. For example, the formation of polyethylene glycol (PEG)-maleimide ligands, which aid in reducing the particle’s hydrophilicity and enhance their biocompatibility. Different ligands, such as carbohydrates, antibodies, oligonucleotides, organic dyes, biotin, peptides and proteins, react with the cysteine residues on the outer surface of the nano-cage28. In addition to these characteristics features, CPMV particles have shown good stability against harsh chemical modifications (e.g., when conjugated with PEG), and a wide range of temperature alteration, solvent and pH fluctuations, which are all vital for drug delivery systems.\n\nMS2 bacteriophage. MS2, the pathogen of the Enterobacteriaceae family, is an icosahedral bacteriophage. Its capsid is formed by self-assembly of 180 identical protein subunits into a 27 nm porous structure (32 pores of 1.8 nm in diameter). This protein nano-cage is stable against harsh environmental changes, such as wide variations in pH range (3–10) and temperature fluctuations29. The porous structure of the MS2 protein nano-cage has good stability against harsh environmental changes, making the particle a very good platform for chemical modifications and functional alterations.\n\nThe Wu research group (1995) was the first to try targeted drug delivery using the MS2 bacteriophage30. The RNA genome of this bacteriophage consists of a specific region known as translational repression RNA stem-loop (TR), which naturally has an affinity to the virus coat protein. The affinity of TR for the viral coat not only prevents the binding of ribosomes and inhibits the translation of the replicase cistron, but also plays an important role in the assembly of viral particles. The Wu research group used this affinity to encapsulate drugs into the MS2 viral cage. They successfully attached the ricin A chain (a highly fatal herbal toxin) to the TR region of the bacteriophage and showed that multiple copies of the TR-RAC conjugate can be incorporated into a single viral shell. They also made some genetic and chemical manipulations on the MS2’s protein coat to add human transferrin to the surface of the shell, in order to establish receptor-mediated endocytosis in target cells30. Later, this work was followed by other researchers worldwide. For example, in 2002 a research group from the University of Leeds, manipulated the MS2 viral shell via a site-directed mutagenesis and made chimeric VLPs, which were strongly immunogenic when carrying either B or T cell epitopes. This group not only made use of a directed immunogenic response of these manipulated particles, but could also protect and effectively deliver nucleic acid-based drug cargoes31,32.\n\nQβ bacteriophage. Qβ bacteriophage is another icosahedral virus with a diameter of 28 nm and a T=3 symmetry. The protein coat of this virus consists of 180 identical proteins, of 14 kDa molecular weight. Therefore, the structure is very similar to the MS2 bacteriophage33. Like other protein nano-particles, many studies have been conducted to make use of the Qβ bacteriophage as an efficient drug vehicle. The onset of these efforts goes back to the Strable et al. (2008) who successfully incorporated unnatural amino acids into the Qβ VLPs and made some notable structural changes from the natural coat, and investigated the stability and properties of the new structure34. Many other researchers have been published since then, which report successful functionalization and/or targeting of Qβ VLPs. For instance, attachment of human transferrin to the bacteriophage protein coat35, modification of Qβ coat so that viral particles can successfully target CD-22 for the treatment of certain autoimmune disorders and cancers36. The most recently published work is by Chen et al. (2016) who reduced the cytotoxicity of Dox by dual functionalization of Qβ VLP by PEG and this chemotherapy agent37.\n\nSimian vacuolating virus 40 (SV40). SV40 is a DNA onco-virus that is found in both monkeys and humans, and belongs to the family of Polyomaviridae that all have an icosahedral structure. Viral proteins that participate in the structure of SV40 varies depending on the early or late translation of the virus genome. The main coat protein of SV40 is viral protein 1 (VP1), which is produced by early transcription of this viral genome with a Svedberg constant of 16. Late transcription of the SV40 genome not only yields VP1, but also two other smaller (19s) viral proteins, VP2 and VP3. A SV40 virus that only consists of VP1 yields a T=7 d (triangulation number 7 dextro) in a 45 nm icosahedral structure composed of 72 pentamers assembled together38,39.\n\nLike the other VLPs, SV40 VLPs can also be used as a drug delivery system. The variable structure of these VLPs, influenced by the types of VPs, provides a specific flexibility in design and applications. Many studies have been focused on specific targeting, as well as functionalization of SV40 VLPs. For instance, genetic modifications of the core proteins for directed targeting40,41, gene delivery and transduction of multiple cell type and so on42–44.\n\nMurine polyomavirus. Like SV40, murine polyomavirus belongs to the family of Polyomaviridae. The protein coat of this virus is composed of an outer layer, which resembles SV40’s structure, and forms via self-assembly of 360 VP1 units, ordered in 72 pentamers and a T=7 icosahedral structure. However, it differs from SV40 due to the presence of an additional inner layer, which is composed of both VP2 and VP3 subunits45.\n\nIn 2005 Brinkman and his colleagues surveyed the beneficial therapeutic effects with different particulate structures of murine polyomavirus VP1-coat protein. They showed that polyomavirus-like-particles (PLPs) represent a highly potent antigen-delivery system for inducing cytotoxic T lymphocyte responses, which can be applied for the treatment of malignant diseases via immunotherapeutic approaches46. Many other notable studies have focused on the genetic manipulation of VP1 subunits of the PLP outer layer in order to render some specific functionalities to these VLPs. For instance, Schmidt et al. added a special protein domain named WW (because of its two conserved tryptophan residues) to PLP’s VP1s46. Via this modification, PLPs gained a specific affinity for proline-rich ligands. The authors showed that this affinity leads to the efficient encapsulation of a variety of ligands to the PLPs47. Attachment of charged groups to the interior layer of the PLPs48, and efficient encapsulation of the foreign cargoes via an anchoring technique for VP1s49 can also be mentioned as some other examples.\n\n\nGeneral strategies to produce protein nano-cages\n\nThere are two main strategies for providing proteins involved in a nano-cage structure. As mentioned, a simple approach is to obtain and purify viral and non-viral proteins from their natural sources. For example, MjHSP from Methanococcus jannaschii I or CCMV from Vigna unguiculata. The other approach is to produce both viral and non-viral proteins by the means of genetic engineering followed by sufficient expression in a suitable host (in most cases E. coli). The expressed proteins will be purified and undergo an assembly/disassembly process by the application of some gentle environmental changes (such as pH changes), in the presence of or absence of the drugs to be delivered. For the latter approach, much more consideration should be applied for constructing viral protein nano-particles due to their hazards and complexity. Using genetic engineering, the possibility for manipulation of the structure for a desired purpose can also be provided by the researcher50–52.\n\n\nConclusions\n\nToday, nano-protein carriers, which are produced and designed via bioengineering and nanotechnology approaches, can be introduced as an effective platform for drug delivery. Since this idea has been modeled from nature, it can be applied in many medical applications, passing many of the adverse effects associated with the other common platforms.\n\nThe perfect and definite structure of nano protein-carriers - low toxicity, biodegradability, biocompatibility, low and/or directed immunogenicity, size variability, ease of functionalization, opportunity of large scale production, water solubility - are the unique characteristics of nano protein-carriers in comparison to other delivery media, such as gold nano-particles and lipid nano-particles. Hence, although nano protein-carriers are in their infancy and have not yet been tested in clinical trials, their characteristic features will lead to a vast demand for exploration of these structures and development of their biomedical applications in the near future.",
"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\nSafari J, Zarnegar Z: Advanced drug delivery systems: Nanotechnology of health design A review. J Saudi Chem Soc. 2014; 18(2): 85–99. Publisher Full Text\n\nPark K: Controlled drug delivery systems: past forward and future back. J Control Release. 2014; 190: 3–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChatterji A, Burns LL, Taylor SS, et al.: Cowpea mosaic virus: from the presentation of antigenic peptides to the display of active biomaterials. Intervirology. 2002; 45(4–6): 362–370. PubMed Abstract | Publisher Full Text\n\nKhan AK, Rashid R, Murtaza G, et al.: Gold Nanoparticles: Synthesis and Applications in Drug Delivery. Trop J Pharm Res. 2014; 13(7): 1169–1177. Publisher Full Text\n\nSuci PA, Varpness Z, Gillitzer E, et al.: Targeting and photodynamic killing of a microbial pathogen using protein cage architectures functionalized with a photosensitizer. Langmuir. 2007; 23(24): 12280–12286. PubMed Abstract | Publisher Full Text\n\nLohcharoenkal W, Wang L, Chen YC, et al.: Protein Nanoparticles as Drug Delivery Carriers for Cancer Therapy. Biomed Res Int. Hindawi Publishing Corporation; 2014; 2014: 180549. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUchida M, Willits DA, Muller K, et al.: Intracellular distribution of macrophage targeting ferritin-iron oxide nanocomposite. Adv Mater. 2009; 21(4): 458–462. Publisher Full Text\n\nDouglas T, Dickson DP, Betteridge S, et al.: Synthesis and Structure of an Iron(III) Sulfide-Ferritin Bioinorganic Nanocomposite. Science. 1995; 269(5220): 54–7. PubMed Abstract | Publisher Full Text\n\nToita R, Murata M, Abe K, et al.: Biological evaluation of protein nanocapsules containing doxorubicin. Int J Nanomedicine. 2013; 8(1): 1989–1999. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchoonen L, van Hest JC: Functionalization of protein-based nanocages for drug delivery applications. Nanoscale. 2014; 6(13): 7124–41. PubMed Abstract | Publisher Full Text\n\nMundra V, Li W, Mahato RI: Nanoparticle-mediated drug delivery for treating melanoma. Nanomedicine (Lond). 2015; 10(16): 2613–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMura S, Nicolas J, Couvreur P: Stimuli-responsive nanocarriers for drug delivery. Nat Mater. 2013; 12(11): 991–1003. PubMed Abstract | Publisher Full Text\n\nFlenniken ML, Liepold LO, Crowley BE, et al.: Selective attachment and release of a chemotherapeutic agent from the interior of a protein cage architecture. Chem Commun (Camb). 2005; (4): 447–449. PubMed Abstract | Publisher Full Text\n\nUchida M, Kosuge H, Terashima M, et al.: Protein cage nanoparticles bearing the LyP-1 peptide for enhanced imaging of macrophage-rich vascular lesions. ACS Nano. 2011; 5(4): 2493–2502. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim KK, Kim R, Kim SH: Crystal structure of a small heat-shock protein. Nature. 1998; 394(6693): 595–599. PubMed Abstract | Publisher Full Text\n\nZhen Z, Tang W, Guo C, et al.: Ferritin nanocages to encapsulate and deliver photosensitizers for efficient photodynamic therapy against cancer. ACS Nano. 2013; 7(8): 6988–96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuresan S, van der Bent A, de Wolf FA: Interaction of beta-lactoglobulin with small hydrophobic ligands as monitored by fluorometry and equilibrium dialysis: nonlinear quenching effects related to protein--protein association. J Agric Food Chem. 2001; 49(5): 2609–18. PubMed Abstract | Publisher Full Text\n\nPanyam J, Labhasetwar V: Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev. 2003; 55(3): 329–47. PubMed Abstract | Publisher Full Text\n\nIzadi Z, Divsalar A, Saboury AA, et al.: β-lactoglobulin-pectin Nanoparticle-based Oral Drug Delivery System for Potential Treatment of Colon Cancer. Chem Biol Drug Des. 2016; 88(2): 209–16. PubMed Abstract | Publisher Full Text\n\nTorchilin VP: Micellar nanocarriers: pharmaceutical perspectives. Pharm Res. 2007; 24(1): 1–16. PubMed Abstract | Publisher Full Text\n\nGhalandari B, Divsalar A, Saboury AA, et al.: β-Lactoglobulin nanoparticle as a chemotherapy agent carrier for oral drug delivery system. Journal of the Iranian Chemical Society. 2015; 12(4): 613–619. Publisher Full Text\n\nMcAlpine AS, Sawyer L: β-lactoglobulin: a protein drug carrier? Biochem Soc Trans. 1990; 18(5): 879. PubMed Abstract | Publisher Full Text\n\nBatrakova EV, Kabanov AV: Pluronic block copolymers: evolution of drug delivery concept from inert nanocarriers to biological response modifiers. J Control Release. 2008; 130(2): 98–106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKratz F: Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Release. 2008; 132(3): 171–183. PubMed Abstract | Publisher Full Text\n\nBairagi U, Mittal P, Mishra B: Albumin: A Versatile Drug Carrier. Austin Therapeutics. 2015; 2(2). Reference Source\n\nGillitzer E, Willits D, Young M, et al.: Chemical modification of a viral cage for multivalent presentation. Chem Commun (Camb). 2002; (20): 2390–2391. PubMed Abstract | Publisher Full Text\n\nGillitzer E, Suci P, Young M, et al.: Controlled ligand display on a symmetrical protein-cage architecture through mixed assembly. Small. 2006; 2(8–9): 962–966. PubMed Abstract | Publisher Full Text\n\nGanta S, Devalapally H, Shahiwala A, et al.: A review of stimuli-responsive nanocarriers for drug and gene delivery. J Control Release. 2008; 126(3): 187–204. PubMed Abstract | Publisher Full Text\n\nPrasuhn DE Jr, Singh P, Strable E, et al.: Plasma clearance of bacteriophage Qbeta particles as a function of surface charge. J Am Chem Soc. 2008; 130(4): 1328–1334. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKawano M, Matsui M, Handa H: SV40 virus-like particles as an effective delivery system and its application to a vaccine carrier. Expert Rev Vaccines. 2013; 12(2): 199–210. PubMed Abstract | Publisher Full Text\n\nWu M, Brown WL, Stockley PG: Cell-specific delivery of bacteriophage-encapsidated ricin A chain. Bioconjug Chem. 1995; 6(5): 587–595. PubMed Abstract | Publisher Full Text\n\nBrown WL, Mastico RA, Wu M, et al.: RNA bacteriophage capsid-mediated drug delivery and epitope presentation. Intervirology. 2002; 45(4–6): 371–80. PubMed Abstract | Publisher Full Text\n\nFu Y, Li J: A novel delivery platform based on Bacteriophage MS2 virus-like particles. Virus Res. 2016; 211: 9–16. PubMed Abstract | Publisher Full Text\n\nGolmohammadi R, Fridborg K, Bundule M, et al.: The crystal structure of bacteriophage Q beta at 3.5 A resolution. Structure. 1996; 4(5): 543–554. PubMed Abstract | Publisher Full Text\n\nSen Gupta S, Kuzelka J, Singh P, et al.: Accelerated bioorthogonal conjugation: a practical method for the ligation of diverse functional molecules to a polyvalent virus scaffold. Bioconjug Chem. 2005; 16(6): 1572–1579. PubMed Abstract | Publisher Full Text\n\nStrable E, Prasuhn DE Jr, Udit AK, et al.: Unnatural amino acid incorporation into virus-like particles. Bioconjug Chem. 2008; 19(4): 866–875. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRhee JK, Baksh M, Nycholat C, et al.: Glycan-targeted virus-like nanoparticles for photodynamic therapy. Biomacromolecules. 2012; 13(8): 2333–2338. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen Z, Li N, Chen L, et al.: Dual Functionalized Bacteriophage Qβ as a Photocaged Drug Carrier. Small. 2016; 12(33): 4563–71. PubMed Abstract | Publisher Full Text\n\nKler S, Asor R, Li C, et al.: RNA encapsidation by SV40-derived nanoparticles follows a rapid two-state mechanism. J Am Chem Soc. 2012; 134(21): 8823–8830. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKawano T, Murata M, Piao JS, et al.: Systemic delivery of protein nanocages bearing CTT peptides for enhanced imaging of MMP-2 expression in metastatic tumor models. Int J Mol Sci. 2014; 16(1): 148–58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmidt U, Günther C, Rudolph R, et al.: Protein and peptide delivery via engineered polyomavirus-like particles. FASEB J. 2001; 15(9): 1646–8. PubMed Abstract | Publisher Full Text\n\nSchmidt U, Rudolph R, Böhm G: Binding of external ligands onto an engineered virus capsid. Protein Eng. 2001; 14(10): 769–74. PubMed Abstract | Publisher Full Text\n\nKimchi-Sarfaty C, Alexander NS, Brittain S, et al.: Transduction of multiple cell types using improved conditions for gene delivery and expression of SV40 pseudovirions packaged in vitro. Biotechniqes. 2004; 37(2): 270–275. PubMed Abstract\n\nSun X, Li W, Zhang X, et al.: In Vivo Targeting and Imaging of Atherosclerosis Using Multifunctional Virus-Like Particles of Simian Virus 40. Nano Lett. 2016; 16(10): 6164–6171. PubMed Abstract | Publisher Full Text\n\nGeorgens C, Weyermann J, Zimmer A: Recombinant virus like particles as drug delivery system. Curr Pharm Biotechnol. 2005; 6(1): 49–55. PubMed Abstract | Publisher Full Text\n\nRamqvist T, Dalianis T: Lessons from immune responses and vaccines against murine polyomavirus infection and polyomavirus-induced tumours potentially useful for studies on human polyomaviruses. Anticancer Res. 2010; 30(2): 279–84. PubMed Abstract\n\nBrinkman M, Walter J, Grein S, et al.: Beneficial therapeutic effects with different particulate structures of murine polyomavirus VP1-coat protein carrying self or non-self CD8 T cell epitopes against murine melanoma. Cancer Immunol Immunother. 2005; 54(6): 611–22. PubMed Abstract | Publisher Full Text\n\nSchmidt U, Rudolph R, Böhm G: Mechanism of assembly of recombinant murine polyomavirus-like particles. J Virol. 2000; 74(4): 1658–1662. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBellini M, Mazzucchelli S, Galbiati E, et al.: Protein nanocages for self-triggered nuclear delivery of DNA-targeted chemotherapeutics in Cancer Cells. J Control Release. Elsevier. 2014; 196: 184–96. PubMed Abstract | Publisher Full Text\n\nAbbing A, Blaschke UK, Grein S, et al.: Efficient intracellular delivery of a protein and a low molecular weight substance via recombinant polyomavirus-like particles. J Biol Chem. 2004; 279(26): 27410–21. PubMed Abstract | Publisher Full Text\n\nHoward Hughes Medical Institute: Large protein nanocages could improve drug design and delivery. 2016. Reference Source\n\nKing NP, Bale JB, Sheffler W, et al.: Accurate design of co-assembling multi-component protein nanomaterials. Nature. 2014; 510(7503): 103–108. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "26080",
"date": "09 Oct 2017",
"name": "John G. Hardy",
"expertise": [
"Reviewer Expertise Chemistry",
"pharmacy",
"biomedical engineering",
"chemical engineering",
"drug delivery",
"tissue engineering",
"neuromodulation"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an interesting overview of protein nanocage-based drug delivery systems, which gives the reader a taster of the interesting properties of this class of nanoscale drug delivery system. The review is on the whole well written, but in need of a little editing of the text (examples of the edits are listed below).\nThe review article would benefit greatly from the addition of figures to help to explain the topic visually to the reader. Please ensure the figures have the appropriate citation and text from the publisher (typically something like: \"This image is reproduced from XXX with permission of the publisher\")\nPlease change the title \"Protein nano-cages: Novel carriers for optimized targeted remedy\" to read \"Protein nanocages: Novel carriers for optimized targeted remedy\"\nThe idea of \"Drug Delivery\" has been around for somewhat longer than 1980, however it is likely the authors intended to include the word \"controlled\" prior to drug delivery to differentiate this from the typical burst release profiles seen with drugs delivered via injections or oral administration.\nPlease change all instances of \"Nano-scale\" to read \"Nanoscale\"\n\"Protein nano-cages are a newly emerging and promising nano-biopolymers.\" should read \"Protein nano-cages are an emerging technology with promise for drug delivery.\"\nPlease change \"and recently they have been applied for the construction of nano-protein cargoes\" to read \"and recently they have been applied for the construction of protein based delivery systems\"\nPlease change \"bio-distribution\" to read \"biodistribution\"\nPlease change \"nano-cage\" to read \"nanocage\"\nPlease change \"nano-particles\" to read \"nanoparticles\"\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": "26311",
"date": "25 Oct 2017",
"name": "Daniela Belletti",
"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\nReview “Protein nano-cages: Novel carriers for optimized targeted Remedy” reviews recent trend in use of protein as nanometric tools for drug delivery and diagnoses. Overall it's a well-written, quite accurate review, but needs some revision before to be indexed.\n\nAbstract is not clear and contains many conceptual error or typos.\n- “Since 1980, when the idea of drug-delivery was proposed, various drug-carriers have been developed, including DNA, proteins, liposomes and several other polymer cages, consisting of many well established natural and synthetic nano-particles.\" This sentence resulted not clear and too confusing: DNA, protein are material for drug delivery systems; liposomes are drug delivery systems composed by lipid.\n- \"All these drug-carriers can self-assemble in the body and can be manipulated for safer delivery into target tissues.\" This concept is wrong, drug carrier not self assemble in the body.\n- The term “carrier’s solubility” is not correct, It is a colloidal system in which carrier is dispersed in the medium.\n- “Science their introduction” is “Since their introduction”.\n\nIntroduction reports many general concepts regarding the drug delivery systems produced during years. This part contains some information not useful for the general content of the review. A comment/comparison between synthetic and natural self assembling drug delivery systems should be more properly.\n\nIntroduction of table or image could make the review more clear.\n\nThe condition for the assembly of protein into nanocage are completely missing.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1541
|
https://f1000research.com/articles/6-1536/v1
|
22 Aug 17
|
{
"type": "Research Note",
"title": "A bibliometric analysis of the global research on sofosbuvir",
"authors": [
"Akram Hernández-Vásquez",
"Diego Rosselli",
"Diego Rosselli"
],
"abstract": "In this article, we examine the research on sofosbuvir with a bibliometric analysis of global research production. The study of sofosbuvir has been a field of intense research in the past few years, with Latin American contributions playing a modest role. With continued drug development and approval of hepatitis C antivirals, research is expected to increase. Our findings will assist scholars and policy makers in their efforts to improve scientific research policies, with the goal of maximizing the access to treatments, especially in low and middle-income countries.",
"keywords": [
"Sofosbuvir",
"Hepatitis C",
"HCV",
"Bibliometric",
"Scientometric",
"Scientific production",
"Bibliographic Database",
"Network Analysis"
],
"content": "Introduction\n\nHepatitis C virus (HCV) has a major impact on public health, with around 170 million people in the world being affected1. In Latin America, the prevalence of hepatitis C has been estimated to be at around 1.6% of the adult population, and the most common genotypes are 1 and 32,3. The new treatments for HCV include direct-acting antivirals (DAAs), which shorten length of therapy, improve sustained virologic response rates, and minimize side effects4.\n\nSofosbuvir is one of the most important DAAs in the market today, but high prices have led to a large increase in spending by health systems and can be a barrier to rapid global treatment, especially in low and middle-income countries5. The identification of global research on DAAs might lead to new insights into the treatments of HCV and suggest research directions.\n\nBased on the above, our study aimed to identify and explore the worldwide development of sofosbuvir research.\n\n\nMethods\n\nWe performed a bibliometric analysis using the original articles indexed in Web of Science. The search strategy used the following MeSH and non-MeSH terms in the title field: Sofosbuvir, Sovaldi, PSI 7977, and GS 7977. The validity of the search strategy was tested by manually reviewing retrieved articles. Bibliometric indicators were investigated by analyzing annual research output, languages, countries, journals, authors, institutions, and citations. Indicators were analyzed with the option “Analyze Results” and “Create Citation Report” in Web of Science. Author co-citation analysis (ACA) was presented as network visualization map using VOSviewer (version 1.6.4) techniques6.\n\n\nResults\n\nA total of 341 publications for the period of 2010–2017 (up to March 31, 2017) were retrieved and assessed. There were a total of 126 journals that published research on sofosbuvir. Twenty-four articles were economics-based. The three most prolific journals were Hepatology (31 articles), Gastroenterology (17), and Journal of Hepatology (17), responsible for 19.1% of the total publication output. The retrieved documents were published by 46 different countries. The largest contributors in absolute number of articles were USA (220), France (47), and Germany (43). Only one article was from Latin America (Brazil), and it was about sofosbuvir and Zika7. The total number of authors for all articles was 2044. John G. McHutchison from Gilead Science (GS) published the most documents in this field (55), followed by William T. Symonds from GS (30), Diana M. Brainard from GS, and Eric Lawitz from Texas Liver Institute/University of Texas Health Science Center (28). Gilead Sciences (131), Bristol Myers Squibb (38), and Merck (27) were the three major funders. A total of 86 articles were from GS, 30 from Inova Fairfax Hospital, and 26 were from University of Texas. The sum of the citations related to the published articles was 10036. Average citations per item were 29.4, with an h-index of 46.\n\nThe number of authors included in the ACA was based on a minimum number of fifteen articles per author. The map produced included 20 authors distributed into three clusters (red, blue, and green), as shown in Figure 1. The red cluster included eleven authors (headed by John G. McHutchison from GS), the blue cluster included five (headed by Eric Lawitz from Texas Liver Institute/University of Texas Health Science Center), and the green cluster included four authors (headed by Zoibar Younossi from Inova Fairfax Hospital).\n\nMinimum of fifteen articles per author, 20 authors were included.\n\n\nConclusions\n\nAccording to our bibliometric analysis, the study of sofosbuvir has been a field of intense research in the past few years. Developed countries have had an enormous impact on the global research in the field. Recently, interest has focused on the use of sofosbuvir to treat Zika infection, and an important contribution to the body of sofosbuvir research has been supported by its manufacturer. With continued drug development and approval of hepatitis C antivirals, research is expected to continue increasing.\n\n\nData availability\n\nDataset 1: Data obtained from Web of Science. CSV contained list of studies included. Doi, 10.5256/f1000research.12314.d1744248",
"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\nde Oliveria Andrade LJ, D'Oliveira A, Melo RC, et al.: Association between hepatitis C and hepatocellular carcinoma. J Glob Infect Dis. 2009; 1(1): 33–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMohd Hanafiah K, Groeger J, Flaxman AD, et al.: Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology. 2013; 57(4): 1333–42. PubMed Abstract | Publisher Full Text\n\nMessina JP, Humphreys I, Flaxman A, et al.: Global distribution and prevalence of hepatitis C virus genotypes. Hepatology. 2015; 61(1): 77–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIm GY, Dieterich DT: Direct-acting antiviral agents in patients with hepatitis C cirrhosis. Gastroenterol Hepatol (N Y). 2012; 8(11): 727–65. PubMed Abstract | Free Full Text\n\nHill A, Simmons B, Gotham D, et al.: Rapid reductions in prices for generic sofosbuvir and daclatasvir to treat hepatitis C. J Virus Erad. 2016; 2(1): 28–31. PubMed Abstract | Free Full Text\n\nvan Eck NJ, Waltman L: Software survey: VOSviewer, a computer program for bibliometric mapping. Scientometrics. 2010; 84(2): 523–538. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSacramento CQ, de Melo GR, de Freitas CS, et al.: The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication. Sci Rep. 2017; 7: 40920. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHernández-Vásquez A, Rosselli D: Dataset 1 in: A bibliometric analysis of the global research on sofosbuvir. F1000Research. 2017. Data Source"
}
|
[
{
"id": "25248",
"date": "04 Sep 2017",
"name": "Juliana Gonçalves Reis",
"expertise": [
"Reviewer Expertise Bibliometric analysis",
"public health",
"VOSviewer."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMethods\nWas it necessary to clean and standardize the data? Describe the option of the bibliographic database wos. Why was it used a high limit for the number of documents per author?\n\nResults\nI suggest a chart of the distribution number of documents per year. I suggest a network of articles keywords with the description of the clusters.\n\nConclusions\nWhat are the main gaps? What are the possibilities for future 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "26177",
"date": "25 Sep 2017",
"name": "Samy A Azer",
"expertise": [
"Reviewer Expertise Medical education",
"bibliometrics",
"clinical pharmacology",
"gastroenterology hepatology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the above titled article. However there are several issues/problems that need to be improved.\n\nAbstract The whole abstract should be rewritten. We need first to know the rationales of the study, or why is this study needed? What is the research question? What is the aim of the study? Then under state the search engine used and key words used in the search, and briefly state how did you process findings. Then state the bibliometric parameters that you aimed to study. State key results that answered your research question and stated under methods (follow the same sequence). Then state 2-3 lines summarizing key lessons learnt from your findings or what we can conclude from your study findings or take home messages. Ask your-self, “did the study answer my research question?” If yes, in what way...\n\nWhat are the bibliometric parameters that you examined? State them under methods and include the findings for each one.\n\nThere are several redundant words in the abstract such as “In this study”, “we examined the research on sofosbuvir with a bibliometric analysis..” but you did not mention, even under methods in the manuscript, which type of research did you include in your analysis. Did you include only human or animal studies as well? Did you include only basic research or clinical research and clinical trials or both? Did you only include randomized controlled studies only or also you did include observational studies, controlled studies, reviews, and meta-analysis? Which of which did you include?\n\nIntroduction\n\nPlease note the following regarding “Second paragraph” under Introduction:\nWhat do you mean by “most important DAAs”? You may omit this We need to know why did you chose Sofosbuvir and not other drugs in this group What are the advantages of Sofosbuvir over other drugs in this group and add references. Not clear about the sentence “high prices have led to a large increase in spending by health systems and can be a barrier to global treatment…” how this is related to the bibliometric analysis you are aiming at? Focus on the study purpose. State the rationale of the study State the bibliometric parameters you aim to analyze. State clearly the research question Add appropriate references for each item The statement “based on the above….” Is meaningless and should be omitted.\n\nMethods:\nState the study design. Add a reference State the date of searching the Web of Science, and who conducted such search. Did both researchers share this responsibility? It is not clear what do you mean by MeSH and non-MeSH? Did you use PubMed as well? Explain why? What do you mean by PSI 7977 and GS7977, if these are the codes for the drug before its marketing. State this and the references for this. What were the inclusion and exclusion criteria? Did you include only basic research or clinical research and clinical trials or both? Did you only include randomized controlled studies only or also you did include observational studies, controlled studies, reviews, and meta-analysis? Which of which did you include? Did you include editorials? Did you include letter to the Editor? Did you include animal studies? Did you include review studies and meta-analysis? All these details are needed. What were the bibliometric parameters that you analyzed? I cannot see any description of how did you analyse the data gathered. How did you reach to agreement to include or not to include an article? Did you calculate the degree of agreement between researchers?\n\nResults\nThis section needs a lot of work The results should mirror the subtitles under methods Provide results for each subitem in the bibliometric analysis you did. The assessment is superficial and more in depth analysis is needed. Use subtitles You may create 2-3 tables summarizing key findings/analysis. What does Figure 1 mean?\n\nDiscussion\nI cannot see a section for “the discussion”. You should add a discussion. Remind the research of your research question, briefly state your key findings Discuss the agreement and disagreement with other researchers. Discuss the meaning of your findings. Discuss the limitation of the study Discuss the significance of your findings Future directions in this area\n\nConclusion\nThis should be rewritten in light of your findings. Your statement about bibliometric analysis is misleading. I cannot see any bibliometric analysis from what I have read.\n\nReferences\nOnly 8 references. Please read the literature in this area and improve this section\n\nSupplementary data I would advice also submitting the raw data of the material collected in a table showing the author (first author and year), title of article, journal detail, type of article (research, review, meta-analysis, article, editorial etc), and name of university/country involved.\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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "28638",
"date": "07 Dec 2017",
"name": "Thiago Moreno L. Souza",
"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 from Hernández-Vásquez and Rosselli reviews the state of art on sofosbuvir and HCV. This analysis leads to the notion that the contribution of Latin American Institutions to the field is marginal, which is likely an anticipated outcome. The authors move out of the main field of interest of the manuscript, HCV, to Zika virus (ZIKV) to highlight a Latin American contribution to the improved comprehension of the pharmacology of sofosbuvir.\nAs sofosbuvir became an antiviral of major interest, not only towards HCV and ZIKV, but also for Dengue virus (DENV) (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524696/) – my suggestion is to compare the studies on the pharmacology of this drug on HCV and Flavivirus members. The study on ZIKV and sofosbuvir cited in this manuscript (https://www.nature.com/articles/srep40920) was preceded by a pre-print, which was the first contribution to open the field of sofosbuvir against flaviviruses (https://www.biorxiv.org/content/early/2016/07/06/061671; https://www.the-scientist.com/?articles.view/articleNo/46585/title/Sofosbuvir-Shows-Anti-Zika-Activity-In-Vitro/) – around six months after the ZIKV outbreak. I presume that the proposed comparison will demonstrate the installed capacity of response to novel (re)emergent viruses in Latin America.\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? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/6-1536
|
https://f1000research.com/articles/5-2881/v1
|
20 Dec 16
|
{
"type": "Research Note",
"title": "Revisiting the phylogeny of phylum Ctenophora: a molecular perspective",
"authors": [
"Luis A. Arteaga-Figueroa",
"Valentina Sánchez-Bermúdez",
"Nicolás D. Franco-Sierra",
"Luis A. Arteaga-Figueroa",
"Valentina Sánchez-Bermúdez"
],
"abstract": "The phylogenetic relationships of deep metazoans, specifically in the phylum Ctenophora, are not totally understood. Previous studies have been developed on this subject, mostly based on morphology and single gene analyses (rRNA sequences). Several loci (protein coding and ribosomal RNA) from taxa belonging to this phylum are currently available on public databases (e.g. GenBank). Here we revisit Ctenophora molecular phylogeny using public sequences and probabilistic methods (Bayesian inference and maximum likelihood). To get more reliable results multi-locus analyses were performed using 5.8S, 28S, ITS1, ITS2 and 18S, and IPNS and GFP-like proteins. Best topologies, consistent with both methods for each data set, are shown and analysed. Comparing the results of the pylogenetic reconstruction with previous research, most clades showed the same relationships as the ones found with morphology and single gene analyses, consistent with hypotheses made in previous research. There were also some unexpected relationships clustering species from different orders.",
"keywords": [
"Ctenophora",
"Phylogenetic reconstruction",
"Ribosomal subunits",
"Non-fluorescent protein (GFP-like)",
"Bayesian Inference",
"Maximum Likelihood",
"Isopenicillin-n-synthase (IPNS)"
],
"content": "Introduction\n\nThe relationships among deep metazoans (Cnidaria and Ctenophora) and Parazoa (Porifera and Placozoa) are not totally clear1. In this paper we try to reconstruct the phylogeny inside the phylum Ctenophora with state of the art methods and compare our results with previous work2,3. Ctenophores are a key phylum for the understanding of the development of organ systems, triploblastic animals and bilateral symmetry4. Our goal was to reconstruct the phylogeny of previous studies using as many sequences as possible available on GenBank. The use of these sequences allowed us to perform a multilocus analysis (MLSA), instead of the single gene analyses previously performed. The sequences selected for this study have never been used for phylogenetic analysis exclusively of this phylum5,6.\n\nOur research consists of 1) the analysis of ribosomal genes (5.8S; 28S; ITS1; ITS2 and 18S) and 2) the analysis of two ortholog genes found in ctenophores (a GFP-like non-fluorescent protein, and isopenicillin-N-synthase FYY1).\n\nThe ribosomal genes were analysed using partitioned nucleotide substitution models while the ortholog genes were analysed using partitioned amino acid substitution models. We compared the findings of our two approaches (ribosomal and ortholog genes) to each other and against the previously reported phylogenetic trees obtained from molecular data3 and morphological data2.\n\n\nMethods\n\nAll sequences corresponding to Ctenophora (Taxonomy ID: 10197) were retrieved from GenBank’s nucleotide database. Short sequences (those shorter than 150 base pairs) or ambiguously labeled sequences (those not assigned to a specific species) were discarded. This criterion was used to obtain an almost complete matrix including most loci available of reported taxa across the phylum.\n\nSeven loci were chosen for analysis; five corresponded to ribosomal RNA regions (5.8S; 28S; ITS1; ITS2 and 18S). The other two corresponded to ortholog genes: a putative non-fluorescent protein (GFP-like protein), and isopenicillin-N-synthase FYY1 (IPNS), a protein involved in the bioluminescence process. The sequences were extracted using the annotation of the retrieved records using Biopython 1.677.\n\nThe taxa present for each analysis is listed in Table 1 and Table 2. All sequences used (with corresponding accession numbers) and scripts used for analysis are available at http://doi.org/10.5281/zenodo.19308016.\n\n(1) Sequence was available on GenBank. (-) sequence was not available on GenBank or not reported.\n\n(1) Sequence was available on GenBank. (-) sequence was not available on GenBank or not reported.\n\nGiven the phylogenetic distance between the different taxa of this phylum, for the protein coding genes, we decided to work at the amino acid sequence level due to high sequence saturation at the nucleotide level.\n\nThe sequences corresponding to the ortholog genes were translated in silico using DNA2PEP 1.18 with standard genetic code, and aligned using MAFFT 7.2229. A MLSA was performed using these two loci. Alignments were concatenated using Python scripts and partitioned by gene to be analyzed for amino acid model and best partition scheme using PartitionFinderProtein 1.1.110 Model adjustment was assessed using Bayesian information criterion (BIC). The best model found by PartitionFinderProtein 1.1.1 for IPNS partition was LG + G + I, and LG + G had better adjustment for GFP-Like partition. Phylogenetic reconstruction for the ortholog genes was carried out by maximum likelihood (ML) and Bayesian inference (BI) methods, using both Garli 2.0111 and MrBayes 3.2.612, with the proper amino acid substitution model parameters for each partition.\n\nFor the ML analysis, using Garli, a total of 5 independent ML searches were performed and supported with 65 bootstrap pseudoreplicates. For BI analysis, using MrBayes, two independent MCMC runs (four chains for each) were carried out for 1.000.000 generations, using a relative burn-in discard of 35% of total sampled trees (sampling frequency of 100 generations).\n\nFor the five rRNA loci, the automated pipeline PhyPipe13 (available at: https://gitlab.com/cibiop/phypipe/) was used for phylogenetic reconstruction by BI and ML methods. A regular PhyPipe run comprises DNA sequence alignment with MAFFT 7.222, partition analysis with PartitionFinder, and phylogenetic reconstruction with RAxML 8.2.814, MrBayes 3.2.612 or Garli 2.0111. For this analysis, MrBayes was executed under the following parameters: two independent MCMC runs, four chains, 1.000.000 generations, 35% of relative burn-in and sampling frequency of 100. For ML analysis, Garli was executed doing first a ML search (5 independent searches), then 1000 bootstrap pseudoreplicates were performed and mapped to the best ML topology using SumTrees from DendroPy 4.1.0 package15.\n\n\nResults\n\nThe majority of the phylum analysed in this study show a standard grouping condition; the organisms that are related in one of the analyses are also related in the other. This is more evident comparing at family level, where the individuals of the same family, and in some cases order, grouped with other organisms of the same order. Exceptions are discussed below.\n\nIn the reported trees there are families represented by several species while complete orders are represented by just one species. For the purpose of clarity, from now on the families represented by several species will be discussed at the family level while the orders represented by just one species will be discussed using the representing species.\n\nThalassocalyce inconstans and Lampocteis cruentiventer are unexpectedly grouped together in both analyses, but the position is not the same in comparison with the other clades. The support values in the ribosomal tree are very low compared to the ortholog genes tree.\n\nThe order Cydippida is divided in five different clades or subgroups, shown in different tones of blue in Figure 1. We confirm that this group is paraphyletic as reported previously in 2,3. The species Bathyctena chuni (Bathyctenidae, Cydippida) is grouped with Ocyropsis maculata (Ocyropsidae, Lobata) in amino acid analysis, but there is no information on the ribosomal sequences of Bathyctena chuni, so it was not possible to compare.\n\nTrees were constructed using Bayesian inference and maximum likelihood methods and consistent topologies were found within methods. Support values are shown at nodes in the form of posterior probability/bootstrap value.\n\nIn the amino acid tree, Pleurobrachiidae, Mertensiidae, Lampeidae, Euplokamididae form a clade; but the relationships between them are not clear, and the bootstrap values and posterior probability are low in this group. In the ribosomal analysis we could include the Platyctenida order, which grouped with high support in the clade formed by the mentioned families and order. In the ribosomal analysis, we see some shared features with one of Harbison’s trees2. Also the Thalassocalyce-Lampocteis clade is related to this group, but the position varies depending of the analysis. Dryodora glandiformis groups with the clade formed by Lobata species, but this result is only evident in the ortholog genes tree, due to the lack of rRNA sequences for this particular taxon.\n\nAll the trees were rooted using Beroida as the outgroup, following the hypothesis that this is the most basal group. The same choice of root was made by Harbison2. Additionally, the Beroe genus is a good outgroup because it belongs to the class Nuda while the other studied species belong to the class Tentaculata (our ingroup).\n\nBathocyroe fosteri is present in an unexpected position in the tree. It should have been included in the Lobata clade. Instead, it was placed outside the subgroup containing the Lobata, Pleurobrachiidae, Mertensiidae, Lampeidae and Thalassocalycidae families. This finding is not compared with rDNA loci analysis since ribosomal sequences for Bathocyroe fosteri were not available.\n\nIn the research performed by Harbison2, the Lobata group was placed below the Cestida group. Later, this finding was not discussed by Podar et al. 20013 as in the ribosomal data they used both groups are in a polytomy. Our finding, using rDNA, is in concordance with the findings of Podar et al. 20013, but using the ortholog genes the finding is contrary to what was proposed by Harbison. We found, on the ortholog genes analysis, that Cestida group is the one derived from Lobata and not vice versa as suggested by Harbison. This finding has a high bootstrap value and posterior probability support.\n\n\nDiscussion\n\nThe Haeckelidae family preserves its position in the phylogenetic trees placed as sister group of all the other Tentaculata taxa analysed, with high support, according to previous studies2.\n\nThe lack of reported DNA sequences of few groups, like the orders Crytolobiferida, Cambodjiida, Ganeshida; several families, like Eurhamplaeidae and some records reported as Ctenophora incertae sedis (Tentaculata incertae sedis), make it harder to have an entire vision of the phylogenetic relationships inside the phylum. The order Ganeshida is grouped with Lobata, according to Harbison2 and the lack of this group may have caused a misplacement of the Thalassocalycida representant.\n\nTo improve the results, Coeloplanidae should be included in the protein phylogenetic analysis. Further studies including more sequences from families such as Leucotheidae, Lampoctenidae and Thalassocalycidae are also needed to solve the resulting polytomies and to obtain better support to confirm the relationship between Thalassocalyce inconstans and Lampoectis cruentiventer in the rRNA analysis.\n\n\nData availability\n\nThe raw data and scripts used for this project are available in Zenodo, DOI 10.5281/zenodo.19308016.",
"appendix": "Author contributions\n\n\n\nLAAF conceived the study, performed the sequence compilation and literature revision. LAAF, VSB and NDFS carried out the phylogenetic reconstructions and analysed the results. All authors were involved in writing the manuscript and have agreed to its final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe specially thank Sergio Pulido-Tamayo for stimulating discussions and critical review of the manuscript, Juan F. Díaz-Nieto, Javier C. Alvarez and Diana Rincón T. for their guidance and valuable comments. We also want to thank Lizette I. Quan-Young and Steve Haddock for providing useful bibliography and sequences for this analysis, respectively.\n\n\nReferences\n\nLynch M: The Age and Relationships of the Major Animal Phyla. Evolution. 1999; 53(2): 319–325. Publisher Full Text\n\nHarbison GR: On the classification and evolution of ctenophora. In: The origin and relationship of lower invertebrates. S Conway Morris, JD George, R Gilson, and HM Platt, ed. 1985.\n\nPodar M, Haddock SH, Sogin ML, et al.: A molecular phylogenetic framework for the phylum Ctenophora using 18S rRNA genes. Mol Phylogenet Evol. 2001; 21(2): 218–230. PubMed Abstract | Publisher Full Text\n\nRyan JF, Pang K, Schnitzler CE, et al.: The genome of the ctenophore Mnemiopsis leidyi and its implications for cell type evolution. Science. 2013; 342(6164): 1242592. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrancis WR, Christianson LM, Powers ML, et al.: Non-excitable fluorescent protein orthologs found in ctenophores. BMC Evol Biol. 2016; 16(1): 167. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrancis WR, Shaner NC, Christianson LM, et al.: Occurrence of Isopenicillin-N-Synthase Homologs in Bioluminescent Ctenophores and Implications for Coelenterazine Biosynthesis. PLoS One. 2015; 10(6): e0128742. 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\nWernersson R, Pedersen AG: RevTrans: Multiple alignment of coding DNA from aligned amino acid sequences. Nucleic Acids Res. 2003; 31(13): 3537–3539. PubMed Abstract | Publisher Full Text | Free 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): 772–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLanfear R, Calcott B, Kainer D, et al.: Selecting optimal partitioning schemes for phylogenomic datasets. BMC Evol Biol. 2014; 14(1): 82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBazinet AL, Zwickl DJ, Cummings MP: A gateway for phylogenetic analysis powered by grid computing featuring GARLI 2.0. Syst Biol. 2014; 63(5): 812–818. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRonquist F, Teslenko M, van der Mark P, et al.: MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012; 61(3): 539–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFranco-Sierra ND, Gómez-Zuluaga M, Díaz-Nieto JF, et al.: PhyPipe: an automated pipeline for phylogenetic reconstruction from multilocus sequences [v1; not peer reviewed]. F1000Res. 2016; 5(ISCB Com): 1609 (poster). Publisher Full Text\n\nStamatakis A: RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9): 1312–1313. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSukumaran J, Holder MT: DendroPy: a Python library for phylogenetic computing. Bioinformatics. 2010; 26(12): 1569–1571. PubMed Abstract | Publisher Full Text\n\nArteaga-Figueroa LA, Sánchez-Bermúdez V, Franco-Sierra ND: Revisiting the phylogeny of phylum Ctenophora: a molecular perspective. Zenodo. 2016. Data Source"
}
|
[
{
"id": "19979",
"date": "08 Feb 2017",
"name": "Martin Dohrmann",
"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\n\n1st paragraph:\n\n- \"deep metazoans\" is an odd term; also \"Parazoa\" is no longer accepted as a valid name. All 4 taxa are clearly metazoans; they are best summarized as \"non-bilaterian metazoans\".\n\n- The citation after the 1st sentence is from 1999; this should be replaced with something more recent as a lot of research in this area has been done since then.\n\n- While the statement of the 1st sentence is true, reconstructing the phylogenetic relationships within Ctenophora does not help much to solve these issues, i.e. finding the position of Ctenophora in the animal tree of life is a separate issue that this study is unable to address.\n\n- The \"previous work\" cited in the 2nd sentence is very old. There is a study from 2015 (Simion et al., Zoology 118: 102-114) that also reconstructed internal relationships of Ctenophora based on multigene analyses. It is crucial that the authors interpret their results in light of that study. It is actually quite puzzling that the paper is not cited, especially because the authors used sequences originally reported in Simion et al. (2015).\n\n- The statement in the following sentence is highly debatable. As long as the phylogenetic position of ctenophores is not resolved (see e.g. Dohrmann & Wörheide 2013 Integr. Comp. Biol. 53: 503-511; Pisani et al. 2015 PNAS 112: 15402-15407), it is totally unclear how relevant they are to answering these questions.\n\n- In the next sentence, \"previous studies\" should be replaced with \"ctenophores\".\n\n- The following 2 sentences suggest that this paper represents the first multilocus analysis addressing internal phylogeny of Ctenophora. As mentioned above, this is not true. In this study, the authors used 2 protein-coding genes and the 28S gene in addition to the 18S and ITS/5.8S markers already used by Simion et al. (2015). This is what sets their study apart from the previous paper, and this has to be clearly communicated. The paper should focus on discussing differences to the results of Simion et al. in light of expanding the set of markers (but also addressing the different taxon sampling in the 2 studies).\n\n- The abbreviation \"MLSA\" is introduced for \"multilocus analysis\" – what does the \"S\" stand for? Maybe it should read \"multilocus sequence analysis\"?\n\n2nd paragraph:\n\n- \"ribosomal genes\" should read \"ribosomal RNA genes\" (also elsewhere in the MS), since there are also genes coding for ribosomal proteins.\n\n- \"ortholog\" should be replaced with \"protein-coding\" (also elsewhere in the MS), since ribosomal RNA genes are also orthologs.\n\n3rd paragraph:\n\n- I think the taxonomic overlap between the protein-coding and the ribosomal RNA datasets is sufficient to conduct a combined analysis, to infer a tree based on all the evidence simultaneously. As far as I recall, using mixed nucleotide and amino-acid data is possible with RAxML and MrBayes; alternatively, the protein-coding partition could be analyzed on nucleotide level (possibly excluding 3rd codon positions if they are oversaturated).\n\nMethods\n\n- It is unclear how ambiguously alignable regions were treated. These have to be excluded prior to analysis, but a quick glance at the concatenated matrices provided in the data supplement (concat_matrix and concat_prot_corrected) suggests otherwise. Difficult-to-align regions can bias phylogenetic inference, so this is an important point to address.\n\n- Information about the lengths of loci and concatenated alignments should be given.\n- Information about how the trees were rooted should be given in this section. In the Results section it is mentioned that Beroida was used as the outgroup to all other ctenophores. However, this is poorly justified. For example, Simion et al. (2015) found this group deeply nested within ctenophores. In general, I suggest following closely the methodological protocol of Simion et al. to make the 2 studies truly comparable.\n\n3rd paragraph:\n\n- Replace \"is\" with \"are\"\n\n- The accession numbers are buried in some text files in the data supplement, which is quite inconvenient for the reader. I suggest providing them directly in Tables 1 and 2.\n\n7th paragraph:\n\n- Information about the substitution models used has to be provided here. I highly recommend using partitioning by gene and incorporating secondary structure information, for full comparability with Simion et al. (secondary structure models are available both in RAxML and MrBayes).\n\nResults, Discussion\n\n- These sections have to be rewritten after reanalysis of the data and comparison with Simion et al. (2015).",
"responses": [
{
"c_id": "2487",
"date": "13 Feb 2017",
"name": "Nicolás D. Franco-Sierra",
"role": "Author Response",
"response": "Thanks for your comments and suggestions on our work, they are really helpful to improve our analysis. We agree with the observations you pointed out above and we are currently working on a revised version of our manuscript. We sincerely apologize for not including the respective comparison with the work performed by Simion et al (2015). We are working on the readjustments in order to make our analysis fully comparable with Simion et al (2015)."
}
]
},
{
"id": "20040",
"date": "13 Feb 2017",
"name": "Steven H.D. Haddock",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article performs a meta-analysis of molecular phylogenetics within the Ctenophores using published sequences. I have previously had amiable correspondence with the authors and sent them data. Although the protein-coding sequences and some of the ribosomal RNA data came from my lab, I nonetheless feel I can give an unbiased assessment of their subsequent use.\nRegrettably, I do not see enough original intellectual contribution or additional scientific value to justify its publication. At best, it is a minor contribution, in which case the interpretation needs to be improved, and at worst, a good portion of it is a re-publishing of work already published by other authors (Simion [1] and Podar [4] in particular).\nA large part of the analysis is building trees using previously published ribosomal RNA datasets. This recapitulation does not add anything to the discussion of ctenophore internal relationships, and in fact, by rooting the tree with Beroe, they obscure the true evolution of the group as shown repeatedly since at least 2001 [4]. They also fail to cite Simion, et al. [1], which is the source of some of the data. Merely adding that citation would not solve the fundamental issue, which is that there is no added value to their re-building of the same phylogeny.\nThe \"novel\" aspect of the paper is building trees based on two protein-coding genes which were also published previously in two separate papers [2,3]. The trees based on their [our] protein datasets do not give any additional insights into ctenophore relationships except in that some species are present in those trees that are not represented in the 18S phylogeny. This taxonomic coverage does not reveal any particular insight. These data also already appeared in trees (albeit not limited to ctenophores only) in the original publication.\n\nThere is some confusion because there is no Hormiphora IPNS gene in their [our] dataset, yet it is listed in the table of genes and that species is present in the tree, apparently based on a GFP-like gene that was found. Furthermore, the IPNS genes are not single-copy[2], so are not reliable for phylogeny building.\nThere is a misspelling of Bathyctena in Table 1 and 2 and of Lampocteis in Table 2.\nIn summary, two gene trees, of which one gene which was found to be absent in a ctenophore lineage, does not seem to be sufficient basis for a paper. The title itself is a vast overstatement of the content of this study.",
"responses": []
},
{
"id": "20287",
"date": "28 Feb 2017",
"name": "Kevin M. Kocot",
"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 study is a metaanalysis of available ctenophore sequence data. My thoughts largely echo those of the previous two reviewers.\nThe methods seem reasonable (with the exception of the methodological problems with analysis of the Homiphora IPNS gene raised by Steve Haddock) but rooting with Beroida as the outgroup is inappropriate as no molecular studies have supported this in the past, key references are absent, and the English of the manuscript needs significant improvement.\nAvailable ctenophore transcriptome data could be used to expand sampling of the protein-coding genes. If that were done, a concatenated analysis of all of the markers used with only taxa sampled for 18S (so all taxa overlap for at least part of the alignment) with the addition of appropriate outgroups would be an interesting improvement.",
"responses": []
},
{
"id": "19434",
"date": "03 Apr 2017",
"name": "D Timothy J. Littlewood",
"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\nUnfortunately I must concur with the major concerns highlighted by other reviewers.\nThe data set is essentially a reassessment (meta-analysis) of previously published data. The phylogeny is functionally rooted against an in-group taxon without explanation. The analyses could have been improved by combining amino acid and nucleotide data (rather than solely treating these data separately). Due reference to similar articles from which these data have been derived was omitted. Other key references are missing. There is confusion within the article over the utility of some multi-copy genes and so interpretation and veracity of results is compromised.\nIn combination and considering the lack of sufficient novelty of data, approach or interpretation the publication falls short of achieving its goal. The phylogeny is revisited and with some investment of time from the authors, but little additional clarity and few insights are forthcoming to merit acceptance in its current state.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-2881
|
https://f1000research.com/articles/6-1527/v1
|
21 Aug 17
|
{
"type": "Research Article",
"title": "Cutaneous vasculitis in children: A nationwide epidemiological study in Spain",
"authors": [
"Leyre Riancho-Zarrabeitia",
"Ana Santurtún",
"Ana Santurtún"
],
"abstract": "Background: Cutaneous vasculitis (CV) are a complex group of conditions in children, of which IgA vasculitis (IgAV) is the most common. The objectives of the current study are to describe the incidence of CV in Spain and to analyze the temporal trend in the last 11 years, as well as it seasonal distribution. Methods: Hospital discharges of patients aged 0-18 years with a diagnosis consistent with CV in Spain from 2005 to 2015 were collected from the Spanish National Institute of Statistics (INE) databases.\n\nResults: A total of 7304 patients from January 2005 to December 2015 were included; 6991 patients (95%) had a diagnosis of IgAV. The yearly incidence in the whole group was 7.7 per 100,000. Mean age at diagnoses was 6±3 years and 52% were male. The highest rate of admissions was found in the 5-9 year-old group, followed by those with 0-4 years of age (15.7 and 9.0 admissions per 100.000, respectively). Admissions due to CV followed an annual cyclic pattern, with the highest number of daily admissions during fall and winter months and the lowest number in summer months. There was an overall downwards trend of the number of hospital admissions during the period of study, in both males and females (p=0.01). Conclusions: We have estimated an incidence of a 7.7 cases per 100,000 CV in children in Spain. CV-related hospitalization rates have a marked seasonal pattern, with a peak in fall and winter and a nadir in summer months. Children between 5 and 9 years of age are most frequently affected. There is a decreasing trend in CV-related hospitalization, the causes of which should be further assessed.",
"keywords": [
"Cutaneous vasculitis",
"IgA vasculitis",
"Children"
],
"content": "Introduction\n\nCutaneous vasculitis (CV) are a complex group of conditions in children. The most common are IgA vasculitis (IgAV) (formerly known as Schonlein-Henoch purpura, (SHP)), which represents more than half of the cases, followed by cutaneous small-vessel vasculitis (formerly known as hypersensitivity vasculitis). Other disorders, such as urticarial vasculitis or ANCA associated vasculitis are poorly represented in children1. The global incidence is not known2, while incidence of IgAV in children range from 3–26.7 per 100,0003. Symptoms vary from a cutaneous-limited disorder to a systemic disease, and the etiology is not fully understood. However in many cases, particularly in IgAV, an external trigger is frequently suspected; IgAV in children has been frequently associated with a preceding upper respiratory infection, but no specific pathogen has been identified. It has also been linked to antibiotics and other medications3. The reported seasonal pattern, with a fall-winter incidence peak, is consistent with the hypothesis of an infectious trigger3.\n\nThe aims of our study are to describe the incidence of CV in Spain and to analyze the temporal trend of CV in the last 11 years, as well as it seasonal distribution.\n\n\nMethods\n\nHospital discharges with a diagnosis consistent with CV (International Classification of Diseases ICD-9 codes hypersensitivity angiitis (446.2) and allergic purpura, including SHP (287.0)) in Spain from January 2005 to December 2015 were collected from the Spanish National Institute of Statistics (INE) databases.\n\nWe calculated the overall average incidence of admission per 100,000 inhabitants during the 11 years in children (from 0 to 18 years). Moreover, we calculated the annual rate of admission in children, and for the temporary trend calculations, a Kendall’s tau correlation coefficient. Monthly admission rates were compared with Krustal-Wallis test. Statistical analysis were performed with R v2.3.\n\n\nResults\n\nA total of 7304 patients from 0 to 18 years of age were discharged from January 2005 to December 2015 with a diagnosis of CV. 6991 patients (95%) had a diagnosis of IgAV and 313 had hypersensitivity angiitis. The yearly incidence in the whole group was 7.7 per 100,000. Mean age at diagnosis was 6±3 years and 52% were male, with a male to female ratio of 1.02:1. The highest rate of admissions was found in the 5-9 year-old group, followed by those aged 0-4 years (15.7 and 9.0 admissions per 100.000, respectively) (Figure 1).\n\nThe highest incidence occurred in children 5–9 years of age.\n\nAdmissions due to CV followed an annual cyclic pattern (Figure 2), with the highest number of daily admissions during fall and winter months and the lowest number in summer months. This pattern was consistent over the 11 years of study, with a 3-fold increase in the number of daily admissions in October compared with August (p<<0.001; Figure 3).\n\nA cyclic pattern was revealed, with a peak during fall and winters months and a nadir in summer.\n\nThe combined analysis confirms the seasonal pattern throughout the period of study.\n\nThe annual analysis showed a downwards trend of the number of hospital admissions during the 11-year period of study, in both men and women (p=0.01; Figure 4).\n\nA downwards trend in the overall incidence during the period of study was observed.\n\n\nDiscussion\n\nThis is the first population-based study of CV among children in Spain. We report the incidence rate of admissions of children with CV, defined as IgAV and hypersensitivity angiitis, over 11 years.\n\nWe estimate a yearly incidence of 7.7 cases per 100.000. Data on incidences rate on CV are scarce, while previous series on IgAV have reported incidences that range from 6.1 in the Dutch population4 to 20.4 in the United Kingdom5. Most published series report incidences between 10 and 20 cases per 100.000, with some discrepancies probably due to the heterogeneity of the criteria used and also by the source of identification of cases (those based exclusively in hospital discharge data fail to identify children not referred to the hospital). The incidence we report in Spanish children keeps in line with previous literature, being in the lower part of the range. Our estimates are based on hospitalized cases, which might somewhat underestimate true incidence. However, we feel our estimated incidence should be close to the true incidence, as most cases are attended to at a hospital, at least in western countries. This idea is supported by a US study reporting that only 10% of children with IgAV were reported exclusively by primary care physicians6 and by a UK study showing that only 3% of IgAV cases were reported by general practitioners5.\n\nIgAV mainly affects children between 3 and 12 years of age3, with a mean age of 5–6 years in most paediatric series6–8. A slightly male predominance has been reported, with a male to female ratio of up to 1.8:15–7, while others reported that cases were equally distributed8, or even a subtle female predominance9. In our case, we found a mean age of 6 years with no differences in sex distribution.\n\nWe found a remarkable seasonal variation in the frequency of CV. This is in line with other studies showing that IgAV has a seasonal distribution, with a peak during fall and winter and a nadir during summer months3,10. This keeps in line with a commonly reported upper respiratory infection preceding the onset of the purpura, and a possible infectious trigger for the disease. Moreover, this increase during fall-winter time could also be related with atmospheric circulation patterns, as recently suggested for Kawasaki disease11.\n\nOur annual analysis showed a downwards trend of the number of hospital admissions during the period of study. A similar trend has been reported previously. Okubo et al.6 found a significant decreasing trend, with a total annual hospitalization rate of 2.45 per 100,000 children in 2003, falling to 1.89 per 100,000 children in 2012. This decrease could indicate a tendency to treat patients with IgAV in outpatient clinics, but also could reflect a real decrease in the incidence of the disease.\n\nIn summary, we have estimated an incidence of a 7.7 cases per 100,000 CV in children in Spain. CV-related hospitalization rates have a marked seasonal pattern, with a peak in fall and winter and a nadir in summer months. Children between 5 to 9 years of age are most frequently affected. There is a decreasing trend in CV-related hospitalization, the cause of which should be further assessed.\n\n\nData availability\n\nData were downloaded freely from the Spanish National Institute of Statistics (INE) databases: http://www.ine.es/prodyser/microdatos.htm.",
"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\nJohnson EF, Wetter DA, Lehman JS, et al.: Leukocytoclastic vasculitis in children: clinical characteristics, subtypes, causes and direct immunofluorescence findings of 56 biopsy-confirmed cases. J Eur Acad Dermatol Venereol. 2017; 31(3): 544–549. PubMed Abstract | Publisher Full Text\n\nTing TV: Diagnosis and management of cutaneous vasculitis in children. Pediatr Clin North Am. 2014; 61(2): 321–346. PubMed Abstract | Publisher Full Text\n\nPiram M, Mahr A: Epidemiology of immunoglobulin A vasculitis (Henoch-Schönlein): current state of knowledge. Curr Opin Rheumatol. 2013; 25(2): 171–178. PubMed Abstract | Publisher Full Text\n\nAalberse J, Dolman K, Ramnath G, et al.: Henoch Schönlein purpura in children: an epidemiological study among Dutch paediatricians on incidence and diagnostic criteria. Ann Rheum Dis. 2007; 66(12): 1648–1650. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGardner-Medwin JM, Dolezalova P, Cummins C, et al.: Incidence of Henoch-Schönlein purpura, Kawasaki disease, and rare vasculitides in children of different ethnic origins. Lancet. 2002; 360(9341): 1197–1202. PubMed Abstract | Publisher Full Text\n\nOkubo Y, Nochioka K, Sakakibara H, et al.: Nationwide epidemiological survey of childhood IgA vasculitis associated hospitalization in the USA. Clin Rheumatol. 2016; 35(11): 2749–2756. PubMed Abstract | Publisher Full Text\n\nTrapani S, Micheli A, Grisolia F, et al.: Henoch Schönlein purpura in childhood: epidemiological and clinical analysis of 150 cases over a 5-year period and review of literature. Semin Arthritis Rheum. 2005; 35(3): 143–153. PubMed Abstract | Publisher Full Text\n\nPiram M, Maldini C, Biscardi S, et al.: Incidence of IgA vasculitis in children estimated by four-source capture-recapture analysis: a population-based study. Rheumatology (Oxford). 2017; 56(8): 1358–1366. PubMed Abstract | Publisher Full Text\n\nGarcía-Porrúa C, Calviño MC, Llorca J, et al.: Henoch-Schönlein purpura in children and adults: clinical differences in a defined population. Semin Arthritis Rheum. 2002; 32(3): 149–156. PubMed Abstract | Publisher Full Text\n\nPenny K, Fleming M, Kazmierczak D, et al.: An epidemiological study of Henoch-Schönlein purpura. Paediatr Nurs. 2010; 22(10): 30–35. PubMed Abstract | Publisher Full Text\n\nRodó X, Curcoll R, Robinson M, et al.: Tropospheric winds from northeastern China carry the etiologic agent of Kawasaki disease from its source to Japan. Proc Natl Acad Sci U S A. 2014; 111(22): 7952–7957. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "25207",
"date": "19 Sep 2017",
"name": "Javier del Pino-Montes",
"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 study about the epidemiological data of cutaneous vasculitis in children in Spain. There is little information on this topic. It is common to find data from hospitals or registries but the value of this paper is that the data comes from all over Spain. It would be interesting to know if there are geographical differences,\nMinor revision:\nIn the results section and Figure 4, children are analyzed by gender as men and women. Being a pediatric population, it is more consistent to use males and females\nI recommend to accept the paper after minor revisions\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": "35432",
"date": "20 Jul 2018",
"name": "Robert Micheletti",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is an interesting epidemiologic study of the incidence of cutaneous vasculitis in children. In particular, the observation of seasonality of the condition, as well as the estimation of the overall incidence and the declining incidence over time, are informative.\nI do have some questions / criticisms that could perhaps be addressed in the discussion / limitations to make this a stronger paper:\nOne limitation is the diagnosis codes used (hypersensitivity angiitis and Henoch-Schonlein purpura). Are these the only codes? The best codes? It seems worth mentioning the limitations of identifying cases in this manner, since not all cases of interest may be identified. If you are not accurately including other types of cutaneous vasculitis with the \"hypersensitivity angiitis\" code, perhaps this is just a study of IgA vasculitis alone and should be described in that manner? The authors argue that their estimate of hospitalized patients is representative of the true incidence of disease based on studies that primary care and general practitioners only handle a subsegment of cases. The inference is that most patients, therefore, end up at the hospital. I don't find this argument particularly persuasive? What about outpatient dermatology? Rheumatology? It is rare for me to manage IgA vasculitis in the hospital, whereas I have many outpatients with the disease. This limitation should be acknowledged (as the authors do), but probably I would eliminate the arguments related to most patients being hospitalized, etc. Additionally, the authors may wish to comment on the changing definition / criteria for IgA vasculitis. Since the Chapel Hill Consensus Conference nomenclature were revised in 2012 (in the middle of the years analyzed in this study), it is worth considering whether case definition affected frequency of diagnosis. Historically, children with palpable purpura were just called Henoch-Schonlein purpura without much effort to determine the presence of IgA, etc. It's possible in recent years some of these patients were classified as having other diagnoses.\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/6-1527
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https://f1000research.com/articles/6-1526/v1
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21 Aug 17
|
{
"type": "Method Article",
"title": "Identification of the ventral occipital visual field maps in the human brain",
"authors": [
"Jonathan Winawer",
"Nathan Witthoft",
"Nathan Witthoft"
],
"abstract": "The location and topography of the first three visual field maps in the human brain, V1-V3, are well agreed upon and routinely measured across most laboratories. The position of 4th visual field map, ‘hV4’, is identified with less consistency in the neuroimaging literature. Using magnetic resonance imaging data, we describe landmarks to help identify the position and borders of ‘hV4’. The data consist of anatomical images, visualized as cortical meshes to highlight the sulcal and gyral patterns, and functional data obtained from retinotopic mapping experiments, visualized as eccentricity and angle maps on the cortical surface.\n\nSeveral features of the functional and anatomical data can be found across nearly all subjects and are helpful for identifying the location and extent of the hV4 map. The medial border of hV4 is shared with the posterior, ventral portion of V3, and is marked by a retinotopic representation of the upper vertical meridian. The anterior border of hV4 is shared with the VO-1 map, and falls on a retinotopic representation of the peripheral visual field, usually coincident with the posterior transverse collateral sulcus. The ventro-lateral edge of the map typically falls on the inferior occipital gyrus, where functional MRI artifacts often obscure the retinotopic data. Finally, we demonstrate the continuity of retinotopic parameters between hV4 and its neighbors; hV4 and V3v contain iso-eccentricity lines in register, whereas hV4 and VO-1 contain iso-polar angle lines in register.\n\nTogether, the multiple constraints allow for a consistent identification of the hV4 map across most human subjects.",
"keywords": [
"visual cortex",
"functional magnetic resonance imaging",
"retinotopic map",
"visual field map",
"HV4",
"population receptive field",
"collateral sulcus",
"fusiform gyrus"
],
"content": "Introduction\n\nThe human brain contains well over a dozen visual field maps1–3. Identification of these maps has been a major success in the history of visual neuroscience. Because researchers can identify the same brain region across multiple measurements and diverse populations, scientific findings can be aggregated across studies to arrive at a better understanding of human brain function. The success of such aggregation, however, is limited by the accuracy with which a given region can be identified across individuals. There is little to no doubt about the position and borders of several maps. Primary visual cortex (V1) always lies on the Calcarine sulcus4–7, and its borders can be identified based on data from a single functional magnetic resonance imaging (fMRI) scanning session with high precision8,9. V2 and V3 can also be identified quite accurately and routinely, and in fact the retinotopy parameters of all three maps can be reasonably estimated from the anatomy alone10,11.\n\nIn contrast, the fourth visual field map has proven more difficult to characterize12, with considerably less consistency in map definition across laboratories, compared to V1–V313–21. There are several reasons. Compared to V1–V3, the fMRI signals in hV4 are less reliably driven by simple contrast patterns22, the homology with animal models is less certain23, imaging artifacts can affect some parts of the map in many subjects15, and anatomical landmarks are less frequently used in map delineation. Recent work, however, has examined the hV4 map in some detail15,20, suggesting that one can identify hV4 and its neighbors (Figure 1) with reasonably good consistency across individuals.\n\nThe locations of 6 medial and ventral visual field maps are shown on a rendering of a subject’s right hemisphere. The mesh underlay is a slightly smoothed rendering of the cortical surface. Sulci are indicated by dark gray and gyri by light gray. Adapted from 2.\n\nIn our view, successful map identification of the hV4 maps is aided by combining several sources of data. First, we consider the anatomy, especially the pattern of sulci and gyri. Second we use the angle map and eccentricity map derived from retinotopy measurements. Third, we use a metric of signal quality such as mean BOLD signal (or signal to noise ratio, or the quality of the retinotopic fit) to identify possible imperfections in the measurements that should be ignored in identifying the map. In the simplest scenarios, all constraints agree and the map is easily identifiable. In some cases, the measurements do not fully agree but we can nonetheless identify the general location of hV4 and at least one or more of the map boundaries based on the mutual constraints.\n\nThe protocol for identifying the hV4 retinotopic map is described in the following text and in a step-by-step video description (Supplementary Movie 1).\n\n\nProtocol\n\nOur procedure for identifying visual field maps involves two kinds of MRI data and several processing steps. The required data are an anatomical image of the subject’s brain (structural MRI) and functional data from a retinotopic mapping experiment. Typically, all data can be collected in less than one hour of scan time on a 3 Tesla scanner using a standard gradient echo pulse sequence for functional data and T1-weighted images for anatomical data. The analysis steps include segmenting gray and white matter to generate a cortical mesh, identifying the sulcal and gyral patterns in ventral occipito-temporal cortex, extracting the eccentricity and angle data from the retinotopic experiments, and then using these measurements to guide the marking of visual field boundaries.\n\nThere is considerable variability in the specific procedures used by different research groups for acquisition of functional and anatomical MRI data and for the derivation of retinotopy parameters from the fMRI scans. Any of the retinotopy methods in common usage is suitable; the only requirements are that one can visualize eccentricity and angle data on a cortical mesh. The methods to produce the images shown in this paper are described in more detail elsewhere20. We summarize these methods briefly and then focus on the specific steps for identifying the hV4 map and its neighbors.\n\n1.1. Anatomical data. Anatomical data are needed in order to render retinotopic data on surface representations. For subjects 1–3 depicted in the video presentation, 2–4 whole brain SPGR T1-weighted MRI images were acquired on a 3T GE scanner at 1 mm isotopic resolution using an 8-channel whole-brain coil. The multiple images for each subject were aligned and averaged. Averaging multiple anatomical images is desirable as it increases the contrast of the boundary between the grey and white matter, and therefore aids segmentation and the creation of an accurate cortical surface.\n\n1.2. Functional data. Functional data were acquired as T2*-weighted gradient echo images with either a spiral24 or rectilinear (EPI) trajectory through k-space, with 2.5 mm isotropic voxels and a TR of either 1.5 s or 2 s. (For details see ref 20.)\n\nRetinotopy was measured with stimuli designed either for travelling wave analysis25 or population receptive field modeling26,27. For traveling wave analysis, alternate scans contained contrast patterns viewed through expanding ring apertures or clockwise rotating wedges with a visual field extent of 14 degrees from fixation. (For details see ref 20.) For population receptive field modeling, stimuli were contrast patterns viewed though moving bar apertures that slowly swept across the visual field, with a maximum eccentricity of 6 degrees from fixation. (For details see refs 15,26.)\n\n3.1. Preprocessing. Several standard fMRI preprocessing steps were used prior to retinotopic analysis, including slice timing correction, motion correction, and high pass temporal filtering with a 1/20 Hz cutoff.\n\n3.2. Travelling wave analysis. Data from traveling wave scans (wedges and rings) were analyzed voxel by voxel by applying the Fourier transform to the time series. The phase at the stimulus frequency measures the polar angle or eccentricity of the stimulus that most effectively drives the BOLD response in that voxel. The coherence of the signal at the stimulus frequency indicates the goodness of fit of the response to the periodic design. Coherence is defined as the power at the stimulus frequency divided by the power across all frequencies. (For details see ref 28.)\n\n3.3. Population receptive fields. A population Receptive Field (pRF) model was solved for each voxel using methods described previously26. In brief, each pRF was defined as a 2-D isotropic Gaussian, parameterized by its center (x, y), size (sd of Gaussian), and amplitude (scale factor). The model prediction is the dot product of the pRF and the stimulus aperture, convolved with a hemodynamic response function. The model was fit through a minimization of least squared error between the predicted and observed time series using a coarse-to-fine approach. The pRF center can be converted to polar coordinates to yield an angle and eccentricity map, similar to that obtained from traveling wave analysis. For the purposes of retinotopic mapping in this paper, other pRF parameters were not used (size and amplitude).\n\nBecause the retinotopic maps in visual cortex are organized on the 2D surface, not the 3D volume, map data is best visualized on a surface representation, typically rendered as an inflated (smoothed) or un-inflated 3D surface mesh, or as a flattened (2D) surface29–31. The data used for the surface rendering is usually the border of the gray and white matter, derived from a segmentation procedure that labels anatomical voxels from the T1 image as gray or white matter, and then finds the boundary that separates the two tissues. For the data shown in this paper, we segment the gray and white matter using the Freesurfer auto-segmentation tools32 followed by manual correction using ITK-GRAY software, modified from ITK-SNAP33. Functional data were then aligned to the whole brain anatomical data and rendered on a smoothed surface using custom software (vistasoft; http://vistalab.stanford.edu/software/).\n\nWe find it helpful to identify several sulci and gyri before drawing the retinotopic maps, and follow the naming conventions used by Duvernoy34. The sulci and gyri labeled in Figure 2 can all be seen in post-mortem brains in Duvernoy‘s text (e.g., his Figure 17). Their locations are described briefly below.\n\nSeveral sulci and gyri are labeled on the ventral and medial surface of a highly smoothed right hemisphere. These sulci and gyri provide useful landmarks in the identification of the visual field maps. Calc: Calcarine sulcus; Ling: Lingual sulcus/gyrus; CoS: Collateral sulcus; Fus: Fusiform gyrus; ptCoS: Posterior transverse collateral sulcus; IOG: Inferior occipital gyrus.\n\n5.1. Calcarine sulcus. The most useful landmark is the calcarine sulcus (sometimes called the calcarine fissure), which locates primary visual cortex (V1). It is the large sulcus in medial occipital cortex separating the lingual gyrus from the cuneus. It can be found unambiguously in every subject and is always the location of V1. It runs on an anterior-posterior axis, with the anterior end marked by the parietal occipital fissure, and the posterior end approximately at the occipital pole, though there is variability in the posterior extent, with the sulcus wrapping around to the lateral surface in some subjects, and terminating on the inferior surface in others35,36.\n\n5.2. Lingual gyrus and lingual sulcus. The lingual gyrus is inferior to the calcarine sulcus and is the location of the ventral portions of V2 and V3. Its inferior boundary is the collateral sulcus. It stretches approximately from the posterior pole to the parahippocampal gyrus. The lingual gyrus contains one or more sulci together, considered the lingual sulcus.\n\n5.3. Collateral sulcus. The collateral sulcus separates the lingual and parahippocampal gyri on the one side from the fusiform gyrus on the other. Its posterior end is usually denoted by a transverse portion of the sulcus, called the posterior transverse collateral sulcus (ptCoS). The ptCoS is an important landmark because it usually marks the division between the hV4 map and the VO-1 map20.\n\n5.4. Fusiform gyrus. The fusiform gyrus is inferior to the lingual and parahippocampal gyrus, and is bounded medially by the collateral sulcus. The foveal representation of the ventral-occipital maps (VO-1/2) lies in the posterior portion of the fusiform gyrus.\n\n5.5. Inferior occipital gyrus. The inferior occipital gyrus lies posterior to the fusiform gyrus and collateral sulcus and inferior to the lingual gyrus. It is generally separated from the posterior portion of the posterior fusiform gyrus by the posterior transverse collateral sulcus. The lateral portion of the hV4 map often lies here. This gyrus is typically in close proximity to the transverse venous sinus, which can lead to fMRI artifacts that obscure this portion of the hV4 map15.\n\n6.1. V1–V3 maps. Identification of the first three visual field maps in humans was one of the first accomplishments in visual neuroscience following the advent of fMRI25,28,37. Delineation of these map boundaries is now routine, although tracing the maps through the foveal confluence can be difficult in the absence of methods developed specifically for this purpose10. In brief, the V1 map is centered on the calcarine sulcus, and its borders with V2 lie on retinotopic representations of the vertical meridian. The peripheral boundary is usually not identified with fMRI because the field of view that can be achieved during scanning is less than the field of view represented in the maps. Hence the peripheral boundary of a region of interest is usually chosen as the most anterior region of the maps where the signal is good, according to a threshold in coherence (traveling wave analysis) or variance explained (pRF analysis). If there are clear reversals in the angle map all the way back to the occipital pole, then it may be possible to trace V2 and V3 in concentric ‘V’ shapes around V1; otherwise the most foveal portion of the maps is typically labeled as a non-specific ‘foveal confluence’, and each map is traced as far into the fovea as the resolution in the angle maps allow, usually one or two degrees from fixation, such that the two arms of the ‘V’ are not connected, and labeled as the dorsal and ventral portions of the map The dorsal and ventral V2/V3 borders are marked by a representation of the horizontal meridian in the angle map.\n\n6.2. The hV4 map. The hV4 map lies on the ventral surface of the occipital lobe and borders at least two other visual field maps, the ventral part of V3 (V3v) and VO-1.\n\n6.2.1. The anterior boundary: hV4/VO-1 The boundary between hV4 and VO-1 can be identified by both anatomical and retinotopic data. The retinotopic feature that defines this border is a reversal in the eccentricity map (Figure 3). This peripheral eccentricity band dividing hV4 from VO-1 usually coincides with a particular anatomical feature, the ptCoS20.\n\nThe color overlay on the smoothed cortical mesh in the main panel shows the stimulus eccentricity that most effectively drives each cortical location. The eccentricity was derived from solving a pRF model, and the stimulus extent was limited to 6 degrees. The asterisk indicates the confluent foveal representation. Black and white lines mark the boundaries between visual field maps. The white line divides hV4 from VO-1 and is the only boundary line in this figure that is derivable from the eccentricity map. This line coincides with the ptCoS (inset) and with the eccentricity reversal (blue in color overlay). Hence the white line also divides the ventral occipital maps into two clusters, one that includes V1-hV4, and one that includes VO-1/217. Data are limited to voxels in which the pRF prediction accounted for at least 10% of the variance explained in the time series and to a posterior mask that includes the 6 labeled visual field maps: V1, V2 and V3 ventral, hV4, VO-1, and VO-2. The inset is the same cortical mesh with labels indicating the major sulcal and gyral patterns, as in Figure 2.\n\n6.2.2. The medial boundary: hV4/V3v One of the borders of hV4 is shared with the ventral portion of V3 (V3v). This border is defined by an upper vertical meridian polar angle reversal (Figure 4). Because the V1–V3 maps are typically well defined by the retinotopic data, it is usually also clear where this boundary is found.\n\nThe color overlay in the main panel indicates the angle in the visual field that most effectively drives responses in each cortical location. The visual field map boundaries in white can be identified from the angle map. Otherwise as Figure 3.\n\n6.2.3. The ventral/lateral boundary: The ventral/lateral boundary of hV4 is more difficult to identify than the other boundaries because there is no unambiguous feature of the retinotopic data to define it. As described in 6.2.1 and 6.2.2, we know what is on the other side of two of the hV4 boundaries, making those boundaries well defined: The anterior boundary is defined by an eccentricity reversal and is shared with VO-1, and the medial boundary is defined by a polar angle reversal and is shared with V3v. In contrast, there is not a well-established map or retinotopic feature abutting the ventral side of hV4. Furthermore, there is often signal dropout in the fMRI measure on the ventral / lateral aspect of the hV4 map due to the transverse venous sinus, which typically runs near the inferior occipital gyrus15. In some cases, the signal dropout defines the most ventral/lateral extent of the map that can be identified (Figure 5 and Figure 6).\n\nThe color overlay in the main panel indicates variance in the voxel time series explained by the pRF model predictions. Otherwise as Figure 3. Some locations with low variance explained are likely due to fMRI artifacts, such as the region indicated by the white line, where the low variance explained is caused by dropout from the transverse sinus. Other regions with low variance explained may have visual field representations outside the stimulus extent, such as the anterior edge of V1, V2, and V3.\n\nThe color overlay in the main panel indicates the mean fMRI signal. The signal is not uniform across cortex. Certain regions have low mean signal due scanning artifacts. The region indicated by the white line lies near the transverse venous sinus which causes signal dropout. Retinotopic data from these locations must be interpreted with caution. Otherwise as Figure 3.\n\n6.2.4. Iso-eccentricity lines shared by hV4 and V3v: The general organization of the hV4 map and its neighbors can be understood by tracing iso-eccentricity and iso-angle lines. The hV4 eccentricity map is in register with the V1–V3 maps, so that iso-eccentricity lines span the hV4/V3v border (Figure 7). Unlike the V1–V3 maps, the hV4 map appears to contain little representation of the far periphery; this may be because the neurons in hV4 do not respond to stimuli in the periphery, or it may be because peripheral representation is highly compressed into a small amount of cortex, such that responses to more foveal stimuli have a much larger effect on the BOLD time course, masking the peripheral representation. Along the posterior-to-anterior axis, hV4 is therefore much shorter than V1–V3, and the most anterior eccentricity band in hV4 crossed V3v near the middle of the length of V3v.\n\nIso-eccentricity lines in visual cortex are continuous within clusters, such as the posterior cluster containing V1, V2, V3, and hV4, and the ventral occipital cluster containing the VO-1/2 maps. The eccentricity overlay and the map boundaries are identical to those in Figure 3.\n\n6.2.5. Iso-angle lines shared by hV4 and VO-1: The iso-angle lines in hV4 are continuous with the VO-1 maps and not the V3v map. The lines usually bend along the hV4/V0-1 border, with the lower meridian representation being the most ventral and the shortest iso-angle line (Figure 8).\n\nThe iso-angle lines in hV4 and VO-1, indicated by white lines, are continuous across the two map clusters. Otherwise as Figure 4.\n\n\nResults\n\nThe sulci and gyri associated on the ventral occipital cortical surface from a representative subject (S1) are shown in Figure 2. The most useful anatomical landmarks for locating the visual field maps are the calcarine sulcus (V1); the lingual gyrus and lingual sulcus (V2v/V3v); the posterior collateral sulcus and inferior occipital gyrus, which bound hV4; and the fusiform gyrus and collateral sulcus, where the VO maps are found.\n\nThe eccentricity map for subject S1 shows the large-scale organization of the maps (Figure 3). The key feature on the eccentricity map for identifying hV4 is the peripheral representation within the ptCoS, which marks the hV4/VO-1 boundary. The angle map (Figure 4) is used to define the V3v/hV4 boundary as well as the VO-1/VO-2 boundary.\n\nFMRI signal quality is not uniform across the cortical surface. Some locations have poor signal due to known measurement artifacts, such as those which arise in regions near large sinuses. In subject S1, the transverse venous sinus corrupts the fMRI signal on the inferior occipital gyrus, resulting in low variance explained by the pRF model (Figure 5) and signal dropout (Figure 6) in this region.\n\nIso-eccentricity and iso-angle lines in subject S1 clarify the internal structure of the hV4 map and the relationship between hV4 and its neighbors. The iso-eccentricity lines are in register across hV4 and V1–V3 (Figure 7). The iso-angle lines are continuous across the hV4/VO-1 boundary (Figure 8).\n\nAngle maps and eccentricity maps from additional subjects show similar patterns to S1. The V1–V3, hV4 and VO-1/2 maps are shown for a representative right hemisphere (S2; Figure 9; Figure 10) and left hemisphere (S3; Figure 11; Figure 12). The large scale organization is similar for all subjects. For example, there is an eccentricity reversal dividing hV4 and VO-1 and an angle reversal dividing hV4 and V3. In all subjects, the eccentricity reversal falls on or near the ptCoS. Some details differ between subjects. For example, the foveal representation in hV4 is clear in S1 and S2 but not S3 (Figure 11), likely due to corrupted signal from draining veins. A second difference across subjects is that the upper meridian angle reversal dividing VO-1 and VO-2 is clear in S1 and S3 but not S2 (Figure 10). Nonetheless, there is sufficient regularity across subjects to identify the principal features defining the hV4 map and its neighbors, V3v and VO-1 (Figure 13).\n\nAn eccentricity map is shown on the partially inflated cortical surface of subject 2’s right hemisphere. The stimulus extent was 14 degrees and the data are from traveling wave analysis of expanding rings. Otherwise as Figure 3.\n\nAn angle map is shown for subject 2’s right hemisphere. Otherwise as Figure 9 and Figure 3.\n\nAn example of a left hemisphere eccentricity map derived from pRF model fitting. Otherwise as Figure 3.\n\nAn example of a left hemisphere angle map derived from pRF model fitting. Otherwise as Figure 4.\n\nFour types of data are shown for three subjects: sulcal and gyral landmarks (column 1), eccentricity (column 2), angle (column 3), and mean fMRI signal (column 4). Comparisons of the datasets reveal regularities across subjects, such as the fact that the ptCoS is well aligned with the hV4/VO-1 boundary defined by an eccentricity reversal (white line in columns 1 and 2). There are also differences across subjects. For example the foveal representation of hV4 is less clear in S3 than in S1 and S2.\n\n\nDiscussion\n\nIdentifying visual field maps is an important component of characterizing the organization and function of visual cortex. It is important to have well-justified and reproducible methods to define the maps. In the case of the V1–V3 maps, the functional and anatomical organization is sufficiently regular and well understood that these maps can be identified using automated procedures (no human intervention)11,12. The hV4 and VO maps are not yet included in automated fitting procedures; however, recent progress suggests that these maps too have a high degree of regularity. Two boundaries are well defined by retinotopic features (sections 6.2.2, 6.2.3), and one of these also coincides with an anatomical landmark, the ptCoS. Moreover, the internal structure of the hV4 map and its neighbors are well understood, such that the iso-eccentricity and iso-angle lines derived from retinotopic mapping follow regular patterns.\n\nWhile most of the large-scale structures in the ventral occipital retinotopic maps are similar across subjects, there are also individual differences. Some of these differences likely reflect quantitative differences between subjects in the size and layout of the maps. Other differences reflect various sources of measurement noise. In no case will a measured map exactly match a template, and in some cases map boundaries will be ambiguous. We believe that the best approach offered is to simultaneously satisfy as many of the constraints from the anatomy, eccentricity, and angle maps as possible.\n\n\nData availability\n\nThe functional and anatomical data for subjects 1–3 have been de-identified and made publicly available on the Open Science Framework (http://doi.org/10.17605/OSF.IO/UYHMX38). Analysis code written in Matlab (Mathworks; Natick, MA) is also available via the same site. The analysis code reproduces the images of the cortical meshes with color overlays as shown in Figure 3–Figure 13.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors acknowledge two funding sources, National Institutes of Health (grant R00-EY022116 to JW) and (grant R01-EY023915 to Kalanit Grill Spector (supporting NW)).\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 Movie 1: Method for identifying the ventral occipital retinotopic maps.\n\nClick here to access the data.\n\n\nReferences\n\nWandell BA, Winawer J: Imaging retinotopic maps in the human brain. Vision Res. 2011; 51(7): 718–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWandell BA, Dumoulin SO, Brewer AA: Visual field maps in human cortex. Neuron. 2007; 56(2): 366–83. PubMed Abstract | Publisher Full Text\n\nWang L, Mruczek RE, Arcaro MJ, et al.: Probabilistic Maps of Visual Topography in Human Cortex. Cereb Cortex. 2015; 25(10): 3911–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHenschen SE: On the visual path and centre. Brain. 1893; 16(1–2): 170–80. Publisher Full Text\n\nInouye T: Die Sehstörungen bei Schussverletzungen der kortikalen Sehsphäre: nach Beobachtungen an Verwundeten der letzten japanischen Kriege. W. Engelmann; 1909. Reference Source\n\nHolmes G: Disturbances of Vision by Cerebral Lesions. Br J Ophthalmol. 1918; 2(7): 353–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHorton JC, Hoyt WF: The representation of the visual field in human striate cortex. A revision of the classic Holmes map. Arch Ophthalmol. 1991; 109(6): 816–24. PubMed Abstract | Publisher Full Text\n\nKirson D, Huk AC, Cormack LK: Quantifying spatial uncertainty of visual area boundaries in neuroimaging data. J Vis. 2008; 8(10): 101–5. PubMed Abstract | Publisher Full Text\n\nSchira MM, Tyler CW, Breakspear M, et al.: The foveal confluence in human visual cortex. J Neurosci. 2009; 29(28): 9050–8. PubMed Abstract | Publisher Full Text\n\nBenson NC, Butt OH, Brainard DH, et al.: Correction of distortion in flattened representations of the cortical surface allows prediction of V1-V3 functional organization from anatomy. PLoS Comput Biol. 2014; 10(3): e1003538. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBenson NC, Butt OH, Datta R, et al.: The retinotopic organization of striate cortex is well predicted by surface topology. Curr Biol. 2012; 22(21): 2081–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWinawer J, Witthoft N: Human V4 and ventral occipital retinotopic maps. Vis Neurosci. 2015; 32: E020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWade AR, Brewer AA, Rieger JW, et al.: Functional measurements of human ventral occipital cortex: retinotopy and colour. Philos Trans R Soc Lond B Biol Sci. 2002; 357(1424): 963–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHansen KA, Kay KN, Gallant JL: Topographic organization in and near human visual area V4. J Neurosci. 2007; 27(44): 11896–911. PubMed Abstract | Publisher Full Text\n\nWinawer J, Horiguchi H, Sayres RA, et al.: Mapping hV4 and ventral occipital cortex: the venous eclipse. J Vis. 2010; 10(5): 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHadjikhani N, Liu AK, Dale AM, et al.: Retinotopy and color sensitivity in human visual cortical area V8. Nat Neurosci. 1998; 1(3): 235–41. PubMed Abstract | Publisher Full Text\n\nBrewer AA, Liu J, Wade AR, et al.: Visual field maps and stimulus selectivity in human ventral occipital cortex. Nat Neurosci. 2005; 8(8): 1102–9. PubMed Abstract | Publisher Full Text\n\nTootell RB, Hadjikhani N: Where is 'dorsal V4' in human visual cortex? Retinotopic, topographic and functional evidence. Cereb Cortex. 2001; 11(4): 298–311. PubMed Abstract | Publisher Full Text\n\nLarsson J, Heeger DJ: Two retinotopic visual areas in human lateral occipital cortex. J Neurosci. 2006; 26(51): 13128–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWitthoft N, Nguyen ML, Golarai G, et al.: Where Is Human V4? Predicting the Location of hV4 and VO1 from Cortical Folding. Cereb Cortex. 2014; 24(9): 2401–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZeki S, Watson JD, Lueck CJ, et al.: A direct demonstration of functional specialization in human visual cortex. J Neurosci. 1991; 11(3): 641–9. PubMed Abstract\n\nKay KN, Naselaris T, Prenger RJ, et al.: Identifying natural images from human brain activity. Nature. 2008; 452(7185): 352–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeadows JC: Disturbed perception of colours associated with localized cerebral lesions. Brain. 1974; 97(4): 615–32. PubMed Abstract | Publisher Full Text\n\nGlover GH, Lai S: Self-navigated spiral fMRI: interleaved versus single-shot. Magn Reson Med. 1998; 39(3): 361–8. PubMed Abstract | Publisher Full Text\n\nEngel SA, Rumelhart DE, Wandell BA, et al.: fMRI of human visual cortex. Nature. 1994; 369(6481): 525. PubMed Abstract | Publisher Full Text\n\nDumoulin SO, Wandell BA: Population receptive field estimates in human visual cortex. Neuroimage. 2008; 39(2): 647–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWandell BA, Winawer J, Kay KN: Computational modeling of responses in human visual cortex. In: Toga A, editor. Brain Mapping: An Encyclopedic Reference. 2015; 1: 651–659. Publisher Full Text\n\nEngel SA, Glover GH, Wandell BA: Retinotopic organization in human visual cortex and the spatial precision of functional MRI. Cerebral Cortex. 1997; 7(2): 181–92. PubMed Abstract | Publisher Full Text\n\nTeo PC, Sapiro G, Wandell BA: Creating connected representations of cortical gray matter for functional MRI visualization. IEEE Trans Med Imaging. 1997; 16(6): 852–63. PubMed Abstract | Publisher Full Text\n\nWandell BA, Chial S, Backus BT: Visualization and measurement of the cortical surface. J Cogn Neurosci. 2000; 12(5): 739–52. PubMed Abstract | Publisher Full Text\n\nCarman GJ, Drury HA, Van Essen DC: Computational methods for reconstructing and unfolding the cerebral cortex. Cereb Cortex. 1995; 5(6): 506–17. PubMed Abstract | Publisher Full Text\n\nDale AM, Fischl B, Sereno MI: Cortical surface-based analysis. I. Segmentation and surface reconstruction. Neuroimage. 1999; 9(2): 179–94. PubMed Abstract | Publisher Full Text\n\nYushkevich PA, Piven J, Hazlett HC, et al.: User-guided 3D active contour segmentation of anatomical structures: Significantly improved efficiency and reliability. Neuroimage. 2006; 31(3): 1116–28. PubMed Abstract | Publisher Full Text\n\nDuvernoy HM: The human brain: surface, three-dimensional sectional anatomy with MRI, and blood supply. 2nd completely rev. and enl. ed. Wien; New York: Springer; 1999; 491. Publisher Full Text\n\nDumoulin SO, Hoge RD, Baker CL Jr, et al.: Automatic volumetric segmentation of human visual retinotopic cortex. Neuroimage. 2003; 18(3): 576–87. PubMed Abstract | Publisher Full Text\n\nStensaas SS, Eddington DK, Dobelle WH: The topography and variability of the primary visual cortex in man. J Neurosurg. 1974; 40(6): 747–55. PubMed Abstract | Publisher Full Text\n\nSereno MI, Dale AM, Reppas JB, et al.: Borders of multiple visual areas in humans revealed by functional magnetic resonance imaging. Science. 1995; 268(5212): 889–93. PubMed Abstract | Publisher Full Text\n\nWinawer J, Witthoft N: Identification of the Ventral Occipital Visual Field Maps in the Human Brain. Open Science Framework. 2017. Data Source"
}
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[
{
"id": "25205",
"date": "23 Aug 2017",
"name": "Ben M. Harvey",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWinawer and Witthoft present a methodological paper describing the practical details that allow researchers to localize the human homologue of V4 (hV4) using fMRI visual field mapping. As described in the Abstract and Introduction, it has become very straightforward to define earlier visual field maps (V1-V3), but hV4 presents particular difficulties that prevented researchers even agreeing hV4’s location for many years. Studies by Winawer and Witthoft themselves addressed this disagreement. Other researchers in the field have typically learned to localize hV4 after demonstrations of the practical details from experienced colleagues. In most studies, many of these practical details are omitted for brevity, and because parts have been described elsewhere. It is becoming increasingly clear that visual field map definitions beyond V1-V3 are not as consistent as they should be across labs. So, the field will benefit from publication of more standardized, practical protocols like those described here to allow consistent localization of hV4. The approach described here is methodologically very strong and clearly described for an inexperienced researcher.\n\nMajor points: The manuscript describes procedures for drawing V1-3 and hV4. However, the anterior boundary of hV4 is described in relation to VO1 (section 6.2.1), and procedures for drawing VO1 are not described. Furthermore, VO1 is fairly far anterior of hV4, so will often not be covered in a scan volume targeted at V1-3 and hV4. I don’t think it is wise to add procedures for drawing VO1-2, because these procedures rely on their relationship to hV4, making quite a circular description. Therefore, this section would benefit from rewriting without assuming that VO1 has been drawn already, i.e. based on the location of an area of maximum preferred eccentricity in an eccentricity gradient, rather than based on an eccentricity reversal to VO1.\n\nProcedures for drawing hV4 are described clearly, but assume the researcher already has good surface renderings and estimates of each voxel’s preferred visual field position. These earlier stages are extensive and complex, but the manuscript describes them only briefly and with reference to previous studies. I don’t believe an inexperienced researcher could work through these stages from the brief descriptions in the manuscript, so effectively the manuscript assumes the reader already has considerable experience. To make a more practical, step-by-step description of the complete procedure, a more complete description of normal segmentation and pRF modelling procedures would be valuable. However, I understand if the authors decide against including this, because it would change the focus of the manuscript considerably.\n\nMinor points: The discussion of differences to expect between subjects is helpful, but the resulting definition of hV4 includes the areas where draining veins introduce artefacts. The manuscript should make clear that subsequent analyses of responses in these areas will be strongly affected by these artefacts. Indeed, for many subsequent analyses these areas give a corrupted view of hV4’s responses, and I would certainly exclude these areas from most subsequent analyses. So, the manuscript should make clear that, while these voxels do lie in hV4 anatomically, it is often preferable to exclude them from our definition of hV4.\n\nIn Figure 7, the iso-eccentricity line with the highest eccentricity is misleading. In this figure, all voxels with preferred eccentricity above 6˚ are labelled in blue. The line running through this blue area therefore links voxels with different eccentricities, all above 6˚.\n\nThe parahippocampal gyrus is mentioned as a landmark in section 5.2, but not shown on Figure 2. Please add this to the figure.\n\nIn section 6.1, the following phrase is ambiguous: “it may be possible to trace V2 and V3 in concentric ‘V’ shapes around V1.” This could be taken to mean that each of the dorsal and ventral quarterfields of V2 and V3 is V-shaped (i.e. ending at a point, outside the foveal confluence), when in fact there are bands passing through the foveal confluence (Schira et al, reference 9). Please re-write this phrase to make it clear that each ‘V’ is a whole hemifield map of V2 or V3.\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": []
},
{
"id": "25204",
"date": "06 Sep 2017",
"name": "Geoffrey K. Aguirre",
"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\nWinawer and Witthoft describe a standard procedure to define human visual area hV4 within retinotopic mapping data obtained using fMRI. The definition of the early, retinotopically organized visual areas is a crucial first step in many studies of the cortical organization for vision. While areas V1-V3 are readily identified within functional MRI mapping data, area hV4 is often challenging due to reduced signal strength, imaging artifacts, and the peculiar organization of this region.\nThe paper documents and supports a supplementary video, which is primarily a presentation of the figures from the paper. This video, narrated in the even, velvet tones of the first author, serves as a very nice introduction to ventral retinotopic maps generally, and to the specific procedure of identifying the borders of area hV4. The video is a sufficiently important resource that I suggest that the authors mention it in the abstract.\nI have no concerns regarding the method or the conclusions of the paper. Instead, I have two things that I would have ideally liked to have seen discussed:\nFirst, for the purposes of this report, I think it is appropriate for the authors to assume that the reader has in hand a retinotopic map that is displayed on an inflated cortical surface. They are therefore justified in providing minimal treatment of the stimulus, MRI acquisition parameters, and initial analysis approach. I would have liked, however, some observations regarding choices one might make in these methods that impact the identification of ventral visual areas. For example, they might observe that retinotopic mapping need not extend into the far periphery to define hV4, as the region has a fairly compressed representation of the periphery. Similarly, it would be helpful to mention that different choices can be made in the content of the mapping stimulus that could enhance the responses from hV4. Finally, they might comment upon choices made in EPI vs. spin-echo imaging that might reduce the influence of the transverse venous sinus upon the hV4 functional data, or MR-V imaging techniques that are useful in the identification of this venous structure. These points could also be addressed in a brief paragraph in the discussion.\nSecond, as the anatomical landmarks are an important guide for the identification of hV4, the authors could comment upon their variability, both within the volumetric space and within a spherical registration space of cortical topology. To my understanding, it is this variability (coupled with variations in structure-function relationships and measurement limitations) that drives the need for individual definition of this region.\nFinally, there is a typo in the introduction: the word \"calcarine\" is unnecessarily capitalized.\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": "27268",
"date": "06 Nov 2017",
"name": "Antony B. Morland",
"expertise": [
"Reviewer Expertise fMRI",
"vision"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 rather like this short paper that reports on how to identify ventral visual field maps. Having had to attempt to do this on many occasions, sometimes with limited success, I find the paper very useful in giving evidence based guidance that could really help. The video and figures that support the paper are excellent and offer a resource for early career researchers to build up confidence in what can be a challenging task.\nOne suggestion I have that might broaden the appeal of the study is to reflect a bit more on the relationship between the layout found in human and that in macaque. V4 is split between dorsal and ventral representations of quadrants, whereas the human data are often consistent with the forth (ventral) map being a representation of a hemifield. The authors touch on this early on, but having presented their data, they may reflect further on this in the Discussion.\nMinor points and suggestions ‘The multiple images for each subject were aligned and averaged. Averaging multiple anatomical images is desirable as it increases the contrast of the boundary between the grey and white matter, and therefore aids segmentation and the creation of an accurate cortical surface.’\nI agree, but the authors might also point out that multiple, relatively short, acquisitions are also probably better than using longer acquisitions during which participant motion could work against benefits in contrast.\nI understood every word of the section that described the functional methods, but I would because I pretty much use the same methods. Are more details required for a naive reader?\n\nAnatomical Figure (Figure 2) I think it would be good to use colour to mark the sulci/gyri and colour the labelling text in line with the sulcal colour. That way repeats of lettering can be avoided and less ambiguity between the location of text and the anatomical feature can be achieved.\nSection on calcarine sulcus. Perhaps a bit more text on the variants observed at the pole - there is a reasonably well-documented ‘Y’ configuration too, I think.\nSection 6.1 'The peripheral boundary is usually not identified with fMRI because the field of view that can be achieved during scanning is less than the field of view represented in the maps.' I assume this refers the the size of the stimulus, not the FOV used in imaging, but some clarification is needed here.\n'and each map is traced as far into the fovea as the resolution in the angle maps allow, usually one or two degrees from fixation'\nI think representation needs to be inserted here a couple of times e.g. ‘as far into the representation of the fovea’ and ‘usually at the representation of one or two degrees from fixation’\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/6-1526
|
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